First isolation of Haemophilus parasuis and other NAD-dependent Pasteurellaceae of swine from European wild boars

Accepted Manuscript

Title: First isolation of Haemophilus parasuis and otherNAD-dependent Pasteurellaceae of swine from Europeanwild boars

Authors: A. Olvera, M. Cerda-Cuellar, G. Mentaberre, E.Casas-Diaz, S. Lavin, I. Marco, V. Aragon

PII: S0378-1135(07)00234-9DOI: doi:10.1016/j.vetmic.2007.05.003Reference: VETMIC 3694

To appear in: VETMIC

Received date: 4-4-2007Revised date: 3-5-2007Accepted date: 10-5-2007

Please cite this article as: Olvera, A., Cerda-Cuellar, M., Mentaberre, G., Casas-Diaz, E.,Lavin, S., Marco, I., Aragon, V., First isolation of Haemophilus parasuis and other NAD-dependent Pasteurellaceae of swine from European wild boars, Veterinary Microbiology(2007), doi:10.1016/j.vetmic.2007.05.003

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DOI : 10.1016/j.vetmic.2007.05.003

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Short communication 1

2

First isolation of Haemophilus parasuis and other NAD-dependent Pasteurellaceae 3

of swine from European wild boars 4

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Running Title: Isolation of H. parasuis from wild boar 6

7

A. Olvera1, M. Cerdà-Cuéllar1, G. Mentaberre2, E. Casas-Diaz2, S. Lavin2, I. Marco2 8

and V. Aragon1* 9

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1 Centre de Recerca en Sanitat Animal (CReSA) - Esfera UAB. Edifici CReSA. Campus 11

de Bellaterra - UAB. 08193-Bellaterra. Barcelona. Spain. 12

2 Servei d'Ecopatologia de Fauna Salvatge, Facultat de Veterinaria, Universitat 13

Autònoma de Barcelona, Campus de Bellaterra. 08193-Bellaterra. Barcelona. Spain. 14

15

* Corresponding author: Virginia Aragon 16

Centre de Recerca en Sanitat Animal (CReSA). Campus de Bellaterra - Universitat 17

Autònoma de Barcelona. 08193-Bellaterra, Barcelona (Spain). 18

Phone: +34 93 581 4494 19

Fax: +34 93 581 4490 20

e-mail: [email protected] 21

Manuscript

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Abstract 22

Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs and the 23

etiological agent of Glässer’s disease, which is characterized by a fibrinous 24

polyserositis, meningitis and arthritis. Glässer’s disease has never been reported in wild 25

boar (Sus scrofa), although antibodies against H. parasuis have been detected. The goal 26

of this study was to confirm the presence of this bacterium in wild boar by bacterial 27

isolation and to compare the strains to H. parasuis from domesticated pigs. Therefore, 28

nasal swabs from 42 hunted wild boars were processed for bacterial isolation and 29

subsequent H. parasuis identification by specific PCR, biochemical tests and 16S rRNA 30

gene sequencing. Two different strains of H. parasuis from two wild boars were 31

isolated. These strains belonged to serotype 2 and were included by 16S rRNA gene 32

sequencing and MLST analysis in a cluster with other H. parasuis strains of nasal origin 33

from domestic pigs. During this study, Actinobacillus minor and Actinobacillus 34

indolicus, which are NAD-dependent Pasteurellaceae closely related to H. parasuis, 35

were also isolated. Our results indicate similarities in the respiratory microbiota of wild 36

boars and domestic pigs, and although H. parasuis was isolated from wild boars, more 37

studies are needed to determine if this could be a source of H. parasuis infection for 38

domestic pigs. 39

40

41

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1. Introduction 42

Haemophilus parasuis is an early colonizer of the respiratory tract of domestic 43

pigs, but it is better known as the etiological agent of Glässer’s disease, which is 44

characterized by a fibrinous polyserositis, meningitis and arthritis (Oliveira and Pijoan, 45

2004; Rapp-Gabrielson et al., 2006). Two studies have attempted to detect the presence 46

of this bacterium in wild boar by serology. The first one was performed in the south-47

central region of Spain (n= 78) and reported no antibodies against H. parasuis (Vicente 48

et al., 2002). However, a recent study reported a prevalence of 18% (n= 178) of wild 49

boars in Slovenia with antibodies against H. parasuis (Vengust et al., 2006). To our 50

knowledge, neither the isolation of H. parasuis nor the existence of Glässer’s disease in 51

wild boar has ever been reported, although it is not rare in domestic pigs weaned 52

outdoors (Docic and Bilkei, 2004). Since domestic pigs and wild boars belong indeed to 53

the same species, it would not be surprising that the microbiota of both animals was 54

similar, although the effect of the different living conditions in modern farms and the 55

wild is not known. The aim of this study was to determine if H. parasuis was indeed 56

present in the upper respiratory tract of wild boars and, if so, which type of strains could 57

be found. 58

59

2. Material and methods 60

2.1. H. parasuis isolation and identification 61

During the hunting season 2005-2006, 42 shot European wild boars (Sus scrofa) from 62

the north eastern of Spain were sampled. Nasal swabs were taken and transported under 63

refrigeration in Amies medium to the laboratory, where they were plated on chocolate 64

agar to isolate single colonies. Isolation and biochemical characterization (Möller and 65

Kilian, 1990) were performed as reported before (Olvera et al., 2006b). DNA was 66

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extracted from pure cultures using a commercial kit (Chelex 100 resin, Bio-Rad 67

Laboratories Inc., CA, USA) following manufacturer’s instructions. Afterwards, H. 68

parasuis-specific PCR (Oliveira et al., 2001) was performed, although final 69

identification was accomplished by 16S rRNA gene sequencing (Olvera et al., 2006a) 70

and database searches using programs based on the Blastn algorithm (Altschul et al., 71

1997) at NCBI (http://www.ncbi.nlm.nih.gov/BLAST). A threshold of 99% sequence 72

identity was used for species identification (Janda and Abbott, 2002). Additionally, a 73

neighbour-joining (NJ) tree with 10,000 bootstraps was constructed with the 16S rRNA 74

gene sequences, including sequences of reference strains from the different serotypes 75

(Kielstein and Rapp-Gabrielson, 1992) currently at the Ribosomal Database Project 76

(http://rdp.cme.msu.edu). Sequences of the type strains of other Pasteurellaceae of 77

swine origin were also included and the tree was rooted using an Escherichia coli 78

sequence. 79

2.2. H. parasuis genotyping and serotyping 80

Genotyping of each isolate was performed by enterobacterial repetitive intergenic 81

consensus (ERIC)-PCR and multilocus sequence typing (MLST) as previously 82

described (Oliveira et al., 2003; Olvera et al., 2006a; Olvera et al., 2006b). 83

Serotype determination was performed by indirect hemagglutination at the Department 84

of Sanidad Animal of the Veterinary School at the University of Leon (Spain) following 85

a previously published protocol (Del Rio et al., 2003). 86

87

3. Results and discussion 88

Selected isolates were genotyped by ERIC-PCR in order to determine if they were 89

different strains (Fig. 1). One representative of each genotype was selected for further 90

characterization, including 16S rRNA gene sequencing (Accession numbers EF396295, 91

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EF396296 and EF396303-EF396307). When a Blastn search was performed with the 92

16S rRNA gene sequences, two isolates (WB21/06-1 and WB24/06-1) were clearly 93

identified as H. parasuis (≥ 99% sequence identity). The geographical origin of these 94

isolates was different: isolate WB21/06-1 came from a 2-3 year-old male, which was 95

captured in Ribes de Freser (Oriental Pyrenees) and isolate WB24/06-1 came from an 96

adult male from Ports de Tortosa i Beseit (Ebre river). Additionally, isolate WB25/06-1 97

showed a 98% 16S rRNA gene sequence identity to H. parasuis strain 131 (Fig 1). On 98

the other hand, isolate WB72/05-2 showed a 99% 16S rRNA sequence identity to 99

Actinobacillus minor NM305 and isolate WB52/06-1 showed 99% 16S rRNA gene 100

sequence identity to Actinobacillus indolicus 37E3. In agreement with the Blastn 101

results, isolates WB21/06-1 and WB24/06-1 clustered with good bootstrap values 102

(>95%) with the H. parasuis reference strains in a neighbour-joining tree constructed 103

with the 16S rRNA gene sequences (Fig 2). As expected, WB52/06-1 clustered with A. 104

indolicus reference strain (bootstrap value >95%) and WB72/05-1 clustered with A. 105

minor (bootstrap value >95%). On the other hand, WB25/06-1 clustered close to A. 106

indolicus (bootstrap value 60%), and not with strain number 131, which was grouped 107

with the H. parasuis sequences, although in a separated branch (65% bootstrap value). 108

Biochemical characterization of the strains supported the identification of WB25/06-1 109

as A. indolicus, since it presented an indol-positive reaction. 110

Isolates WB21/06-1 and WB24/06-4 represented two different strains, firstly indicated 111

by their different ERIC-PCR profile (Fig 1) and confirmed by their different sequence 112

type (ST) in MLST. Isolate WB21/06-1 was ST 157; allelic profile 7, 20, 22, 8, 12, 1, 6, 113

and isolate WB24/06 was ST 147; allelic profile 13, 20, 7, 8, 14, 1, 11, (Accession 114

numbers EF424377-EF424390). Both STs were singletons and shared a maximum of 115

four alleles with STs from isolates of domestic pigs. Nevertheless, no allele was found 116

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to be specific of these wild boar isolates. In addition, these isolates were grouped in a 117

MLST cluster not related to strains isolated from systemic lesions (Olvera et al., 2006b). 118

In agreement, the H. parasuis 16S rRNA gene sequences were separated into two 119

branches as reported in recent works (Olvera et al., 2006a; Angen et al., 2007). One 120

branch included virulent reference strains, such as the Nagasaki strain, while the other 121

branch included non-virulent reference strains. Our two isolates from wild boars were 122

clustered with the non-virulent strains, pointing out that these isolates could be part of 123

the respiratory microbiota of wild boar. Further characterization of WB21/06-1 and 124

WB24/06-4 determined that both strains belonged to serotype 2, which has been 125

described as a moderately virulent serovar in experimental infections (Rapp-Gabrielson 126

et al., 2006), although in some studies no disease could be reproduced (Nielsen, 1993). 127

The low prevalence found in our study could be explained by the fastidious growth 128

requirements of H. parasuis and its difficult isolation from dead animals. However, a 129

previous study performed in Spain based on serology reported 0% prevalence. This 130

incongruence with our results can be explained by the different geographical origin of 131

the animals or by the fact that an animal can be colonized by bacteria without 132

developing circulating antibodies against them. 133

Although the lack of reports of Glässer’s disease in wild boar might be explained by a 134

lack of highly virulent strains, the existence of those in wild boar can not be discarded. 135

In fact, one of the main factors that triggers the development of disease is the early 136

weaning of piglets. Thus, it is probable that wild piglets spent enough time with their 137

mothers to allow the development of natural immunity through a balance between 138

colonization and protection by maternal immunity. On the other hand, another 139

possibility is that wild boars are not susceptible animals for H. parasuis infection and 140

represent solely carriers of the bacterium. 141

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In summary, this study reported the isolation of H. parasuis, A. indolicus and A. 142

minor from nasal swabs from wild boars. This is an indication that the microbiota from 143

the upper respiratory tract of wild boar could be similar to the one of domestic pigs. 144

Although the H. parasuis strains found in wild boars were not different from those of 145

domestic pig, if wild boar could be a source of H. parasuis infection for domestic pigs 146

needs further study. 147

148

Acknowledgements 149

We thank Núria Galofré for technical support. This work was funded by grant 150

AGL2004-07349 from the Ministerio de Educación y Ciencia of Spain. Fellowship 151

support for A. O. from CReSA is also acknowledged. 152

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References 153

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, 154

D.J., 1997, Gapped BLAST and PSI-BLAST: a new generation of protein 155

database search programs. Nucleic Acids Res 25, 3389-3402. 156

Angen, O., Oliveira, S., Ahrens, P., Svensmark, B., Leser, T.D., 2007, Development of 157

an improved species specific PCR test for detection of Haemophilus parasuis. 158

Vet Microbiol 119, 266-276. 159

Del Rio, M.L., Gutierrez, C.B., Rodriguez Ferri, E.F., 2003, Value of indirect 160

hemagglutination and coagglutination tests for serotyping Haemophilus 161

parasuis. J Clin Microbiol 41, 880-882. 162

Docic, M., Bilkei, G., 2004, Prevalence of Haemophilus parasuis serotypes in large 163

outdoor and indoor pig units in Hungary/Romania/Serbia. Berl Munch Tierarztl 164

Wochenschr 117, 271-273. 165

Janda, J.M., Abbott, S.L., 2002, Bacterial identification for publication: when is enough 166

enough? J Clin Microbiol 40, 1887-1891. 167

Kielstein, P., Rapp-Gabrielson, V.J., 1992, Designation of 15 serovars of Haemophilus 168

parasuis on the basis of immunodiffusion using heat-stable antigen extracts. J 169

Clin Microbiol 30, 862-865. 170

Möller, K., Kilian, M., 1990, V factor-dependent members of the family 171

Pasteurellaceae in the porcine upper respiratory tract. J Clin Microbiol 28, 172

2711-2716. 173

Nielsen, R., 1993, Pathogenicity and immunity studies of Haemophilus parasuis 174

serotypes. Acta Vet Scand 34, 193-198. 175

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Oliveira, S., Blackall, P.J., Pijoan, C., 2003, Characterization of the diversity of 176

Haemophilus parasuis field isolates by use of serotyping and genotyping. Am J 177

Vet Res 64, 435-442. 178

Oliveira, S., Galina, L., Pijoan, C., 2001, Development of a PCR test to diagnose 179

Haemophilus parasuis infections. J Vet Diagn Invest 13, 495-501. 180

Oliveira, S., Pijoan, C., 2004, Haemophilus parasuis: new trends on diagnosis, 181

epidemiology and control. Vet Microbiol 99, 1-12. 182

Olvera, A., Calsamiglia, M., Aragon, V., 2006a, Genotypic Diversity of Haemophilus 183

parasuis Field Strains. Appl Environ Microbiol 72, 3984-3992. 184

Olvera, A., Cerdà-Cuéllar, M., Aragon, V., 2006b, Study of the population structure of 185

Haemophilus parasuis by multilocus sequence typing. Microbiol 152, in press. 186

Rapp-Gabrielson, V., Oliveira, S., Pijoan, C., 2006, Haemophilus parasuis, 9th Edition. 187

Iowa State University Press, Iowa, 1153 p. 188

Vengust, G., Valencak, Z., Bidovec, A., 2006, A serological survey of selected 189

pathogens in wild boar in Slovenia. J Vet Med B Infect Dis Vet Public Health 190

53, 24-27. 191

Vicente, J., Leon-Vizcaino, L., Gortazar, C., Jose Cubero, M., Gonzalez, M., Martin-192

Atance, P., 2002, Antibodies to selected viral and bacterial pathogens in 193

European wild boars from southcentral Spain. J Wildl Dis 38, 649-652. 194

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Figure legends 197

198

Fig 1. UPGMA clustering of enterobacterial repetitive intergenic consensus (ERIC)-199

PCR patterns of isolates from nasal swabs from wild boar. One representative isolate for 200

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each strain was selected and it is indicated by an asterisk. 16S rRNA gene of these 201

representative isolates was sequenced and used for confirmation of species 202

identification. The best Blastn species hit and the length of 16S rRNA gene sequence 203

are indicated in the figure. 204

205

Fig. 2. Neigbour-Joining tree of 16S rRNA gene partial sequences using 10,000 206

bootstraps. Sequences obtained in this study are highlighted by black squares. 207

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1473H. parasuis Nº 131WB 25/06-1*

WB 25/06-3

1393A. indolicus 37E3WB 52/06-1

1416A. minor NM305

WB 72/05-2*

WB 72/05-4

WB 21/06-3

WB 21/06-4

1473H. parasuis SW114

WB 24/06-1*

WB 24/06-2

WB 24/06-3

WB 21/06-2

ERIC-PCR Length (bp)Best Blastn HitIsolate

1473H. parasuis SW140

WB 21/06-1*

Pearson correlation (Opt:0.74%) [0.0%-100.0%]

100

80604020

Figure

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Hparasuis SW124 MAFF 911037Hparasuis SW140 MAFF911034Hparasuis No.4 MAFF911032Hparasuis SW114 MAFF911035Hparasuis CCUG 3712Hparasuis Bakos A9Hparasuis NCTC 4557Hparasuis 174Hparasuis D74Hparasuis H465Hparasuis C5

WB21/06-1 WB24/06-1

Hparasuis H367Hparasuis Nagasaki MAFF911038Hparasuis 5D-84-15995Hparasuis 1A-84-22113

Hparasuis No.131Hparasuis ME4

Aindolicus (T) 46KC2Aindolicus 37E3

WB52/6-1

WB25/06-1

Aporcinus sp62Aporcinus 27KC10Aporcinus CCUG38924Aporcinus (T) NM319

Aporcinus B-20Taxon C CAPM5113

Aminor (T) NM305

WB72/05-2

Hsp 202Aporcitonsillarum 99-536-55H-FAporcitonsillarum RF0347

Asuis ATCC33415Asuis CCM5586

Apleuropneumoniae Shope4074Apleuropneumoniae N273

Apleuropneumoniae HS143

5459

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65

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8966

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58

9897

9199

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5199

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