J Vet Diagn Invest 1:223-226 (1989) Control of Johne’s disease in four commercial dairy herds in Iowa Charles O. Thoen, Larry A. Moore Abstract. A 6-year study was conducted in 4 dairy herds in Iowa in which Johne’s disease was diagnosed previously. Fecal specimens were collected at 6-month intervals from animals 2 years of age and over for mycobacteriologic examination. Serum samples were obtained at 3-month intervals and tested by enzyme- linked immunosorbent assay (ELISA). The antigen used in the ELISA was a potassium chloride extract of a field strain of Mycobacterium paratuberculosis. The ELISA reactions were observed in 87% of the cows from which M. paratuberculosis was isolated. Dairy producers that participated in the Johne’s control program reported reduced economic losses. Increased income was attributed to improved milk production, increased value of vaccinated animals sold as replacements to other dairy herds in which Johne’s disease had been diagnosed, and the increased market value of slaughter animals removed from the herd. Johne’s disease (paratuberculosis), a chronic enteric disease in cattle that causes important economic losses, is reported to be widespread in Iowa and other areas in the United States. 10,17 Fecal culture examination has been used widely for confirming a diagnosis of para- tuberculosis in cattle and other animals 14 These pro- cedures are laborious and expensive. Although nu- merous serologic procedures have been described for use in detecting cattle exposed to Mycobacterium para- tuberculosis, the efficacy of these methods are of limited value for identifying animals with subclinical dis- ease. 5,14 The usefulness of serologic tests has been lim- ited by the failure of cattle in early stages of infection to develop detectable levels of humoral antibody. At- tempts to eradicate paratuberculosis from herds by use of fecal culture examinations and/or serologic tests have been reported unsuccessfu1. 12,18 Efforts to control Johne’s disease in a dairy herd without calfhood vaccination revealed that infection persisted during the 5-year study period. l8 No clinical signs of Johne’s disease were reported in cows in the herd during the last year. Results of cultural exami- nation on tissues collected at necropsy revealed that M. paratuberculosis was isolated from 11 of 82 (14%) cows. Positive enzyme-linked immunosorbent assay (ELISA) reactions were detected in sera from 5 of 11 culture-positive animals; 2 animals had positive re- actions on a complement fixation (CF) test, and none were positive on agar-gel immunodiffusion (AGID) From the Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Thoen), and Postville Veterinary Clinic, Postville, IA (Moore). Presented at the 31st Annual Meeting of the AAVLD, Little Rock, AR, October 15-18, 1988. Received for publication November 7, 1988. tests. Similar findings were obtained in 2 other herds in which Johne’s disease was diagnosed. The purpose of this study was to develop additional procedures for use in the diagnosis and control of paratuberculosis in cattle. Materials and methods Protocol for pilot study. An extension of preliminary in- vestigations involved a pilot study on 4 commercial dairy herds in Iowa in which Johne’s disease was diagnosed by cultural examinations of feces. During Phase I of this study (years l-3), the following practices were initiated: 1) fecal cultural examinations were conducted on cows 2 yr of age and over at 6-mo intervals, 2) animals from which M. paratu- berculosis was isolated were immediately removed and sold, 3) replacement heifer calves were vaccinated with heat-killed M. paratuberculosis in oil a ; calves were separated from dams at birth and maintained in a separate unit from the adult cow herds, 4) ELISA was conducted on sera collected at 3-mo intervals on cows, 5) no fecal material was spread on pastures or on land from which hay was to be harvested, and 6) pens and facilities were thoroughly cleaned and disinfected using a cresylic compound at 6-12-mo intervals. During Phase II (years 4-6) the following practices were initiated: 1) all replacement heifers were vaccinated, 2) calves were separated immediately from dams at birth, 3) pasture rotation was followed, 4) fecal culture examinations were conducted annually, 5) calves from M. paratuberculosis- in- fected cows were not kept as herd replacements, 6) culture- positive cows were sold immediately, and 7) fecal material was removed from lots and facilities were disinfected pe- riodically. Mycobacteriological examination. Fecal samples were collected and shipped to the laboratory and processed using 0.3% benzalkonium chloride as previously described. 14 My- cobacteriological examinations conducted on tissues collect- ed at necropsy were processed using 0.2% zephiran. Slants of Middlebrook’s 7H10 egg yolk agar medium with or with- 223
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UnknownJ Vet Diagn Invest 1:223-226 (1989)
Control of Johne’s disease in four commercial dairy herds in Iowa
Charles O. Thoen, Larry A. Moore
Abstract. A 6-year study was conducted in 4 dairy herds in Iowa in which Johne’s disease was diagnosed previously. Fecal specimens were collected at 6-month intervals from animals 2 years of age and over for mycobacteriologic examination. Serum samples were obtained at 3-month intervals and tested by enzyme- linked immunosorbent assay (ELISA). The antigen used in the ELISA was a potassium chloride extract of a field strain of Mycobacterium paratuberculosis. The ELISA reactions were observed in 87% of the cows from which M. paratuberculosis was isolated. Dairy producers that participated in the Johne’s control program reported reduced economic losses. Increased income was attributed to improved milk production, increased value of vaccinated animals sold as replacements to other dairy herds in which Johne’s disease had been diagnosed, and the increased market value of slaughter animals removed from the herd.
Johne’s disease (paratuberculosis), a chronic enteric disease in cattle that causes important economic losses, is reported to be widespread in Iowa and other areas in the United States. 10,17 Fecal culture examination has been used widely for confirming a diagnosis of para- tuberculosis in cattle and other animals14 These pro- cedures are laborious and expensive. Although nu- merous serologic procedures have been described for use in detecting cattle exposed to Mycobacterium para- tuberculosis, the efficacy of these methods are of limited value for identifying animals with subclinical dis- ease.5,14 The usefulness of serologic tests has been lim- ited by the failure of cattle in early stages of infection to develop detectable levels of humoral antibody. At- tempts to eradicate paratuberculosis from herds by use of fecal culture examinations and/or serologic tests have been reported unsuccessfu1.12,18
Efforts to control Johne’s disease in a dairy herd without calfhood vaccination revealed that infection persisted during the 5-year study period.l8 No clinical signs of Johne’s disease were reported in cows in the herd during the last year. Results of cultural exami- nation on tissues collected at necropsy revealed that M. paratuberculosis was isolated from 11 of 82 (14%) cows. Positive enzyme-linked immunosorbent assay (ELISA) reactions were detected in sera from 5 of 11 culture-positive animals; 2 animals had positive re- actions on a complement fixation (CF) test, and none were positive on agar-gel immunodiffusion (AGID)
From the Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Thoen), and Postville Veterinary Clinic, Postville, IA (Moore).
Presented at the 31st Annual Meeting of the AAVLD, Little Rock, AR, October 15-18, 1988.
Received for publication November 7, 1988.
tests. Similar findings were obtained in 2 other herds in which Johne’s disease was diagnosed. The purpose of this study was to develop additional procedures for use in the diagnosis and control of paratuberculosis in cattle.
Materials and methods
Protocol for pilot study. An extension of preliminary in- vestigations involved a pilot study on 4 commercial dairy herds in Iowa in which Johne’s disease was diagnosed by cultural examinations of feces. During Phase I of this study (years l-3), the following practices were initiated: 1) fecal cultural examinations were conducted on cows 2 yr of age and over at 6-mo intervals, 2) animals from which M. paratu- berculosis was isolated were immediately removed and sold, 3) replacement heifer calves were vaccinated with heat-killed M. paratuberculosis in oila; calves were separated from dams at birth and maintained in a separate unit from the adult cow herds, 4) ELISA was conducted on sera collected at 3-mo intervals on cows, 5) no fecal material was spread on pastures or on land from which hay was to be harvested, and 6) pens and facilities were thoroughly cleaned and disinfected using a cresylic compound at 6-12-mo intervals.
During Phase II (years 4-6) the following practices were initiated: 1) all replacement heifers were vaccinated, 2) calves were separated immediately from dams at birth, 3) pasture rotation was followed, 4) fecal culture examinations were conducted annually, 5) calves from M. paratuberculosis- in- fected cows were not kept as herd replacements, 6) culture- positive cows were sold immediately, and 7) fecal material was removed from lots and facilities were disinfected pe- riodically.
Mycobacteriological examination. Fecal samples were collected and shipped to the laboratory and processed using 0.3% benzalkonium chloride as previously described.14 My- cobacteriological examinations conducted on tissues collect- ed at necropsy were processed using 0.2% zephiran. Slants of Middlebrook’s 7H10 egg yolk agar medium with or with-
223
224 Thoen, Moore
Table 1. Results of cultural examinations and other herd infor- mation during Phase I (years l-3) in 4 dairy herds in which Johne’s disease was diagnosed.
Herd number
1 2 3 (n = 53)† (n = 56) (n = 42) (n =28)
Initial infection rate (%) 12 15 25 9 Number sold per year for 3 yr 8 10 12 2 Number of animals that died‡ 1 2 24 0 Number of vaccinees 47 42 10 12 Number of non-vaccinees 6 14 32 16
* Results for 1 yr. † n = number of cows in herd. ‡ Includes cows and heifers.
out mycobactin were inoculated. The cultures were incubated at 37 C for 20 wk; cultures were observed for appearance of colonies at 2-wk intervals. Mycobacterium paratuberculosis was identified by subculture on media with or without my- cobactin.
ELISA. An indirect ELISA was conducted using a non- fractionated potassium chloride (KCl) extract of a field strain of M. paratuberculosis containing phenylmethylsulfonyl- fluoride, a protease inhibitor; the antigen was diluted in 0.1 M Na2CO2 (pH 9.6) .6,16 An aliquot of antigen (0.05 ml cor- responding to 500 ng protein) was added to each well of a microtiter plate.b Carbodiimidec (50 µ1) diluted in 0.1 M Na2CO3 (1 mg/ml) was added to each well. The microtiter plates were placed in plastic bags, incubated for 16 hr at 4 C, and washed 3 times with phosphate-buffered saline so- lution (PBSS) (pH 7.2). Then 0.01 M NH4Cl (0.1 ml/well) was added to each well, and the plates were incubated for 30 min at 22 C. The plates were washed 3 times with wash solution (0.5 M NaCl containing 0.5% Tween 80, adjusted to pH 7.5 with 1 N NaOH).
Sera obtained from cattle, 2 yr of age and older, were serially diluted (1:40 to 1:640) in ELISA diluent (0.5 NaCl containing 1% bovine serum albumin and 1% Tween 80, pH 7.5), and 0.1 ml was added to wells of the plates. Serum samples were incubated for 30 min at 22 C on a horizontal shaker.d The plates were then washed 8 times with wash solution and allowed to stand inverted 30 min. An affinity- purified goat anti-bovine immunoglobulin G, heavy and light chain specific, labeled with horseradish peroxidase,e was added to each well (0.05 ml, 20 ng/well), and the wells were incu- bated for 30 min at 22 C on a horizontal shaker. Microtiter plates were again washed 8 times and allowed to stand 30 min. A substrate-indicator solution of hydrogen peroxide and 2,2'-azino-di-(3-ethyl benzthiazoline-6-sulfonate)f in 0.05 M citric acid (pH 4.0) was added to each well (0.15 ml) and incubated for 1 hr. The color intensity of the reactions was determined at a wavelength of 405 nm.b
Results Results of laboratory examinations, calf vaccina-
tion, and relevant information on history in the 4 herds, in which paratuberculosis was diagnosed during Phase
I (3-year period), are shown in Table 1. The initial infection rate (number of animals positive on fecal culture divided by the total number of cows) was from 9 to 25%. Twenty-four deaths were reported during a 3-year period in herd 3, 2 cows died in herd 2, and 1 cow died in herd 1. The number of cows sold in which Johne’s disease was confirmed by culture or suspect due to presence of clinical disease varied for each of the 4 herds. The number of calves vaccinated with heat-killed M. paratuberculusis in oil in each herd was as follows: herd 1, 47 of 53 (88%); herd 2, 42 of 56 (75%); herd 3, 10 of 42 (24%); and herd 4, 12 of 28 (43%).
A comparison of results of ELISA of sera and bac- teriologic examination on fecal specimens from cows in 4 herds is shown in Table 2. Positive ELISA reac- tions were detected in sera of 3 1 of 46 (67%) cows from which M. paratuberculosis was isolated. Twenty-two of 28 cows with clinical Johne’s disease had positive ELISA reactions, and 5 had suspicious ELISA reac- tions. Nine of 18 (50%) cows with subclinical Johne’s disease had positive ELISA reactions, 4 were suspi- cious, and 5 were negative. Positive ELISA reactions were detected in 10 cows (3 vaccinees) from which M. paratubercuhsis was not isolated from feces; 9 of 23 which had suspicious ELISA reactions were vaccinees. No ELISA reactions were detected in sera of 149 cows negative on fecal culture. Serum dilutions of 1:160 and 1:320 provide the most reliable information in iden- tifying cattle infected with M. paratuberculosis. The ELISA identified 87% of the culture-positive cows tested in herd 1, 78% of positive cows in herd 2, 92% of positive cows in herd 3, and 100% of positive cows in herd 4.
The results of cultural examinations and related in- formation for each of the 4 herds during Phase II (years 4-6) are shown in Table 3. All replacement heifers in each of the herds were vaccinated. The number of fecal culture-positive cows decreased in each of the 4 herds. Two of 3 cows that developed clinical Johne’s disease in herd 2 had positive ELISA reactions on sera col- lected previously. One of 2 cows in herd 3 had a pos- itive ELISA reaction on sera. Mycobacterium paratu- berculosis was not isolated from these animals.
Discussion
Efforts to eradicate Johne’s disease in cattle herds have been limited by the lack of reliable serologic tests for use in detecting cattle with subclinical Johne’s dis- ease. l7 Although serologic tests have been developed to detect mycobacterial antibodies in sera of cattle with advanced clinical disease, these procedures are of little value in identifying cattle in early stages of infection with M. paratuberculosis. 12,18 Results obtained in this study provide further evidence that ELISA is a more
Control of Johne’s disease 225
Table 2. Comparison of results of ELISA and fecal culture ex- Table 3. Results of cultural examination and other herd infor- aminations on cows in 4 herds in which Johne’s disease was diag- mation during Phase II (years 4-6) in 4 dairy herds in which Johne’s nosed. disease was diagnosed.
ELISA reactions Herd number
1 2 3 4
Total number of heifers in last 3 yr Replacements per year Number vaccinated per year Number of culture-positive cows in
63 54 30 45 21 18 10 15 21 18 10 15
last 3 yr 2 1 0 0 Number of clinical cases in last 3 yr 0 3* 2 0
* None in last 2 yr.
sensitive test than CF or AGID tests for detecting cows exposed to M. paratuberculosis. 2,4,20 Apparently, mac- rophages of some animals engulf and retain M. para- tuberculosis in an intracellular environment, thus min- imizing the release of antigens and subsequent stimulation of antibody-forming cells. The importance of M cells in the ileum as a site for antigen availability in development of immunologic responses remains to be elucidated.11
The efficiency of serologic procedures in detecting antigen-antibody reaction may be influenced by the specificity of antibodies against determinants on the antigens.13 Studies indicate absorption of sera from cattle with Mycobacterium phlei may improve speci- ficity.20 However, other investigations indicate absorp- tion fails to provide definitive results.4 In this study, KC1 extract of a field strain of M. paratuberculosis was used as the antigen in the ELISA. The preparations of this antigen from live cells differs from other antigens prepared from autoclaved cells or culture filtrates. De- naturation of proteins by heat treatment is well doc- umented. The KC1 extract of M. paratuberculosis used in this study was prepared from live M. paratubercu- losis recently isolated from a cow with clinical disease, therefore circumventing alterations of the protein an- tigens due to heat inactivation.
The increased susceptibility of young calves as com- pared with that of adult cattle has been reported previously8; therefore, the segregation of calves as soon after birth as possible is important in reducing expo- sure to M. paratuberculosis. l5 Control measures should emphasize the importance of thoroughly cleaning fa- cilities in which cattle are maintained and disinfection with a cresylic compound.
Considerable economic losses due to Johne’s disease in dairy cattle have been reported.’ Economic benefits were realized by each of the herd owners participating in this study. Moreover, continued benefits could be anticipated in their herds, because it is expected the losses due to Johne’s disease will be reduced markedly.
Acknowledgements
The authors acknowledge the technical assistance of Kyle Waite. This work was supported in part by Special Grant No. 82-CRSR-2-2022, US Department of Ag- riculture, Washington, DC.
Sources and manufacturers
Because the isolation of M. paratuberculosis often requires 6 to 8 weeks, the development of a DNA probe to detect M. paratuberculosis in fecal or tissue speci- mens would allow for a more rapid diagnosis. Avail- able information indicates M. paratuberculosis and certain strains of Mycobacterium avium complex iso- lated from animals are closely related; therefore, it will be necessary to identify “species-specific” segments of DNA.3,7,9,19
Fromm Laboratories, Grafton, WI. Dynatec Laboratories, Inc., Alexandria, VA. Sigma Chemical Company, St. Louis, MO. Arthur H. Thomas Co., Philadelphia, PA. Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD. National Veterinary Services, US Department of Agriculture, Ames, IA.
References
Sporadic death loses are often reported in herds in which Johne’s disease persists.17 It was of interest that 24 heifers and cows died in herd 3 during Phase I of this study. Since no other etiologic agents were asso- ciated with these deaths, the losses were attributed to M. paratuberculosis infection by the veterinarian re- sponsible for the herds’ health.
Benedictus G, Dijkhuizen AA, Stelwagen J: 1987, Economic losses due to paratuberculosis in dairy cattle. Vet Record 121: 142-146. Colgrove GS, Thoen CO, Blackburn BO, Murphy CD: 1989, Paratuberculosis in cattle: a comparison of three serologic tests with results of fecal culture. Vet Microbiol 19:183-l87. Collins DM, DeLisle GW: 1986, Restriction endonuclease analysis of Mycobacterium paratubercu1osis isolated from cattle. Am J Vet Res 47:2226-2229. Eingereicht AK: 1988, Vergleichende Untersuchungen zur di- agnose der Paratuberkulose des Rindes Mittels KBR, ELISA, und AGIDT. Inaugural-Disseration zur Eriangung des Doktor-
226 Thoen, Moore
grades beim Fachbereich Veterinarmedizin der Justus-Liebig- Universitat Geiben.
5. Goudswaard J, Gilmour NJL, Dijkstra RG, et al.: 1976, Di- agnosis of Johne’s disease in cattle: a comparison of five sero- logic tests under field conditions. Vet Rec 98:461462
6. Hall MR, Thoen CO: 1983, Preparation of biologically active extracts of Mycobacterium bovis, using Triton X-100 or potas- sium chloride. Am J Vet Res 44:1602-l604.
7. Labidi A, Thoen CO: 1989, Genetic relatedness of Mycobac- terium paratuberculosis and of M. avium. Acta Leprol (in press).
8. Larsen AB, Merkal RS, Cutlip RC: 1975, Age of cattle as related to resistence to infection with Mycobacterium paratuberculosis. Am J Vet Res 36:225-257.
9. McFadden J, Hull E, Taylor JH: 1988, DNA probes to identify and detect Mycobacterium paratuberculosis in clinical and vet- erinary samples. Proc 2nd Int Colloq Paratuberculosis, pp. 20l- 205. Sept. 22-23, 1988.
10. Merkal RS, Whipple DL, Sacks JM, Snyder GR: 1987, Prev- alence of Mycobacterium paratuberculosis in ileocecal lymph nodes of cattle culled in the United States. J Am Vet Med Assn 190:676-680.
11. Momotani E, Whipple DL, Thiermann AB, Cheville NF: 1988, Role of M cells and macrophages in the entrance of Mycobac- terium parutuberculosis into domes of ileal Peyer’s patches in calves. Vet Pathol 25:13l-l37.
12. Ringdal G: 1965, Studies on Johne’s disease in a single herd during a five-year period. Nord Vet Med 17:73-96.
13. Roitt I, Brosstoff J, Male D: 1985, Immunology. The C. V. Mosby Co., St. Louis, MO.
14. Thoen CO: 1989, Mycobacterium. In: Diagnostic procedures in veterinary bacteriology and mycology, ed. Carter GC, Cole JR, 5th ed. Charles C Thomas Publ., Springfield, IL (in press).
15. Thoen CO, Baum K: 1988, Current knowledge on paratuber- culosis. J Am Vet Med Assn 192:1609-l611.
16. Thoen CO, Hall MR, Petersburg TA, Angus RD: 1983, De- velopment of a modified enzyme-linked immunosorbent assay for detecting mycobacterial antibodies in sera of cattle from which Mycobacterium paratuberculosis was isolated. Proc 3rd Int Symp Vet Lab Diagn 1: 141-150.
17. Thoen CO, Muscoplat CC: 1979, Recent developments in di- agnosis of paratuberculosis (Johne’s disease). J Am Vet Med Assn 174:838-840
18. Thoen CO, Stumpff CD, Tupper D, et al.: 1983, Some obser- vations on naturally occurring paratuberculosis (Johne’s dis- ease) in cattle herds in Nebraska, Colorado, and Minnesota. Proc 1st Int Colloq Res Paratuberculosis. Pp. 127-135.
19. Whipple DL, Le Febvre RB, Andrews RE, Thierman AB: 1987, Isolation and analysis of restriction endonuclease digestive pat- terns of chromosomal DNA from Mycobacterium paratuber- culosis and other Mycobacterium species. J Clin Microbiol 25: 1511-1515.
Control of Johne’s disease in four commercial dairy herds in Iowa
Charles O. Thoen, Larry A. Moore
Abstract. A 6-year study was conducted in 4 dairy herds in Iowa in which Johne’s disease was diagnosed previously. Fecal specimens were collected at 6-month intervals from animals 2 years of age and over for mycobacteriologic examination. Serum samples were obtained at 3-month intervals and tested by enzyme- linked immunosorbent assay (ELISA). The antigen used in the ELISA was a potassium chloride extract of a field strain of Mycobacterium paratuberculosis. The ELISA reactions were observed in 87% of the cows from which M. paratuberculosis was isolated. Dairy producers that participated in the Johne’s control program reported reduced economic losses. Increased income was attributed to improved milk production, increased value of vaccinated animals sold as replacements to other dairy herds in which Johne’s disease had been diagnosed, and the increased market value of slaughter animals removed from the herd.
Johne’s disease (paratuberculosis), a chronic enteric disease in cattle that causes important economic losses, is reported to be widespread in Iowa and other areas in the United States. 10,17 Fecal culture examination has been used widely for confirming a diagnosis of para- tuberculosis in cattle and other animals14 These pro- cedures are laborious and expensive. Although nu- merous serologic procedures have been described for use in detecting cattle exposed to Mycobacterium para- tuberculosis, the efficacy of these methods are of limited value for identifying animals with subclinical dis- ease.5,14 The usefulness of serologic tests has been lim- ited by the failure of cattle in early stages of infection to develop detectable levels of humoral antibody. At- tempts to eradicate paratuberculosis from herds by use of fecal culture examinations and/or serologic tests have been reported unsuccessfu1.12,18
Efforts to control Johne’s disease in a dairy herd without calfhood vaccination revealed that infection persisted during the 5-year study period.l8 No clinical signs of Johne’s disease were reported in cows in the herd during the last year. Results of cultural exami- nation on tissues collected at necropsy revealed that M. paratuberculosis was isolated from 11 of 82 (14%) cows. Positive enzyme-linked immunosorbent assay (ELISA) reactions were detected in sera from 5 of 11 culture-positive animals; 2 animals had positive re- actions on a complement fixation (CF) test, and none were positive on agar-gel immunodiffusion (AGID)
From the Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Thoen), and Postville Veterinary Clinic, Postville, IA (Moore).
Presented at the 31st Annual Meeting of the AAVLD, Little Rock, AR, October 15-18, 1988.
Received for publication November 7, 1988.
tests. Similar findings were obtained in 2 other herds in which Johne’s disease was diagnosed. The purpose of this study was to develop additional procedures for use in the diagnosis and control of paratuberculosis in cattle.
Materials and methods
Protocol for pilot study. An extension of preliminary in- vestigations involved a pilot study on 4 commercial dairy herds in Iowa in which Johne’s disease was diagnosed by cultural examinations of feces. During Phase I of this study (years l-3), the following practices were initiated: 1) fecal cultural examinations were conducted on cows 2 yr of age and over at 6-mo intervals, 2) animals from which M. paratu- berculosis was isolated were immediately removed and sold, 3) replacement heifer calves were vaccinated with heat-killed M. paratuberculosis in oila; calves were separated from dams at birth and maintained in a separate unit from the adult cow herds, 4) ELISA was conducted on sera collected at 3-mo intervals on cows, 5) no fecal material was spread on pastures or on land from which hay was to be harvested, and 6) pens and facilities were thoroughly cleaned and disinfected using a cresylic compound at 6-12-mo intervals.
During Phase II (years 4-6) the following practices were initiated: 1) all replacement heifers were vaccinated, 2) calves were separated immediately from dams at birth, 3) pasture rotation was followed, 4) fecal culture examinations were conducted annually, 5) calves from M. paratuberculosis- in- fected cows were not kept as herd replacements, 6) culture- positive cows were sold immediately, and 7) fecal material was removed from lots and facilities were disinfected pe- riodically.
Mycobacteriological examination. Fecal samples were collected and shipped to the laboratory and processed using 0.3% benzalkonium chloride as previously described.14 My- cobacteriological examinations conducted on tissues collect- ed at necropsy were processed using 0.2% zephiran. Slants of Middlebrook’s 7H10 egg yolk agar medium with or with-
223
224 Thoen, Moore
Table 1. Results of cultural examinations and other herd infor- mation during Phase I (years l-3) in 4 dairy herds in which Johne’s disease was diagnosed.
Herd number
1 2 3 (n = 53)† (n = 56) (n = 42) (n =28)
Initial infection rate (%) 12 15 25 9 Number sold per year for 3 yr 8 10 12 2 Number of animals that died‡ 1 2 24 0 Number of vaccinees 47 42 10 12 Number of non-vaccinees 6 14 32 16
* Results for 1 yr. † n = number of cows in herd. ‡ Includes cows and heifers.
out mycobactin were inoculated. The cultures were incubated at 37 C for 20 wk; cultures were observed for appearance of colonies at 2-wk intervals. Mycobacterium paratuberculosis was identified by subculture on media with or without my- cobactin.
ELISA. An indirect ELISA was conducted using a non- fractionated potassium chloride (KCl) extract of a field strain of M. paratuberculosis containing phenylmethylsulfonyl- fluoride, a protease inhibitor; the antigen was diluted in 0.1 M Na2CO2 (pH 9.6) .6,16 An aliquot of antigen (0.05 ml cor- responding to 500 ng protein) was added to each well of a microtiter plate.b Carbodiimidec (50 µ1) diluted in 0.1 M Na2CO3 (1 mg/ml) was added to each well. The microtiter plates were placed in plastic bags, incubated for 16 hr at 4 C, and washed 3 times with phosphate-buffered saline so- lution (PBSS) (pH 7.2). Then 0.01 M NH4Cl (0.1 ml/well) was added to each well, and the plates were incubated for 30 min at 22 C. The plates were washed 3 times with wash solution (0.5 M NaCl containing 0.5% Tween 80, adjusted to pH 7.5 with 1 N NaOH).
Sera obtained from cattle, 2 yr of age and older, were serially diluted (1:40 to 1:640) in ELISA diluent (0.5 NaCl containing 1% bovine serum albumin and 1% Tween 80, pH 7.5), and 0.1 ml was added to wells of the plates. Serum samples were incubated for 30 min at 22 C on a horizontal shaker.d The plates were then washed 8 times with wash solution and allowed to stand inverted 30 min. An affinity- purified goat anti-bovine immunoglobulin G, heavy and light chain specific, labeled with horseradish peroxidase,e was added to each well (0.05 ml, 20 ng/well), and the wells were incu- bated for 30 min at 22 C on a horizontal shaker. Microtiter plates were again washed 8 times and allowed to stand 30 min. A substrate-indicator solution of hydrogen peroxide and 2,2'-azino-di-(3-ethyl benzthiazoline-6-sulfonate)f in 0.05 M citric acid (pH 4.0) was added to each well (0.15 ml) and incubated for 1 hr. The color intensity of the reactions was determined at a wavelength of 405 nm.b
Results Results of laboratory examinations, calf vaccina-
tion, and relevant information on history in the 4 herds, in which paratuberculosis was diagnosed during Phase
I (3-year period), are shown in Table 1. The initial infection rate (number of animals positive on fecal culture divided by the total number of cows) was from 9 to 25%. Twenty-four deaths were reported during a 3-year period in herd 3, 2 cows died in herd 2, and 1 cow died in herd 1. The number of cows sold in which Johne’s disease was confirmed by culture or suspect due to presence of clinical disease varied for each of the 4 herds. The number of calves vaccinated with heat-killed M. paratuberculusis in oil in each herd was as follows: herd 1, 47 of 53 (88%); herd 2, 42 of 56 (75%); herd 3, 10 of 42 (24%); and herd 4, 12 of 28 (43%).
A comparison of results of ELISA of sera and bac- teriologic examination on fecal specimens from cows in 4 herds is shown in Table 2. Positive ELISA reac- tions were detected in sera of 3 1 of 46 (67%) cows from which M. paratuberculosis was isolated. Twenty-two of 28 cows with clinical Johne’s disease had positive ELISA reactions, and 5 had suspicious ELISA reac- tions. Nine of 18 (50%) cows with subclinical Johne’s disease had positive ELISA reactions, 4 were suspi- cious, and 5 were negative. Positive ELISA reactions were detected in 10 cows (3 vaccinees) from which M. paratubercuhsis was not isolated from feces; 9 of 23 which had suspicious ELISA reactions were vaccinees. No ELISA reactions were detected in sera of 149 cows negative on fecal culture. Serum dilutions of 1:160 and 1:320 provide the most reliable information in iden- tifying cattle infected with M. paratuberculosis. The ELISA identified 87% of the culture-positive cows tested in herd 1, 78% of positive cows in herd 2, 92% of positive cows in herd 3, and 100% of positive cows in herd 4.
The results of cultural examinations and related in- formation for each of the 4 herds during Phase II (years 4-6) are shown in Table 3. All replacement heifers in each of the herds were vaccinated. The number of fecal culture-positive cows decreased in each of the 4 herds. Two of 3 cows that developed clinical Johne’s disease in herd 2 had positive ELISA reactions on sera col- lected previously. One of 2 cows in herd 3 had a pos- itive ELISA reaction on sera. Mycobacterium paratu- berculosis was not isolated from these animals.
Discussion
Efforts to eradicate Johne’s disease in cattle herds have been limited by the lack of reliable serologic tests for use in detecting cattle with subclinical Johne’s dis- ease. l7 Although serologic tests have been developed to detect mycobacterial antibodies in sera of cattle with advanced clinical disease, these procedures are of little value in identifying cattle in early stages of infection with M. paratuberculosis. 12,18 Results obtained in this study provide further evidence that ELISA is a more
Control of Johne’s disease 225
Table 2. Comparison of results of ELISA and fecal culture ex- Table 3. Results of cultural examination and other herd infor- aminations on cows in 4 herds in which Johne’s disease was diag- mation during Phase II (years 4-6) in 4 dairy herds in which Johne’s nosed. disease was diagnosed.
ELISA reactions Herd number
1 2 3 4
Total number of heifers in last 3 yr Replacements per year Number vaccinated per year Number of culture-positive cows in
63 54 30 45 21 18 10 15 21 18 10 15
last 3 yr 2 1 0 0 Number of clinical cases in last 3 yr 0 3* 2 0
* None in last 2 yr.
sensitive test than CF or AGID tests for detecting cows exposed to M. paratuberculosis. 2,4,20 Apparently, mac- rophages of some animals engulf and retain M. para- tuberculosis in an intracellular environment, thus min- imizing the release of antigens and subsequent stimulation of antibody-forming cells. The importance of M cells in the ileum as a site for antigen availability in development of immunologic responses remains to be elucidated.11
The efficiency of serologic procedures in detecting antigen-antibody reaction may be influenced by the specificity of antibodies against determinants on the antigens.13 Studies indicate absorption of sera from cattle with Mycobacterium phlei may improve speci- ficity.20 However, other investigations indicate absorp- tion fails to provide definitive results.4 In this study, KC1 extract of a field strain of M. paratuberculosis was used as the antigen in the ELISA. The preparations of this antigen from live cells differs from other antigens prepared from autoclaved cells or culture filtrates. De- naturation of proteins by heat treatment is well doc- umented. The KC1 extract of M. paratuberculosis used in this study was prepared from live M. paratubercu- losis recently isolated from a cow with clinical disease, therefore circumventing alterations of the protein an- tigens due to heat inactivation.
The increased susceptibility of young calves as com- pared with that of adult cattle has been reported previously8; therefore, the segregation of calves as soon after birth as possible is important in reducing expo- sure to M. paratuberculosis. l5 Control measures should emphasize the importance of thoroughly cleaning fa- cilities in which cattle are maintained and disinfection with a cresylic compound.
Considerable economic losses due to Johne’s disease in dairy cattle have been reported.’ Economic benefits were realized by each of the herd owners participating in this study. Moreover, continued benefits could be anticipated in their herds, because it is expected the losses due to Johne’s disease will be reduced markedly.
Acknowledgements
The authors acknowledge the technical assistance of Kyle Waite. This work was supported in part by Special Grant No. 82-CRSR-2-2022, US Department of Ag- riculture, Washington, DC.
Sources and manufacturers
Because the isolation of M. paratuberculosis often requires 6 to 8 weeks, the development of a DNA probe to detect M. paratuberculosis in fecal or tissue speci- mens would allow for a more rapid diagnosis. Avail- able information indicates M. paratuberculosis and certain strains of Mycobacterium avium complex iso- lated from animals are closely related; therefore, it will be necessary to identify “species-specific” segments of DNA.3,7,9,19
Fromm Laboratories, Grafton, WI. Dynatec Laboratories, Inc., Alexandria, VA. Sigma Chemical Company, St. Louis, MO. Arthur H. Thomas Co., Philadelphia, PA. Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD. National Veterinary Services, US Department of Agriculture, Ames, IA.
References
Sporadic death loses are often reported in herds in which Johne’s disease persists.17 It was of interest that 24 heifers and cows died in herd 3 during Phase I of this study. Since no other etiologic agents were asso- ciated with these deaths, the losses were attributed to M. paratuberculosis infection by the veterinarian re- sponsible for the herds’ health.
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