OF Master of Veterinary Science - Semantic Scholar€¦ · COLLEGE OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY ANAND AGRICULTURAL UNIVERSITY ANAND-388001 (GUJARAT) 2007 Reg. No. 04-0213-2005.

SEROLOGICAL, CULTURAL AND MOLECULAR DETECTION

OF BRUCELLA INFECTION IN BOVINES INCLUDING

QUANTIFICATION IN MILK BY REAL-TIME PCR

A

THESIS

SUBMITTED TO THE

ANAND AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE

OF

Master of VeterinaryScience

INVETERINARY MICROBIOLOGY

BY

TANMAY J. PATEL

B. V. Sc. & A. H.

DEPARTMENT OF VETERINARY MICROBIOLOGY

COLLEGE OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY

ANAND AGRICULTURAL UNIVERSITY

ANAND-388001 (GUJARAT)

2007

Reg. No. 04-0213-2005

SEROLOGICAL, CULTURAL AND MOLECULAR DETECTION OFBRUCELLA INFECTION IN BOVINES INCLUDING QUANTIFICATION

IN MILK BY REAL-TIME PCR

Student: Tanmay J. Patel Major Advisor: Dr. J. H. Purohit

Department of Veterinary Microbiology College of Veterinary Science and Animal Husbandry

Anand Agricultural University, Anand-388 001

A B S T R A C T

Brucellosis is a widespread and economically important infectious disease of

animals and humans caused by members of the genus Brucella. The transmission of the

disease is by direct or indirect contact with infective excretors. Brucella contaminated

milk presents a potential threat to the new born calves and human beings as it can spread

through ingestion and causes infectious abortion and undulant fever. The correct and

prompt diagnosis is important in controlling and eradicating the disease in animals.

The present study was under taken to detect the Brucella antibodies in serum as

well as in milk and for detection of Brucella organisms in bovine milk. The ELISA was

used for detection of Brucella antibodies in serum in conjunction with RBPT and STAT

to detect their efficacy as compared to ELISA, whereas, ELISA and MRT were employed

for detection of antibodies in milk. To detect the presence of Brucella organisms in milk,

cultural and PCR methods were used. Comparison among three genus specific primer

pairs was made to detect their efficacy to detect Brucella DNA by PCR. The real-time

was used for quantification of Brucella in milk. A comparison was also made among

antibody detection, cultural and molecular methods to detect Brucella infection in

bovines.

A total of 231 bovine serum samples were screened for presence of Brucella

antibodies. Of these, 67 (29.00%) serum samples were found positive for Brucella

antibodies by ELISA. The much higher seropositivity (38.29%) was found in cattle than

in buffaloes (26.63%). Sensitivity of RBPT and STAT were found to be of 25.37% and

61.19%, respectively, with considering ELISA as a gold standard test while specificity

was found to be of 99.39% and 98.78%, respectively.

Out of 53 bovine milk samples screened 15 (28.30%) and 08 (15.09%) were

found positive for Brucella antibodies by ELISA and MRT, respectively. Considering

ELISA as a gold standard test, the sensitivity and specificity of MRT were found to be

40.00% and 94.73%, respectively.

In comparison of serum antibody and milk antibody detection tests on total 53

serum and milk samples from common individual animals, the ELISA detected antibodies

in more number of serum samples (37.73%) than that of milk (28.30%). Considering

serum ELISA as a gold standard test, the sensitivity of RBPT, STAT, MRT and milk-

ELISA were found to be of 40.00%, 70.00%, 30.00% and 55.00%, respectively, while

specificity was found to be of 100.00%, 100.00%, 93.93% and 87.87%, respectively. The

overall agreement of 77.34% for RBPT, 88.67% for STAT, 69.81% for MRT and 75.47%

for milk-ELISA were found with the serum ELISA.

Brucella could be recovered only from four milk samples (2 each from cows and

buffaloes) of the 53 cultivated on Brucella agar medium. The isolates were identified as

Abstract…

ii

Brucella organisms by cultural, morphological and biochemical characteristricts and

further confirmed by PCR using different genus specific primer pairs.

For DNA extraction from milk the method described by Romero and Lopez-Goni

(1996) was found most suitable. Of the 53 milk samples tested by three Brucella genus

specific primer pairs, 9 were found positive by B4/B5 primer pair, 1 by JPF/JPR primer

pair and 2 by F4/R2 primer pair. The B4/B5 primer pair was found more suitable than

other two as the same resulted in highest positive numbers as well as gave all the samples

positive that were positive by other two primer pairs.

Real-time PCR assay based on intercalating dye SYBR Green I using B4/B5

primer pair was found suitable for quantifying the load of Brucella from the milk. On the

basis of melt curve analysis, a detection limit of real-time PCR assay was found

50 fg DNA or 10 CFU/ml of milk (considering 5 fg is equal to one Brucella cell) using 5

µl of template DNA. While, load of Brucella organisms in milk of bovines were ranged

from 1.128 x 104 CFU/ml to 172.800 x 104 CFU/ml of milk.

On comparison of serum antibody detection, milk antibody detection, cultural and

molecular methods for detection of Brucella infection in 53 bovines, in serum antibody

detection tests RBPT, STAT and ELISA detected 15.09%, 26.41% and 37.73% of positive

bovines in serum, respectively. Whereas, MRT and ELISA detected 15.09% and 28.30%

of positive bovines in milk, respectively. While, 7.54% of bovines were found culturally

positive. Among the PCR assays 16.98%, 1.88% and 3.77% of bovines were found

positive by B4/B5, JPF/JPR and F4/R2 primer pairs, respectively. The highest numbers of

bovines were found positive by serum ELISA whereas, cultural method detected the least

number of positive bovines. Among the four, tests detected Brucella antibodies in serum

Abstract…

iii

were resulted in highest number of positive bovines (20, 37.73%) followed by tests

detected Brucella antibodies in milk (17, 32.07%), PCR assays (09, 16.98%) and

cultural isolation (04, 7.54%). The over all agreement between these methods was found

58.49%.

Finally, the study revealed presence of Brucella antibody in serum as well as in

milk and presence of Brucella organisms in the bovine milk. Simultaneously the

seronegative bovine also revealed the presence of Brucella organisms and vice versa.

Thus under Health Control Programme to eradicate the brucellosis from animals as well

as for public health point of view proper measures must be taken at State level for

controlling brucellosis. Therefore all animals must be tested periodically for detection of

both Brucella antibody and presence of organisms.

Abstract…

iv

CERTIFICATE

This is to certify that, I have no objection for supplying copy of any

part of this thesis at a time through reprographic process, if necessary for

rendering reference service in a library or documentation centre.

Place: Anand (TANMAY J. PATEL)

Date: 18/05/2007 Research Scholar

(J. H. Purohit)

Major Advisor

Dr. J. H. Purohit Ph.D.Professor and Head (retd.)Department of Veterinary MicrobiologyCollege of Veterinary Science and Animal HusbandryAnand Agricultural UniversityAnand - 388 001.Gujarat State (India)

C E R T I F I C A T E

This is to certify that the thesis entitled “SEROLOGICAL, CULTURAL

AND MOLECULAR DETECTION OF BRUCELLA INFECTION IN BOVINES

INCLUDING QUANTIFICATION IN MILK BY REAL-TIME PCR” submitted

by Dr. TANMAYKUMAR JAYRAMBHAI PATEL (Reg. No. 04-0213-2005) in

partial fulfilment of the requirements for the award of the degree of MASTER OF

VETERINARY SCIENCE in the subject of VETERINARY MICROBIOLOGY

of the Anand Agricultural University, Anand is a record of bona fide research work

carried out by him under my guidance and supervision and the thesis has not

previously formed the basis for the award of any degree, diploma or other similar title.

Place: Anand (J. H. Purohit)Date: 18 /05/ 2007 MAJOR ADVISOR

ACKNOWLEDGEMENT

On the accomplishment of the present study, I would like to take

this opportunity to extend my deepest sense of gratitude and words of

appreciation towards those, who dedicated their today for my

tomorrow. I deem it a proud privilege and feel immense pleasure to

acknowledge all those who are directly or indirectly involved.

I consider myself fortunate and greatly privileged to have

worked under the supervision and guidance of Dr. J. H. Purohit, Ph.D.,

Professor and Head (retd.), Department of Veterinary Microbiology,

College of Veterinary Science and Animal Husbandry, A.A.U., Anand.

Words can never express the high regards and love I have towards my

guide. He treated me more like a son than as a student. The critical

advices given have helped me to overcome many tough moments. He

provided me invaluable and critical suggestions, scientific acumen

perspicacious remarks, scholarly guidance, active persuasion and

supervision, which served as a constant source of inspiration

throughout the course of my study and research work. He provided full

support even after his retirement. Without his support, I could not have

completed my work in time. I pray that he gets all the best things in

life.

I express my heart-felt gratitude to Minor Advisor Dr. B. P. Joshi,

Professor, Department of Veterinary Pathology and to the members of

Advisory Committee Dr. M. K. Jhala, Associate Professor, Department of

Veterinary Microbiology and Dr. P. H. Vataliya, Professor and Head,

Department of Animal Genetics and Breeding for their consistent and

invaluable inspirations, prolific and introspective guidance with

constructive suggestions, deliberative discussions and active

persuasion throughout the course of my study.

I also owe my sincere thanks to Dr. Ashish Roy, Professor and

Head, Department of Veterinary Microbiology, Dr. I. H. Kalyani,

Assistant Professor, Department of Veterinary

Microbiology, AAU, Anand for Indispensable suggestions during my

entire research work and ever willing help and goodwill.

Words are inadequate in the available lexicon to avouch the

excellent cooperation and suggestion given by my senior and like elder

brother Dr. Amit N. Kanani, Ph. D. His dedication to research,

meticulous planning, consecutive counsel and unreserved help served

as a beacon light throughout the course of research work. I feel

indebted for his encouragement that kept me patient in all the odds

during my sojourn in research work.

I am really falling short of words to express my gratitude to Dr.

Lata Jain who served as a source of motivation to work hard without

weary and remain pleasured even in odd circumstances and for their

unreserved help, continuous motivation and well wishes for me. I

extend sincere thanks to Dr. Niraj for his constant motivation during

my initial time of degree.

I extend sincere thanks to Dr. C. G. Joshi, Professor, Department

of Animal Biotechnology and Dr. D. N. Rank, Associate Research

Scientist, Department of Animal Genetics and Breeding for their even

willing help, edifying, criticism and conductive advice throughout the

course of study. I also owe my sincere thanks to Dr. P. G. Koringa,

Assistant Professor, Department of Animal Biotechnology, for his

constant help and support during the entire work.

I am equally grateful to Dr. J. V. Solanki, Principal, College of

Veterinary Science and Animal Husbandry, Anand for his generous

attitude in providing necessary facilities to carry out the research

work.

I am profoundly thankful to Dr. J. G. Sarvaiya, Director,

Information Technology Center, for providing me the world class

information technology services whenever needed during the entire

study period.

I will always thankful to the farm management for the collection

of samples. I also remain thankful to veterinary officers of the region

for their unreserved help for collection and processing of samples

during my research work. I also remain thankful to Assistant Director,

and staff members of ADIO, Ahmedabad, for their unreserved help for

processing of samples during my research work.

I am really falling short of words to express my gratitude to my

colleagues Drs. Bharat, Pranay, Supriya, Channabasayya, for their

unreserved help, continuous motivation and well wishes as sources of

constant inspiration. I owe my special thanks to my departmental

colleagues Drs. Mahesh, Mahendra, Ashutosh, Vandana, Gayatri,

Sanjay, Mathakiya for their support and unreserved help during the

course of study.

I am immensely thankful to Drs. Kamlesh, Umed, Rajni, Basanti,

Trupti, Ashvin, Gramsci, Chandrakant, Shadma and Mr. Mehta for their

constant help and support during the entire work.

On the way of completion, the friends who have shared the

moments of laughter and sorrow can never be forgotten. I thank my

colleagues, Drs. Nilesh, Rajesh, Jayesh, Prabhat, Mucchara, Subhash,

Pinakin, Sharma, Sajid, Paresh, Manan, Kavani, Mittul, Goandaliya,

Rahul, Girish, Amit, Shivkumar and other friends for their constant

inspiration and for whole hearted co-operation.

I am highly thankful to departmental staff, Smt. Jayaben,

Khodbhai, Mohanbhai, Surabhai and Harishbhai for their co-operation

throughout the course of study.

Finally no words of admiration can reply to all those dump

animals who have contributed their blood and milk for this study.

Last but not the least my vocabulary utterly fails in expressing

my accolade to my revered parents who brought me to this stage. I

deeply express my sincere thanks to my brother whose continuous

inspiration, encouragement and affection, boosted up my moral during

the period of study. I apologize for the faux pass of the persons who

have extended the help in a way or other and deserved such thanks.

Place: Anand

(Tanmay J. Patel)

Date: 18/05/2007

CONTENTS

CHAPTERNO.

CHAPTER PAGE NO.

I INTRODUCTION 1-4

II REVIEW OF LITERATURE 5-55

III MATERIAL AND METHODS 56-80

IV RESULTS AND DISCUSSION 81-119

V SUMMARY AND CONCLUSIONS 120-128

REFERENCES i-xix

APPENDIX I-VIII

LIST OF TABLES

Sr.No.

Table No.

TitlePageNo.

1 Table 3.1 Details of samples collected from bovines 56

2 Table 3.2 Contents of Brucella ELISA Kit 60

3 Table 3.3 List of primers 70

4 Table 3.4Quantity and concentration of various components used in PCR

70

5 Table 3.5Steps and conditions of thermal cycling for different primer pairs in PCR

71

6 Table 3.6 Contents of the QIAamp DNA Mini Kit 74

7 Table 3.7Quantity and concentration of various componentsused in real-time PCR based on SYBR Green I

79

8 Table 3.8Steps and conditions of thermal cycling for real-timePCR based on SYBR Green I

79

9 Table 4.1Serodetection of brucellosis in cattle and buffaloes byELISA

83

10 Table 4.2Serodetection of brucellosis in cattle and buffaloes by RBPT and STAT

85

Cont…Cont…

LIST OF TABLES

Sr.No.

TableNo.

TitlePageNo.

11 Table 4.3Sensitivity and specificity of RBPT and STAT bycomparing with ELISA (gold standard test) fordetection of Brucella antibodies

85

12 Table 4.4Detection of Brucella antibodies in milk of cattle and buffaloes by ELISA and MRT

87

13 Table 4.5Sensitivity and specificity of MRT by comparing withELISA (gold standard test) for detection of Brucellaantibodies

89

14 Table 4.6Comparison of serum antibody and milk antibodydetection tests

90

15 Table 4.7Sensitivity and specificity of RBPT, STAT, MRT andmilk-ELISA by comparing with serum ELISA fordetection of Brucella antibodies

92

16 Table 4.8 Isolation of Brucella from milk samples 94

17 Table 4.9 Biochemical characters of Brucella isolates 95

18 Table 4.10 Confirmation of Brucella isolates by PCR 97

19 Table 4.11Brucella detection in milk of bovines by PCR usingdifferent primer pairs

99

20 Table 4.12 Load of Brucella in bovine milk 104

Cont…Cont…

LIST OF TABLES

Sr.No.

TableNo.

TitlePageNo.

21 Table 4.13Comparison of antibody detection, cultural andmolecular methods for detection of brucellosis inbovines

107

22 Table 4.14 Summary of Table No. 4.13 109

LIST OF PLATES

Sr.No.

TableNo.

TitleAfterPageNo.

1 Plate 2.1Real-time PCR assay based on non-specific intercalation ofSYBR Green I in double-stranded DNA (ds DNA)

42

2 Plate 3.1 ELISA kit for detection of Brucella antibodies 60

3 Plate 4.1ELISA module showing positive and negative reactions for Brucella antibodies

83

4 Plate 4.2Brucella colonies in five day old culture on Brucella agarmedium

94

5 Plate 4.3 MZN staining of Brucella isolates (1000X) 94

6 Plate 4.4Agarose gel electrophoresis pattern of Brucella bcsp31 gene223 bp specific PCR product amplified with primer B4/B5

97

7 Plate 4.5Agarose gel electrophoresis pattern of Brucella omp2 gene 193bp specific PCR product amplified with primer JPF/JPR

101

8 Plate 4.6Agarose gel electrophoresis pattern of Brucella gene encodinga 16S rRNA of B. abortus 905 bp specific PCR productamplified with primer F4/R2

101

9 Plate 4.7

Agarose gel electrophoresis pattern of Brucella bcsp31 gene 223 bp specific PCR product of serially diluted template DNA amplified with primer B4/B5 in SYBR green I based real-timePCR

105

LIST OF FIGURES

Sr. No.Figure

No.Title

AfterPageNo.

1 Figure 4.1 Serodetection of brucellosis in bovines by ELISA 83

2 Figure 4.2Comparison of RBPT, STAT and ELISA for detection of Brucella antibodies in bovines

85

3 Figure 4.3Comparison of MRT and ELISA for detection of Brucella antibodies in bovine milk

87

4 Figure 4.4 Comparison of serum and milk antibody detection tests 90

5 Figure 4.5 Real-time PCR amplification/cycle graph for SYBR-490 104

6 Figure 4.6Real-time PCR melt (dissociation) curve graph for SYBR-490

104

7 Figure 4.7 Real-time PCR standard curve graph for SYBR-490 104

8 Figure 4.8 Comparison of serum antibody detection, milk antibody detection, molecular and cultural methods

110

ACRONYMS

% Per centl Microliter°C Degree Celsiusµg Microgram (s)AB-ELISA Avidin-biotin ELISAABR Antigen Abortus Bang Ring AntigenACF Automated Complement FixationAI Artificial InseminationBA Blood agarBAM Brucella agar medium bp Base pairBPAT Buffered Plate Antigen TestBSM Brucella selective mediumc- ELISA Competitive Enzyme-linked immunosorbent assayCFT Complement Fixation TestCFU Colony Forming Unit CO2 Carbon dioxideCt threshold cycleCT Card TestDNA Deoxyribonucleic aciddNTPs Deoxynucleoside triphosphatedot- ELISA dot Enzyme-linked immunosorbent assayDIA dot-immunobinding assayEDTA Ethylene diamine tetra acetateELISA Enzyme linked immunosorbent assayEIA Enzyme Immunoassayet al. et aliietc et cet·er·afg femtogramFM Farrell's mediumFPSR False positive serological reactionsg Acceleration due to gravityh hourI-ELISA indirect ELISAi.e. id est (that is)I.U. International UnitIgG Immunoglobulin GIgM Immunoglobulin MkDa KilodaltonM MolarMA MacConkey agarMAb Monoclonal antibody

Cont…

ACRONYMS

MCF Micro Complement FixationME 2-Mercaptoethanol Agglutination M-ELISA Milk-ELISAmg Milligrammin Minute (s)ml MilliliterMM Mair's mediummM MillimolarMRT Milk Ring TestMZN Modified Ziehl-Neelsen NC Negative controlng nanogramsnm Nanometernt nucleotideOD Optical densityOIE Office International des EpizootiesPA Plate Agglutination PBS Phosphate buffer salinePCR Polymerase Chain ReactionPI Performance indexpmole Picomole (s)RBPT Rose Bengal Plate TestRM Ryan's medium RNase Ribonucleaserpm Rotation per minuteRiv RivanolSDA Serum dextrose agarSDS Sodium dodecyl sulphateSDS Sequence Detection Softwaresec SecondsSTAT Standard Tube Agglutination TestTaq Thermus aquaticusTBE Tris borate EDTA bufferTE Tris-EDTA BufferU UnitUV Ultra violetV Voltsviz. Videlicet (namely)W Watt

Cont…

CHAPTER – I

INTRODUCTION

Brucellosis is an infectious disease caused by Gram negative facultative

intracellular bacterial organisms of the genus Brucella that are pathogenic for a wide

variety of animals and human beings. It is an emerging disease since the discovery of

Brucella melitensis as the cause of Malta Fever by Bruce in 1887 and the isolation of

B. abortus from aborted cattle by Bang in 1897 (Mcmahan, 1944). The disease has a

considerable impact on human and animal health, as well as socioeconomic impacts,

especially, in which rural income relies largely on livestock breeding and dairy products.

The genus Brucella currently contains six nomen species: Brucella melitensis,

B. abortus, B. suis, B. ovis, B. canis and B. neotomae which vary in their ability to infect

host animals. Brucella produces generalized infection with a bacteremic phase followed

by localization in the reproductive organs and reticuloendothelial system. Brucellosis is

essentially a disease of sexually matured animals and have predilection for ungulates

placentae, foetal fluids, mammary gland, joints and testes of bulls, rams, boars and male

dogs. The disease is manifested by reproductive failure, which includes abortion, birth of

unthrifty calves and retained placentae in female animals. Localization may also occur in

mammary tissues with excretion in the milk (Corbel, 1988). Lesions in Brucella infected

male are largely confined to the genital organs including testicles, seminal vesicles and

epididymes (Morgan and MacKinnon, 1979).

Bovine brucellosis is found worldwide however it has been eradicated from many

countries but it is one of the most serious diseases in developing countries. The rates of

infection vary greatly from one country to another and between regions within a country.

The highest prevalence is seen in dairy cattle. In India, brucellosis was first recognized in

1942 and is now endemic throughout the country. The disease has been reported in cattle,

buffaloes, sheep, goats, pigs, dogs and humans.

Despite the advances made in the diagnosis and therapy, brucellosis is still wide

spread and its prevalence in many developing countries is increasing. Economic losses by

brucellosis in animals are due to abortions, premature births, decreased milk production

and due to repeat breeding and may lead to temporary or permanent infertility in infected

livestock. Economic losses due to brucellosis in livestock are considerable in an agrarian

country like India.

Probably the main route of portal of entry is the ingestion. Transmission via the

teat canal has also been suggested as a route of infection but laboratory results and

extensive field experience have not confirmed this as an important route. The practice of

sharing equipment between various farms is also a potential danger. It has also been

observed that calves fed on infected milk harbour infection and excrete Brucella

organisms in their faeces for up to 4 weeks after the cessation of feeding. The high rate of

isolation of the Brucella from the udder and the supra mammary lymph nodes is reflected

in the numbers excreted in milk which can vary from a few hundreds up to

2, 00,000 organisms/ml of milk (Corbel, 1988). Thus, the milk is an important material to

be processed for knowing the prevalence of brucellosis in particular area.

Conventionally, serological tests are used to screen for, or to confirm the disease.

These screening tests are inexpensive, fast and highly sensitive but not necessarily highly

specific. The most widely used serological tests for diagnosis of brucellosis in animals

Introduction…

2

are Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (STAT) and

Enzyme Linked Immunosorbent Assay (ELISA). The diagnostic value may be

questionable on individual basis because of cross-reacting antibodies but for screening of

herd these tests remain ideal. Other than serum, Brucella antibodies are also excreted in

milk. The Milk Ring Test (MRT) is often used as a herd test to know the prevalence of

Brucella infection & screening of herd. The MRT can also be used to test individual milk

samples to identify the infected animal in the herd but, it may give false-posiitve results

shortly after parturition, near the end of lactation and when mastitis is present

(Alton et al., 1988). To overcome this problem Milk-ELISA is employed on individual

milk samples to detect Brucella antibodies.

In such situation, isolation of the causative agent is most accepted tool for

confirmatory diagnosis of brucellosis. It has the advantage of detecting the organisms

directly, but it is time consuming since it takes about 10 days or longer for proper

identification of the causative agents and it has reduced sensitivity in chronic infection.

Besides, the culture materials must be handled carefully, as the organisms are class III

pathogens (Alton et al., 1988). For these reasons, genetic characterization using

molecular DNA technique has been pursued. Numerous PCR based assays have been

developed for the rapid identification of Brucella.

PCR has been applied to detect Brucella DNA in varieties of clinical samples

including tissues (mainly aborted foetuses and associated maternal tissues), blood, milk

and semen (Fekete et al., 1992; Leal-Klevezas et al., 1995; Amin et al., 2001;

Kanani, 2007). PCR based tests are also found more sensitive than traditional culture

method (Amin et al., 2001; Kanani, 2007).

Introduction…

3

More recently, real-time PCR has been used for detection of Brucella, offering

improvement in detection times and specificity. The real-time PCR is also useful for

quantifying the load of microorganisms in samples.

Looking to the present scenario of high seroprevalence of brucellosis (8.8%) in

India and (8.7%) in Gujarat (Renukaradhya et al., 2001), it is utmost necessary to know

the status of Brucella infection in bovine animals to prevent the spread of economically

as well as zoonotically important disease.

Keeping the above facts in view, the present study was under taken to assess the

status of brucellosis in cattle and buffaloes, major milch animal species, by examining

serum and milk with following objectives:

1. To compare the conventional tests for detection of Brucella antibodies in

serum and milk.

2. To isolate and identify the Brucella organism from milk and confirmation

by molecular methods.

3. To standardize and apply PCR for detection of Brucella from milk.

4. To standardize and apply real-time PCR for quantification of Brucella

DNA from milk.

5. To compare the efficacy of serum antibody detection, milk antibody

detection, cultural and molecular methods for detection of Brucella

infection.

Introduction…

4

CHAPTER – II

REVIEW OF LITERATURE

Brucellosis is an important disease of livestock species and wild animals widely

prevalent in most of the developing countries. The disease causes a variety of

reproductive disorders, viz., infertility, retained placenta, abortions, endometritis, etc. and

resulting in to heavy economic losses due to interrupted lactation and also due to loss of

calves, wool, meat and milk production which are the main impediments to profitability.

The disease has a significant health hazard in contact human beings.

Brucellosis is named after David Bruce, a British army medical doctor, who

isolated Brucella melitensis (Micrococcus melitensis) from the spleen of a dead British

soldier on the island of Malta in 1887. After that in 1897 Bang isolated B. abortus from

aborted cattle (Mcmahan, 1944). Many historical accounts of the disease before this time

could actually be describing brucellosis including abortion epidemics in animals and

fever in humans. In the 20th century, brucellosis became recognized as a clinical entity

and pasteurization was implemented to prevent human infection by Brucella abortus

(Bacillus abortus).

Brucella organisms are coccobacilli or short rods, arranged singly and less

frequently in pairs or small groups. They are Gram negative and partially acid-fast as do

not decolorize by 0.5% acetic acid in the modified Ziehl-Neelsen (MZN) stain. The stain

carbol fuchsin is retained and the Brucellae appear as red-stained coccobacilli

(Alton et al., 1988).

Review of Literature…

5

The genus Brucella comprises of six recognized species: Brucella melitensis,

B. abortus, B. suis, B. ovis, B. canis and B. neotomae (Corbel and Brinley-Morgan,

1984). In addition, a new species proposed B. maris, has been isolated from marine

mammals (Jahans et al., 1997). This nomenclature was established on the basis of

difference in pathogenicity, host preference, growth and biochemical characteristics

(Corbel and Brinley-Morgan, 1984). The subcommittee on Taxonomy of Brucella had

proposed that this genus could contain a single species, B. melitensis (Corbel, 1988). This

proposal was based on DNA-DNA hybridization studies conducted on 51 strains

representing all known species and biovars (Verger et al., 1985) and was fully supported

by the recent genomic sequence data. The single species B. melitensis could then be

divided into several biovars corresponding with the former species (Corbel, 1988).

However, for practical purpose the older nomenclature is generally preferred.

Brucellic mastitis is chronic and often clinically unapparent. Because infected

female excretes large numbers of viable Brucellae in milk for months to years, apparently

normal glands represent important source of infection not only to other lactating cows but

also to calves and humans who consume raw milk.

Control of brucellosis depends upon reliable methods for detection of the

infection in livestock, wildlife and humans. Several diagnostic strategies have been

developed which when used in concert, have been instrumental in decreasing the

incidence of the disease.

At present mainly serological methods are used for diagnosis of this infection in

India. The long-term serological studies at national level have indicated that 5% of cattle

and 3% of buffaloes could be infected with brucellosis (Renukaradhya et al., 2002).


6

2.1 DETECTION OF BRUCELLA ANTIBODIES IN SERUM AND MILK

2.1.1 Serum

The serological tests depend on a reaction between Brucella antigen and

antibodies produced in response to the infection. A number of classes and subclasses of

antibody (isotypes) may occur in positive sera. The various serological tests vary in their

ability to detect different isotypes.

Historically, the Standard Tube Agglutination Test (STAT) has been recognized as

the principal serological test used for the diagnosis of brucellosis. IgM isotypes of

antibody is the most active agglutinin at neutral pH (Rice and Boyes, 1971; Corbel, 1972;

Nielsen et al., 1984). Therefore the STAT is susceptible to false positive reaction by

cross-reacting antibodies (Corbel, 1988; Nielsen, 2002). Due to low specificity by

original tube agglutination test, a large number of modifications have been made to

destroy or inactivate IgM agglutinins. Of these modifications, the acidified antigen,

rivanol precipitation and 2-mercaptoethanol are in common use in various laboratories

for inactivating IgM.

In Rose Bengal Plate Test (RBPT) antigen is used at a pH of 3.65. The low pH

prevents some agglutination by IgM and encourages agglutination by IgG1 thereby

reducing non-specific interactions (Corbel, 1972 and 1973; Allan et al., 1976). The RBPT

is considered to be suitable for screening of individual animals, however, some cross-

reacting antibodies have been detected by this test and false negative reaction may occur

mostly due to prozoning (OIE, 2004).

Numerous variations of the Indirect Enzyme Linked Immunosorbent Assay

(I-ELISA) have been described employing different antigen preparations, antiglobulin-


7

enzyme conjugates and substrates/chromogens. Several commercial I-ELISAs are

available that have been validated in extensive field trials and are in wide use. The

I-ELISA is a highly sensitive test but sometimes not capable of differentiating between

antibody resulting from S 19 vaccination or other false positive serological reactions

(FPSR) and that induced by pathogenic Brucella strains. The I-ELISA should therefore be

considered more as a screening test rather than a confirmatory test for testing of

vaccinated cattle or herds affected by FPSR problems (OIE, 2004).

Thus no single test appears to be free from demerits. This has prompted many

workers to carry out comparative studies and to determine the efficacy of different tests.

Nielsen (2002) and Gall and Nielsen (2004) after reviewing various serological tests

concluded that no individual test found perfect, however, error could be minimized using

the most reliable test.

Serological evidence suggested that brucellosis is highly endemic in most parts of

the India (Maiti et al., 1980; Chandramohan et al., 1992; Isloor et al., 1998; Mehra et al.,

2000; Shringi et al., 2002; Sarumathi et al., 2003a; Barbuddhe et al., 2004; Mahato et al.,

2004; Singh et al., 2004; Mittal et al., 2005). The seroprevalence rate of brucellosis in

cattle ranged from 0.3% in Himachal Pradesh (Renukaradhya et al., 2002) to 56.2% in

Assam (Chakraborty et al., 2000). In the states of Uttar Pradesh and Delhi Sharma et al.

(1979) carried out sero-epidemiologic investigation on brucellosis by testing 361 (cattle)

and 551 (buffaloes) sera. They revealed seropositivity of 6.37 % in cattle and 4.9 % in

buffaloes.

Byrd et al. (1979) found that ELISA was comparable to the Complement Fixation

Test (CFT) and Rivanol (Riv) test, but less sensitive than the STAT.


8

Heck et al. (1979) tested 4551 serum samples from 863 B. abortus strain 19

vaccinated and non-vaccinated adult cattle independent of disease status. Five serological

methods viz., STAT, Buffered Brucella Antigen or Card Test (CT), CFT, ELISA and

Rivanol (Riv) for detecting antibodies to B. abortus were carried out. They found 95%

probability for agreement among CT negative samples, between serological methods, for

all groups of vaccinated and non-vaccinated cattle.

Magee (1980) compared ELISA with direct agglutination, complement fixation

and Coombs tests for detection of Brucella antibody by testing 112 sera. Of these,

15 (13.39%) were found positive by ELISA which included the 13 that were found

positive by other tests. Thus, he considered ELISA as more sensitive, more rapid and

simpler method than the other tests.

Ruppanner et al. (1980) collected serum samples from 24 unvaccinated heifers

before challenge and 11 times between 12 and 102 days after conjunctival sac challenge

with B. abortus strain 2308. Antibody titers were determined by ELISA, STAT, 2-

Mercaptoethanol Test (2 MET), Micro Complement Fixation (MCF) and Automated

Complement Fixation (ACF). Agreement between ELISA and other tests were found to

be 100% (STAT), 75.7% (ME), 97.8% (MCF) and 95.2% (ACF).

Stemshorn et al. (1985) compared six agglutination and two complement fixation

tests by using 1051 sera from brucellosis free herds, with respect to specificity, sensitivity

and relative sensitivity for the sero diagnosis of bovine brucellosis. They revealed the

specificity of 98.9%, 99.2% and 99.3% for the Buffered Plate Antigen Test (BPAT), STAT

and Plate Agglutination Test (PAT), respectively, and 99.8% for the 2 MET. Whereas,

RBPT, CT and the CFT were failed to detect antibody against brucellosis.


9

Turilli et al. (1986) tested 1188 serum samples from cattle and revealed that the

specificity and sensitivity of ELISA for brucellosis were about the same as those of STAT

and CFT. They found more than 99% agreement between ELISA and each of the other

tests.

Kim et al. (1988) compared serological tests in 84 brucellosis reactors and 44

healthy controls. The PAT resulted in 3.1% of false positive and 1.6% of false negative

reactions in comparison with that of STAT. The agreement between both the tests was

found to be 61.7%. Among the Riv, 2 MET, ELISA, RBPT and CFT, the ELISA and CFT

resulted in to very sensitive reactions, while the Riv test revealed the most specific

reactions.

Sharma et al. (1990) tested breedable murrah buffaloes by applying various

serological tests for brucellosis in Vietnam having history of abortion in buffaloes at 6-9

months of pregnancy and suspected for brucellosis. Of the 59 animals examined, 33 were

found positive reactors.

Seroprevalence study of brucellosis was carried out by Lodhi et al. (1995) by

collecting 208 serum samples of adult buffaloes and cows in and around Faisalabad by

RBPT and STAT. They found 12.98% and 2.40% of seroprevalence by RBPT and SAT,

respectively. They also revealed that 5 animals positive to STAT, 3 (60%) gave positive

results with RBPT.

Kerby et al. (1997) evaluated an I-ELISA for detection of brucel1osis in

comparison to the CFT on sera from a non-vaccinated negative population, B. abortus

strain 19 vaccinated negative population and a brucellosis positive population of

unknown vaccination status and concluded that, against sera from the positive population,


10

the ELISA gave many more positive reactions than that of CFT, probably a combination

of both higher sensitivity and lower specificity.

A total of 878 selected serum samples from cattle and buffaloes in the Amazonian

region were examined by 5 serological tests (BPAT, STAT, CFT, I-ELISA, competitive

ELISA) by Molnar et al. (1998). The I-ELISA yielded the highest number of positive

results, except in samples derived from the Marajo Island, for which the competitive

ELISA was found to be the most sensitive. They found sensitivity of the classical tests

(agglutination and complement fixation) markedly lower than that of the ELISAs.

Prahlad et al. (1999) carried out seroprevalence study of brucellosis in buffaloes.

Of the 296 serum samples collected at an abattoir in Delhi 7.09%, 2.70%, 11.14% and

8.10% were found to be positive by RBPT, STAT, CFT and dot-ELISA, respectively.

Seroprevalence of brucellosis in Punjab was found higher (21.39%) than in Uttar Pradesh

(11.32%). RBPT showed the highest relative sensitivity (33.33%) using CFT as an

indicator test. All the tests showed relative specificity of >90%.

Rao et al. (1999) collected 160 serum samples (80 Murrah buffaloes and 80

crossbred cows) with a history of frequent abortions. The samples were subjected to PAT,

STAT and dot-ELISA. They noticed that dot-ELISA gave a higher percentage of positive

results (16.25% and 31.25%) followed by RPAT (11.5% and 16.25%) and STAT (8.75%

and 15.00%) in graded Murrah buffaloes and cross bred cows, respectively. They

concluded that dot-ELISA was found to be a good screening test for detecting bovine

brucellosis.

A total of 141 bovine sera in Assam were screened for brucellosis by RBPT, STAT

and I-ELISA by Chakraborty et al. (2000). Out of these 79 (56.02%), 71 (50.35%) and 47


11

(33.33%) were found positive by ELISA, STAT and RBPT, respectively. The relative

sensitivity and specificity of STAT and RBPT for bovine brucellosis classified on basis of

ELISA results which were found to be 88.61% and 98.59% for STAT, respectively and

56.96% and 96.77% for RBPT, respectively.

Mehra et al. (2000) tested serum samples of 877 cows, 349 heifers, 70 buffaloes,

from organized farms using STAT and compared out with serum samples of 135 cows, 95

buffaloes, from unorganized farms in Madhya Pradesh, India to determine the magnitude

of bovine brucellosis in Satpura and Madhya. The seroprevalence of organized farms in

cows, heifers and buffaloes were 9.6%, 12.6% and 11.4%, respectively, whereas

seropositive cows of unorganized farms were only 2.2% vs. 9.4% buffaloes against

Brucella.

Pati et al. (2000) tested 23 sera from buffaloes (male 2, female 21) of the village

Danpur, Distt. Moradabad (U.P) by applying RBPT, STAT and ELISA. They concluded

that ELISA was more sensitive than the RBPT and STAT.

Sandhu et al. (2001) studied 666 cows and 750 buffaloes to determine the

seroprevalence of brucellosis in Punjab, India. Of these, 67 cows and 70 buffaloes were

positive for brucellosis with a prevalence of 10.06 and 9.33%, respectively.

Paweska et al. (2002) analyzed 4,803 cattle sera from South Africa (n=3,643),

Canada (n=652), Germany (n=240), France (n=73) and USA (n=195). In study the

diagnostic sensitivity of I-ELISA was found 100% and of CFT 83.3%, whereas the

diagnostic specificity of I-ELISA was 99.8% and of CFT 100%. Finally, they revealed

that the I-ELISA performed on an automated ELISA work station provided a rapid,

simple, highly sensitive and specific diagnostic system for large-scale detection of


12

antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported, the

authors suggested that it could replace not only the currently used confirmatory CFT, but

also other two routine screening tests, namely the RBPT and STAT.

Renukaradhya et al. (2002) found seroprevalence of brucellosis in Gujarat at the

rate of 6.6% (247out of 3750) in cattle and 6.3% (14 out of 222) in buffaloes.

Rajesh et al. (2003) assessed seroprevalence of brucellosis in 719 cattle of Kerala

(India) using RBPT and STAT. Of these samples, 9 were found positive by RBPT but 5

gave a doubtful reaction, whereas all 14 samples were positive in STAT. They found that

the overall seroprevalence was 1.95% and greater in adult cattle. They also concluded

that seropositivity was higher in heifers and pregnant animals.

The efficacy of AB-ELISA, RBPT and STAT in detecting antibodies to Brucella

of 1541 serum samples from cattle with a history of reproductive failures and in healthy

cattle from farms in Andhra Pradesh was compared by Sarumathi et al. (2003b).

AB-ELISA, RBPT and STAT gave specificities of 100%, 88.22% and 90.59%,

respectively. They also found AB-ELISA as a reliable screening test for detecting

antibodies to Brucella in cattle.

Varasada (2003) tested 344 cattle and 251 buffaloes for brucellosis. The results

revealed that 68(19.76%), 57(16.57%) and 83(24.12%) of cattle were positive by RBPT,

STAT and I-ELISA, respectively. Whereas 32(12.75%), 28(11.16%) and 48(19.12%) of

buffaloes were positive by RBPT, STAT and I-ELISA, respectively. In an overall

seroprevalence study of brucellosis in cattle and buffaloes of central Gujarat, 16.80%,

14.03% and 22.01% of animals were found positive by RBPT, STAT and I-ELISA,

respectively.


13

Prevalence of brucellosis in organized farms with abortion storms in Goa region

was investigated by Barbuddhe et al. (2004). Out of 107 serum samples tested for

brucellosis, 40 (37.38%), 39 (36.45%) and 43 (40.18%) were found positive for

antibodies against Brucella, by RBPT, STAT and AB-ELISA, respectively.

Chand and Sharma (2004) screened out the serum samples of cattle for brucellosis

by ELISA, RBPT and STAT in three states viz., Haryana, Uttar Pradesh and Madhya

Pradesh. Overall prevalence rate of brucellosis in the cattle farms was found to be

26.50% by ELISA, 20.47% by RBPT and 18.89% by STAT. The use of ELISA in

comparison to RBPT and STAT for assessing the situation of brucellosis in cattle was

advocated by researchers, to have better results because chances of non detection of an

infected animal in ELISA are minimum.

Erdenebaatar et al. (2004) used ELISA to eliminate false positive amongst RBPT

positive sera. They collected 697 serum samples in Mongolia from human and animals in

23 nomadic herds which classified in to three groups as brucellosis endemic (BE),

brucellosis suspected (BS) or Brucella vaccinated (BV). The 295 sera (43.0%) were

found positive by RBPT, but 206 (69.8%) of these were positive according to ELISA;

therefore, 30.2% of the RBPT positive sera found to be false positive. The false positive

samples for RBPT represented 4.1%, 27.4% and 68.2% of the animals from the BE, BS

and BV herds, respectively.

Mahato et al. (2004) tested serum samples of 67 cows and 141 heifers by STAT

and ELISA. They found 43.28% and 47.76% positivity in cows by STAT and ELISA,

respectively, whereas 14.89% and 17.02% positivity in heifers by STAT and ELISA,

respectively and concluded that ELISA was found more sensitive than the STAT.


14

Nasir et al. (2004) performed seroprevalence of brucellosis using RBPT and

STAT in 1473 cattle and 481 buffaloes and 286 cattle and 223 buffaloes from various

Government and private livestock farms, respectively. The RBPT recorded the

seroprevalence as 14.70% and 15.38% in cattle and buffaloes, respectively, at

Government farms and 18.53% and 35.40% in cattle and buffaloes, respectively, at

various private livestock farms. Of these RBPT positive animals, 7.19% of cattle and

2.91% of buffaloes at Government farms, whereas, 9.0% of cattle and 23.70% of

buffaloes at private livestock farms were found seropositive by STAT.

A serological survey was performed by Singh et al. (2004) in 6 organized dairy

farms in Punjab using RBPT, STAT and AB-ELISA. To compare the sensitivity and

specificity of RBPT and STAT, AB-ELISA was used as the gold standard. The study

revealed that the sensitivity of RBPT (88.46%) was higher when compared with STAT

(46.15%), while specificity of STAT (98.31%) was slightly higher than RBPT (97.75%).

In a comparative study for detection of Brucella antibodies, AB-ELISA detected

antibodies in 43(11.94%), RBPT in 37(10.28%) and STAT in 29(8.05%) samples out of

360 bovine serum samples tested in Assam (Bhattacharya et al., 2005).

Genc et al. (2005) collected and tested sera from 163 aborted dairy cattle that had

no history of vaccination against brucellosis. They detected B. abortus antibodies in these

serum samples as 68.1, 65.6, 58.9 and 55.2%, respectively, by the C-ELISA, CFT, RBPT

and STAT.

A total of 859 cattle and 133 buffaloes of organized sector (Goshala and Tabela)

and unorganized sector of Jodhpur region were screened by Kachhawaha et al. (2005)

using RBPT. The positive samples were subjected to STAT. The prevalence of brucellosis


15

was found much higher in cattle (41.79%) than in buffaloes (25.56%) and also more in

cattle of organized sector (Goshala) in comparison to unorganized sector.

Mishra et al. (2005) found 1.55% of cows and 1.97% of buffaloes seropositive for

brucellosis by STAT, while 3.11% of cows and 4.18% of buffaloes by I-ELISA out of the

total 579 of cows and 407 of buffaloes serum samples tested.

Mittal et al. (2005) compared three serological tests namely RBPT, STAT and

ELISA by testing 217 cattle sera and 67 buffalo sera from the district Udham Singh

Nagar, Uttranchal. They found that ELISA was more sensitive followed by RBPT and

STAT when applied to cattle sera, whereas RBPT was more sensitive followed by STAT

and ELISA when applied to buffalo sera.

Sunder et al. (2005) found 13.83% of the samples positive in RBPT while 10.4%

of the samples positive in AB-ELISA in sero screening analysis of cattle belonging to

Andaman and Nicobar islands.

Ganesan and Anuradha (2006) compared dot-ELISA and RBPT for diagnosis of

bovine brucellosis. Out of the total 81 samples tested 11.11% and 13.59% were found

positive by RBPT and dot-ELISA, respectively.

Agrawal et al. (2007) compared three serological tests namely RBPT, STAT and

ELISA by applying to the 142 cattle and 61 buffalo sera of Bageshwari district of the

state Uttaranchal. They found that ELISA was more sensitive followed by RBPT and

STAT.

A total of 194 serum samples from breeding bulls of different AI Centres of

Gujarat were screened for presence of Brucella antibodies by Kanani (2007). Of these,

16 serum samples were found positive for Brucella antibodies by ELISA yielding an


16

overall seroprevalence of 8.25%. They also found that the much higher seroprevalence

(16.13%) was found in cattle than in buffalo bulls (0.99%). Sensitivity of RBPT and

STAT was found to be of 50% and 62.5%, respectively, with considering ELISA as a gold

standard test while specificity was found to be of 98.31% and 97.75%, respectively.

Sharma et al. (2007) screened 2988 animals in 62 dairy farms/gaushalas of Punjab

for brucellosis and 540 (18.07%) were found positive by STAT.

2.1.2 Milk

Milk ring test (MRT) has been used for many years for detection of dairy cows

infected with B. abortus, since milk constitutes a highly desirable source of antibody for

routine screening purpose and for the identification of infected individuals as sample

collection is simple and noninvasive (Roepke et al., 1950 and 1974; Alton et al., 1975). It

is particularly useful on bulk milk samples and is effective for screening and monitoring

small dairy herds for brucellosis. In large herds (>100 lactating animals) and in early

infection the MRT does not show the sufficient sensitivity. Furthermore, the MRT when

performed using undiluted whole milk from individual may give false positive results

shortly after parturition, near the end of lactation and when mastitis is present

(Alton et al., 1988).

Considering the limitations of MRT several workers reported the use of various

enzyme antibody immuno-assays for detection of antibodies to B. abortus in milk or

serum.

Thoen et al. (1979) developed an enzyme immunoassay (EIA) for detecting

Brucella antibodies in milk of cows infected with B. abortus. They found that EIA has

more sensitivity and specificity than MRT for detecting antibodies in milk of cow,


17

experimentally infected with B. abortus strain 2308 and in milk of 16 naturally infected

cows from which B. abortus was isolated and no detectable EIA reactions were present in

milk of 11 non infected controls.

Boraker et al. (1981) used BrucELISA (I-ELISA) for the detection of antibody to

B. abortus in cow's milk of Florida (U.S.A.) herd. They found that I-ELISA was highly

correlated with positive MRT reactions and culture positivity. The method also eliminated

false positive MRT reactions and detected antibody in some MRT negative samples. The

milk-ELISA system described in their report detected antibody to B. abortus in

cow's milk at levels far lower than those detected in the MRT and provided for rapid as

well as inexpensive screening of both individual and bulk milk samples. The results

indicated that the milk-ELISA was not only sensitive and specific, but was able to

distinguish between infected and vaccinated animals and was of diagnostic value in

predicting which animals were shedding or will eventually shed cultivable B. abortus.

Mikolon et al. (1998) found milk-ELISA significantly more sensitive and more

specific than MRT. The milk-ELISA also had the advantage of objectivity and ease of

interpretation.

Nazem et al. (1998) compared milk-ELISA with MRT by testing 54 milk samples

from Friesian cows belonging to a herd with a history of brucellosis. Correlation of MRT

with milk-ELISA was 48.10%. The sensitivity and specificity of MRT as compared to

milk-ELISA were 48.15% and 72.22%, respectively. Finally they concluded that

milk-ELISA was found more sensitive and specific as compared to MRT.

Kang'ethe et al. (2000) tested 212 and 222 raw (unpasteurized) milk consuming

households, 262 and 246 informal markets during dry and wet seasons, respectively, in


18

Kenya. They also tested 110 formally (pasteurized) marketed milk samples using MRT

and Indirect milk-ELISA. The overall prevalence of brucellosis at consumer level by

ELISA and MRT were 4.9% and 3.9%, respectively, whereas, at the informal market

level, positivity by ELISA and MRT were 2.4% and 3.4%, respectively.

Vanzini et al. (2001) evaluated I-ELISA for the detection of B. abortus antibodies

in bovine bulk milk samples. About 31 individual milk samples from B. abortus infected

cows were diluted in to bulk milk from a brucellosis free herd. Individual milk samples

obtained from 96 negative or positive herds to ELISA or MRT were tested by ELISA.

Four samples which were negative in the MRT were found positive in the ELISA. Using

bulk milk samples, the sensitivity of the ELISA (98.1%) was found higher than the MRT

(72.2%).

Bonfoh et al. (2002) checked specificity of milk-ELISA to detect Brucella

antibodies by applying to the fermented cow milk and compared to the MRT. They

revealed that positive results were highly correlated with the positive results of MRT.

They also compared sensitivity of the test on fermented milk and fresh milk of the same

animals and found that sensitivity was not significantly decreased.

Rivera et al. (2003) collected 1,523 milk samples from individual animals and

bulk milk belonging to 200 herds in the province of Cundinamarca, Colombia for

detection of B. abortus antibody and comparative evaluation of the MRT and I-ELISA in

cattle milk. They found that the I-ELISA was highly sensitive and specific with 95.3%

sensitivity and 95.1% specificity and recommended the use of I-ELISA for testing a large

number of herds or individual samples, for enhancing the efficiency of surveillance

programmes and control campaigns.


19

Chand et al. (2004) compared out ELISA with MRT for detection of Brucella

antibodies in sheep milk by testing 165 and 114 milk samples from ewes belonging to an

organized farm which had endemic B. melitensis infection and unorganized farm,

respectively. From organized farm 22 samples were found positive by MRT and 45 by

milk-ELISA whereas 9 were found positive by MRT and 15 by milk-ELISA from an

unorganized farm. They also tested 10 clotted milk samples from an organized farm by

ELISA that were unsuitable for MRT and found 3 of the milk samples were positive by

ELISA and also by serum ELISA and concluded that ELISA appeared to be a useful

assay for detecting Brucella antibodies in the milk of sheep and can also be conducted on

milk unsuitable for MRT.

Gumber et al. (2004) analyzed 970 bulk milk samples by AB milk-ELISA and

MRT to know the status of bovine brucellosis in Punjab. Of the samples, 218 and 115

were found positive by AB milk-ELISA and MRT, respectively. They also found that the

prevalence of brucellosis at village level was 22.5% by milk-ELISA. The MRT showed

lower sensitivity (68.8%) but had comparable specificity (98.9%) than that of

milk-ELISA.

Mahato et al. (2004) used MRT to detect Brucella antibody in individual milk

samples of 67 cows and found 24 (35.82%) positive.

Funk et al. (2005) applied I-ELISA to detect B. melitensis specific antibodies in

goat milk by using Brucella salt-extractable protein extract as an antigen and a

horseradish peroxidase labeled polyclonal anti-goat antibody as an anti-species conjugate.

They found positivity thirteen of 13 (100%) when applied to individual infected goat


20

milk samples, whereas, 134 of 134 (100%) negativity when applied to uninfected bulk

milk samples.

2.1.3 Comparison of Antibody Detection Tests for Serum and Milk

Heck et al. (1980) evaluated an ELISA for the detection of antibodies to

B. abortus in cow’s milk from seropositive or negative cows and determined ELISA to be

an appropriate method for detecting antibodies to B. abortus in bovine milk.

Al-Khalaf and El-Khaladi (1989) investigated the presence of Brucella antibodies

in serum and milk of camels in Kuwait by applying three serological tests for serum,

namely RBPT, STAT and CFT, whereas, MRT for milk. The prevalence rate was 14.8%

from serum by CFT and RBPT and 10.8% by STAT. For milk prevalence rate was 8.0%.

Barman et al. (1989) tested 129 serum and 42 milk samples from cattle of

organized dairy cattle farms from Assam, India, with a history of abortion, retention of

placenta, mastitis, swelling of joints and repeat breeding, of which 44.9% were positive

for the STAT and 54.7% for the MRT.

Zowghi et al. (1990) tested 6,472 cows from eight infected herds by collecting

serum and milk simultaneously for serological and bacteriological testings. Of these

1,056 (16.31%) were serologically positive and 1632 (25.21%) were positive to MRT.

Milk-ELISA was established by Biancifiori et al. (1996) to know the specificity

and sensitivity to detect low levels of Brucella antibodies in ewe milk by means of

reference standards and compared with conventional screening and confirmatory tests

under field conditions. They found that the specificity of M-ELISA was 65% and 83%

when compared to RBPT and CFT, respectively, and 92% relative to culture positive

animals, whereas, specificity was 100% when applied to Brucella free herds. They also


21

monitored Brucella antibodies in milk of positive sheep in colostrum and in mature milk

for 30 days after parturition and it appeared that concentrations of immunoglobulins in

milk was having tend to decrease sharply soon after parturition while in serum they

remained constantly high and concluded that M-ELISA for Brucella antibodies in ewe

milk can be regarded as a complementary diagnostic tool for individual testing but it

would be unsuitable for use as a screening test applied to pooled flock milks.

Vanzini et al. (1998) evaluated an I-ELISA for B. abortus antibodies detection in

bovine milk and serum samples by collecting 2646 sera and 2119 milk samples from

cows older than 24 months from 12 brucellosis free herds of Argentina for at least the

previous 5 years and they found that the I-ELISA was a highly sensitive and specific test

and thus advocated to process a large number of samples.

Gurturk et al. (1999) detected Brucella antibodies in bovine sera and milk by

dot-immunobinding assay (DIA), STAT, RBPT and MRT on 116 paired blood and milk

samples from 56 aborted and from 60 healthy dairy cows. Of these, 24 were found

positive and 72 were negative by all the tests. Serum samples of six aborted cows were

found positive by DIA, STAT and RBPT but the milk samples were negative by DIA and

MRT, whereas, four aborted cows gave positive reaction only by DIA when applying to

serum and milk samples. The remaining six aborted cows were negative only by MRT

and two of them were negative by both RBPT and MRT. Four sera of healthy cows were

found to be positive only by STAT.

Milk-ELISA was evaluated and validated by comparing with the RBPT, STAT,

CFT, Coombs anti-globulin test (Coombs), 2-ME and serum-ELISA by testing individual

milk samples from cows by Szulowski (1999). Seventy nine RBPT positive milk


22

samples, 14 CFT, Coombs or 2 MET positive milk samples, 530 milk samples from cows

considered free of brucellosis and 309 milk samples from cows from herds suspected for

brucellosis were tested. He observed no correlation between the milk-ELISA and RBPT

results; among 79 samples from cows in which sera reacted positively in the RBPT, only

22 were positive or doubtful in the ELISA. He also revealed that there was 100% (14 out

of 14 samples) correlation between milk-ELISA and CFT, Coombs and/or 2-ME. All 530

milk samples from cows free of brucellosis were negative in the milk-ELISA. Among

309 milk samples from cows from herds suspected of brucellosis, 11 positive and 20

doubtful results were obtained with the milk-ELISA and concluded that milk-ELISA was

more sensitive than traditional methods and serum-ELISA, in which only 3 samples

reacted positively and 2 doubtfully.

A total of 150 individual samples of blood and raw milk of cows collected from

El-Behera Governorate were subjected for STAT, MRT and cultural isolation of Brucella

organisms by Abdel Hakiem (2000). The results showed that MRT was found to be

reliable and sensitive, as it gave positive results in 8% of samples, as compared with the

results of STAT (10.8%) for serum.

Chand et al. (2004) tested 165 milk and blood samples from ewes belonging to an

organized farm that had endemic B. melitensis infection. Of these 45 and 48 samples

were positive by milk-ELISA and serum-ELISA, respectively. They also tested 10 clotted

milk samples by milk-ELISA that was unsuitable for MRT. Three of the milk samples

were positive by milk-ELISA and also by serum-ELISA when tested the blood samples

from the same animals.


23

Chand et al. (2005) examined milk and blood samples from 704 lactating ewes

from brucellosis free sheep flock, brucellosis infected sheep flock and private sheep

flocks of which status for brucellosis was not known for the diagnosis of B. melitensis

infection by milk-ELISA, serum-ELISA, RBPT, STAT and culture of milk. They revealed

that the specificity of milk-ELISA in brucellosis free flock was 100% and sensitivity and

positive predictive value were 96.11% and 94.28%, respectively, in infected flocks. The

Brucella antibody levels in milk and serum samples as determined by milk-ELISA and

serum-ELISA correlated significantly and the milk-ELISA for brucellosis appears to be

an attractive alternative of serum-ELISA particularly in the lactating ewes.

2.2 ISOLATION AND IDENTIFICATION OF BRUCELLA

Conventional method of bacterial isolation is still the only absolute method for

establishing the infection status. Isolation from a single animal is sufficient evidence to

establish the infection status of a herd and is considered to be the gold standard test.

Brucella species have been recovered from foetal membranes, vaginal secretions,

milk, semen, arthritis or hygroma fluids and the stomach content, spleen and lungs from

aborted foetuses. From the carcasses, the bacteria could sometimes be isolated from the

lymph nodes, spleen, uterus, udder, testes, epididymes, joint exudate, abscesses and other

tissues (Alton et al., 1988, Quinn et al., 1994).

A wide range of selective media can be used for cultivation. The suitable media

include Brucella agar medium (BAM) base, trypticase soy agar, modified Thayer-Martin

medium, Farrell’s medium, serum dextrose agar (SDA), glycerol dextrose agar and

Castaneda’s medium (OIE, 2004).


24

In a four day old culture, colonies of Brucella appeared to be in smooth form and

appear pale honey colour when viewed through a transparent medium, 1-2 mm in

diameter, translucent and round, with smooth margins. The colonies are convex and

pearly white when seen from the above. In rough form, the colonies are much less

transparent and have a more granular, dull, matte white to brown surface. In nature,

B. abortus, B. melitensis, B. suis and B. neotomae usually occur in smooth form while

B. ovis and B. canis are found in rough form. Identification up to the genus level is done

by biochemical tests and slide agglutination test (Quinn et al., 1994, OIE, 2004).

As such scanty literature is available on the isolation of Brucella particularly from

the milk hence the literature pertaining to the isolation from various materials including

the use of different media for isolation has been reviewed.

In India, Polding (1942) first reported the recovery of 46 isolates of Brucella from

cattle, buffalo, goat, horse and man. Mathur (1963) isolated 16 strains of B. abortus from

placenta of 14 cows having history of abortion as well as from 2 buffaloes and 8 strains

were recovered from 23 milk samples.

Farrell and Robertson (1972) compared Ryan's medium (RM) and Farrell's

medium (FM) with that of Mair's medium (MM) and SDA medium for isolation of

Brucellae from milk. Brucella organisms could be isolated from milk of 111 (21%) of

516 MRT positive animals.

Brodie and Sinton (1972) examined 500 herd milk samples, which were MRT

positive and 41.8% samples yielded B. abortus using modified Farrell’s medium.


25

Shin et al. (1978) found Schaedler agar medium containing cysteine as a reducing

agent better than Brucella (albimi) agar for isolation of Brucella. In the study an isolation

rate of 69% was obtained from milk samples with a titre of 1:100 or greater in STAT.

Pal and Jain (1985) isolated B. abortus from clinical specimens like placenta,

foetal lung, foetal abomasum and vaginal discharge of 17 buffaloes in West Delhi region.

They used tryptose agar and serum dextrose agar for isolation. During the study only

9 specimens of the 43 yielded the growth of Brucella organisms and remaining plates

were contaminated with fast growing microorganisms.

Halder and Sen (1986) recovered six isolates of B. abortus biotype I from milk of

MRT positive cows using dehydrated tryptose agar. They used cyclohexamide, brilliant

green and crystal violet in the medium for selective isolation.

Corner et al. (1987) processed tissues from 3 infected bulls for isolation. They

found that 11 of the 12 tissues examined were found infected in at least one of the bulls.

The most frequently infected tissues were the mandibular, prescapular, subiliac and

scrotal lymph nodes.

Al-Khalaf and El-Khaladi (1989) attempted for isolation of Brucella from

sediment and cream of milk that were positive by MRT. They failed to isolate Brucella

from milk, but able to isolate from foetus and confirmed as B. abortus biovar 1.

Milk, vaginal discharge and cervical swabs from 47 cows having abortions were

cultured on tryptose agar containing crystal violet 1:500,000, brilliant green 1:250,000

and cyclohexamide for isolation of Brucella organism by De et al. (1989). No Brucella

organisms could be isolated from these samples, even though the 10 cows were

seropositive.


26

Brucellosis was investigated in aborting buffaloes (126) and cows (55) belonging

to 15 farms from Bombay in between 1983-85 by Das et al. (1990). They used Brucella

selective medium (BSM) (Himedia, Bombay), gas-generation kit (carbon dioxide system)

and oxoid jar to support isolation. The occurrence was found to be 38.18% in cows and

14.28% in buffaloes. They also compared isolation efficacy of BSM with the biological

method (guinea pig inoculation) using 3 culturally positive cervical mucus and found

BSM superior to biological method.


milk for serological and bacteriological testings. They recovered 397 isolates of Brucella

from 1,632 MRT positive milk samples of which, 119 came from 5,686 seronegative

cows.

Hadad and Al-Azawy (1992) isolated 13(42%) isolates of B. melitensis out of 31

samples examined. These isolates were recovered from 7 aborted foetuses, 4 vaginal

swabs and one milk sample from serologically positive recently aborted ewes and 1

synovial fluid from a ram.

Nicoletti and Tanya (1993) processed udder secretions for cultural isolation of

Brucella organisms from 828 cows which were vaccinated with B. abortus strain 19.

They were able to isolate field strain of B. abortus or strain 19 from 278 cows.

Milk, vaginal swab, hygroma fluid and semen samples from 177 cows and bulls

having Brucella agglutinins at positive diagnostic level (80 IU/ml) were subjected to

cultural isolation for isolation of Brucella sp. by Chatterjee et al. (1995). They revealed

the overall isolation rate of Brucella was 6.2%.


27

Hadad et al. (1997) isolated 3 Brucella strains from 65 kishfa (prepared by

collecting the thick upper layer of ewe milk after boiling for 10 min and cooling to room

temperature) and 8 Brucella strains from 85 fresh soft cheese samples obtained from

local markets of Mosul City, Iraq.

Hadad (1998) examined 160 samples (80 buffalo milk and 80 gaymar-a buffalo

milk fat product) from Mosul City (Iraq) for isolation of Brucella. Of these, 5% of the

samples were found positive for Brucella.

Jeyaprakash et al. (1999) processed 64 milk samples from cows having symptoms

of abortion and retained placenta for isolation of Brucella by using SDA. They revealed

10 (15.62%) of the samples positive to brucellosis.

Shome et al. (1999) processed samples of the stomach content of foetuses and

cotyledons of the placenta from 4 cases of abortion in 3 cattle herds from the Andaman

and Nicobar Islands. The Brucella could be isolated from 3 cases.

Botelho et al. (2000) used Brucella agar incorporating antimicrobial agents and

could isolate Brucella spp. from raw milk of seropositive cows.

Langoni et al. (2000) analyzed 49 milk samples from seropositive animals for

brucellosis by inoculating pellet and the supernatant in Farrel and Brodie-Sinton (BS)

medium supplemented with antimicrobial agents and incubated at 37oC for 7 days, with

10% CO2 atmosphere. The suspected bacterial growth in BS medium was immediately

cultivated on Brucella agar medium, under the same conditions. The isolates were

identified on the basis of Gram staining, CO2 requirement, H2S production, urease

activity and growth in the presence of thionin and fuchsin. Of the 49 analyzed samples,

15 (30.61%) contained B. abortus.


28

Rathore et al. (2002) processed 82 samples (79 cattle and 3 buffalo) of aborted

foetuses comprising of foetal stomach contents, lungs, liver etc. from Uttar Pradesh.

Samples were plated on blood agar, kept in MacIntosh and Fildes jar containing 10% CO2

tension and jar was incubated at 37oC for 5 days and identified up to genus level by

biochemical tests such as catalase, oxidase, nitrate, indole, urease activity, CO2

requirement and H2S production. Of the 79 cattle samples processed 32 (40.05%) yielded

Brucella organisms where as none out from buffalo.

Chahota et al. (2003) collected samples from five aborted cows comprising of

placenta, vaginal swabs and samples from aborted foetus (abomasal contents, heart blood

and peritoneal cavity fluid). They attempted isolation of Brucella by inoculation of

morbid materials/swabs on 8% sheep blood agar and enriched Brucella agar plates. After

processing the samples B. abortus biotype I was isolated from the morbid materials from

all cows.

Manterola et al. (2003) smeared semen swabs on plates of modified

Thayer-Martin medium and incubated under 10% CO2 tension for at least 7 days at 37oC

and could isolate B. ovis from experimentally as well naturally infected rams.

Joshi et al. (2005) tried modified cold ZN staining on the broth cultures for early

presumptive identification of Brucella growth. In the study five blood cultures positive

for Brucella, acid-fast coccobacilli were seen in broth smears stained with modified cold

ZN stain, thus providing presumptive identification of Brucella growth. Acid-fast bacteria

were not seen in the broth smears of the 17 broths negative for Brucella growth. Modified

cold ZN staining methods was found simple, reliable and reproducible by authors.


29

Tryptose soya agar medium was used for isolation of Brucella organism from

5 semen samples and all were found to be negative. However, one samples from stomach

content of aborted foetus yielded Brucella organism (Anonymous, 2006).

Kaur et al. (2006) inoculated 61 samples comprising of 9 vaginal mucous,

15 foetal membranes and 37 foetal stomach content from aborted cattle and buffaloes on

selective medium consisting of BHI agar with 7-10% defibrinated sheep blood and

antimicrobial agents for isolation of B. abortus. They recovered 17 (27.86%) isolates of

B. abortus and also conducted RBPT and STAT on sera of 51 of these animals and found

10 and 11 positive cases, respectively. They were also able to isolates B. abortus from

RBPT and STAT negative animals. Thus, isolation was found more sensitive as compared

to RBPT and STAT by them.

Kanani (2007) recovered 8 isolates out of 101 semen samples from five AI

Centres of Gujarat state, by using Brucella agar medium. Out of these, six were from

cattle bulls while two were from buffalo bulls. All the eight isolates were identified as

Brucella organisms by cultural, morphological, serum agglutination and biochemical

characteristricts and the isolates were further confirmed by PCR using different genus

specific primer pairs.

2.3 MOLECULAR DETECTION OF BRUCELLA

The Brucella genome is encoded on two circular chromosomes with sizes close to

2.05 Mb and 1.15 Mb for each species (Michaux-Charachon et al., 1997). Only the small

chromosomes of B. suis, B. canis and B. neotomae are 50 kb longer. The

guanine/cytosine (G + C) contents in the DNA of various members of the genus Brucella

are very similar, 55 to 58% (Hoyer and McCullough, 1968; Verger et al., 1987). Both


30

chromosomes contain almost identical proportions of potential coding regions (1028 and

1035, respectively). Housekeeping genes are evenly distributed all over the genome,

which makes a long coexistence highly probable (Moreno and Moriyon, 2002).

Chromosomal mapping revealed a high conservation of restricted sites and gene order.

Variability is localized to certain regions, most often on the small chromosome. The

nucleotide sequence similarity between all Brucella species is also high and DNA-DNA

homology exceeds 90%. The six species are so closely related that a monospecies genus

has been suggested (Verger et al., 1985). This hypothesis was also confirmed by

16S rRNA gene sequence analysis and was reflected in the biochemical characteristics of

the organisms (Moreno et al., 1990).

Nevertheless, remarkable differences are found in host specificity and pathogenic

properties of the six nomen-species and each Brucella species seems to be genetically

isolated. Virulence is restricted to a small number of specific hosts, active multiplication

is not possible in the environment and genetic exchange, e.g. through plasmid, temperate

bacteriophages or transformation, does not occur naturally in Brucella

(Michaux-Charachon et al., 1997).

Culturing has advantage of detecting the organisms directly but it is time

consuming as it takes about 10 days or longer for identification of the causative agents

and has reduced sensitivity in chronic infection. Besides, the culture materials must be

handled carefully, as Brucella organisms are class III pathogens (Alton et al., 1988).

Amplification of DNA by PCR has currently been used for the diagnosis of several

infectious diseases caused by fastidious or slowly growing bacteria. Different target


31

genes, primer pairs, PCR techniques and extraction procedures have been used by

different scientists for detection of Brucella DNA.

Various regions of the Brucella genome have been identified and used in PCR

assays; for example, the IS711-genetic element, also known as IS6501 (Bricker and

Halling, 1994; Ouahrani-Bettache et al., 1996), 16S rRNA (Herman and Herman, 1992;

Romero et al., 1995), 31 kDa outer membrane protein (Baily et al., 1992; Gallien et al.,

1998; Sreevatsan et al., 2000), bcsp31 (Guarino et al., 2000), 43 kDa outer membrane

protein (Fekete et al., 1990) and omp2 gene (Leal-Klevezas et al., 1995), using crude cell

lysates and DNA extracted from cell lysates of Brucella spp. The method has been

optimized for a number of Brucella spp., using tissues, blood or milk samples.

2.3.1 Extraction of DNA from Bacterial Culture

Various methods of DNA extraction from bacteria have been described and used

by various workers around the globe.

Fekete et al. (1992) extracted DNA from methanol-killed Brucella cells after

washing and incubating on ice in the presence of lysozyme. The solution was incubated

to 50°C after the addition of proteinase K and cell lysis was brought by the addition of

N-lauryl sarcosyl. The lysates were treated with RNase A and extracted repeatedly with

phenol.

Herman and Herman (1992) used a crude cell lysate in a final reaction mixture of

PCR. Crude cell lysates were obtained by sonication of 109 bacterial cells in 100 μl of

H2O for 60 sec. and then by heating for 2 min. at 100°C. Before starting the sonication,

Brucella cells were killed by heating for 2 h at 80°C.


32

Leal-Klevezas et al. (1995) obtained DNA from isolated colonies for use in PCR

by adding 400 μl of lysis solution (2% Triton X-100, 1% sodium dodecyl sulfate, 100

mM NaCl, 10 mM Tris-HCl, pH 8.0) and 10 ml of proteinase K (10 mg/ml) and the

contents were mixed thoroughly and incubated for 30 min at 50ºC. Finally DNA was

extracted by a standard protocol with phenol-chloroform-isoamyl alcohol, precipitating

with 95% ethanol, washing with 70% ethanol and drying.

Romero et al (1995) modified the method described by Wilson (1990) for

extraction of genomic DNA from Brucella cultures. Method included making suspension

of cells in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) followed by sodium

dodecyl sulfate (SDS) and proteinase K digestion. Cell wall debris, denatured proteins

and polysaccharides were removed by precipitation with 5 M NaCl and CTAB-NaCl

solution. DNA was extracted by a standard protocol with phenol-chloroform-isoamyl

alcohol, precipitating with isopropanol, washing with 70% ethanol and drying.

Rijpens et al. (1996) prepared crude cell lysates of Brucella strains for PCR

detection by addition of 100 ml of 0.1 M NaOH-0.25% SDS to the pellet obtained from a

2 ml volume of a pure culture and subsequent heating at 90ºC.

Vizcaino et al. (1997) extracted DNA by mixing the TE buffer (50 mM Tris, 50

mM EDTA, 100 mM NaCl, pH 8±0), 10% SDS solution, and 2% (w/v) proteinase K to

the pellet of the Brucella cells and incubated for 1 h at 37°C. The lysed cell suspension

was extracted twice with phenol-chloroform and nucleic acids were precipitated by

gently mixing the aqueous phase with 2 volumes chilled ethanol.

Navarro et al. (2002) extracted DNA from B. melitensis Rev 1 and from

B. abortus B 19. Bacterial strains were resuspended in phosphate-buffered saline (PBS),


33

pH 7.4, then an equal volume of propanol was added and the recovered cells were stored

at 4ºC. Immediately before use, 400 μl of bacteria were pelleted by centrifugation and

resuspended in TE buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0). The cells

were incubated at 50ºC for 30 min with 400 μl of lysis solution (2% Triton X-100, 1%

SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0) and 10 μl of proteinase K (10 mg/ml).

Cell wall debris, denatured proteins and polysaccharides were removed by precipitation

with 5 mM NaCl and CTAB-NaCl solution and incubating at 65ºC for 10 min. DNA was

extracted with organic solvents by standard protocol and precipitating with 95% ethanol.

Bogdanovich et al. (2004) prepared template DNA by placing one loopful of

bacteria (= one small colony) directly from blood agar plates into an Eppendorf tube

containing 100 μl of double-distilled water and keeping it at 95°C for 10 min before

centrifuging it for 5 min at 13,000 X g. Three microliter supernatant was used as the

template in the final PCR assay.

Probert et al. (2004) prepared crude nucleic acid extracts by resuspending a

loopful of bacteria into 100 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8), boiling the

suspension for 10 min and pelleting the cellular debris by centrifugation. The supernatant

was collected as the crude DNA extract. In some cases, nucleic acids were purified using

DNeasy spin columns (Qiagen Inc., Valencia, Calif.) according to the manufacturer’s

recommendations.

Kanani (2007) prepared genomic DNA from colony according to Wilson (1987)

with minor modifications for confirmation by PCR. He successfully extracted DNA from

colony and confirmed by PCR.


34

2.3.2 Extraction of DNA from Milk

Leal-Klevezas et al. (1995) obtained DNA from 400 μl samples from the fatty top

layer of raw milk by adding 400 μl of lysis solution (2% Triton X-100, 1% sodium

dodecyl sulfate, 100 mM NaCl, 10 mM Tris-HCl [pH 8.0]) and 10 ml of proteinase K

(10 mg/ml) were added and contents were mixed thoroughly and incubated for 30 min at

50oC. Finally DNA was extracted by a standard protocol with phenol-chloroform-isoamyl

alcohol, precipitating with 95% ethanol, washing with 70% ethanol and drying.

Romero et al. (1995) extracted DNA from milk by mixing 100 μl of NET buffer

(50 mM NaCl, 125 mM EDTA, 50 mM Tris-HCl, pH 7.6) to the 500 μl of milk after

incubating at 80ºC for 15 min, and cooled on ice for 2 min. The mixture was digested

with RNase at 50ºC for 1 h followed by SDS and proteinase K digesion at 50ºC for 3 h.

Cell wall debris was removed by precipitation with 5 M NaCl and a CTAB-NaCl solution

at 65oC for 10 min. Finally DNA was extracted by a standard protocol with phenol-

chloroform-isoamyl alcohol, precipitating with isopropanol, washing with 70% ethanol

and drying.

Rijpens et al. (1996) extracted DNA from 1 ml of milk by breaking fat and

proteins with 200 ml of a lipase-phospholipase solution (17,600 U of lipase) and 73 U of

phospholipase A2 (for fat) and 700 ml of a trypsin solution, 0.02 M EDTA,

2.5% Triton X-100 (for proteins) after incubating at 37oC for 1 h. After that the pellet was

washed three times with 1 ml of H2O, resuspended in 35 ml of 0.15 M NaOH–0.5% SDS

and subjected to microwave treatment for 4 min. Finally the DNA was extracted with

phenolchloroform-isoamyl alcohol (24:24:2) in the presence of 0.5 M guanidinium


35

thiocyanate, precipitating with 100% ethanol in the presence of 0.4% Etachinmate and

0.03 M Na acetate and drying.

Romero and Lopez-Goni (1999) evaluated different methods of extraction of

bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR.

They used lysis buffer with high concentrations of Tris, EDTA, and NaCl, high

concentrations of SDS and proteinase K, with high temperatures of incubation for the

efficient extraction of Brucella DNA.

Evangelista et al. (2005) extracted DNA from milk sediment and cream layer after

(Centrifuging milk at 12,000 X g for 1 min) by commercially available kit designed for

soil samples (MO BIO laboratory, Inc, CA.) following manufacturer’s directions.

Leary et al. (2006) extracted DNA from whole milk, sediment and cream layer

using the QIAampe DNA mini kit, glass bead method (for maximum cell disruption) and

PBS-Tween method. They found QIAampe DNA mini kit fit for routine use to extract

DNA from fresh milk as it yielded good quality DNA consistently. They also successfully

extracted DNA from whole blood and lymph node tissue using QIAampe DNA mini kit.

2.3.3 Detection of Brucella in Cultures, Blood, Semen and Clinical Samples by PCR

Romero et al. (1995) amplified a 905 bp fragment, by using a primer pair F4/R2.

As little as 80 fg of Brucella DNA was detected by this method. DNAs from all of the

representative strains of the species along with the biovars of Brucella and from 23

different Brucella isolates were analyzed and yielded exclusively 905 bp fragment. No

amplification was detected with DNAs from 10 phylogenetically related strains to

Brucella, 5 Gram negative bacteria showing serological cross-reactions with Brucella and

36 different clinical isolates of non-Brucella species. Only Ochrobactrum anthropi


36

biotype D yielded a PCR product of 905 bp, suggesting a closer relationship between

Brucella and O. anthropi biotype D.

Matar et al. (1996) developed a PCR based assay for the rapid and specific

laboratory diagnosis of human brucellosis directly from whole blood using specific

primers for the PCR amplification of a 223 bp region on the sequence encoding the

31 kDa immunogenic B. abortus protein (bcsp31). It also determined specificity of

amplification by Southern hybridization and restriction endonuclease analysis. It was

concluded that the test was rapid and specific for the laboratory confirmation of acute

human brucellosis.

Gallien et al. (1998) applied PCR assay to detect Brucella species in the uterus,

udder, spleen, lymph nodes, kidney and liver of 3 cows which had been naturally infected

in an outbreak of brucellosis. They applied a pre-enrichment procedure for the PCR. They

revealed that PCR was more sensitive than the bacteriological method. They also found

that PCR with DNA from 8 Yersinia strains gave no amplification product.

JungSuckChan et al. (1998) developed and evaluated a PCR assay for the

detection of Brucella in bovine semen. They designed and synthesized genus specific

primers from the sequence of a gene encoding a 31 kDa bcsp31 and a 36 kDa ompB of

B. abortus. As little as 1 pg of B. abortus genomic DNA could be detected by this PCR

method. They screened out semen samples from 185 bulls from herds on Cheju Island by

PCR and Seminal Plasma Agglutination Test. The results suggested that the PCR was

found better than the agglutination test for detection of Brucella infection from semen of

bulls.


37

The diagnostic PCR test was standardised using 31 kDa gene and evaluated the

same on clinical samples (Anonymous, 1999). Twenty two, out of 23 field isolates were

confirmed as Brucella positive by PCR.

The PCR was also applied for detecting 31 kDa diagnosis gene sequences in

semen of seropositive bulls. In a comparative study it was observed that the 31 kDa gene

could be detected in the semen of 9 buffaloes but only six corresponding blood samples

were found positive by PCR. They also noticed that, the omp2 gene was only detected in

2 semen samples and 3 blood samples. In one bull the omp2 gene was simultaneously

detected in blood as well as in semen whereas, in other cases it was either from semen or

from blood. This study indicated that the Brucella gene target might have located in

various tissues at a given time that might have affected the detection by PCR. In another

study (Anonymous, 2000), out of 88 semen samples of buffalo, 36 were found positive

by PCR and non-radioactive DNA probe technology employing bcsp31 kDa gene target.

Of the positive samples, 17 were ELISA positive and 19 were ELISA negative bulls.

Amin et al. (2001) in Egypt evaluated PCR for detection of B. melitensis in

bovine and ovine semen. The semen was separated into a seminal fluid, non-sperm cells

and sperm head. Analysis of separated fractions of naturally Brucella contaminated

semen by the PCR revealed that in most of the cases B. melitensis DNA was present in

the seminal fluid and non-sperm fractions. They also observed inhibition of PCR

amplification of the sperm head fraction in control template. Finally they concluded that

inhibition observed in the sperm head fraction and whole semen samples might have been

preliminary due to high DNA concentration.


38

Casanas et al. (2001) evaluated the specificity of a PCR assay for detecting

Brucella DNA using primers specific for the amplification of a 223 bp region of the

sequence encoding a 31 kDa immunogenic bcsp31 and concluded that it had very good

degree of specificity. Together with its high yield demonstrated in previous clinical

studies, this PCR assay could be a useful tool for the diagnosis of human brucellosis.

Morata et al. (2001) evaluated the diagnostic yield of a PCR assay for patients

with focal complications of brucellosis, against conventional microbiological techniques

and concluded that the PCR assay was found far more sensitive than conventional

cultural method. This coupled with its speed and reduction in risk to the laboratory

workers, made this technique a very useful tool for the diagnosis of focal complications

of brucellosis.

Navarro et al. (2002) compared three pairs of primers amplifying three different

fragments (i) a gene encoding a 31 kDa B. abortus antigen (primers B4/B5),

(ii) a sequence 16S rRNA of B. abortus (primers F4/R2) and (iii) a gene encoding an

omp2 (primers JPF/JPR). The three primer assay showed a difference in sensitivity for

detecting purified Brucella DNA. The sensitivity of the primers F4/R2 and B4/B5 was

affected by the presence of human DNA but not of the primers JPF/ JPR. The most

sensitive primers were F4/R2 as they amplified 8 fg of purified B. melitensis Rev 1 DNA.

On the other hand, primers B4/B5 and JPF/JPR amplified 5 pg and 20 pg, respectively.

Varasada (2003) tested 77 human blood samples (50 from occupationally exposed

group and 27 from patients of pyrexia of unknown origin) by PCR targeting sequence of

223 bp of bcsp31 gene using B4/B5 primer set. Twenty (25.97%) of them were found

positive by PCR.


39

Vaid et al. (2004) applied PCR for detection of brucellosis using primers derived

from the 43 kDa outer membrane protein gene of B. abortus, the 16S rRNA gene,

insertion sequence IS711, BCSP31 (Brucella Cell Surface Protein) gene. This technique

provides a promising option in Brucella diagnosis with high sensitivity in detecting

Brucella from pure cultures.

Kanani (2007) compared three pairs of primers amplifying three different

fragments (i) a gene encoding a 31 kDa B. abortus antigen (primer B4/B5),

(ii) a sequence 16S rRNA of B. abortus (primer F4/R2) and (iii) a gene encoding an omp2

(primer JPF/JPR) by testing 101 semen samples from breeding bulls of AI Centers of

Gujarat. He found that B4/B5 primer was more sensitive followed by F4/R2 primer and

JPF/JPR primer.

2.3.4 Detection of Brucella in Milk by PCR

Leal-Klevezas et al. (1995) developed a single step PCR for detection of Brucella

from blood and milk of infected animals. Two oligonucleotides homologous to regions of

the gene encoding for an outer membrane protein (omp) were designed to detect Brucella

in milk and blood of infected goats, cattle and human. The sensitivity of the test and its

ability to detect Brucella in field samples were promising in the diagnosis of brucellosis

in animals and man.

Serpe et al. (1998) developed a rapid PCR method for detection of Brucella in

milk. They used a single step procedure based on freezing and thawing for DNA

extraction and for amplification two oligonucleotides primer homologous to the regions

of the gene coding for a 31 kDa omp characteristic of the genus Brucella. They found the

method was fast and specific for detection.


40

Vesco et al. (2000) applied PCR for the diagnosis of brucellosis in milk by using

two specific target sequences, the first one was a fragment within the 16S rRNA region

and the second one was the 3'-end fragment of 1S711 element for identification of the

species. Oligonucleotides to these regions allowed amplification of species-specific

products with different sizes. They concluded that the PCR could be used in

epidemiological studies to determinate the prevalence of different Brucella biovars.

Evangelista et al. (2005) developed PCR for detection of Brucella DNA from

milk by using the eryD and wboA genes. They found that PCR was more sensitive as

compared to bacteriological method, but less sensitive than the serological methods.

Gupta et al. (2005) developed PCR assay for the detection of Brucella in milk of

22 Barbari, Jamunapari and non-descript goats from nearby villages of the Central

Institute for Research on Goat, Makhdoom, India. The goats had history of abortions and

12 of the 22 goats were serologically positive by STAT. The developed PCR assay

amplified 720bp sequence of the gene encoding the omp 31 antigens. Of these 22 milk

samples tested, 18 samples (82%) were found positive by PCR, which includes the 12

samples positive by STAT. Finally they concluded that PCR assay was faster, safe to use

and had higher sensitivity and specificity.

The PCR kit for detecting omp 25 gene of B. abortus in raw milk was tested by

XiaoAn et al. (2005) to ckeck its storage life, specificity and stability. Total 98 and 350

milk samples were sampled from 98 STAT positive and negative dairy cows from

Ningxia, Gansu, Nei Monggol, Shanxi, Heilongjiang and Xinjiang provinces, China,

separately. They revealed that the positive coincident rate of the PCR kit detecting result

with STAT was 100% (98/98) and the negative coincident rate of the PCR kit detecting


41

result with STAT was 97.71% (342/350) showed that the sensitivity of the PCR kit was

higher than that of the STAT.

Leary et al. (2006) applied PCR assay for detection of B. abortus infection in

blood, milk and lymph tissues by using different primers that amplify various regions of

the Brucella genome, IS711 genetic element, 31 kDa outer membrane protein and

16S rRNA. They found that there was no any amplification when PCR assays applied to

the blood samples, but was detected in a proportion of the culture positive milk (44%)

and lymph tissue samples by the same methods.

2.4 REAL-TIME PCR

Real-time PCR assay renders post amplification manipulations unnecessary.

Sample processing is automated minimizing the risk of carry-over contamination and

reducing hands-on-time. Additionally, real-time PCR allows detecting and quantifying

DNA targets by online monitoring the accumulation of PCR amplification products

during cycling, getting first results before the whole run is ended.

A fluorescence signal can be measured during the PCR process which is obtained

by different approaches, relying on the cleavage of fluorogenic probes, e.g. by

double-stranded DNA intercalating dye (SYBR Green I), by enzymatically released

fluorophores (5’exonuclease assay) or by fluorescence resonance energy transfer

(hybridization probes).

Using SYBR Green I assay, probe design is not needed because of the

non-specific intercalation of the dye in double-stranded DNA followed by fluorescence

emission (Plate 2.1). However melting curve analysis is mandatory and sequencing of the

amplicon can be necessary sometimes.


42

Redkar et al. (2001) developed real-time PCR based assays specific for

B. abortus, B. melitensis and B. suis. The assays utilized an upstream primer that was

derived from 3' end of the genetic element IS711, whereas the downstream primers and

probes were designed from signature sequences specific to a species or a biovar. The

assays were tested on known strains as well as field isolates and were found to be specific

for all known biovars of B. abortus, B. melitensis and biovar 1 of B. suis.

Colmenero et al. (2003) used the LightCycler detection system and

SYBR Green I to develop a rapid diagnostic tool for human brucellosis. This quantitative

real-time PCR assay detected a 223 bp target sequence in a gene, which was highly

conserved at genus level encoding an immunogenic 31 kDa protein of the external

membrane of B. abortus. They examined serum samples of 60 patients suffering from

active brucellosis and the assay reached a sensitivity of 91.9% and a specificity of 96.4%.

A bcsp31 based real-time PCR assay also tested by Debeaumont et al. (2003) and

compared to a real-time PCR assay targeting the B. melitensis omp31 gene encoding a

31 kDa omp found in all Brucella species except for B. abortus. Specificity was

determined by negative amplification results of DNA samples from serologically cross-

reacting bacteria, clinically relevant and potentially sample contaminating bacteria. Both

methods proved to be highly specific and sensitive. One Colony Forming Unit (CFU) per

5 μl of DNA extract was detected.

Newby et al. (2003) evaluated three approaches viz. SYBR Green I (a double-

stranded DNA intercalating dye), 5 -exonuclease (enzymatically released fluors) and

hybridization probes (fluorescence resonance energy transfer) for use in a real-time PCR

assay to detect B. abortus. These assays utilized the same amplification primers to


43

produce an identical amplicon. This amplicon spanned a region of the B. abortus genome

that included portions of the alkB gene and the IS711 insertion element. All three assays

were of comparable sensitivity, providing a linear assay over 7 orders of magnitude

(from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the

hybridization probe assay.

Queipo-Ortuno et al. (2003) used LightCycler technology, SYBR Green I and

primer targeting 223 bp sequence of bcsp31. After optimization of the real-time PCR

protocol as little as two genomic equivalents (about 10 fg) of purified bacterial DNA

could be detected. The assay showed a high reproducibility ranging from 96 to 99%.

Tomaso et al. (2003) established a single tube duplex LightCycler PCR assay

targeting bcsp31 for the detection of Brucella with common pair of primer B4/B5 and

including a PCR system for the detection of bacteriophage lambda DNA as an internal

amplification control. The internal amplification control can be monitored on a separate

channel with a melting temperature clearly different from that of the Brucella specific

target. Template controls and positive controls containing DNA of B. melitensis biovar 1

were included in each run to detect contamination or amplification failure. This

LightCycler PCR assay detected Brucella isolates of all species with a detection limit of

one genome equivalent. No false positive reactions were seen in large panel of bacteria

known to cross-react immunologically with Brucella or with organisms causing similar

syndrome.

Queipo-Ortuno et al. (2005) developed a LightCycler based real-time PCR

(LC-PCR) assay and evaluated its diagnostic use for the detection of Brucella DNA in

serum samples. Following amplification of a 223 bp gene sequence encoding an


44

immunogenetic membrane protein (bcsp31) specific for the Brucella genus, melting

curve and DNA sequencing analysis were performed to verify the specificity of the PCR

products. The LC-PCR assay was found to be 91.9% sensitive and 95.4% specific when

tested with 65 negative control samples and 62 serum samples from 60 consecutive

patients with active brucellosis. They opined that the assay was reproducible, easy to

standardize, having minimum risk of infection in laboratory workers and had a total

processing time of <2 h.

Kanani (2007) quantified the load of Brucella infection in semen of breeding bulls

by real-time PCR using SYBR Green I assay using B4/B5 primer. He found the detection

limit of the assay was 50 CFU/ml of semen. He also tried the TaqMan assay for

confirmation of Brucella isolates, but failed to standardize the assay.

2.5 COMPARATIVE STUDY ON ANTIBODY DETECTION,

CULTURAL AND MOLECULAR METHODS

Prompt and accurate diagnosis is the key to prevent the spread and control of the

diseases so for brucellosis. However, diagnosis of brucellosis is frequently difficult to

establish. This is not only because clinically, the disease can mimic any infectious and

noninfectious disease, but also because the established diagnostic methods are not always

successful. Various research workers have tried to evaluate PCR techniques in the

diagnosis of brucellosis in comparison to conventional techniques like cultural isolation

and serological methods.

Al-Khalaf and El-Khaladi (1989) investigated the presence of Brucella antibodies

in serum and milk of camels in Kuwait by applying three serological tests for serum,

namely RBPT, STAT and CFT, whereas, MRT for milk. The prevalence rate was 14.8%


45

from serum by CFT and RBPT and 10.8% by the STAT. For milk, prevalence rate was

8.0%. They were unable to isolate the Brucella organisms from sediment and cream of

milk, however, B. abortus could be isolated from two of the 5 foetuses.


serum and milk simultaneously for serological and bacteriological testings. Of these,

1,056 (16.31%) were serologically positive and 1632 (25.21%) were positive to MRT.

They also isolated 397 Burcella isolates from MRT positive milk samples, of which 119

came from 5,686 seronegative cows.

El-Gibaly (1993) correlated serological tests and isolation of B. melitensis in an

infected sheep farm. They retested 60 STAT positive reactors sheep at the time of

slaughter (3 months later) and different tissue specimens were collected for cultural

isolation. The testing showed that 41 (68.3%), 53 (83.3%), 35 (58.3%) and 30 (50%)

sheep were positive in STAT, BPAT, RBPT and Riv. test, respectively. Bacteriological

examination confirmed the isolation of 34 strains of B. melitensis biovar 3. The organism

could be isolated from 7 animals out of 19, 1 out of 7, 11 out of 25 and 12 out of 30

serologically negative cases to STAT, BPAT, RBPT and Riv. test, respectively.

Ferris et al. (1995) compared results of 6 serological tests (Particle Concentration

Fluorescence Immunoassay, ACF Assay, Card Test, Buffered Acidified Plate Antigen

Assay, STAT and Rivanol Test) for diagnosis of brucellosis with the results of bacterial

cultivation from 221 pigs of 39 naturally infected herds. They observed that the

sensitivities varied from 57% (ACF Assay) to 83% (STAT) and specificities ranged from

62% (STAT) to 95% (Rivanol Test). In this study B. suis could be isolated from 46 of 221

(21%) pigs. Out of thses 46 culturally positive pigs, 8 were found negative by all the six


46

serological tests. Thus, antibodies could not be detected in some of the culturally positive

pigs by various serological tests. The results illustrated the difficulty of eliminating

brucellosis by a “test-and-removal programme” and supported the policy of depopulating

infected herds.

Milk samples from 56 Brucella milk culture positive cattle and 37 cattle from

Brucella-free herds were examined for Brucella DNA by PCR and for specific antibodies

by an I-ELISA by Romero et al. (1995). The specificities of both the tests were found

100% on testing the milk samples from Brucella-free cattle. The 49 milk samples from

infected cattle were found positive by PCR (87.5% sensitivity) and 55 were found

positive by ELISA (98.2% sensitivity). One PCR positive sample was found negative by

ELISA and 7 ELISA positive samples were found PCR-negative, yielding an observed

proportion of agreement of 0.91 for the 2 tests. Although the results suggested that

ELISA considered being a better screening test than PCR, the combined sensitivity of

both the assays was 100% and simultaneous application could be more useful than one

test alone for rapid screening of brucellosis in dairy cattle.

Gallien et al. (1998) used PCR assay to detect Brucella species from the uterus,

udder, spleen, lymph nodes, kidney and liver of 3 cows, which had been naturally

infected in an outbreak of brucellosis and compared their results with the result of

bacteriological investigations. All 18 samples reacted positively in the PCR, but

5 samples had weak bands after the electrophoretic separation of PCR mixtures. Brucella

could not be isolated from the 5 samples.

JungSuckChan et al. (1998) compared PCR assay with conventional methods by

collecting semen samples from 185 bulls from serologically negative herds for


47

brucellosis on Cheju Island. They found that 5 bulls were positive by cultural and PCR

methods whereas, one was positive and 5 were suspicious by the semen plasma

agglutination test. In another study, semen samples obtained from 177 bulls were

negative by semen plasma agglutination, culture and PCR methods. Finally the results of

comparative tests suggested that the PCR was a better test than the agglutination test on

semen from bulls.

A total of 150 individual samples of blood and raw milk of cows collected from

El-Behera Governorate were subjected for STAT, MRT and cultural isolation of Brucella

organisms by Abdel Hakiem (2000). The results showed that MRT was found to be

reliable and sensitive, as it gave positive results in 8% of the samples, as compared with

the results of STAT (10%) for serum. Whereas, only one (0.7%) of the samples yielded

Brucella.

Guarino et al. (2000) collected 44 blood samples from buffaloes belonging to

several herds in the province of Caserta, Italy, during routine monitoring of Brucella

infections. They also collected supramammary lymph nodes for bacteriological culture.

The DNA was extracted from whole blood and employed PCR to detect Brucella. All

blood sera were tested for the presence of Brucella antibodies using I-ELISA as a

screening method and CFT for confirmation. In the study Brucella or other organisms

could not be detected in whole blood samples by cultural isolation. However, they found

13 blood samples from different naturally infected buffalo herds were positive by gene

specific PCR (29.5%) for detection of Brucella species. PCR and serological assays

agreed in 19 cases and 5 samples that were negative by CFT and ELISA found positive

by PCR. Six borderline samples were found negative by CFT but near to the limit of


48

detection for ELISA, two of these were found positive by PCR. In one case B. abortus

could be isolated from lymph gland by culture, showed positive by PCR but negative by

CFT and ELISA. The study indicated that PCR analysis can be complementary to

classical serological tests for the detection of the etiological agent of Brucella infections

in living buffaloes, especially in the initial phase when the immune response of the

animal is not detectable.

Leal-Klevezas et al. (2000) compared PCR, serological and bacteriological

techniques to diagnose goat brucellosis. Milk and blood samples were taken from

randomly chosen 22 females and one male out of 300 clinically healthy mixed breed of

goats. They processed milk and blood samples for bacteriological cultures and DNA of

the pathogen whereas sera were tested by RBPT. Results showed that 86% of the blood

samples were positive on the PCR test, while 60% were positive on the serological test.

The pathogen could be isolated from only one blood culture. Sixty four percent of the

milk samples were positive on PCR tests, but failed to yield bacteria in the culture. This

study demonstrated the higher sensitivity of PCR over RBPT and blood culture.

Amin et al. (2001) in Egypt compared PCR for detection of Brucella melitensis

DNA in bovine and ovine semen by culture isolation. Semen samples were collected from

serologically RBPT positive animals and their reciprocal titers were varied between 1/80

to 1/640 by the STAT. They evaluated PCR as more sensitive than the traditional culture

methods since it detected Brucella DNA in 12 (10%) out of 120 semen samples while

direct culture detected only 7 (5.8%) from the same semen samples. The limit of

detection by PCR was found to be 100 CFU/ml of semen in their study. They found


49

superiority of PCR over a traditional culture method and recommended the use of PCR as

routine assay.

Leyla et al. (2003) evaluated detection of Brucella DNA directly from the

stomach contents of aborted sheep foetuses with culture isolation. From 38 of 39 culture

positive foetal stomach contents B. melitensis-specific DNA was detected by PCR and

found negative in all of the culture negative samples. Compared with culture, sensitivity

and specificity of PCR were determined as 97.4 and 100%, respectively. The results

indicated that this PCR procedure had a potential for use in routine diagnosis of sheep

brucellosis.

Manterola et al. (2003) compared sensitivity and specificity of PCR assay using

primers derived from the insertion sequence IS6501 with that of bacteriological culture

and serological tests for the diagnosis of B. ovis infection in rams. The comparison of the

semen culture and PCR was done at 4th, 5th, 6th and 8th week post inoculation in 14

experimentally infected rams with B. ovis. The study revealed 23 samples positive in

culture and 19 samples positive in PCR. Although PCR was able to detect all 10 rams that

were positive by culture on at least one sampling date during the experiment period.

Further they evaluated PCR with 101 semen swabs from field rams, belonging to flocks

naturally infected with B. ovis, whose serological and semen culture status was known. A

total of 52 rams showed serological evidence of B. ovis infection, but only 26 (50%) of

the seropositive were found positive in semen culture. Out of theses 26 positive rams, 22

(84.6%) were positive in the PCR test. In the same study five PCR positive samples were

obtained in 26 seropositive but culturally negative rams, whereas 3 semen samples from

49 rams serologically and culturally both negative, were found positive in the PCR. On


50

considering serological results as gold standard, relative sensitivity for the diagnosis of

B. ovis infections were found 50% for semen culture and 51.9% for PCR by them. The

comparison of the semen culture and PCR results from 192 semen samples tested,

showed a proportion of agreement of 0.91 between both the tests. They concluded that

PCR based test was having sensitivity similar to that of semen culture and could be used

as a complementary test for the direct diagnosis of B. ovis in semen samples of rams.

Nimri (2003) tested blood specimens from brucellosis suspected cases of human

patients by serology, culture and PCR. Study included peripheral blood specimens from

50 healthy control subjects and 165 seropositive patients having compatible signs and

symptoms that were clinically diagnosed to have brucellosis. These specimens were

tested by blood culture and by PCR using genus specific primers from the conserved

region of the 16S rRNA sequence. Diagnosis of Brucella was established by PCR in 120

cases (72.7%). All of them were seropositive and 20 were positive by culture. Forty-eight

from 58 (82.8%) of the relapsed cases two months after completing the treatment with an

increase in the previous serological titers were positive by PCR. The assay had 85.7%

positive predicative value, 100% sensitivity and specificity since it correctly identified all

cases that were positive by blood cultures, 95.8% by serology and none of the control

group was positive. It was concluded that PCR assay can be applied with serology for the

diagnosis of brucellosis suspected and relapsed cases regardless of the duration or type of

the disease without relying on the blood cultures, especially in chronic cases.

While comparing PCR, RBPT and STAT for diagnosis of brucellosis in human

being (50 from occupationally exposed group and 27 from patients of pyrexia of


51

unknown origin), Varasada (2003) found highest number of positive results by PCR (20)

followed by RBPT (6) and STAT (3).

Gall and Nielsen (2004) reviewed over 50 publications in which the sensitivity

and specificity values of assays used for the detection of exposure to B. abortus had been

examined. Mean sensitivity of culture, RBPT, STAT, PCR and I-ELISA were found to be

46.1%, 81.2%, 75.9%, 82.0% and 96.0%, respectively, while mean specificity were found

100.0%, 86.3%, 95.7%, 98.6% and 93.8%, respectively. The sum of the sensitivity and

specificity values for each test was averaged to give a performance index (PI) and

allowed for a comparison between the different methodologies. A score of 200 was

considered perfect. Based on the PI, they found the buffered antigen plate agglutination

test (BPAT) rated highest (PI=193.1) indicating better accuracy than the other

conventional tests including the RBPT (PI=167.6) and the CFT (PI=172.5). They

observed, the primary binding assays, including the fluorescence polarization assay

(PI=196.4), the I-ELISA (PI=189.8) and the competitive ELISA (PI=188.2), were overall

more accurate than the conventional tests, except for the BPAT.

Lavaroni et al. (2004) compared diagnosis of bovine brucellosis, using PCR in

blood, I-ELISA, in vitro isolation in milk and CFT and competitive ELISA in serum.

Serological tests showed 100% sensitivity related to PCR. The specificity for CFT,

competitive ELISA and I-ELISA were 100%, 99% and 95%, respectively. It was

concluded that PCR could be useful to identify Brucella biotypes and to complement

serologic tests.

Scarcelli et al. (2004) analysed samples of abomasal contents, organs and/or

foetal annexes of 67 aborted bovine foetuses by means of bacteriological methods and by


52

the PCR multiplex for Brucella and Leptospira. PCR-multiplex showed 50.7% (34/67) of

the samples positive for Brucella. However, the Brucella could be isolated from

38.8% (26/67) of the samples, which showed an 88% agreement rate between the two

methods used. Results showed that PCR found more sensitive than culture in bovine

brucellosis cases.

Gupta et al. (2005) developed PCR assay for the detection of Brucella in milk of

22 Barbari, Jamunapari and non-descript goats from nearby villages of the Central

Institute for Research on Goat, Makhdoom, India. The goats had history of abortions and

12 of the 22 goats were serologically positive by STAT. The developed PCR assay

amplified 720bp sequence of the gene encoding the omp 31 antigens. Of these 22 milk

samples tested, 18 samples (82%) were found positive by PCR, which included the 12

samples positive by STAT. Finally they concluded that PCR assay was faster, safe to use

and had higher sensitivity and specificity.

Rahman (2005) evaluated serological and cultural methods for diagnosis of

B. abortus biotype 1 infection in experimentally infected Sprague-Dawley rats. By RBPT,

STAT, Mercapto Ethanol Test and Plate Agglutination Test, the antibodies titres were

remained constant at 1st and 2nd weeks post-infection and increased at 4th week post-

infection then gradually decreased. Further, no antibody titres were found in sera of

24 weeks post-infection both through Plate Agglutination Test and STAT despite the

presence of bacteremia. However, RBPT and Mercapto Ethanol Test revealed the

antibody titres at 24th week post-infection.

XiaoAn et al. (2005) tested PCR kit for detecting omp25 gene of B. abortus in

raw milk from dairy cows in different provinces of China. Out of total 448 samples


53

tested, 98 milk specimens were from 98 STAT positive dairy cows separately and the 350

milk specimens from STAT negative dairy cows separately. It was observed that the

positive coincident rate of the PCR kit detecting result with STAT was 100% (98/98)

while negative coincident rate was 97.71% (342/350). However, 8 samples showing

negative by STAT were proved to be Brucella positive by the kit, showing higher

sensitivity than that of the STAT.

Gupta et al. (2006) calculated sensitivity and specificity of the tissue PCR in

comparison to serology by collecting samples from culturally positive and negative goats.

They employed STAT and dot-ELISA for antibodies detection and amplified a target

sequence of 720 bp on omp31 gene specific for B. melitensis in PCR. In the study PCR

showed 83% sensitivity and 100% specificity in comparison to 67% and 83% sensitivity

and specificity, respectively, in serology. The PCR detected 12 samples exclusively

positive, which were not detected in serology.

Leary et al. (2006) assessed the viability of using conventional and real-time PCR

assays as potential diagnostic tools for the detection of B. abortus in naturally infected

cows. In this study, PCR assays that amplified various regions of the Brucella genome,

IS711 genetic element, 31 kDa omp and 16S rRNA, were optimised using nine known

Brucella strains. They also used real-time PCR for examining the detection efficiency of

the IS711 assay and was estimated at 10 gene copies. B. abortus could not be detected in

blood samples collected from naturally infected cows by conventional or real-time PCR,

but was detected in a proportion of the culture positive milk (44%) and lymph tissues

(66% – retropharyngeal, 75% – supramammary) samples by the same methods. They

found no difference between PCR and bacteriological detection methods. On the basis of


54

their results they suggested that there was no advantage of using PCR methods over

standard serological and bacteriological methods.

Kanani (2007) compared serological, cultural and molecular methods for

detection of Brucella infection in serum and semen of 101 bulls of AI Centers of Gujarat.

He revealed 5.94% (6), 9.90% (10) and 9.90% (10) of bulls found positive by RBPT,

STAT and ELISA, respectively. While, 7.92% (8) of bulls found culturally positive.

Among the PCR assays 18.81% (19), 1.98% (2) and 4.95% (5) of bulls found positive by

B4/B5, JPF/JPR and F4/R2 primer pairs, respectively. Finally he concluded that a PCR

assay was more sensitive as compared to other two serological and cultural methods.


55

CHAPTER - III

MATERIAL AND METHODS

The present work on antibody detection, cultural examination and molecular

detection of Brucella infection in bovines (cattle and buffaloes) was carried out at the

Department of Veterinary Microbiology, College of Veterinary Science and Animal

Husbandry, Anand Agricultural University, Anand. A total of 231 serum and 53 milk

samples from bovines were collected for the study (Table 3.1).

Table 3.1 Details of samples collected from bovines

Animal

Samples

Serum Milk

Cattle 47 13

Buffalo 184 40

Total 231 53

3.1 GENERAL MATERIALS

3.1.1 Glasswares and Plasticwares

During the course of this study, properly cleaned, neutral and standard glasswares

and plasticwares compatible with molecular biology work were used.

3.1.2 Media, Stains, Chemicals, Buffers, Reagents etc.

The details of media, stains, chemicals, buffers and molecular biological reagents

used during the study were as per the Appendix.

Material and Methods…

56

3.2 COLLECTION OF SAMPLES

3.2.1 Serum

About 9 ml of blood was collected aseptically from the jugular vein of individual

animal in a vacuette with serum clot activator (Greiner bio-one, Austria). The vacuettes

were kept in upright position at room temperature for about 2 h. The separated serum was

collected in a screw capped plastic vials and transported to the laboratory. The serum

samples were heat inactivated at 56ºC for 30 min and merthiolate (1:10,000) was added

in all vials as a preservative. The sera were stored at -20ºC till further use. Collected

serum samples were subjected to Rose Bengal Plate Test (RBPT), Standard Tube

Agglutination Test (STAT) and Enzyme Linked Immunosorbent Assay (ELISA).

3.2.2 Milk

The udder was thoroughly washed and cleaned with potassium permanganate

solution (1:1000) and dried with clean cloth. Teat openings were disinfected with 70% of

ethyl alcohol. After discarding few drops of milk, approximately 10 ml of milk from each

quarter was collected in two sets of sterile screw capped plastic vials and transported on

the ice to the laboratory. One set was used for cultural isolation and another was used for

Milk Ring Test (MRT), ELISA and DNA extraction. The milk samples were stored at

-20ºC for future use.

3.3 REFERENCE BACTERIAL STRAINS

The Brucella abortus biovar 1 strain 544 (Biotechnology Laboratory,

National Dairy Development Board, Anand) and Brucella abortus live vaccine strain 19

(Bruvex, Indian Immunologicals Limited, Hyderabad) were used as reference bacterial

strains for cultural and molecular work.


57

3.4 RBPT

3.4.1 RBPT Antigen

The antigen obtained from the Indian Veterinary Research Institute (I.V.R.I.),

Izatnagar, Uttar Pradesh was used for the test.

3.4.2 Procedure

The test was performed according to the manufacturer's literature.

Serum samples and RBPT antigen were brought to the room temperature and then

one drop (0.03 ml) of serum was taken on a clean, dry and non greasy glass slide by

micropipette. The antigen bottle was shaken well to ensure homogenous suspension and

then one drop (0.03 ml) of the antigen was added. The antigen and serum were mixed

thoroughly with the spreader and then the slide was rotated for four min. The result was

noted immediately after four min.

3.4.3 Observation of Result

Definite clumping/agglutination was considered as positive reaction, where as no

clumping/agglutination was considered as negative.

3.5 STAT

3.5.1 Brucella STAT Antigen

The antigen obtained from the I.V.R.I., Izatnagar was used for the test.

3.5.2 Procedure


All serum samples were tested up to minimum of five dilutions. For high titre

sera, more dilutions were prepared in order to achieve end point titre.

Five agglutination tubes were placed in a rack. 0.8 ml of 0.5 % phenol saline was

taken in a first tube and 0.5 ml in rest of the tubes. 0.2 ml of serum was added in the first


58

tube, mixed well and transferred 0.5 ml of diluted serum to the second tube. The process

was continued up to the fifth tube and 0.5 ml was discarded from the last tube after

mixing. 0.5 ml of antigen was added to each tube and mixed thoroughly. This provided

final dilutions of 1:10, 1:20, 1:40, 1:80 and 1:160 and so on. Considering the special

significance of 50 per cent end point, a control tube was set up to simulate 50 per cent

clearing by mixing 0.5 ml of antigen with 1.5 ml of 0.5 % phenol saline in an

agglutination tube. All the tubes were incubated at 37ºC for 20 h before result was

recorded.


The degree of agglutination was judged by opacity of the supernatant fluid. The

highest serum dilution showing 50 per cent or more agglutination (50 % clearing) was

considered as the titre of the serum. The titre so obtained was expressed in unit system by

doubling of the serum titre as International Unit (I.U.) per ml of serum.

3.5.4 Interpretation of Result

80 I.U. per ml or above was considered positive for brucellosis.

3.6 ELISA

Brucella Antibody Test Kit, ELISA along with the user manual was procured from

VMRD, Inc., U.S.A and the test was performed as per the protocol outlined in the user

manual. The contents of the kit were as in Table 3.2 and Plate 3.1.


59

Table 3.2 Contents of Brucella ELISA Kit

Sr.No. Items Quantity Storage

1.Antigen-Coated Plates 5 plates 2-7º C

2.Positive Control 1 ml 2-7º C

3.Negative Control 1 ml 2-7º C

4.100X Antibody-Peroxidase Conjugate 1 ml 2-7º C

5.Conjugate Diluting Buffer 75 m1 2-7º C

6.Substrate Solution 75 m1 2-7º C

7.Stop Solution 75 m1 2-7º C

8. 10X Wash/Diluent SolutionConcentrate

400 m1 2-7º C

3.6.1 Preparation of Reagents

i Warming up of reagents: Samples, reagents and plate(s) were brought to room

temperature prior to starting the test.

ii Preparation of wash/diluent solution: 1X wash/diluent solution was prepared by

diluting one part of the l0X wash/diluent solution concentrate with nine parts of

distilled water. Approximately 3 ml was prepared per well.

iii Preparation of serum samples: Serum samples were diluted 1:100 with 1X

wash/diluent solution.

iv Preparation of controls: Positive and negative controls were diluted 1:100 in 1X

wash/diluent solution.

v Preparation of plates: The plate(s) was removed from the foil pouch (es).

The strips to be used were placed in the frame and numbered the top of each

strip to maintain orientation with the setup record.


60

vi Preparation of conjugate: 1X antibody-peroxidase conjugate was prepared by

diluting one part of 100X antihody-peroxidase conjugate with 99 parts of

conjugate diluting buffer.

3.6.2 Test Procedure

a) Loading of samples and controls: Using a pipettor set at 100 μl, samples and

controls were transferred to the antigen-coated plate. The loaded assay plate was

gently mixed by tapping the side of the plate several times by taking care not to

spill samples from well to well. The plate was incubated 30 min at room

temperature (21-25°C).

b) Washing of wells: After incubation, the plate was washed four times by manual

washing. For manual washing, the contents of the wells were dumped into a

sink and the remaining sera and controls were removed by striking sharply the

inverted plate four times on a clean paper towel. Each well was filled

immediately with 1X wash/diluent solution using a multichannel pipettor, then

the solution was dumped and struck the inverted plate sharply on a clean paper

towel as above. This washing procedure was repeated thrice (total four washes).

c) Adding conjugate: 100 μl of diluted (1X) antibody-peroxidase conjugate was

added to each well. The plate contents were gently mixed by tapping the side of

the plate several times. The plate was incubated uncovered for 30 min at room

temperature (21-25°C).

d) Washing of wells: After incubation, the washing procedure was repeated as per

Step b (total four washes).

e) Adding of substrate solution: 100 μl of substrate solution was added to each

well. The contents were gently mixed by tapping the side of the plate several


61

times. The plate was incubated 10 min at room temperature (21-25°C).

Exposure of plate to the direct sunlight was avoided.

f) Adding of stop solution: 100 μl of stop solution was added to each well. The

contents were gently mixed by tapping the side of the plate several times.

g) Reading and recording of the test result: Immediately after adding the stop

solution, the plate was read on a plate reader. The optical density (OD) reading

wavelength was set to 620 nm.

3.6.3 Validation of Test

The test was considered valid when mean OD of the negative controls was

< 0.250 and the mean OD of the positive controls was ≥ 0.500 and ≤ 1.800.


SP Ratio was calculated as follows:

SP = [(Sample OD - Mean NC) ÷ (Mean PC - Mean NC)] x 100

Where:

SP = Sample/Positive Control Ratio

Sample OD = OD value of sample

Mean NC = Mean OD value of Negative Control

Mean PC = Mean OD value of Positive Control

Samples producing SP ratio <25 were considered negative, where as with SP

ratios ≥ 25 were considered positive.


62

3.7 MILK RING TEST (MRT)

3.7.1 Abortus Bang Ring Antigen (ABR Antigen)

The antigen obtained from the I.V.R.I., Izatnagar was used for the test.

3.7.2 Procedure


MRT was performed on individual milk samples.

Antigen and milk samples were brought to the room temperature prior to

performing the test. About 30-50 μl of antigen was added to the 2 ml of milk in a narrow

test tube and mixed thoroughly. The tubes then were incubated at 37°C for 1 h together

with positive and negative working standards.


A strongly positive reaction was indicated by formation of dark pink ring above a

white milk column. The test was considered to be negative if the pink colour of the

underlying milk exceeds that of the cream layer.

3.8 ELISA

The same Brucella Antibody Test Kit, ELISA (VMRD, Inc., USA) which was

used for serum was also used for milk. The test was performed as per the protocol

outlined in the user manual.

The contents of the kit were as in Table 3.2 and Plate 3.1.

Preparation of reagents and procedure were same as for serum (3.6.1 & 3.6.2)

except that milk samples were tested undiluted after removing milk fat. The validation of

the test and observation of the result were also same as per 3.6.3 & 3.6.4.


63


For isolation and identification of Brucella from bovine milk, the standard

procedures (Alton et al., 1988, Quinn et al., 1994 and OIE, 2004) were followed. The

isolates were further confirmed by molecular techniques.

3.9.1 Isolation

For isolation of Brucella from milk, about 100 µl of milk pellet and milk fat were

separately inoculated by spreading on Brucella agar medium (BAM) plates. Milk pellet

was prepared by centrifuging milk at 6000-7000 rpm for 15 min. The plates were

incubated at 37oC for minimum 15 days under 10% CO2 tension (Carbon dioxide

incubator). The plates were observed at every 24 h for observation of the growth. The

suspected colonies of Brucella were picked up and transferred to another BAM plates and

incubated under 5% CO2 tension to obtain pure culture.

The isolates so obtained were streaked on plates of blood agar (BA) and

MacConkey agar (MA). The isolates producing haemolysis on BA and

lactose-fermenting colonies on MA were eliminated considering non Brucella.

The isolates suspected for Brucella were subjected to Gram staining and modified

Ziehl-Neelsen (MZN) staining for checking the purity of cultures and morphological

characters.

3.9.2 Identification

The isolates suspected to be of Brucella were subjected to agglutination and

biochemical tests as described below.

3.9.2.1 Rapid slide agglutination test: One drop (0.03 ml) of Brucella positive field

serum was taken on a glass slide by micropipette. The loopful culture from single


64

suspected colony was mixed thoroughly with the spreader and then the slide was rotated

for four min. The result was read immediately. Definite clumping/agglutination was

considered as positive reaction, where as no clumping/agglutination was considered as

negative.

3.9.2.2 Acriflavine test: One drop of acriflavine solution (1:1000 diluted in distilled

water) was placed on a glass slide. The loopful of culture was mixed thoroughly and

observed for agglutination. Smooth colonies were remained in suspension while rough

colonies were agglutinated.

3.9.2.3 Oxidase test: Standard oxidase discs (HiMedia Laboratories Ltd., Mumbai)

were used to perform the test. The loopful culture from single colony was just touched on

the disc. Immediate development of blue colour was considered as positive test.

3.9.2.4 Catalase test: This test was performed by taking 2-3 drops of 3 per cent H2O2 on

clean grease-free glass slide and single colony from BAM plate was mixed with the help

of a wire loop. Immediate formation of gas bubbles was considered as positive test.

3.9.2.5 Nitrate reduction: Few drops of 4 days old broth culture were added in peptone

water containing 0.1 per cent Potassium nitrate and then incubated under 5% CO2 tension

at 37°C for 2 days. Presence of nitrate was detected by adding approximately 1.0 ml of

sulfanilic acid and 1.0 ml of α-naphthylamine reagent to nitrate broth culture.

Development of a distinct red colour (which may turn to brown rapidly) was considered

as positive test.

3.9.2.6 Urease test: Urea agar slants were inoculated and incubated under 5% CO2

tension at 37°C and observed up to 7 days. A positive reaction was observed by

development of pink colour in the slant.


65

3.9.2.7 Indole test: Few drops of xylene were added in a 4 days old growth of the isolate

in two ml of tryptone water and mixed thoroughly to dissolve indole and about 0.2 ml of

Kovac’s reagent was added from side of the test tube. Pink layer of xylene was

considered as positive reaction.

3.9.2.8 Motility and production of H2S: Motility Sulphide Medium (HiMedia

Laboratories Ltd., Mumbai) was used for detection of motility and H2S production. The

loopful culture from single colony was stabbed in to the tube and incubated under 5%

CO2 tension at 37ºC. H2S production was indicated by blackening of the medium. Non-

motile organisms revealed growth along stabbed line while motile revealed diffused

growth.

3.9.3 Confirmation by Polymerase Chain Reaction (PCR)

PCR was used for confirmation of the Brucella isolates. The template DNA from

colony was prepared according to Wilson (1987) with minor modifications.

3.9.3.1 Preparation of material for nucleic acid extraction from colony

Suspected colonies from BAM plates were streaked on BAM slants. Slants were

incubated at 37°C for 4 to 5 days at 5% CO2 tension. After visible growth on the slant,

colonies were washed with 2 to 4 ml of PBS (pH 6.4). The suspension in PBS was

centrifuged at 10,000 rpm for 10 min at 50°C. The supernatant was discarded and the

pellet was used for extraction of nucleic acid.


66

3.9.3.2 Solutions used for extraction

i) Tris-EDTA (pH 8.0)

ii) 10 mM Tris-HCL

iii) 1 mM EDTA

iv) SDS (10% w/v)

v) Proteinase K solution (20mg/ml, w/v) (MBI Fermentas)

vi) 5 M Sodium chloride

vii) CTAB (Hexadecyl trimethyl ammonium bromide, 10% solution in 0.7M

NaCl)

viii) Tris saturated phenol (pH 8.0)

ix) Chloroform

x) Isoamyl alcohol

xi) 7.5 M Ammonium acetate

xii) Chilled absolute ethanol

xiii) 70% Ethanol

xiv) 0.3X TE (Appendix)

3.9.3.3 Isolation of genomic DNA (template DNA) by proteinase K-SDS method

Pellet containing bacterial cells was suspended in 2 ml of Tris-EDTA (pH

8.0), 250 μl of SDS (10% w/v) and 10 μl of proteinase K solution (20

mg/ml, w/v) and incubated for 1 h at 37ºC.

Subsequently, 500 μl of 5 M NaCl followed by 100 μl CTAB (10% solution in

0.7 M NaCl) were added and incubated in water bath for 10 min at 65ºC.


67

The solution was spun at 8,000 rpm for 10 min after mixing with equal volume

of chloroform : isoamyl alcohol (24:1). The upper phase was transferred to a

clean microfuge tube.

Equal volume of phenol : chloroform : isoamyl alcohol (25:24:1) was added,

mixed well by inverting, then spun for 10 min at 10,000 rpm. The upper

aqueous phase was transferred to a clean microfuge tube.

In the collected supernatant, the DNA was precipitated with equal volume of

chilled absolute ethanol in the presence of one-tenth volume of 7.5 M

ammonium acetate.

Tube was centrifuged for 10 min at 11,000 rpm and supernatant was discarded.

The pellet was washed in 70% ethanol and again spun for 10 min at 11,000 rpm.

Supernatant was discarded and the step was repeated twice.

The pellet was dried at room temperature for overnight.

DNA was resuspended in 200 μl sterile distilled water or 0.3X TE and kept in

water bath at 65ºC for one hour and stored at –20ºC till use.

3.9.3.4 Quantitation and quality assessment of DNA

a) Materials

i) Gel loading buffer 6X (Appendix)

ii) Agarose gel (0.8%)

iii) 0.5X TBE (Appendix)

iv) Ethidium bromide

b) Method


68

Quality and purity of DNA were checked by submarine agarose gel

electrophoresis using 0.8% agarose in 0.5X TBE (pH 8.0) buffer (Sambrook et al., 1989).

Ethidium bromide (1%) was added @ 5µl/100ml. The wells were charged with 5µl of

DNA preparations mixed with 1µl of 6X gel loading buffer dye. Electrophoresis was

carried out at 5V/cm for 20 min at room temperature and then the DNA was visualized

under UV transilluminator.

Quantity of DNA was calculated by nano drop spectrophotometric method. OD at

260 and 280 nm was taken in nano drop spectrophotometer with distilled water as

reference. Purity of DNA was judged on the basis of optical density ratio at 260:280 nm.

The samples with acceptable purity (i.e. ratio 1.7-2.0) were used for PCR.

3.9.3.5 PCR

a) Materials

i) 2X Master Mix (Catalog No.K0710, MBI Fermentas)

ii) Nuclease free distilled water

iii) Extracted DNA

iv) Thin walled PCR tubes of 200 µl capacity (Bio-Rad)

v) Tubes of 500µl capacity (Axygen)

vi) Primers: three pairs of primers (synthesized by MWG-Biotech AG,

Germany) were used for PCR amplification as per the details given in

Table 3.3.


69

Table 3.3 List of Primers

Name of

primers

Sequence

5’─ 3’

Product

length

(bp)

References

B4 (F) TGG CTC GGT TGC CAA TAT CAA223

Baily

et al. (1992)B5 (R) CGC GCT TGC CTT TCA GGT CTG

JPF (F) GCG CTC AGG CTG CCG ACG CAA193

Leal-Klevezas

et al. (1995)JPR (R) ACC AGC CAT TGC GGT CGG TA

F4 (F) TCG AGC GCC CGC AAG GGG905

Romero

et al. (1995)R2 (R) AAC CAT AGT GTC TCC ACT AA

(F) = Forward primer; (R) = Reverse primer

b) Method

PCR of suspected colonies was carried out in final reaction volume of 25 µl in

thermal cycler (MyCycler, Bio-Rad, USA). Quantity and concentration of various

components used for colony PCR were as per Table 3.4. Steps and conditions of thermal

cycling for different primer pairs in PCR were as per Table 3.5.

Table 3.4 Quantity and concentration of various components used in PCR

Sr. No. Components Colony PCR

1. PCR Master Mix (2X) 12.5 µl

2. Forward Primer (10 pmol/l) 1 µl

3. Reverse Primer (10 pmol/l) 1 µl

4. Template DNA 3 µl

5. Distilled water 7.5 µl


70

Table 3.5 Steps and conditions of thermal cycling for different primer pairs in PCR

Primers

(Forward

and

Reverse)

Cycling conditions

Initial

denaturationDenaturation Annealing Extension

Final

extension

B4 (F)

B5 (R)

93°C

5 min

90°C

1 min

64°C

30 sec

72°C

1 min

72°C

10 min

Repeated for 35 cycles

JPF (F)

JPR (R)

94°C

4 min

94°C

1 min

60°C

1 min

72°C

1 min72°C

3 minRepeated for 35 cycles

F4 (F) 95°C

5 min

95°C

30 sec

54°C

90 sec

72°C

90 sec72°C

6 minR2(R)

Repeated for 30 cycles

c) Visualization of PCR products by agarose gel electrophoresis

i) Materials

1. Gel loading buffer 6X

2. Agarose gel (2%)

3. 0.5X TBE (Appendix)

4. Ethidium bromide

ii) Method

To confirm the targeted PCR amplification, five l of the PCR products from each

tube was mixed with one l of 6X gel loading buffer and electrophoresed along with

DNA molecular weight marker (Gene Ruler, MBI Fermentas) on 2.0% agarose gel

containing ethidium bromide (at the rate of 0.5 g/ml) at constant 80V for 30 min in


71

0.5 X TBE buffer. The amplified product was visualized as a single compact band of

expected size under UV light and documented by gel documentation system (SynGene,

Gene Genius BioImaging System,UK).

3.10 MOLECULAR DETECTION OF BRUCELLA FROM MILK

3.10.1 DNA Extraction from Milk Spiked with Reference Bacteria

DNA extraction from milk spiked with reference bacteria was carried out by

following different methods.

I. By boiling method

In this method the milk spiked with reference bacteria was heated at 95°C in

water bath for 1 h and chilled on ice prior to processing for PCR.

II. DNA extraction as per the procedure described by Romero and Lopez-Goni

(1999)

a) Materials

i) NET buffer (Appendix)

ii) 24% Sodium dodecyl sulfate (SDS) solution

iii) Proteinase K solution (MBI Fermentas, 20 mg/ml, w/v)

iv) Phenol

v) Phenol: Chloroform: Isoamyl alcohol (Appendix)

vi) Chloroform: Isoamyl alcohol (Appendix)

vii) Isopropanol

viii) 3 M Sodium acetate ( pH 5.5) (Appendix)

ix) Absolute ethanol


72

x) 70% ethanol

xi) 0.3X TE (Appendix)

b) Method

500 µl of milk was thoroughly mixed with 100 µl of NET buffer.

After that 85 µl of 24% SDS was added, thoroughly mixed by vortexing and

incubated at 80 ºC for 10 min.

Mixture was immediately cooled on ice for 10 min.

After that 12 µl proteinase K solution (20 mg/ml, w/v) were added and

incubated for 2-3 hours at 50°C.

The solution was spun at 10,000 rpm for 10 min after mixing with equal volume

of phenol: chloroform: Isoamyl alcohol (25:24:1). The upper phase was

transferred to a clean microfuge tube.

Equal volume of chloroform: Isoamyl alcohol (24:1) was added, mixed well by

inverting, then spun for 10 min at 10,000 rpm. The upper aqueous phase

was transferred to a clean microfuge tube.

In the collected supernatant, the DNA was precipitated with equal volume of

Isopropanol in the presence of one-tenth volume of 3 M sodium acetate.

Tube was centrifuged for 15 min at 12,000 rpm and supernatant was discarded.

The pellet was washed in 70% ethanol and again spun for 15 min at 12,000 rpm.

Supernatant was discarded and the step was repeated twice.

The pellet was dried at room temperature for overnight.

DNA was resuspended in 100 μl sterile distilled water or 0.3X TE and kept in

water bath at 65ºC for one hour and stored at –20ºC till use.


73

III. Using QIAamp DNA Mini Kit as per the manufacturer’s protocol

a) Materials

QIAamp DNA Mini Kit (Catalog No. 51304, Qiagen Pvt. Ltd.) was obtained for

extraction of DNA from milk samples. The contents of the kit were as in Table 3.6.

Table 3.6 Contents of the QIAamp DNA Mini Kit

Contents Quantity

QIAamp Spin Columns in 2ml Collection Tubes 50

Collection Tubes (2ml) 150

Buffer ATL 10 ml

Buffer AL 12 ml

Buffer AW1 (concentrate) 19 ml

Buffer AW2 (concentrate) 13 ml

Buffer AE 22 ml

Proteinase K 1.25 ml

Handbook 1

b) Method

Buffer AW1 and AW2 (both concentrated) were diluted with 25 ml and 30 ml

of absolute ethanol, respectively, to make the final working solution of

volume 44 ml and 43 ml, respectively.

To the bottom of a 2 ml microcentrifuge tube 20 µl of Qiagen proteinase K

was pipetted.

Then 200 µl of sample was added in the microcentrifuge tube and mixed by

pulse vortexing for 15 sec.

Then 200 µl of buffer AL was added and mixed by pulse vortexing for 15 sec.

The tube was incubated at 56ºC for 10 min.


74

The microcentrifuge tube was briefly centrifuged to remove the drops from

inside of the lid.

To the sample, 200 µl of ethanol (96-100%) was added and mixed by pulse

vortexing for 15 sec. After mixing, the microcentrifuge tube was briefly

centrifuged to remove drops from the inside of the lid.

The mixture was carefully applied from the previous step to the QIAamp spin

column with a 2 ml collection tube without wetting the rim, the cap was

closed and centrifuged at 6000 x g (8000 rpm) for 1 min. The QIAamp

spin column was placed in a clean 2 ml collection tube (provided) and the

tube containing filtrate was discarded.

The QIAamp spin column was opened and 500 µl of buffer AW1 was added

without wetting the rim. The cap was closed and centrifuged at 6000 x g

(8000 rpm) for 1 min. The QIAamp spin column was placed in a clean 2

ml collection tube (provided) and the collection tube with filtrate was

discarded.

The QIAamp spin column was opened carefully and 500 µl of buffer AW2

was added without wetting the rim. The cap was closed and centrifuged at

20000 x g (14000 rpm) for 3 min.

The QIAamp spin column was placed in a new 2 ml collection tube (not

provided with the kit) and the collection tube with the filtrate was

discarded. It was centrifuged at full speed for 1 min.

The QIAamp spin column was placed in a new 2 ml collection tube (not

provided with the kit) and the collection tube with the filtrate was


75

discarded. QIAamp spin column was opened carefully and 50 µl of buffer

AE was added. The tube was incubated at room temperature for 5 min and

centrifuged at 6000 x g (8000 rpm) for 1 min.

The filtrate in the collection tube contained the eluted DNA. The eluted DNA

was stored at -20ºC for long term use.

3.10.2 DNA Extraction from Milk Samples

Extraction of DNA from milk samples was carried out with the procedure

described by Romero and Lopez-Goni (1999) as described earlier (3.10.1, II).

3.10.3 Quantitation and Quality Assessment of DNA

Quality and quantity of DNA were checked as per the method described earlier

(3.9.3.4).


76

3.10.4 PCR

PCR for detection of Brucella from milk was carried out as per the method

described earlier (3.9.3.5) using three different primer pairs and template DNA prepared

as described earlier (3.10.1, II mentioned in 3.10.2).

3.11 QUANTIFICATION OF BRUCELLA IN MILK USING REAL-TIME

PCR

To determine the load of Brucella organisms in the milk real-time PCR based on

SYBR Green I was employed.

3.11.1 Preparation of Standard Template DNA

B. abortus strain 544 was inoculated in Brucella broth and incubated at 37ºC for 4

days at 5% CO2 tension. About 5 ml of broth was taken and centrifuged at 8000 rpm for

10 min to get pellet. DNA was extracted from this pellet as per the procedure of Romero

and Lopez-Goni (1999). This DNA was diluted in 100 fold dilution series to get final

concentration of 50 ng , 0.5 ng , 0.005ng , 0.00005 ng per reaction.

3.11.2 Template DNA of Milk Samples

Extracted DNA of field milk samples, found positive in PCR by primer pair

B4/B5, were subjected to the quantification by real-time PCR based on SYBR Green I

using same primer pair.

3.11.3 Real-time PCR based on SYBR Green I

a) Materials

i) 2X QuantiTectTM SYBR green PCR master mix (Cat. No. 204143, Qiagen)

ii) Nuclease free distilled water (Qiagen)

iii) Extracted DNA


77

iv) MicroAmpTM Optical 96-well Reaction Plate with Barcode (PN. 4306737,

Applied Biosystems)

v) MicroAmpTM Optical Adhesive Film (PN.4311971, Applied Biosystems)

vi) Tubes of 500 µl capacity (Axygen)

vii) Primers: B4/B5 primer pair was used for real-time PCR amplification

(Table 3.3)

b) Method

Real-time PCR based on SYBR Green I was carried out in final reaction volume

of 25 µl in 7500 Real Time PCR systems, Applied Biosystem, USA. Quantity and

concentration of various components used for assay were as per Table 3.7. Steps and

conditions of thermal cycling were as per Table 3.8. Fluorescence was measured once

every cycle after the extension step using filters for SYBR Green (excitation at 492 nm

and emission at 530 nm). The normalized fluorescence data were converted to a log scale

and the threshold was determined to calculate the threshold cycle value (Ct; the cycle at

which the threshold line crosses the amplification curve). In every run, the threshold was

set above the background (0.01) normalized fluorescence value. Upon completion of

real-time PCR run, data were automatically analyzed for melt curve and quantification by

7500 system Sequence Detection Software (SDS).


78

Table 3.7 Quantity and concentration of various components used in

real-time PCR based on SYBR Green I

Sr. No. Components

Real-time

PCR based on

SYBR Green I

1. QuantiTectTM SYBR Green PCRmaster mix (2X)

12.5 µl

2. Forward Primer (10 pmol/l) 1.0 µl

3. Reverse Primer (10 pmol/l) 1.0 µl

4. Template DNA 5 µl

5. QuantiTect DNAse Free water 5.5 µl

Table 3.8 Steps and conditions of thermal cycling for real-time PCR based on SYBR

Green I

Primers

(forward

and

reverse)

Cycling conditions

Initial

denaturationDenaturation Annealing Extension Dissociation curve

B4 (F)

B5 (R)

93°C

5 min

90°C

1 min

64°C

30 sec

72°C

1 min

Starting from 60°C to

95°C

Repeated for 35 cycles Repeated for 1 cycle

c) Visualization of PCR products by agarose gel electrophoresis

Visualization of PCR products by agarose gel electrophoresis was carried as

described earlier (3.9.3.5, c).


79

3.12 STATISTICAL ANALYSIS

To compare the sensitivity, specificity and overall agreement between the various

tests, the statistical formula given by Samad et al. (1994) was used as described below.

------------------------------------------------------------------------------------------------------------ Gold standard test Total

positive negative------------------------------------------------------------------------------------------------------------The test Positive a b a+bto becompared Negative c d c+d------------------------------------------------------------------------------------------------------------

Total a+c b+d a+b+c+d = N------------------------------------------------------------------------------------------------------------The notations used above are defined as under.

a = Number of samples positive to both conventional and the gold standard tests

b = Number of samples positive to conventional but negative to the gold standard test

c = Number of samples negative to conventional but positive to the gold standard test

d = Number of samples negative to both conventional and the gold standard tests

a + b + c + d = Total number of samples (N)

Definitions and formulae of the indices used for comparing the different assays

are described follows.

Sensitivity: It is the capacity of the test to detect diseased animals, when

compared with the gold standard test (a/a+c x 100).

Specificity: It is the capacity of the test to detect non-diseased animals, when

compared with the gold standard test (d/b+d x 100).

Overall agreement: Is the proportional similarity of the results of both the tests

(a+d/N x 100).


80

CHAPTER – IV

RESULTS AND DISCUSSION

To prevent the transmission of Brucella infection to new born calf as well as in

humans by ingestion of milk of infected animals it is utmost essential that milking

animals must be free from brucellosis. For diagnosis of brucellosis various serological

tests are employed with varying degree of sensitivity and specificity. Isolation of

organisms is tedious, cumbersome, time consuming and also health hazardous to the

laboratory workers thus it is generally not being followed as routine diagnostic

procedure. Moreover, attempts to isolate Brucella from individual animals and in chronic

cases may not be always successful. Simultaneously the Polymerase Chain Reaction

(PCR) technique has also been applied for direct detection of Brucella DNA in clinical

specimens. The widespread success of PCR as a technique comes from the fact that it is

rapid, automated, efficient, sensitive and specific. Recently, real-time PCR has been

developed which is likely to be more sensitive and specific for detection of Brucella

DNA, as well as also useful for quantification of the microbial load in the samples to

know whether animal has chronic or acute infection.

The present study was carried out by employing Rose Bengal Plate Test (RBPT),

Standard Tube Agglutination Test (STAT) and ELISA to detect Brucella antibodies in

bovine serum while Milk Ring Test (MRT) and Milk-ELISA were performed to detect

antibodies in bovine milk whereas culture and PCR techniques were used for detection of

Brucella organisms and Brucella DNA, respectively, in the bovine milk. Real-time PCR

was used for quantifying the load of Brucella excreted in the milk.

Results and Discussion…

81

The present study on brucellosis was concentrated on following points.

1. Serodetection in bovines (cattle and buffaloes) employing ELISA and comparing it

with RBPT and STAT.

2. Detection of antibodies in milk employing ELISA and comparing it with MRT.

3. Comparison between tests detecting antibodies in serum and milk.

4. Isolation and identification of Brucella from milk and confirmation by PCR.

5. Detection of Brucella DNA in milk by PCR assay using different pairs of primer

and comparing the efficacy of them.

6. Quantifying the load of Brucella organisms in the milk using real-time PCR.

7. Comparative efficacy of antibody detection, cultural and molecular methods for

detection of Brucella infection.

4.1 SERODETECTION

In the present study the serodetection in bovines was assessed by RBPT, STAT

and ELISA. A total of 231 sera comprising of 47 cattle and 184 of buffaloes were

processed for antibody detection.

4.1.1 Serodetection by ELISA

A total of 231 bovine serum samples were screened by ELISA. Of these, 67

(29.00%) serum samples were found positive for Brucella antibodies. The details of the

samples with results are given in Table 4.1 and Fig. 4.1. The Plate 4.1 is showing the

photograph of microtitre strips with the result of ELISA.


82

Table 4.1 Serodetection of brucellosis in cattle and buffaloes by ELISA

Animal

No. of serum samples

Tested Positive

Cattle 47 18 (38.29)

Buffalo 184 49 (26.63)

Total 231 67 (29.00)

Figures in parentheses indicate percentage

In this study using ELISA overall seropositivity of 29.00% was found with

38.29% in cattle and 26.63% in buffaloes. While in bovines of Central Gujarat using

ELISA comparative lower values, overall seroprevalence of 22.01% with 24.12% in

cattle and 19.12% in buffaloes, were reported by Varasada (2003). Whereas, much lower

seroprevalence (3.85%) was observed in bulls of Gujarat (Anonymous, 1999). Similarly,

Kanani (2007) also recorded lower seroprevalence of 8.25% in bulls of Gujarat with

16.13% in cattle and 0.99% in buffalo bulls. The lower values of 6.37% in cattle and

4.9% in buffaloes were also observed by Sharma et al. (1979). Magee (1980) also found

less serum samples (13.39%) positive by ELISA.

In the serodetection study applying serological tests other than ELISA, supporting

results for presence of Brucella antibodies in bovines were obtained by different workers.

Lodhi et al. (1995) found 12.98% (cattle) and 2.40% (buffaloes) of seropositivity by

RBPT and STAT, respectively. Prahlad et al. (1999) found 7.09%, 2.70%, 11.14% and

8.10% seropositivity by RBPT, STAT, CFT and dot-ELISA, respectively. Mahato

et al. (2004) revealed 43.28% and 47.76% positivity in cows by STAT and ELISA,

respectively, whereas, 14.89% and 17.02% positivity in heifers by STAT and ELISA,

respectively. However, Pati et al. (2000) found all the bulls negative out of six tested by

ELISA and RBPT from semen station of Uttar Pradesh.


83

In present study much higher seroprevalence (38.29%) was found in cattle than

(26.63%) in buffaloes (Table 4.1 and Fig. 4.1). In Gujarat species-wise higher

seroprevalence of 9.8% in cattle bulls was found in comparison to 5.8% in buffalo bulls

by ELISA (Anonymous, 1999). Varasada (2003) in bovines of Central Gujarat reported

higher seroprevalence (24.12%) in cattle than (19.12%) in buffaloes. Similarly, Kanani

(2007) also reported higher seroprevalence (16.13%) in cattle than (0.99%) in buffalo

bulls of Gujarat. However, in Gujarat irrespective of sex Renukaradhya et al. (2002)

found more or less equal proportion of seroprevalence (6.6%, 247 out of 3750) in cattle

and (6.3%, 14 out of 222) buffaloes. In Punjab Sandhu et al. (2001) also revealed more or

less equal seroprevalence of 10.06% and 9.33% in cattle and buffaloes, respectively.

4.1.2 Comparative Efficacy of Serological Tests

In the present study ELISA in conjunction with other serological tests was

employed to compare the efficacy. All 231 serum samples were tested for the presence of

Brucella antibodies by RBPT and STAT in addition to ELISA. The details of the samples

with results are given in Table 4.2 and Fig. 4.2.


84

Table 4.2 Serodetection of brucellosis in cattle and buffaloes by RBPT and STAT

No. of serum samples

AnimalTested RBPT Positive STAT Positive

Cattle 47 03 (6.38) 14 (29.78)

Buffalo 184 15 (8.15) 29 (15.76)

Total 231 18 (7.79) 43 (18.61)


To find out relative sensitivity and specificity of RBPT and STAT, cross tabulation

of results of RBPT and STAT with that of ELISA, considering ELISA as a gold standard

test, are given in Table 4.3.

Table 4.3 Sensitivity and specificity of RBPT and STAT by comparing with ELISA

(gold standard test) for detection of Brucella antibodies

TestELISA

TotalSensitivity

(%)Specificity

(%)

OverallAgreement

(%)Positive Negative

RBPTPositive 17 01 18

25.37 99.39 77.92Negative 50 163 213Total 67 164 231

STATPositive 41 02 43

61.19 98.78 87.87Negative 26 162 188 Total 67 164 231

In comparison to 29.00% of seropositivity in ELISA, 7.79% and 18.61% of the

samples were found seropositive in RBPT and STAT, respectively. Thus, ELISA resulted

in highest number of seropositive animals than both the tests. Varasada (2003) also found

higher seropositivity by ELISA (22.01%) as compared to RBPT (16.80%) and STAT

(14.03%) in cattle and buffaloes of Central Gujarat. Similarly, higher seropositivity by

ELISA as compared to RBPT and STAT were also recorded by Rao et al. (1999),


85

Chakraborty et al. (2000), Sarumathi et al. (2003b), Barbuddhe et al. (2004), Chand and

Sharma (2004) and Bhattacharya et al. (2005) in cattle and buffaloes. Kanani (2007) also

recorded higher seropositivity by ELISA (8.25%) as compared to RBPT (5.67%) and

STAT (7.22%) in breeding bulls of Gujarat. This might have been due to the ability of

ELISA to detect all types of immunoglobulins (Quinn et al., 1994).

The sensitivity of RBPT and STAT was found to be of 25.37% and 61.19%,

respectively, considering ELISA as a gold standard test while specificity was found to be

of 99.39% and 98.78%, respectively. Thus, STAT was found to be more sensitive but

slightly less specific than that of RBPT. Similarly, Chakraborty et al. (2000) also found

higher sensitivity of STAT (88.61%) over RBPT (56.96%), however, they reported higher

specificity of the STAT (98.59%) than that of RBPT (96.77%). In contrast Singh et al.

(2004) revealed sensitivity of RBPT (88.46%) much higher than STAT (46.15%), while

specificity of STAT (98.31%) was found slightly higher than RBPT (97.75%) considering

ELISA as gold standard. Similarly, Sarumathi et al. (2003b) also found higher specificity

of STAT (90.59%) than RBPT (88.22%). While reviewing Gall and Nielsen (2004) came

across higher sensitivity of ELISA as compared to RBPT and STAT. Between, RBPT and

STAT they reviewed higher sensitivity of RBPT while higher specificity of the STAT. Pati

et al. (2000) also concluded that ELISA was more sensitive than that of RBPT and STAT.

Agrawal et al. (2007) and Kanani (2007) also realized ELISA as more sensitive than that

of RBPT and STAT.

Paweska et al. (2002) suggested that ELISA could replace not only the currently

used confirmatory CFT, but also other two routine screening tests, namely the RBPT and

STAT. Nielsen (2002) and Gall and Nielsen (2004) after reviewing various serological


86

tests, concluded that no individual test found perfect, however, error could be minimized

using the most reliable test. Chand and Sharma (2004) advocated the use of ELISA in

comparison to RBPT and STAT for assessing the situation of brucellosis in cattle to have

better results because chances of non detection of an infected animal in ELISA are

minimum. As per OIE (2004) the I-ELISA should be considered more as a screening test

rather than a confirmatory test for testing of vaccinated cattle or herds affected by FPSR

problems.

In this study overall agreement of RBPT and STAT with ELISA was found

77.92% and 87.87%, respectively. Hence for serodetection of Brucella, ELISA was found

to be a better serological test as compared to RBPT and STAT thus it could be advocated

for screening of brucellosis in large number of animals.

4.2 ANTIBODY DETECTION IN MILK

A total of 53 milk samples comprising of 13 cows and 40 buffaloes from

individual animals were screened for detection of Brucella antibodies using ELISA and

MRT. The details of the samples with results are given in Table 4.4 and Fig. 4.3.

Table 4.4 Detection of Brucella antibodies in milk of cattle and buffaloes

by ELISA and MRT

No. of milk samples Animal Tested MRT Positive ELISA Positive

Cow 13 05 (38.46) 04 (30.76)Buffalo 40 03 (07.50) 11 (27.50)Total 53 08 (15.09) 15 (28.30)


Out of 53 milk samples, 15 (28.30%) were found positive using ELISA for

Brucella antibodies, whereas, 08 (15.09%) were found positive by MRT. Thus, ELISA

detected higher number of positive animals than that of MRT for presence of Brucella


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antibodies, however, reverse was true in case of cow milk. The results of MRT and

ELISA both revealed the higher percentage of Brucella antibodies in cattle milk than that

of buffalo milk.

Heck et al. (1980) detected Brucella antibodies in cow milk by ELISA from

seropositive or negative cows and determined to be an appropriate method for detecting

antibodies in bovine milk.

Boraker et al. (1981) found that I-ELISA was highly correlated with positive

MRT reactions and also eliminated false positive MRT reactions and detected antibody in

some MRT negative samples. They also revealed that milk-ELISA was not only sensitive

and specific, but was able to distinguish between infected and vaccinated animals.

Vanzini et al. (1998) used an I-ELISA for detection of B. abortus antibodies detection in

bovine milk and serum samples and found that the I-ELISA was a highly sensitive and

specific test.

Kang'ethe et al. (2000) found more positivity by ELISA (4.9%) than that of MRT

(3.9%), however, it was lower than the present study. Chand et al. (2004) also found more

numbers of samples positive by ELISA (27.27% and 9.09%) than the MRT (19.29% and

7.89%) when applied to organized and unorganized farms, respectively. Gumber et al.

(2004) also found higher 218 (22.47%) number of animals positive by AB milk-ELISA

than the MRT 115 (11.85%) out of 970 tested bulk milk samples. Mahato et al. (2004)

found 24 (35.82%) cows positive by MRT out of 67 cows.

In present study the slight higher positivity by MRT in cow milk might have been

due to false positivity of MRT because of recent parturition, end of lactation and due to

sub-clinical mastitis (Alton et al., 1988).


88

To find out relative sensitivity and specificity of MRT, cross tabulation of results

of MRT with that of ELISA, considering ELISA as a gold standard test, are given in

Table 4.5.

Table 4.5 Sensitivity and specificity of MRT by comparing with ELISA (gold

standard test) for detection of Brucella antibodies

TestELISA

TotalSensitivity

(%)Specificity

(%)

OverallAgreement


MRTPositive 06 02 08

40.00 94.73 79.24Negative 09 36 45Total 15 38 53

The sensitivity of MRT was found to be of 40.00% when compared to ELISA,

while specificity was found to be of 94.73% and overall agreement between these two

tests was found to be 79.24%. However, Nazem et al. (1998) found lower value (48.10%)

of overall agreement. In their study, the sensitivity and specificity of MRT as compared to

ELISA were 48.15% and 72.22%, respectively, thus, ELISA was found more sensitive

than MRT. Vanzini et al. (2001) also found higher sensitivity of the milk-ELISA (98.1%)

than that of the MRT (72.2%). Bonfoh et al. (2002) revealed that results of milk-ELISA

were highly correlated with the positive results of MRT. Gumber et al. (2004) also

revealed lower sensitivity (68.8%) of MRT but had comparable specificity (98.9%) than

that of milk-ELISA.

The higher sensitivity and specificity of ELISA than MRT were also recorded by

Thoen et al. (1979), Mikolon et al. (1998), Vanzini et al. (1998), Rivera et al. (2003) and

Chand et al. (2004).


89

Thus, no correlation could be observed between ELISA and MRT for detection of

Brucella antibodies in milk, however, more number of milk samples were found positive

by ELISA.

4.3 COMPARISON OF ANTIBODY DETECTION TESTS FOR SERUM

AND MILK

The serum and milk both from 53 same milking animals were subjected for

detection of antibodies. The antibodies in serum were detected by RBPT, STAT and

ELISA whereas MRT and ELISA were used for detection of antibodies in milk. The

details of the samples with results are given in Table 4.6 and Fig. 4.4.

Table 4.6 Comparison of serum antibody and milk antibody detection tests

TestedAnimals

Serum samples Milk samples

RBPTPositive

STATPositive

ELISAPositive

MRTPositive

ELISAPositive

5308

(15.09)14

(26.41)20

(37.73)08

(15.09)15

(28.30)Figures in parentheses indicate percentage

Out of 53 serum samples tested, the ELISA showed highest number of positive

samples (37.73%) followed by STAT (26.41%) and RBPT (15.09%). The same way the

ELISA also detected antibodies in more number of milk samples (28.30%) than that of

MRT (15.09%). Thus, the ELISA detected more number of positive samples both in

serum and milk.

On comparison, the ELISA detected antibodies in more number of serum samples

(37.73%) than that of milk (28.30%). Chand et al. (2004) recorded slightly higher

positive percentage (29.09%) of serum samples than (27.27%) in milk when tested

165 serum and milk samples by ELISA. Chand et al. (2005) detected Brucella antibody


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levels in milk and serum samples by ELISA and revealed significant correlation between

both the tests thus the milk-ELISA for brucellosis was appeared to be an attractive

alternative of serum-ELISA particularly in the lactating ewes.

In the present study the positivity by STAT (26.41%) was found higher as

compared to MRT (15.09%). Abdel Hakiem (2000) found slightly higher (10.8%)

positivity by STAT than (8%) by MRT. Gurturk et al. (1999) also found higher positivity

by STAT than that of MRT. Whereas, the reverse was true in case of Barman et al. (1989)

who reported 44.9% positivity by STAT and 54.7% by MRT.

In present study when compared detection of antibody in serum as well as in milk

by any of the tests, the serum ELISA showed more number of positive samples.

Al-Khalaf and El-Khaladi (1989) found 14.8% prevalence rate by RBPT and 10.8% by

the STAT with 8.0% milk prevalence rate. Whereas, Zowghi et al. (1990) recorded the

reverse result than that of the present study.

To find out relative sensitivity and specificity of RBPT, STAT, MRT and milk-

ELISA cross tabulation of results of RBPT, STAT, MRT and milk-ELISA with that of

serum ELISA, considering serum ELISA as a gold standard test, are given in Table

4.7.


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Table 4.7 Sensitivity and specificity of RBPT, STAT, MRT and milk-ELISA

by comparing with serum ELISA for detection of Brucella antibodies

TestsSerum ELISA

TotalSensitivity

(%)Specificity

(%)

OverallAgreement


RBPTPositive 08 00 08

40.00 100.00 77.34Negative 12 33 45Total 20 33 53

STATPositive 14 00 14

70.00 100.00 88.67Negative 06 33 39Total 20 33 53

MRTPositive 06 02 08

30.00 93.93 69.81Negative 14 31 45Total 20 33 53

Milk-ELISA

Positive 11 04 1555.00 87.87 75.47Negative 09 29 38

Total 20 33 53

The sensitivity of RBPT, STAT, MRT and milk-ELISA were found to be of

40.00%, 70.00%, 30.00% and 55.00%, respectively, with considering serum ELISA as a

gold standard test while specificity were found to be of 100.00%, 100.00%, 93.93% and

87.87%, respectively. The overall agreement of 77.34% for RBPT, 88.67% for STAT,

69.81% for MRT and 75.47% for milk-ELISA were found with the serum ELISA. Thus,

STAT was found to be more sensitive than that of the tests detected Brucella antibodies

both in serum and milk. Szulowski (1999) observed no correlation between the

milk-ELISA and RBPT results; among 79 samples from cows in which sera reacted

positively in the RBPT, only 22 were positive or doubtful in the ELISA. He also observed

100 per cent correlation between milk-ELISA and seronegative animals. He revealed 11

positive and 20 doubtful cases by the milk-ELISA among 309 milk samples which were

suspected of brucellosis and concluded that milk-ELISA was more sensitive than

traditional methods and serum-ELISA, in which only 3 samples reacted positive and only


92

2 were doubtful. Biancifiori et al. (1996) found that the specificity of M-ELISA was 65%

and 83% when compared to RBPT, whereas, 100% when applied to Brucella free herds.

They found that milk-ELISA was less sensitive than the tests detected antibodies in

serum and revealed that concentrations of immunoglobulins in colostrum and in mature

milk tend to decrease sharply soon after parturition while in serum they remained

constantly high. They concluded that M-ELISA for Brucella antibodies in ewe milk can

be regarded as a complementary diagnostic tool for individual testing but it would be

unsuitable for use as a screening test applied to pooled flock milks.

Thus, in present study variable results were obtained by different tests for

detection of antibodies in serum and milk, however, more number of samples were

detected positive using serum ELISA.


4.4.1 Isolation

Brucella agar medium (BAM) was used as a primary culture medium for

isolation of Brucella organisms. A total of 53 milk samples from bovines were cultured

using plates of BAM for isolation of Brucella organisms. The round, glistening and

smooth or mucoid colonies on plates of BAM were suspected to be of Brucella

(Plate 4.2). The isolates were streaked on blood agar (BA) and MacConkey agar (MA)

plates. The non-haemolytic isolates on BA as well as non-lactose fermenting isolates on

MA were preliminary presumed to be of Brucella.

Of the 53 milk samples processed, only four (each from 2 cows and 2 buffaloes)

yielded the isolates presumed to be of Brucella (Table 4.8). A total of six such isolates of

Brucella, two from only milk pellets of two cows and four from both milk pellets and


93

cream layer of same two buffaloes were recovered. Thus, Brucella could be recovered

from both pellets and cream layer of milk of same 2 buffaloes, whereas, the same could

not be recovered from the cream layer of milk of two cows which yielded the recovery

from the pellet.

Table 4.8 Isolation of Brucella from milk samples

No. of milk samples processed

Animal Tested Positive

Cattle 13 2 (15.38 )

Buffalo 40 2 (05.00)

Total 53 4 (07.54)


4.4.2 Identification

The isolates presumptive to be of Brucella were subjected to Gram staining and

modified Ziehl-Neelsen (MZN) staining. On the basis of Gram staining, the isolates were

found to be Gram negative, coccobacillary rods whereas by MZN staining they appeared

to be red (Plate 4.3). All the isolates were found positive by Rapid Slide Agglutination

Test.

The isolates were further identified by biochemical tests (Table 4.9). All the

isolates were found in smooth form by acriflavine test. Oxidase, catalase, urease and H2S

were produced by all the isolates but none of the isolates produced indole and all were

found non-motile. All the isolates were found to reduce nitrate.


94

Table 4.9 Biochemical characters of Brucella isolates

Sr.No.

Name of tests

IsolatesReference

strains

C1Pellet

C9Pellet

B19Pellet

B19Cream

B53Pellet

B53Cream

B.abortusstrain544

B.abortusstrain

191. Acriflavine test - - - - - - - -

2. Oxidase + + + + + + + +

3. Catalase + + + + + + + +

4. Nitrate reduction + + + + + + + +

5. Urease test + + + + + + - +

6. Indole test - - - - - - - -

7. Motility - - - - - - - -

8. Production of H2S + + + + + + + +

+ = Positive, - = Negative, C = Cow, B = Buffalo. Numerical indicates sample number

Based on the growth charcters on BAM, BA and MA as well as considering the

Rapid Slide Agglutination Test and biochemical tests all six presumtive isolates were

identified as Brucella organisms. Brucella agar medium was also used by various

workers for isolation of Brucella from clinical and tissue samples (Shin et al., 1978;

Das et al., 1990; Botelho et al., 2000; Chahota et al., 2003).

During this study out of 53 milk samples, Brucella could only be recovered from

four (7.54%) samples. Out of these two were from cattle while two were from buffaloes.

Thus, it showed the presence of Brucella in milk of bovines. Similarly, Mathur (1963)

also recovered 8 isolates from the 23 milk samples of MRT positive cows. Halder and

Sen (1986) recovered six isolates of B. abortus biotype I from milk samples. Similarly,

Farrell and Robertson (1972), Brodie and Sinton (1972), De et al. (1989), Zowghi et al.

(1990), Nicoletti and Tanya (1993), Chatterjee et al. (1995), Jeyaprakash et al. (1999),


95

Botelho et al. (2000) and Langoni et al. (2000) were able to isolate Brucella organisms

from milk of bovines with lower or higher isolation rate than the present study. However,

no Brucella organism could be isolated by Al-Khalaf and El-Khaladi (1989) from

sediment and cream of milk.

During the study pure colony of Brucella were recovered and no any

contaminating organisms were grown except fast growing organisms especially fungal

organisms were observed in many plates at the last stage of incubation, which might have

been due to either very poor microbial quality of milk or inefficiency of Brucella

selective supplement to prevent the growth of fungi. Similar problems were also faced by

Pal and Jain (1985) while isolating Brucella using tryptose agar and serum dextrose agar

and by Kanani (2007) using BAM.

4.4.3 Confirmation by PCR

PCR technique was used for confirmation of the Brucella isolates. The template

DNA from colony was prepared using proteinase K–SDS method followed by CTAB and

phenol : chloroform : isoamyl alcohol according to Wilson (1987) with minor

modifications. Using comparable methods Brucella DNA was successfully extracted

from the cultures by Leal-Klevezas et al. (1995), Romero et al. (1995), Navarro et al.

(2002) and Kanani (2007).

DNA extracted from reference Brucella strains and from the Brucella isolates

were subjected to PCR using three different Brucella genus specific primer pairs

(i) B4/B5 (ii) JPF/JPR and (iii) F4/R2. Desired sized products were obtained in reference

strains by all the three primer pairs and results of PCR amplifications of Brucella isolates

are depicted in Table 4.10.


96

Table 4.10 Confirmation of Brucella isolates by PCR

Sr.

No.Isolates

Primer pairs

B4/B5 JPF/JPR F4/R21. C1(Pellet) + + +

2. C9(Pellet) + - +

3. B19(Pellet) + + +

4. B19(Cream) + + +

5. B53(Pellet) + + +

6. B53(Cream) + + +

+ = Positive, - = Negative

Earlier Navarro et al. (2002) and Kanani (2007) used the same three primer pairs

for detection of Brucella DNA from Brucella isolates. The desired product of 223 bp

using B4/B5 primer pair was amplified in all the six isolates (Plate 4.4). However, isolate

C9 (pellet) could not produce a desired product of 193 bp using primer pair JPF/JPR even

after repeated trials. Hence, the non-amplification might be due to probable mutation in

primer attachments sights particularly at 3’ end. Further, in absence of sequence

information of the annealing site of field isolate, no conclusive inference could be drawn

about the behaviour of this primer pair. Navarro et al. (2002) observed a slightly different

sensitivity of these three primer pairs with conclusion that difference in sensitivity might

be due to samples pretreatment methods and extraction methods of DNA. Kanani (2007)

also observed the difference in the sensitivity of the same three primer pairs with high

sensitivity by B4/B5 primer.

In past the research workers (Anonymous, 1999; Casanas et al. 2001) also

succeeded to amplify the 223 bp region of bcsp31 for identification of Brucella while,


97

Romero et al. (1995) amplified a 905 bp fragment by using a primer pair F4/R2 and

detected as little as 80 fg of Brucella DNA by this method.

From cow milk Brucella could be isolated from the pellet only, whereas, in case

of buffalo milk they could be isolated both from pellet as well as cream. Thus, for

isolation of Brucella from the milk, pellet as well as cream both should be cultured on

suitable appropriate medium.

4.5 MOLECULAR DETECTION OF BRUCELLA IN MILK

4.5.1 DNA Extraction

For standardization, DNA was extracted from milk spiked with reference strains

using three different methods as described earlier in section 3.10.1. The quality and

quantity of extracted DNA were checked and subjected for PCR using different primers.

Out of these three methods, the PCR fragment of expected size could not be obtained

from DNA extracted through boiling method and QIAamp Mini Kit yielded less

concentration of DNA as compared to method described by Romero and Lopez-Goni

(1996) from same milk samples spiked with reference strains. Therefore in present study

DNA from all the 53 field milk samples was extracted using procedure described by

Romero and Lopez-Goni (1996) and subjected to PCR using different primer pairs.

However, Leary et al. (2006) successfully extracted Brucella DNA from whole milk,

sediment and cream layer using the QIAamp DNA Mini Kit while Kanani (2007)

extracted Brucella DNA from semen using the same method.

Failure of getting PCR by boiling method, as stated by Romero and Lopez-Goni

(1996), might have been due to difficulty associated with lysing the microorganisms or

due to the presence of PCR inhibitors in milk. Further they also mentioned that getting


98

less concentration of DNA, extracted by QIAamp Mini Kit might heve been due to

inefficiency of the kit to lyse the Brucella cell envelope (CE) that was more resistant to

non ionic detergents, EDTA and Tris than those of other Gram negative bacteria because

Brucella cell envelope is held by forces stronger than those acting in the CE of other

bacteria.

4.5.2 Detection of Brucella DNA in Milk Samples by PCR

In the present study Brucella DNA was detected by using three primer pairs viz.,

B4/B5, JPF/JPR and F4/R2 in PCR. The results are depicted in Table 4.11.

Table 4.11 Brucella detection in milk of bovines by PCR using different primer pairs

Animal

Positive by Primer pairs

Tested B4/B5 JPF/JPR F4/R2

Cattle 13 05 (38.46) 01 (07.69) 02 (15.38)

Buffalo 40 04 (10.00) 00 (00.00) 00 (00.00)

Total 53 09 (16.98) 01 (01.88) 02 (03.77)


4.5.2.1 B4/B5 primer pair

B4/B5 primer pair amplified a 223 bp region of the sequence encoding a 31 kDa

immunogenic bcsp31. Reference strains as well as nine (16.98%) of the 53 milk samples

produced 223 bp amplicon. The remaining 44 samples failed to produce the targeted

amplification. Thus, out of 53 milk samples, 9 (16.98%) were found positive for Brucella

infection by bcsp31 gene based primer with 5 (38.46%) positive in cattle and 4 (10.00%)

in buffaloes (Table 4.11).


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This bcsp31 gene based primer had also been successfully used by JungSuckChan

et al. (1998), Anonymous (1999 and 2000) and Kanani (2007) for detection of Brucella

DNA in semen of bulls. Matar et al. (1996) also used this primer pair for diagnosis of

human brucellosis directly from whole blood and in their study PCR assay was found

rapid and specific. Similar results were also reported by Morata et al. (2001),

Casanas et al. (2001), Navarro et al. (2002) and Varasada (2003) using same primer pair

for diagnosis of human brucellosis.

4.5.2.2 JPF/JPR primer pair

JPF/JPR primer pair amplified a 193 bp region of the sequence encoding an outer

membrane protein (omp2). Reference strains as well as one (1.88%) of the 53 milk

samples produced 193 bp amplicon (Plate 4.5). The remaining 52 samples failed to

produce the targeted amplification. Thus, out of 53 mik samples, one (1.88%) was found

positive for Brucella infection by omp2 gene based primer and the positive sample was

from cattle (Table 4.11).

Leal-Klevezas et al. (1995) and Serpe et al. (1998) also used primer homologus to

regions of the gene coding for an omp2 for detection of Brucella in blood and milk of the

infected animals and obtained promising results. Navarro et al. (2002) and Kanani (2007)

also used the same primer for detection of Brucella in Blood of infected human and

semen of bulls, respectively. Leary et al. (2006) also used the same primer for detection

of Brucella. They could not found amplification when applied to blood samples, but was

detected in a proportion of the culturally positive milk (44%) and lymph tissue samples

by the same methods.


100

4.5.2.3 F4/R2 primer pair

F4/R2 primer pair amplified a 905 bp region of the sequence 16S rRNA of

B. abortus. Reference strains as well as two (3.77%) of the 53 milk samples produced

905 bp amplicon (Plate 4.6). The remaining 51 samples failed to produce the targeted

amplification. Thus, out of 53 milk samples, 2 (3.77%) found positive for Brucella

infection by F4/R2 primer were from cattle (Table 4.11).

Romero et al. (1995) applied this primer pair to DNA extracted from all of the

representative strains of the species, biovars of Brucella and from 23 different Brucella

isolates and yielded exclusively the 905 bp fragment, however, they also obtained same

size amplicon from Ochrobactrum anthropi biotype D due to its closer relationship with

Brucella. Vesco et al. (2000) applied PCR for the diagnosis of brucellosis in milk by

using the same primer to identify the species. They concluded that the PCR could be used

in epidemiological studies to determinate the prevalence of different Brucella biovars.

Navarro et al. (2002) found the same primer as most sensitive primer because they

amplified 8 fg of purified B. melitensis Rev 1 DNA. Leary et al. (2006) also used the

same primer for detection of Brucella infection from blood, milk and lymph tissues and

found that no any amplification could be obtained when applied to blood samples, but

detected from milk and lymph tissues. Kanani (2007) also used the same primer for

detection of Brucella DNA from semen of breeding bulls of Gujarat and able to detect in

5 of the 101 semen samples.


101

4.5.2.4 Comparative efficacy of different primer pairs

In present study a total of three primer pairs amplifying three different fragments

(i) a gene encoding a 31 kDa immunogenic bcsp31 (primer pair B4/B5), (ii) a gene

encoding an omp2 (primer pair JPF/JPR) and (iii) a sequence 16S rRNA of B. abortus

(primer pair F4/R2) were compared for their efficiency for detection of Brucella DNA

from the field milk samples. These three PCR assays showed a difference in sensitivity

for detecting Brucella DNA in milk. Primer pair B4/B5 could produce the desired

amplicons of 223 bp in nine out of the 53 milk samples tested. However, primer pair

JPF/JPR could produce the desired amplicons of 193 bp in one sample only and F4/R2

could produce the desired amplicons of 905 bp in two samples out of the 53. One sample

found positive by primer pair JPF/JPR was also found positive by primer pairs B4/B5 and

F4/R2. The two samples found positive by primer pair F4/R2 were also found positive

by primer pair B4/B5. Thus variation could be observed between primers in detection of

Brucella DNA, however, it prooved the existence of the Brucella infection in the milk of

bovines.

The variation was also noticed in detection ability of two primer pairs in

comparative study (Anonymous, 1999). They obseverd that the 31 kDa gene could be

detected in the semen of 9 buffaloes while the omp2 gene was only detected in 2 semen

samples of seropositive buffalo bulls by PCR. When they used these primer pairs for

detection from blood samples of seropositive buffalo bulls by PCR, 31 kDa gene based

primer could detect 6 positive while omp2 gene based primer could detect 3 positive

samples.


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Navarro et al. (2002) also compared the same three primer pairs for detection of

Brucella DNA. The study showed a difference in sensitivity for detecting purified

Brucella DNA. In their study F4/R2 was found most sensitive primer pair while in

present study primer pair B4/B5 yielded highest number of positive samples. They also

studied the sensitivity of the primers in the presence of human DNA and observed that

F4/R2 and B4/B5 were affected by the presence of human DNA but not the primer pair

JPF/ JPR. Amin et al. (2001) also observed inhibition of PCR amplification of the sperm

head fraction in control template, which might have been due to high DNA concentration.

Kanani (2007) also compared the same three primer pairs for detection of Brucella DNA

in semen of breeding bulls of Gujarat. The study showed a difference in sensitivity for

detecting purified Brucella DNA. In their study B4/B5 was found most sensitive primer

pair. In present study same primer pair also yielded highest number of positive samples.

Thus, variation in detection ability by different primer pairs in PCR assays

observed in present study might be due to the presence of large amounts of genomic

DNA and low proportionate presence of Brucella DNA. This could be as a result of

competitive non-specific hybridization of the large amounts of bovine genomic DNA

with these primer pairs.

4.6 QUANTIFICATION OF BRUCELLA IN MILK USING REAL-TIME

PCR

Real-time PCR was employed for quantifying the load of Brucella from milk. For

quantifying the load in milk, standard template DNA was prepared from pure colony of

Brucella reference strain 544 as described in 3.11.1. In real-time PCR, intercalating dye

SYBR Green I was used for monitoring the amplification as well as quantifying the load.


103

For amplification, primer pair B4/B5 was used as it resulted in maximum number of

positive samples in PCR. Fluorescence was measured once every cycle after the

annealing step using filters for SYBR Green I (excitation at 492 nm and emission at

530 nm). All the nine samples found positive by B4/B5 primer pair were quantified by

real-time PCR. The normalized fluorescence data were converted to a log scale and the

threshold was determined and calculated the threshold cycle (Ct) value (Fig. 4.5). Upon

completion of real-time PCR run, data were automatically analyzed for melt curve

(Fig. 4.6) and quantification by SDS software with the help of standard curve (Fig. 4.7).

After getting DNA quantity of field samples, the DNA quantity was converted in to

CFU/ml of milk considering 5 fg of DNA quantity per one Brucella organism. The results

are depicted in Table 4.12.

Table 4.12 Load of Brucella in bovine milk

Sr.No.

SampleLabel

Brucella load in milk(CFU/ml)

1. C1 1.136 x 104

2. C5 34.000 x 104

3. C9 172.800 x 104

4. C10 74.480 x 104

5. C13 2.296 x 104

6. B19 14.480 x 104

7. B42 1.128 x 104

8. B46 1.408 x 104

9. B53 7.368 x 104

In present study, load of Brucella organisms in milk ranged from 1.128 x 104

CFU/ml to 172.800 x 104 CFU/ml of milk. On the basis of melt curve analysis, detection

limits of this real-time PCR assay was found up to 50 fg DNA or 10 CFU/ml milk

(considering 5 fg is equal to one Brucella cell) using 5 µl of template DNA. However,

when PCR products of serially diluted DNA were visualized by agarose gel


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electrophoresis at 15 min of electrophoresis run no band was observed in sample having

concentration of 50 fg of Brucella DNA or 10 CFU/ml (Plate 4.7), however,

real-time PCR detected the amplification and melt curve of the same sample. These

observations indicated the higher sensitivity of the real-time PCR especially when

microbial load is low.

Colmenero et al. (2003) used the LightCycler detection system and

SYBR Green I for diagnosis of human brucellosis using 223 bp target sequence of a gene

encoding an immunogenic 31 kDa protein and found sensitivity and specificity of the

assay 91.9% and of 96.4%, respectively. Debeaumont et al. (2003) detected one CFU per

5 μl of DNA extract from Brucella by a bcsp31 based real-time PCR assay. Newby et al.

(2003) also used SYBR Green I based real-time PCR assay along with 5–exonuclease

and hybridization probes to detect B. abortus. They found that all three assays were of

comparable sensitivity, however, the greatest specificity was achieved with the

hybridization probe assay. Queipo-Ortuno et al. (2003) also used LightCycler technology,

SYBR Green I and primer targeting 223 bp sequence of bcsp31. They found that these

real-time PCR protocol could detect as little as two genomic equivalents (about 10 fg) of

purified bacterial DNA. Queipo-Ortuno et al. (2005) used these bcsp31 based real-time

PCR for diagnosis of human brucellosis from serum samples and found to be 91.9%

sensitive and 95.4% specific. Kanani (2007) also used the same primer to quantify the

load of Brucella in semen of breeding bulls of Gujarat by real-time PCR using SYBR

Green I assay. He found the detection limit of the assay 50 CFU/ml of semen.

Thus, the use of real-time PCR for quantification of Brucella in milk seems to be

the first time as no literature could be available for the same.


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The real-time PCR could detect the Brucella in milk even the microbial load is

very low, therefore, the use of real-time PCR can be advocated for quantification of

Brucella in milk being the milk as potential source of Brucella infection.

4.7 COMPARISON OF ANTIBODY DETECTION, CULTURAL AND

MOLECULAR METHODS

The 53 bovines were such from those both serum (for antibodies) and milk

(for antibodies, cultural isolation and PCR) were tested for presence of brucellosis. The

comparison was made, considering the samples from these 53 bovines, to detect the

Brucella infection. For detection of Brucella antibodies in serum three serological

methods (RBPT, STAT and ELISA) and in milk two methods (MRT and ELISA) were

employed, whereas, for isolation of Brucella organisms BAM was used and for detection

of Brucella DNA by PCR three different primer pairs (B4/B5, JPF/JPR and F4/R2) were

used. For comparison of these methods, animal showing the presence of Brucella

antibodies in serum by any one of the RBPT, STAT or ELISA and in milk either by MRT

or by ELISA was considered to be positive. The same way, detection of Brucella DNA by

any one of the primer pairs (B4/B5, JPF/JPR and F4/R2) in PCR assay, the animal was

considered to be PCR positive. The results are depicted in Tables 4.13, 4.14 and Fig. 4.8.


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107


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Table 4.14 Summary of Table No. 4.13

No. of bovines positive in all four methods viz., tests detected Abs in seruma,

tests detected Abs in milkb, cultural and PCRc04

No. of bovines positive in tests detected Abs in milk, cultural and PCR but

negative in tests detected Abs in serum00

No. of bovines positive in tests detected Abs in serum, cultural and PCR but

negative in tests detected Abs in milk00

No. of bovines positive in tests detected Abs in serum, tests detected Abs in milk

and PCR but negative in cultural 03

No. of bovines positive in tests detected Abs in serum, tests detected Abs in milk

and cultural but negative in PCR00

No. of bovines positive only in tests detected Abs in serum 07

No. of bovines positive only in tests detected Abs in milk 05

No. of bovines positive only in cultural isolation 00

No. of bovines positive only in PCR 01

No. of bovines positive in cultural and PCR but negative in tests detected Abs in

serum and tests detected Abs in milk 00

No. of bovines positive in tests detected Abs in milk and PCR but negative in

tests detected Abs in serum and cultural 00

No. of bovines positive in tests detected Abs in serum and cultural but negative

in tests detected Abs in milk and PCR00

Cont……..

Cont……..


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No. of bovines positive in tests detected Abs in milk and cultural but negative in

tests detected Abs in serum and PCR00

No. of bovines positive in tests detected Abs in serum and tests detected Abs in

milk but negative in cultural and PCR05

No. of bovines positive in at least one method 26

No. of bovines negative in all four methods 27

a = positive by any one of the serological tests detecting Brucella antibodies in

serum

b = positive by any one of the tests detecting Brucella antibodies in milk

c = positive by any one of the primer pairs in PCR considered as PCR positive

Considering 53 bovines, RBPT, STAT and ELISA detected 15.09%, 26.41% and

37.73% of positive bovines in serum, respectively. Whereas, MRT and ELISA detected

15.09% and 28.30% of bovine positive in milk, respectively. While, 7.54% of bovines

were found culturally positive. Among the PCR assays 16.98%, 1.88% and 3.77% of

bovines were found positive by B4/B5, JPF/JPR and F4/R2 primer pairs, respectively.

The highest numbers of bovines were found positive by serum ELISA whereas, cultural

method detected the least number of positive bovines.


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4.7.1 Comparison of Serum Antibody Detection and Cultural Methods

Of the 53 bovines tested, 20 revealed presence of Brucella antibodies in serum,

while 4 bovines yielded recovery of Brucella in milk by cultural isolation. Agreement

between these two methods was found 69.81%. In present study Brucella could be

isolated from milk of 4 bovines which all were seropositive. Thus, in present study

Brucella could be isolated only from seropositive animals.

Shin et al. (1978) found 69% isolation rate from milk samples having a titre of

1:100 or greater in STAT. However, De et al. (1989) failed to isolate Brucella organisms

from milk, vaginal discharge and cervical swabs of seropositive cows. Zowghi et al.

(1990) recovered 397 isolates of Brucella from 1,632 MRT positive milk samples, of

which 119 came from 5,686 seronegative cows. Hadad and Al-Azawy (1992) isolated 13

(42%) isolates of B. melitensis out of 31 samples examined. These isolates were

recovered from 7 aborted foetuses, 4 vaginal swabs and one milk sample from

serologically positive recently aborted ewes. El-Gibaly (1993) isolated Brucella

organisms from different tissues of 7 animals out of 19, 1 out of 7, 11 out of 25 and 12

out of 30 serologically negative cases to STAT, BPAT, RBPT and Riv. test, respectively.

Chatterjee et al. (1995) revealed 6.2% of isolation rate from milk, vaginal swab, hygroma

fluid and semen samples of 177 cows and bulls having Brucella agglutinins at positive

diagnostic level (80 IU/ml). Ferris et al. (1995) could recover B. suis from 8 seronegative

pigs tested by six different serological methods. Similarly, B. abortus could be isolated by

Guarino et al. (2000) from lymph glands of CFT and ELISA negative buffaloes. Langoni

et al. (2000) recovered 15 (30.61%) isolates of B. abortus from 49 milk samples of

seropositive animals. Whereas, Kaur et al. (2006) isolated Brucella from vaginal mucus,


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foetal membranes and foetal stomach content of aborted cattle and buffaloes which were

RBPT and STAT positive. They were also able to isolate B. abortus from RBPT and

STAT negative animals and concluded that the isolation method was most sensitive in

comparison to RBPT and STAT.

4.7.2 Comparison of Milk Antibody Detection and Cultural Methods

Of the 53 bovines tested, 17 revealed presence of Brucella antibodies in milk,

while 4 bovines yielded recovery of Brucella in milk by cultural isolation. Agreement

between these two methods was found 75.47%. In present study Brucella could be

isolated from 4 bovines which all showed the presence of Brucella antibodies in milk.

Thus in present study Brucella could only be isolated from Brucella antibody positive

bovine milk.

Farrell and Robertson (1972) isolated Brucella organisms from milk of 111 (21%)

of 516 MRT positive animals. Likely, Brodie and Sinton (1972) recovered

B. abortus from 41.8% samples out of 500 MRT positive milk samples. Boraker et al.

(1981) detected Brucella antibody to B. abortus in cow's milk by BrucELISA (I-ELISA)

and found that it was highly correlated with culture positivity. Their results showed that

the I-ELISA was not only sensitive and specific, but was also able to distinguish between

infected and vaccinated animals and was of diagnostic value in predicting which animals

were shedding or will eventually shed cultivable B. abortus. Halder and Sen (1986) also

recovered six isolates of B. abortus biotype I from MRT positive cows. However, Al-

Khalaf and El-Khaladi (1989) failed to isolate Brucella from milk of MRT positive

animals. Zowghi et al. (1990) recovered 397 isolates of Brucella from 1,632 MRT

positive milk samples of which, 119 of which came from 5,686 seronegative cows.


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Biancifiori et al. (1996) used ELISA for detection of Brucella antibodies in ewe milk and

revealed that the specificity of ELISA was 92% relative to culture positive animals. Abdel

Hakiem (2000) tested 150 individual raw milk of cows for MRT and cultural isolation of

Brucella organisms. They revealed that MRT was found to be reliable and sensitive, as it

revealed positive results in 8% of the samples, whereas, only one (0.7%) of the samples

yielded Brucella. Funk et al. (2005) applied I-ELISA to detect B. melitensis

specific antibodies in goat milk and revealed positivity thirteen of 13 (100%)

when applied to individual infected goat milk samples, whereas, 134 of 134 (100%)

negativity when applied to uninfected bulk milk samples.

4.7.3 Comparison of Serum Antibody Detection and PCR Methods

Of the 53 bovines tested, 20 revealed presence of Brucella antibodies in serum of

bovines, while 09 bovines showed presence of Brucella DNA in milk. Agreement

between these two methods was found 79.24%. In present study 8 out of 20 seropositive

bovines revealed presence of Brucella DNA in PCR assay thus Brucella DNA could not

be detected in milk of 12 seropositive bovines. However, Brucella DNA could be

detected from 1 of the 33 seronegative bovines.

XiaoAn et al. (2005) found 98 milk samples positive in PCR from 98 STAT

positive cows, however, Brucella DNA could also be detected by PCR in 8 milk samples

of the 350 STAT negative cows. Gupta et al. (2005) tested 22 milk samples and found 18

(82%) of the samples positive by PCR, which included the 12 samples positive by STAT

thus from the six STAT negative samples Brucella DNA could also be detected. Finally,

they concluded that PCR assay was faster, safe to use and had higher sensitivity and

specificity. The variable results were also observed by different workers using materials


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other than milk for PCR assay. JungSuckChan et al. (1998) found 5 bulls positive by

PCR assay of the185 seronegative bulls. Brucella DNA could be detected in 36 semen

samples from 17 ELISA positive and 19 ELISA negative buffalo bulls (Anonymous,

2000). Guarino et al. (2000) found 13 blood samples positive by PCR (29.05%) out of 44

blood samples that were positive for Brucella antibodies by I-ELISA and CFT. However,

they found 5 samples positive by PCR that were negative by CFT and ELISA. Finally,

they indicated that PCR analysis can be complementary to classical serological tests for

the detection of the etiological agent of Brucella infections in buffaloes, especially in the

initial phase when the immune response of the animal is not detectable. Leal-Klevezas

et al. (2000) found 86% of the blood samples positive on the PCR, while 60% were

positive by RBPT when tested 22 females and one male out of 300 clinically healthy

mixed breed of goats. Amin et al. (2001) found only 12 semen samples positive in PCR

assay for detection of B. melitensis when tested 120 animals which were RBPT positive.

Out of 120 bovine and ovine semen samples collected from seropositive animals

Manterola et al. (2003) could detect 27 semen samples positive in PCR out of 52

seropositive rams. However, they also found 3 semen samples positive in PCR from 49

seronegative rams. While, Varasada (2003) found highest number of positive results by

PCR (20) followed by RBPT (6) and STAT (3) from 77 blood samples of human.

Lavaroni et al. (2004) revealed 100% sensitivity of serological tests related to PCR using

blood for diagnosis of bovine brucellosis. Gupta et al. (2006) found higher sensitivity and

specificity of the PCR than serological methods in a study conducted for diagnosis of

brucellosis in goat. They found 12 samples exclusively positive in PCR, which were not

detected in serology. Kanani (2007) revealed 11 bulls positive for presence of Brucella


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antibodies in serum, while 19 bulls showed presence of Brucella DNA in semen of 101

bulls. He found 84.16% agreement between these two methods. He detected Brucella

DNA from seropositive as well as seronegative bulls.

4.7.4 Comparison of Milk Antibody Detection and PCR Methods

Of the 53 bovines tested, 17 revealed presence of Brucella antibodies in milk of

bovines, while 09 bovines showed presence of Brucella DNA in milk. Agreement

between these two methods was found 77.35%. In present study 7 out of 17 animals

positive for milk antibodies revealed presence of Brucella DNA in PCR assay thus,

Brucella DNA could not be detected in 10 positive animals. However, Brucella DNA

could be detected by PCR from 2 of the 36 bovines negative for mik antibodies. Brucella

DNA could also be detected by PCR from one animal that was not having Brucella

antibodies both in serum and milk.

Romero et al. (1995) found higher sensitivity of milk-ELISA (98.2%) as

compared to PCR (87.5%) for detection of Brucella infection when tested 56 Brucella

milk culture positive cattle. They found one PCR positive sample to be negative by

milk-ELISA and 7 milk-ELISA positive samples turned out to be PCR negative, yielding

an observed proportion of agreement of 0.91 for the 2 tests. They also found 100%

specificities of both the tests when testing the milk samples from Brucella-free cattle.

Evangelista et al. (2005) found that PCR was less sensitive than the serological methods.

4.7.5 Comparison of Cultural and PCR Methods

Of the 53 milk samples of bovine tested, 4 and 9 were found positive in cultural

isolation and in PCR, respectively. All 4 culturally positive animals were also found


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positive in PCR assay but 5 PCR positive failed to yield Brucella in the culture.

Agreement between these two methods was found 90.56%.

Similarly, Romero et al. (1995) found 49 milk samples positive by PCR (87.5%

sensitivity) from 56 Brucella milk culture positive cattle. Leal-Klevezas et al. (2000)

revealed 64% of the milk samples positive on PCR tests, but failed to yield bacteria in the

culture. Leary et al. (2006) found no difference between PCR and bacteriological

methods for detection of Brucella infection in cows.

Gallien et al. (1998) used PCR assay to detect Brucella species from the uterus,

udder, spleen, lymph nodes, kidney and liver of 3 cows. They revealed that all 18 samples

reacted positively in the PCR, whereas, Brucella could not be isolated from the 5

samples. JungSuckChan et al. (1998) found 5 bulls positive by cultural and PCR methods

out of 185 bulls from serologically negative herds for brucellosis. Guarino et al. (2000)

from blood and Amin et al. (2001) from semen revealed more number of animals positive

by PCR as compared to cultural isolation. Leyla et al. (2003) found sensitivity and

specificity of PCR compared to cultural isolation as 97.4% and 100%, respectively, for

detection of B. melitensis specific DNA from stomach contents of aborted foetuses of

sheep. Manterola et al. (2003) found a proportion of agreement of 0.91 between semen

culture and PCR results after testing 192 semen samples of rams. Scarcelli et al. (2004)

detected Brucella by PCR from 50.7% (34/67) of the samples of aborted bovine foetuses

while Brucella could be isolated only from 38.8% (26/67) of the samples with agreement

rate of 88% between the two methods. Kanani (2007) revealed 19 bulls positive by PCR

assay and 8 bulls by culturally out of 101 bulls. The agreement between these two tests

was 89. 11%.


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4.7.6 Overall Comparison

Out of the total 53 bovines tested for detection of Brucella infection only four

were found positive employing all the four methods and 27 were found negative by all

the four methods (Table 4.14). The over all agreement between these methods was found

58.49%. Among the four, tests detected Brucella antibodies in serum were resulted in

highest number of positive (20, 37.73%) followed by tests detected Brucella antibodies in

milk (17, 32.07%), PCR assays (09, 16.98%) and cultural isolation (04, 7.54%).

Similarly, Manterola et al. (2003) found higher sensitivity of serological tests than

that of semen culture and semen based PCR assay. Leary et al. (2006) found no

advantage of using PCR methods over standard serological and bacteriological methods

for diagnosis of brucellosis. On the contrary, Guarino et al. (2000) and Leal-Klevezas

et al. (2000) from blood whereas, Kanani (2007) from semen found higher sensitivity of

PCR over serological and culture methods.

While comparing all the four methods, 26 bovines revealed the presence of

Brucella infection by any one of the methods while 27 bovines did not found to carry the

infection by any of the methods employed during present study (Table 4.14). Thus,

possibly indicating the presence of Brucella infection with previous or recent exposure in

26 bovines while no infection and no previous exposure in 27 bovines.

Further, one of the animal found negative for antibodies both in serum as well as

in milk was found positive in PCR indicating the possibilities of the vanishing antibodies

due to long lasting infection (Rahman, 2005) and also due to recent exposure to low dose

of Brucella thus not getting sufficient period for development of immune response or

might be animal born to infected dam and harbouring the infection and retained it in to


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adult life (Corbel, 1988). Similarly, two animals negative for Brucella antibodies in milk

were found positive in PCR. Romero et al. (1995) found one PCR positive sample

negative by milk-ELISA in milk of cattle. JungSuckChan et al. (1998) found 5 bulls

positive both by cultural and PCR methods of 185 seronegative bulls. Guarino et al.

(2000) also found the same result as 5 samples that were negative by CFT and ELISA

found positive by PCR. Kanani (2007) also revealed the same results that PCR and/or

culturally positive bulls were seronegative.

Likewise, some of the bovines showing antibodies both in serum as well as in

milk could not reveal the presence of Brucella in milk either by PCR or cultural isolation.

This might be due to the previous exposure and possibility of periodic shedding or no

shedding of the Brucella in milk (Corbel, 1988). Brucella gene target might have located

in various tissues at a given time might have affected the detection of Brucella in semen

by PCR (Anonymous, 1999). Similarly, Al-Khalaf and El-Khaladi (1989) detected

Brucella antibodies in serum and milk of camel but unable to isolate Brucella organisms

from sediment and cream of milk, however, B. abortus could be isolated from two of the

5 foetuses. Amin et al. (2001) detected Brucella DNA only in 12 semen samples out of

120 RBPT positive animals. Manterola et al. (2003) also failed to detect B. ovis by PCR

as well as by cultural isolation of 4 semen out of the 14 experimentally infected rams.

However, seropositivity due to possibility of cross-reacting antibodies could not be

overlooked (Nielsen, 2002).

Brucella could not be recovered in cultural isolation from 5 PCR positive

samples, which might be due to the slow growth and fastidious nature of the organisms.

Even, types of cultural medium and selective supplements may affect the recovery rate of


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the Brucella from the specimens (Farrell and Robertson, 1972; Shin et al., 1978). In

present study overgrowth of fungi at the last stage of incubation was also observed during

cultural isolation, which might have affected the recovery of Brucella organisms.

The available literature as well as the results of this study indicated the variability

among the different methods for detection of Brucella infection, however, the study

revealed prevalence of Brucella infection in bovine.


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CHAPTER – V

SUMMARY AND CONCLUSIONS

5.1 SUMMARY

Brucellosis is an infectious disease caused by Gram negative facultative

intracellular bacterial organisms of the genus Brucella that are pathogenic for a wide

variety of animals and human beings. The disease is manifested by reproductive failure,

which includes abortion, birth of unthrifty calves and retained placentae in female

animals. Lesions in Brucella infected male are largely confined to the genital organs

including testicles, seminal vesicles and epididymes. The disease has a considerable

impact on human and animal health, as well as socioeconomic impacts, especially, in

which rural income relies largely on livestock breeding and dairy products.

Brucellosis is essentially a disease of sexually matured animals and have

predilection for ungulates placentae, supra mammary lymph nodes, foetal fluids, joints

and testes of bulls, rams, boars and male dogs.

Brucellosis in cattle seems to be associated primarily with intensive farming

practices in large organized dairy farms. Risk behaviours such as unrestricted trade and

movement of animals, increasing demand for dairy products and protein, changing

agricultural methods, use of local cattle yards and fairs for trading, sending dry animals

back to villages for maintenance, use of semen from unscreened bulls for artificial

insemination and poor farm hygiene probably all contribute to the spread and

transmission of the infection and caused concerns that the prevalence may increase. Free

grazing and movement with frequent mixing of flocks of sheep and goats also contribute

to the high prevalence and wide distribution of brucellosis in these animals in India.

Summary and Conclusions…

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The mode of transmission of Brucella from the bovine to the human constitutes a

most important public health factor in connection with infectious abortion and undulant

fever. If the organism remains viable through the manufacturing processes used in the

preparation of the common dairy products and can remain viable in these products over

considerable holding periods, then the use of dairy products made from unpasteurized

milk from infected herds constitutes a channel of infection. Therefore, there is an urgent

need for the strict implementation of a control policy.

Given the complexity of the epidemiology of brucellosis involving various animal

species, the effective control will require a long lasting and carefully controlled and

monitored effort. Measures that may need consideration include improved farm hygiene,

restriction and control of trade and movement of animals, improved food hygiene

including the pasteurization of milk and protection from infection of high risk groups

such as milkers and other people working in the dairy industry. Health education of risk

groups through community participation and health education programmes could play an

important role to increase the acceptance and use of preventive measures. However, for

controlling and eradication of disease the best measure is testing of animals and isolation

and removal of infected animals by routine diagnostic tests that can detect Brucella

infection in biological clinical samples quickly, economically and reliably.

The present study was undertaken to detect Brucella antibodies in serum and

milk, presence of Brucella organisms in milk of bovines and detection of Brucella DNA

by different laboratory procedures for effective diagnosis of brucellosis. For detection of

Brucella antibodies in serum, three serological methods viz., ELISA, RBPT and STAT

were employed, whereas, in milk ELISA and MRT were employed. For isolation of

Brucella organisms Brucella agar medium (BAM) was used and for detection of Brucella


121

DNA by PCR three different genus specific primer pairs viz., B4/B5, JPF/JPR and F4/R2

were used. For quantification of Brucella in milk real-time was employed.

A total of 231 serum samples from bovines were screened for presence of

Brucella antibodies. Of these, 67 serum samples were found positive for Brucella

antibodies by ELISA yielding an overall seropositivity of 29.00% with higher in cattle

(38.29%) than in buffaloes (26.63%).

Among the three serological tests employed for antibody detection in serum, the

highest positive results were obtained by ELISA (67, 29.00%) followed by STAT

(43, 18.61%) and RBPT (18, 7.79%). Considering ELISA as a gold standard test, the

sensitivity of RBPT and STAT were found to be of 25.37% and 61.19%, respectively,

while specificity were found to be of 99.39% and 98.78%, respectively.

A total of 53 milk samples from bovines were screened for presence of Brucella

antibodies. Of these, 15 milk samples were found positive for Brucella antibodies by

ELISA yielding an overall seropositivity of 28.30% with higher in cattle (30.76%) than in

buffaloes (27.50%).

Among the two tests applied to milk for detection of Brucella antibodies ELISA

detected higher number (15, 28.30%) of positive results by ELISA followed by MRT

(08, 15.09%). Considering ELISA as a gold standard test, the sensitivity and specificity

of MRT were found to be 40% and 94.73%, respectively, whereas, agreement between

these two tests was 79.24%.

In comparison of serum antibody and milk antibody detection tests on total 53

serum and milk samples from common individual animals, the ELISA detected antibodies

in more number of serum samples (20, 37.73%) than that of milk (15, 28.30%).

Considering serum ELISA as a gold standard test, the sensitivity of RBPT, STAT, MRT


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and milk-ELISA were found to be of 40.00%, 70.00%, 30.00% and 55.00%, respectively,

while specificity was found to be of 100.00%, 100.00%, 93.93% and 87.87%,

respectively. The overall agreement of 77.34% for RBPT, 88.67% for STAT, 69.81% for

MRT and 75.47% for milk-ELISA were found with the serum ELISA.

Out of 53 milk samples, Brucella could be recovered from four samples on BAM.

Out of these, two were from cattle while two were from buffaloes. A total of six such

isolates of Brucella, two from only milk pellets of two cows and four from both milk

pellets and cream layer of same two buffaloes were recovered.

Based on the growth characters on BAM, blood agar and MacConkey agar,

morphological characters as well as considering the Rapid Slide Agglutination Test and

biochemical tests, all six isolates were identified as Brucella organisms and they were

further confirmed by PCR using three different genus specific primer pairs.

In the process of standardization of methods for DNA extraction from milk,

method described by Romero and Lopez-Goni (1996) was found most suitable among all

the three methods. Of the 53 milk samples tested by three Brucella genus specific primer

pairs, 9 were found positive by B4/B5 primer pair, 1 by JPF/JPR primer pair and 2 by

F4/R2 primer pair. The B4/B5 primer pair found more suitable than other two as they

resulted in highest positive number of samples as well as gave all the samples positive

that were positive by other two primer pairs.

Real-time PCR assay based intercalating dye SYBR Green I using B4/B5 primer

pair was found suitable for quantifying the load of Brucella from the milk. On the basis

of melt curve analysis, detection limits of real-time PCR assay was found up to 50 fg

Brucella DNA or 10 CFU/ml of milk (considering 5 fg is equal to one Brucella cell)


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using 5 µl of template DNA. While, load of Brucella organisms in milk of bovine was

ranged from 1.128 x 104 CFU/ml to 172.800 x 104 CFU/ml of milk.

Comparing the Serum antibody detection, Milk antibody detection, cultural and

molecular methods for detection of Brucella infection in 53 bovines, RBPT, STAT and

ELISA detected 15.09%, 26.41% and 37.73% of positive bovines in serum, respectively.

Whereas, MRT and ELISA detected 15.09% and 28.30% of positive bovines in milk,

respectively. While, 7.54% of bovines were found culturally positive. Among the PCR

assays 16.98%, 1.88% and 3.77% of bovines were found positive by B4/B5, JPF/JPR and

F4/R2 primer pairs, respectively. The highest numbers of bovines were found positive by

serum ELISA whereas, cultural method detected the least number of positive bovines.

Among the four methods, serum antibody detection tests were resulted in highest

number of positive bovines (20, 37.73%) followed by milk antibody detection tests

(17, 32.07%), PCR assays (09, 16.98%) and cultural isolation (04, 7.54%). The over all

agreement between these methods was found 58.49%. Brucella could be isolated from 4

of the 20 and 17 bovines which could detected Brucella antibodies in serum and milk,

respectively. While, Brucella could not be isolated from bovines which were negative for

Brucella antibodies in serum and milk. The over all agreement between serum antibody

detection tests and cultural method was found 69.81%, whereas, 75.47% between milk

antibody detection tests and cultural method. In present study 8 out of 20 seropositive

bovines revealed presence of Brucella DNA in PCR assay thus Brucella DNA could not

be detected in 12 seropositive bovines. However, Brucella DNA could be detected by

PCR from 1 of the 33 seronegative bovines. The over all agreement between serum

antibody detection tests and PCR was found 79.24%. In comparison of PCR with milk

antibody detection tests, 7 out of 17 animals revealed presence of Brucella DNA in PCR


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assay thus, Brucella DNA could not be detected in 10 animals which detected antibodies

in milk. However, Brucella DNA could be detected by PCR from 2 of the 36 bovines

which could not detected Brucella antibodies in mik. The over all agreement between

milk antibody detection tests and PCR was found 77.35%. Brucella DNA could be

detected by PCR from 1 bovines which could not detetced Brucella antibodies both in

serum and milk. All the culturally positive bovines were also found positive by PCR

assay. However, from 5 PCR positive bovines Brucella could not be recovered by cultural

isolation. The over all agreement between cultural and PCR methods was found 90.56%.

The study indicated the variability among the different methods for detection of Brucella

infection, however, the study revealed prevalence of Brucella infection in bovines.


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5.2 CONCLUSIONS

The analysis of the findings from the present study implies following conclusions.

1. The overall seropositivity of 29.00%, 18.61% and 7.79% for Brucella antibodies

by ELISA, STAT and RBPT, respectively.

2. Comparatively higher seropositivity (38.29%) of Brucella antibodies was

observed in cattle than in buffaloes (26.63%) by ELISA.

3. Sensitivity and specificity of RBPT were found 25.37% and 99.39%, respectively,

while that of STAT were found 61.19% and 98.78%, respectively, considering the

ELISA as gold standard test.

4. Out of 53 milk samples, 15 (28.30%) milk samples were found positive using

ELISA for Brucella antibodies, whereas, 08 (15.09%) were found positive by

MRT.

5. Sensitivity and specificity of MRT were found 40.00% and 94.73%, respectively,

considering the ELISA as gold standard test.

6. Milk-ELISA detected higher number of positive samples (15, 28.30%) than the

MRT (08, 15.09%) from 53 milk samples.

7. In comparison of serum antibody and milk antibody detection tests ELISA

revealed more number of positive samples in serum than in milk.

8. The four of the 53 bovines yielded Brucella in milk by cultural isolation

9. From cow milk Brucella could be isolated from the pellet only, whereas, in case

of buffalo milk they could be isolated both from pellet as well as cream. Thus, for

isolation of Brucella from the milk, pellet as well as cream both should be

cultured on suitable appropriate medium.


126

10. For DNA extraction method described by Romero and Lopez-Goni (1996) was

found better in comparison to other two methods indicating the necessity of high

concentration of lysis buffer and high temperature for incubation because the

Brucella cell envelope (CE) was more resistant to non ionic detergents, EDTA and

Tris than those of other Gram negative bacteria because Brucella cell envelope is

held by forces stronger than those acting in the CE of other bacteria.

11. Among the three different genus specific primer pairs used for amplification by

PCR, B4/B5 primer pair detected Brucella DNA in the highest number of milk

samples (16.98%) followed by F4/R2 (3.77%) and JPF/JPR (1.88%) primer pairs.

12. Proper selection of primer pair is essential, as it was affected by the presence of

bovine genomic DNA and finally PCR efficiency.

13. The real-time PCR can be useful for quantifying the load of microorganisms in

milk.

14. Real-time PCR was more sensitive than the conventional PCR.

15. Load of Brucella organisms in milk ranged from 1.128 x 104 CFU/ml to 172.800

x 104 CFU/ml of milk.

16. Agreement between serum antibody detection tests and cultural method, milk

antibody detection tests and cultural method, serum antibody detection tests and

PCR, milk antibody detection tests and PCR, cultural and PCR methods and all

the four methods were found 69.81%, 75.47%, 79.24%, 77.35%, 90.56% and

58.49%, respectively.

17. While comparing antibody detection methods with PCR for 53 bovines, 15.09%

were found positive and 50.94% were negative by both the methods. However,

bovine negative for Brucella antibodies revealed the presence of Brucella DNA


127

and vice versa. Thus in control and eradication programme, it is necessary to test

the bovines both for the presence of antibody and detection of Brucella DNA,

respectively.

18. Comparatively PCR was found more suitable method for detection Brucella in

milk as compared to cultural methods because more numbers of bovines were

found positive by this method as well as all culturally positive bovines also found

positive in PCR.

19. It is further required to identify strains and biotypes of Brucella isolates as well as

to study the molecular characterization of Brucella isolates especially by DNA

sequencing.

20. Finally, the study revealed presence of Brucella antibody in serum and milk as

well as presence of Brucella organisms in the milk of bovines. The seronegative

bovine also revealed the presence of Brucella organisms in milk and vice versa.

Thus under Health Control Programme to eradicate the brucellosis from animals

as well as for public health point of view proper measures must be taken at State

level for controlling brucellosis. Therefore all animals must be tested periodically

for detection of both Brucella antibody and presence of organisms.


128

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APPENDIX

A) DETAILS OF MEDIA, STAINS AND BUFFERS

1. Brucella Agar Medium (BAM)

Brucella agar base (Dehydrated, HiMedia)

Ingredients Grams/liter

Casein enzyme hydrolysate 10.00Dextrose 1.00Peptic digest of animal tissue 10.00Sodium bisulphite 0.10Sodium chloride 5.00Yeast extract 2.00

Brucella selective supplement (HiMedia)

Ingredients 1 Vial

Cycloheximide 50.00 mgNalidixic acid 2.50 mgVancomycin 10.00 mg Nystatin 50000.00 I.U.Bacitracin 12500.00 I.U.Polymyxin B sulphate 2500.00 I.U

Rehydrated the contents of 1 vial with 5ml of 50% methanol.

Brucella agar medium was prepared by suspending 21.55 gm of

dehydrated Brucella agar base in 500 ml of distilled water and sterilized by

autoclaving at 15 psi pressure, 121oC temperature for 15 minutes. The molten

medium was cooled to about 45oC temperature and aseptically added 5% v/v

sterile inactivated horse serum (HiMedia) and rehydrated contents of one vial of

Brucella selective supplement. The above medium was mixed well and poured

into sterile petri plates.

2. Blood Agar (BA)

Blood agar base (Dehydrated, HiMedia)


Beef heart, infusion form 500.00Tryptose 10.00Sodium chloride 5.00Agar 15.00 Final pH (at 25oC) 7.3 + 0.2

Suspended 40 gm of dehydrated blood agar base in 1000 ml distilled water

and sterilized by autoclaving at 15 psi pressure, 121oC temperature for 20

minutes. The molten medium was cooled to about 50oC temperature and

aseptically 5% v/v sterile defibrinated sheep blood was added. The above medium

was mixed well and poured into sterile petri plates.

3. MacConkey Agar (MA) (Dehydrated, HiMedia)


Peptic digest of animal tissue 20.00Lactose 10.00Bile salt 5.00Sodium chloride 5.00Neutral red 0.07Agar 15.00Final pH (at 25oC) 7.5 + 0.2

Suspended 55.07 gm of dehydrated MCA in 1000 ml distilled water and

sterilized by autoclaving at 15 psi pressure, 121oC for 20 minutes. The molten

medium was cooled to about 50oC temperature and poured into sterile petri plates.

4. Nitrate Reduction Test

Test medium

Peptone 10.00 gSodium chloride 5.00 gDistilled water 1000.00 mlAdjusted pH to 7.4 then addedPotassium nitrate 1.00 g

The above medium was sterilized by autoclaving at 10 psi pressure, 115oC

temperature for 30 minutes.

II

Test reagents

a) Sulfanilic acid: 8.0 gm of sulphanilic acid was dissolved in 1000 ml of 5 M

acetic acid.

b) Alpha naphthylamine: 5 gm of alpha naphthylamine was dissolved in 1000 ml

of 5 M acetic acid.

5. Urea Agar

Solution: 1Peptone 1.0 gNacl 1.5 gGlucose 1.0 gPotassium dihydrogen phosphate 2.0 gPhenol red 0.012 g (or 0.2% soln 6 ml)Agar 15.0 gDistilled Water 900.0 mlpH 6.8, Autoclave at, 15 psi pressure for 15 min

Solution: 2Urea 2.0 gDistilled water 100.0 mlpH 6.8 to 6.9, Sterilize by seitz filter

Total 1000 ml

Note: To constitute the medium Solution 2 was added in Solution 1, temperature

of Solution 1 was brought down to 50oC in a water bath maintained at 50oC and

then distributed in test tubes.

6. Indole Test

a) Kovac’s reagents

Paradimethylaminobenzaldehyde 50 gPure amyl or Isoamyl alcohol 75 mlConcentrated pure hydrochloric acid 25 ml

The aldehyde was dissolved in the alcohol by gentle warming in water

bath, cooled and then hydrochloric acid was added. Protected from light and was

stored at 4oC temperature.

III

b) Tryptone water

Tryptone 10 gSodium chloride 5 gDistilled water 1000 ml

The ingredients were dissolved in distilled water by gentle warming and

then sterilized at 15 psi pressure, 121oC temperature for 20 minutes.

7. Motility Sulphide Medium (MSM)


Proteose peptone 10.00Beef extract 3.00L-Cystine 0.20Ferrous ammonium citrate 0.20Sodium citrate 2.00Sodium chloride 5.00Gelatine 80.00Agar 4.00Final pH (at 25oC) 7.3 + 0.2

Suspended 10.44 gm of dehydrated MSM in 100 ml distilled water. The

medium was boiled with constant agitation and dissolved completely. The 4 ml

medium was dispensed in tubes and sterilized by autoclaving at 15 psi pressure,

121ºC for 20 minutes. The tubes were cooled in an upright position.

8. Modified Ziehl-Neelsen (MZN) Stain

a) Diluted carbol fuchsin (HiMedia)

b) Acetic acid (decolourizer)Concentrated acetic acid 1 ml

Distilled water 100 ml

c) Methylene blue (counter stain) Methylene blue 8.0 gEthanol 95% (v/v) 300 ml Distilled water 1300 mlPotassium hydroxide 0.13 g

IV

9. Gram’s Stain

a) Ammonium oxalate crystal violet

Solution 1: Crystal violet 2.0 g

Ethyl alcohol (95 per cent) 20.0 ml

Solution 2: Ammonium oxalate 0.8 g

Distilled water 80.0 ml

Solution 1 and 2 was mixed well and then filtered.

b) Gram’s iodine solution

Iodine 1.0 g

Potassium iodide 2.0 g

The ingredients were dissolved in distilled water to make total volume 300 ml

and then filtered.

c) Acetone or Ethyl alcohol (decolorizer)

d) Safranin (counter stain)

Safranin-O (2.5 per cent solution)

in 95 per cent alcohol 10 ml

Distilled water 100 ml

10. Phenol Saline

NaCl 8.55 g

DI/dH2O Q.S. to 1 liter

Mixed well than add 0.5 ml phenol.

11. Phosphate Buffer Saline Solution (pH 6.4)

Na2HPO4 3.0 g

KH2PO4 6.7 g

NaCl 8.5 g


V

12. Peptone Saline (pH 7.0)

Peptone 10.0 g

NaCl 5.0 g


The ingredients were dissolved and sterilized by autoclaving at 15 psi

pressure, 121ºC for 20 minutes.

13. NET Buffer

NaCl 0.07305 g

EDTA 1.1625 g

Tris-HCl (pH 7.6) 0.197 g

Deionized water up to 25 ml

14. TE Buffer (pH 8.0)

Tris base 0.06 g

EDTA 0.0075 g


15. Chloroform:isoamyl alcohol

Chloroform 24 ml

Isoamyl alcohol 1 ml

16. Phenol:Chloroform:isoamyl alcohol

Phenol 25 ml

Chloroform 24 ml

Isoamyl alcohol 1 ml

17. 3 M Sodium Acetate

Sodium acetate 8.1648 g


18. 70% Ethanol

Absolute ethanol 70 ml

Autoclaved distilled water 30 ml

VI

19. 0.3 X TE

1 X TE 30 ml

Autoclaved distilled water 70 ml

20. TBE (5X)

Tris base 54 g

Boric acid 27.5 g

0.5 M EDTA (pH 8.0) 20 ml


21. Agarose Gel Loading Buffer (6X)

Bromophenol blue 0.25% (w/v)

Xylene cyanol FF 0.25% (w/v)

Ficoll 15% (w/v)

(Type 400; Pharmacia)

Dissolved in appropriate volume of deionized water.

22. Ethidium Bromide (1%)

Ethidium bromide 10 mg

Distilled water 1.0 ml

VII

B) LIST OF EQUIPMENTS

Some of the important equipments used for the present study were as below.

i) Automated ELISA plate washer Labsystems Autowash II,

Labsystem, Sweden

ii) Bench top refrigerated centrifuge Universal 30RF

Hettich Zentrifugen, Germany

iii) CO2 Incubator Binder, Germany

iv) ELISA plate reader Multiskan plus, Labsystems AB,

Stockholm, Sweden

v) Gel documentation system SynGene, Gene Genius Bio Imaging

System, UK

vi) Microcentrifuge Minispin, Eppendorf, Germany

vii) Micropipettes Finnpipette, Thermo Electron

Corporation, USA

viii) Orbital Shaker S-03, Stuart Scientific,UK.

ix) Power pack Power pack 1000, BioRad, USA

ATTO, Japan

x) 7500 Real Time PCR systems Applied Biosystem, USA

xi) Spectrophotometer UV/VIS Spectrophotometer

Unicam, UK

xii) Submarine gel electrophoresis apparatus Atto Corporation, Japan

xiii) Thermocycler MyCycler, Bio-Rad, USA

xiv) UV transilluminator Mini Transilluminator,

Bio-Rad, USA

xv) Weighing balance BP 210 D, Sartorius, Germany

VIII

Table 4.13 Comparison of antibody detection, cultural and molecular methods for detection of brucellosis in bovines

Sr.No

SampleLabel

Species

Detection of Antibody Milk

Serum Milk CulturalIsolation

PCR

RBPT STAT ELISA MRT ELISA B4/B5 JPF/JPR F4/R2

1 C1 Cattle Positive Positive Positive Positive Positive Positive Positive Positive Positive2 C2 Cattle Negative Positive Positive Negative Negative Negative Negative Negative Negative3 C3 Cattle Negative Negative Negative Negative Negative Negative Negative Negative Negative4 C4 Cattle Negative Negative Negative Positive Negative Negative Negative Negative Negative5 C5 Cattle Negative Positive Positive Positive Negative Negative Positive Negative Negative6 C6 Cattle Negative Negative Negative Negative Negative Negative Negative Negative Negative7 C7 Cattle Negative Negative Negative Negative Negative Negative Negative Negative Negative8 C8 Cattle Negative Positive Positive Negative Positive Negative Negative Negative Negative9 C9 Cattle Positive Positive Positive Positive Positive Positive Positive Negative Positive10 C10 Cattle Positive Positive Positive Positive Positive Negative Positive Negative Negative11 C11 Cattle Negative Negative Negative Negative Negative Negative Negative Negative Negative12 C12 Cattle Negative Negative Negative Negative Negative Negative Negative Negative Negative13 C13 Cattle Negative Positive Positive Negative Negative Negative Positive Negative Negative14 B1 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative15 B2 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative16 B3 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative17 B4 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative18 B5 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative19 B6 Buffalo Negative Positive Positive Positive Positive Positive Positive Negative Negative20 B7 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative21 B8 Buffalo Negative Positive Positive Negative Negative Negative Negative Negative Negative22 B9 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative23 B10 Buffalo Negative Negative Positive Negative Positive Negative Negative Negative Negative24 B11 Buffalo Negative Negative Negative Negative Positive Negative Negative Negative Negative25 B12 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative26 B13 Buffalo Negative Positive Positive Negative Negative Negative Negative Negative Negative

Cont…

Sr.No

SampleLabel

Species

Detection of Antibody Milk

Serum Milk CulturalIsolation

PCR

RBPT STAT ELISA MRT ELISA B4/B5 JPF/JPR F4/R2

27 B14 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative28 B15 Buffalo Negative Negative Negative Negative Positive Negative Negative Negative Negative29 B16 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative30 B17 Buffalo Negative Negative Positive Negative Positive Negative Negative Negative Negative31 B18 Buffalo Negative Negative Negative Positive Positive Negative Negative Negative Negative32 B19 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative33 B20 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative34 B21 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative35 B22 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative36 B23 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative37 B24 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative38 B25 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative39 B26 Buffalo Negative Negative Positive Negative Negative Negative Negative Negative Negative40 B27 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative41 B28 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative42 B29 Buffalo Negative Negative Positive Negative Positive Negative Positive Negative Negative43 B30 Buffalo Negative Negative Positive Negative Negative Negative Negative Negative Negative44 B31 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative45 B32 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative46 B33 Buffalo Negative Negative Negative Negative Negative Negative Positive Negative Negative47 B34 Buffalo Negative Negative Negative Negative Positive Negative Negative Negative Negative48 B35 Buffalo Negative Negative Negative Negative Negative Negative Negative Negative Negative49 B36 Buffalo Positive Positive Positive Negative Positive Negative Negative Negative Negative50 B37 Buffalo Positive Positive Positive Positive Positive Negative Negative Negative Negative51 B38 Buffalo Positive Positive Positive Negative Negative Negative Negative Negative Negative52 B39 Buffalo Positive Negative Positive Negative Negative Negative Negative Negative Negative53 B40 Buffalo Positive Positive Positive Negative Positive Positive Positive Negative NegativeTOTAL POSITIVE 08 14 20 08 15 04 09 01 02

% POSITIVE 15.09 26.41 37.73 15.09 28.30 7.54 16.98 1.88 3.77

Cont…

OF Master of Veterinary Science - Semantic Scholar€¦ · COLLEGE OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY ANAND AGRICULTURAL UNIVERSITY ANAND-388001 (GUJARAT) 2007 Reg. No. 04-0213-2005.

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