Seroprevalence and risk factors associated with Haemophilus parasuis infection in Tibetan pigs in Tibet

Acta Tropica 132 (2014) 94–97

Contents lists available at ScienceDirect

Acta Tropica journal homepage: www.elsevier.com/locate/actatropica

Seroprevalence and risk factors associated with Haemophilus parasuis infection in Tibetan pigs in Tibet Nian-Zhang Zhang a , Dong-Hui Zhou a,∗ , Si-Yang Huang a , Meng Wang a , Xin-Chun Shi a,b , Danba Ciren c , Xing-Quan Zhu a,d,∗∗ a State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, People’s Republic of China b College of Veterinary Medicine, Northwest A & F University, Yangling, People’s Republic of China c College of Animal Science, Tibet College of Agriculture and Animal Husbandry, Nyingchi, People’s Republic of China d College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 12 October 2013 Received in revised form 24 December 2013 Accepted 27 December 2013 Available online 5 January 2014 Keywords: Haemophilus parasuis Tibetan pigs Seroprevalence Indirect hemagglutination assay (IHA) China

a b s t r a c t Haemophilus parasuis is the causative agent of Glässer’s disease, an important emerging infectious disease, but little is known of H. parasuis infection in Tibetan pigs in Tibet. The objective of the present investigation was to examine H. parasuis seroprevalence in Tibetan pigs in Tibet, China. Serum samples from 423 Tibetan pigs in Nyingchi, Tibet, China from April to December in 2010 were examined independently for the presence of antibodies against H. parasuis. A total of 147 (34.75%, 95% confidence interval [CI] 30.21–39.29) Tibetan pigs were tested positive for H. parasuis antibodies by the indirect hemagglutination assay (IHA) using a kit commercially available. 80 of 231 in Nyingchi (34.63%, 95% CI 28.50–40.77) and 67 of 192 in Mainling (34.89%, 95% CI 28.15–41.64) were tested positive, but the difference was not statistically significant (P > 0.05, 2 = 0.003). The prevalence ranged from 19.72% (95% CI 10.46–28.97) to 75.00% (95% CI 32.57–100) varying in different age groups, with higher prevalence in breeding boars than in piglets, and the difference was statistically significant (P < 0.05). The prevalence of H. parasuis infection in males (45.03%, 95% CI 37.57–52.49) was significantly higher than that in the female (30%, 95% CI 22.41–37.59) pigs (P < 0.05, 2 = 7.361). Gender of Tibetan pigs was the main risk factor associated with H. parasuis infection. The results of the present survey indicated a wide distribution of H. parasuis among Tibetan pigs in Tibet, China and further investigation should better assess circulation of H. parasuis in Tibetan pigs. To the best of our knowledge, the present study represents the first report of H. parasuis infection in Tibetan pigs in China. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Haemophilus parasuis is the causative agent of Glässer’s disease, which is characterized by the systemic symptom including fibrinous polyserositis, polyarthritis and meningitis (Hoefling, 1991; Oliveira and Pijoan, 2004). This disease is recognized as an important emerging infectious disease that has been paid more and more attention due to the considerable economic losses it caused to the pig industry worldwide (Cai et al., 2005; Rapp-Gabrielson et al., 2006). By far, fifteen different serovars have been recognized using traditional serotyping, and large numbers of the isolated

∗ Corresponding author. Tel.: +86 931 8342813; fax: +86 931 8340977. ∗∗ Corresponding author at: State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, People’s Republic of China. Tel.: +86 931 8342813; fax: +86 931 8340977. E-mail addresses: [email protected] (D.-H. Zhou), [email protected] (X.-Q. Zhu). 0001-706X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.12.021

non-typeable H. parasuis strains have been assessed the heterogeneity at the molecular level (Kielstein and Rapp-Gabrielson, 1992; Olvera et al., 2006). In China, a large number of field isolates have been studied previously (Cai et al., 2005; Li et al., 2009; Chu et al., 2011) and serovars 4 and 5 were considered the most prevalent H. parasuis strains (Cai et al., 2005). However, no information is available regarding H. parasuis infection in Tibetan pigs, China. Tibetan pig is a local pig breed most distributed in the southeastern Tibet Plateau and the surrounding areas (with an average altitude of more than 3000 m). The pig is mainly fed by the freerange system combined with the drylot husbandry. Under the special breeding system, this animal is characterized by disease resistance, easy breeding and carcass lean high-quality. Its meat is delicious with high proteins, tender texture and rich amino acids, and is an important source of income for Tibetans. In the present investigation, the indirect hemagglutination assay (IHA) was used to investigate the most prevalent serovars 4, 5

N.-Z. Zhang et al. / Acta Tropica 132 (2014) 94–97

95

Table 1 Prevalence of Haemophilus parasuis infection in pigs in China. Province

No. tested

Positive (%)

Test methoda

Time tested (year)

Reference

Fujian

1104 200 231

18.00 6.50 11.00

LAT IHA LAT

2005 2008 UNb

Lin et al. (2006) He et al. (2010) Zheng (2006)

Guangxi

305 86

4.59 30.20

PCR PCR

2006–2010 2009–2010

Ma et al. (2010) Xuan et al. (2011)

Hubei

332 450

32.50 21.70

IHA IHA

2010 UN

Wan et al. (2010) Gao et al. (2009)

Jiangxi Ningxia Heilongjiang

150 403 400

44.00 25.31 48.75

IHA IHA ELISA

UN UN UN

Zhao et al. (2009) Bai et al. (2010) Luo et al. (2010)

Qinghai

639 209

25.19 24.40

IHA IHA

UN UN

Lu et al. (2009b) Lu et al. (2009a)

Sichuan

1062

10.00

IHA

2006–2008

Feng et al. (2009)

a b

LAT, latex agglutination test; PCR, polymerase chain reaction; IHA, indirect hemagglutination assay; ELISA, enzyme-linked immunosorbent assay. UN, unknown.

and 12 of H. parasuis in Tibetan pigs in Tibet, China. The prevalences of H. parasuis infection in other kinds of pigs in China, which were originally published in local Chinese journals (hardly accessible to international readers), are summarized in Table 1. To the best of our knowledge, this is the first report of the H. parasuis seroprevalence in Tibetan pigs. 2. Materials and methods 2.1. The study site The present study was carried out in Nyingchi and Mainling counties of the Nyingchi Prefecture, southeast Tibetan. This area was geographically isolated from Tibet and Sichuan Provinces by Himalayas, Nyainqentanglha Range and Hengduanshan Mountain and shares borders with Yunnan Province and Qamdo Prefecture in the east and northeast, Naqu Prefecture in the north, Lhasa city in the west, Shannan Prefecture in the southwest, and India and Myanmar in the south. The average elevation of the surveyed area is more than 3000 m above sea level. 2.2. Collection of serum samples Tibetan pigs are bred in the cage-free system in rural areas. A total of 423 blood samples (including 231 from Nyingchi county and 192 from Mainling county) representing 171 males, 140 females and 112 unknown genders were collected from April to December, 2010. All the operations were guided by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2010-010). Before sample collection, the general characters of Tibetan pigs were examined thoroughly. These blood samples were centrifuged at 3000 × g for 5 min. The separated sera were stored at −20 ◦ C until further analysis. 2.3. Serological examination Antibodies against H. parasuis were examined by IHA using a commercially marked kit (Lanzhou Veterinary Institute, Chinese Academy of Agriculture Sciences). The detection procedure was performed according to the manufacturer’s instructions. Briefly, each test serum was screened by two-fold serial dilutions starting from 1:2 to 1:1024. In the eighth hole, discard 25 ␮L mixture to maintain 50 ␮L system. Positive and negative controls were always included in the same plate. After addition of 25 ␮L of H. parasuis antigen sensitized glutaraldehyde-fixed sheep erythrocytes

(serovars 4, 5 and 12) to each well, the plates were shaken gently for 1–2 min and then incubated at 37 ◦ C for 2–3 h. The tests were considered positive for H. parasuis antibodies when the sera IHA titers were 1:8 or higher. Dilution of 1:4 was suspicious and should be retested. 2.4. Statistical analysis Data were statistically analyzed using the procedure of SAS (Statistical Analysis System, Version 8.0). Chi-square analysis was used to compare the difference and association between pairs of interesting measures. Logistic regression models were constructed for each pig population to calculate odds ratios for seropositivity among areas and ages while controlling for gender. All tests were 2sided, and the level of significant difference was defined as P < 0.05. 3. Results Antibodies against H. parasuis were detected in 147 of 423 (34.75%, 95% confidence interval [CI] 30.21–39.29) serum samples by IHA; 80 of 231 in Nyingchi (34.63%, 95% CI 28.50–40.77) and 67 of 192 in Mainling (34.89%, 95% CI 28.15–41.64) were tested positive, but the difference was not statistically significant (P > 0.05, 2 = 0.003). However, male Tibetan pigs from Mainling county were >2 × more than females to be seropositive (odds ratio [OR] 2.42, 95% CI 0.20–1.19) (Table 2). The same association between male pigs and positive serologic test result was also observed in Nyingchi County. Statistical analysis revealed that the prevalence of H. parasuis in male pigs (45.03%, 95% CI 37.57–52.49) was significantly higher than that in the female pigs (30%, 95% CI 22.41–37.59) (P < 0.05, 2 = 7.361) (Table 2). The proportion of seropositive animals was not equal among ages (P < 0.05, 2 = 12.614). The highest seroprevalence was found in breeding boars (75%, 95% CI 32.57–100) and the lowest in piglets (19.72%, 95% CI 10.46–28.97) (Table 3). The antibody titers of the confirmed positive samples were 1:1024 in one, 1:512 in one, 1:256 in three, 1:128 in one, 1:64 in six, 1:32 in seven, 1:16 in 33 and 1:8 in 95 in Tibetan pigs (Table 3). 4. Discussion H. parasuis can reside as commensal flora in the upper respiratory tract of pigs and is known as an opportunistic microorganism. However, under certain conditions, these organisms can cause disease as the secondary pathogens probably associated with

96

N.-Z. Zhang et al. / Acta Tropica 132 (2014) 94–97

Table 2 Relationship between gender and seroprevalence of Haemophilus parasuis relative to Tibetan pigs’ ages and geographical locations in Tibet, China. Statistical characteristic

Male

Female

P value

Odds ratio (95% CI)

No. positive/total no.

% (95% CI)

No. positive/total no.

% (95% CI)

Age, year <1 year ≥1 year ≥2 years ≥3 years

2/7 28/57 22/47 25/60

28.57 (0–62.04) 49.12 (36.14–62.1) 46.81 (32.54–61.07) 41.67 (29.19–54.14)

2/10 10/26 18/56 14/48

20.00 (0–44.79) 38.46 (19.76–57.16) 32.14 (19.91–44.38) 29.17 (16.31–42.03)

0.69 0.37 0.13 0.18

1.60 (0.07–0.17) 1.54 (0.25–0.60) 1.86 (0.24–0.83) 1.73 (0.25–0.77)

Total

77/171

45.03 (37.57–52.49)

42/140

30 (22.41–37.59)

<0.01

1.91 (0.33–1.19)

Areas Mainling Nyingchi

34/76 43/95

44.74 (33.56–55.92) 45.26 (35.25–55.27)

17/68 25/72

25 (14.71–35.29) 34.72 (23.73–45.72)

<0.05 0.17

2.43 (0.20–1.19) 1.55 (0.34–0.83)

Total

77/171

45.03 (37.57–52.49)

42/140

30 (22.41–37.59)

<0.01

1.91 (0.33–1.19)

severity lesions (Rapp-Gabrielson et al., 2006). Clinically, acute disease was usually found in the immunological naïve herds and pigs that were co-infected with H. parasuis and other pathogens, such as porcine reproductive and respiratory syndrome virus (SolanoAguilar et al., 1997), Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae (Vengust et al., 2006), Streptococcus suis, and Pasteurella multocida (Cai et al., 2005). In China, serological and genetic investigations of H. parasuis in other pigs have been widely reported (Table 1). As shown in Table 1, commercial pigs in nearly a quarter of China’s provinces have been reported to be infected with H. parasuis, while the prevalences were varied markedly among regions and over time. Diagnosis of H. parasuis infections is usually based on herd history, clinical signs, presence of lesions and bacterial isolation. However, isolation of the bacterium from clinical samples is hampered by the fragility and fastidious growth requirements (Rapp-Gabrielson et al., 2006) and the pathogen in the host usually results in subclinical infection of carrier animals (Oliveira and Pijoan, 2004). Serological methods such as the IHA and the gel diffusion (GD) have been widely used to classify the H. parasuis strains (Olvera et al., 2007). In this study, the IHA method was used to investigate H. parasuis seroprevalence in Tibetan pigs in Tibet, China for the first time. A total of 147 (34.75%, 95% CI 30.21–39.29) Tibetan pigs were tested positive for H. parasuis antibodies, which was higher than that reported for the intensively kept pigs in Fujian (He et al., 2010), Hubei (Gao et al., 2009), Ningxia (Bai et al., 2010), Qinghai (Lu et al., 2009a,b) and Sichuan (Feng et al., 2009) using the same serological test kit and in Guangxi (Ma et al., 2010; Xuan et al., 2011) by PCR, but lower than that in Jiangxi (Zhao et al., 2009) and Heilongjiang (Luo et al., 2010) provinces in China, and wild boars in Germany (Gerald et al., 2010). The difference in H. parasuis seroprevalence may be due to diagnostic methods, immune capacities, feeding conditions, stresses and socioeconomic, ecological conditions as well as animal husbandry practices and animal welfares. H. parasuis is also commonly observed in farms with high sanitary standards (Rubies et al., 1999; Cai et al., 2005). Under the

extensive feeding system, Tibetan pigs could expose more to infectious agents than the pigs in the intensive farms. These investigated Tibetan pigs had high T. gondii prevalence (Wu et al., 2012). H. parasuis may easily infect Tibetan pigs following such immunosuppression pathogens, which may also contribute to the high seropositivity of H. parasuis in Tibetan pigs. The fact that Nyingchi Prefecture is geographically isolated from other parts of China raises the question as to from where H. parasuis in Tibetan pigs in Nyingchi Prefecture was originated. The first possibility is that H. parasuis was introduced by commercial pigs from mainland China to supplement the meat consumed by Tibetans in Nyingchi area, and the second possibility is that H. parasuis was spread from other countries or areas by wild animals or monsoon in view of the ability of H. parasuis to survive for a long time in the appropriate environment (MacInnes and Desrosiers, 1999). Obviously, further studies are required to elucidate the origins of H. parasuis infection in Tibetan pigs in Tibet. Previous studies have indicated that gender was not a significant factor during H. parasuis spread (Gerald et al., 2010; He et al., 2010). However, in the present study, male Tibetan pigs were >1 × more likely than females to be seropositive (OR 1.91, 95% CI 0.33–1.19). After controlling for age, the association was also noted. The more epidemiological surveys should show the relationship between gender of Tibetan pigs and the H. parasuis infection. H. parasuis infection in early-weaning piglets may increase the outcome of Glässer’s disease demonstrated that maternal antibodies appeared to be a critical factor in protecting against H. parasuis infection in piglets (Solano-Aguilar et al., 1999). The H. parasuis seroprevalence in piglets in the present investigation was the lowest among different age groups of Tibetan pigs and was similar to the breeding sows, which might indicate: (1) the maternal antibodies level, (2) piglets were naturally infected with H. parasuis. Both hypotheses indicated the absence of necessary control and prevention of H. parasuis infection in Tibetan pigs. Control of H. parasuis prevalence was suggested using commercial, autogenous vaccines, or some effective subunit vaccines (Oliveira and Pijoan, 2004; Frandoloso et al., 2011; Tian et al.,

Table 3 Antibody titers to Haemophilus parasuis infection in Tibetan pigs in Tibet, China. Types of pig

No. examined

IHA titers

% (95% CI)

≤1:4

1:8

1:16

1:32

1:64

1:128

1:256

1:512

1:1024

Breeding boar Breeding sow Slaughter pig Fattening pig Growing pig Piglet

4 5 109 103 131 71

1 4 72 63 79 57

2 1 21 26 37 8

1 0 10 9 11 2

0 0 3 3 0 1

0 0 0 2 2 2

0 0 1 0 0 0

0 0 0 0 2 1

0 0 1 0 0 0

0 0 1 0 0 0

75.00 (32.57–100) 20.00 (0–55.06) 33.94 (25.06–42.84) 38.83 (29.42–48.25) 39.69 (31.32–48.07) 19.72 (10.46–28.97)

Total

423

276

95

33

7

6

1

3

1

1

34.75 (30.21–39.29)

N.-Z. Zhang et al. / Acta Tropica 132 (2014) 94–97

2011), especially in the breeding sows and boars, as well as efficient management measures, improved animal welfare and some other integrated strategies. Although antimicrobial treatment was also effective for the prevention and control of infections caused by this pathogen, antimicrobial therapy was not suggested as the major method to eradicate H. parasuis, because of the presence of antibiotic resistance (Zhou et al., 2010; Xu et al., 2011), the sudden onset of the disease (Kielstein and Rapp-Gabrielson, 1992) and the transfer of antibiotic resistance genes to other human and animal pathogens (Lancashire et al., 2005; San et al., 2007). In conclusion, the present survey firstly demonstrated a high seroprevalence of H. parasuis infection in Tibetan pigs in Tibet, China. Although control of H. parasuis was suggested using vaccines, the integrated and improved strategies and measures are necessary for the effective prevention and control of H. parasuis prevalence in Tibetan pigs. Competing interests The authors declare that they have no competing interests. Acknowledgements Project support was provided, in part, by the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006) and the Program for Outstanding Scientists in Agricultural Research to XQZ. References Bai, G., He, Y., Zhao, P., Chu, Y.F., Gao, P.C., Zhang, N.Z., Feng, X.M., Guo, H., Fan, H.Q., Wu, R., Lu, Z.X., 2010. Serology investigation of Haemophilus parasuis in partial area of Ningxia. Gansu Animal and Veterinary Sciences 40, 3–4 (in Chinese). Cai, X., Chen, H., Blackall, P.J., Yin, Z., Wang, L., Liu, Z., Jin, M., 2005. Serological characterization of Haemophilus parasuis isolates from China. Veterinary Microbiology 111, 231–236. Chu, Y.F., Gao, P.C., Zhao, P., He, Y., Zhang, N.Z., Liu, Y.S., Liu, J.X., Lu, Z.X., 2011. Genotyping of Haemophilus Parasuis isolated from northwest China using PCRRFLP based on the ompA gene. Journal of Veterinary Medical Science 73, 337–343. Feng, X.M., Xue, H.W., Chu, Y.F., He, Y., Gao, P.C., Zhao, P., Guo, H., Fan, H.Q., Lu, Z.X., 2009. Serologic investigation on pig Haemophilus parasuis in some regions of Sichuan Province. Guizhou Agricultural Sciences 37, 135–136 (in Chinese). Frandoloso, R., Martínez, S., Rodríguez-Ferri, E.F., García-Iglesias, M.J., PérezMartínez, C., Martínez-Fernández, B., Gutiérrez-Martín, C.B., 2011. Development and characterization of Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin and comparison with other subunit and commercial vaccines. Clinical and Vaccine Immunology 18, 50–58. Gao, P.C., He, Y., Chu, Y.F., Zhao, P., Lu, Z.X., Zhao, H.Y., 2009. Epidemiological investigation of Haemophilus parasuis in some areas of Hubei Province. Hubei Agricultural Sciences 48, 926–927 (in Chinese). Gerald, R., Christina, F., Sebastian, B., Ingo, H., Hermann, W., 2010. Prevalence of Haemophilus parasuis infection in hunted wild boars (Sus scrofa) in Germany. European Journal of Wildlife Research 56, 815–818. He, Y., Zhao, P., Chu, Y.F., Gao, P.C., Lu, Z.X., 2010. Epidemiological investigation of Haemophilus parasuis in some areas of Fujian Province. Guizhou Agricultural Sciences 38, 130–131 (in Chinese). Hoefling, D.C., 1991. Acute myositis associated with Haemophilus parasuis in primary SPF sows. Journal of Veterinary Diagnostic Investigation 3, 354–355. Kielstein, P., Rapp-Gabrielson, V.J., 1992. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. Journal of Clinical Microbiology 30, 862–865. Lancashire, J.F., Terry, T.D., Blackall, P.J., Jennings, M.P., 2005. Plasmid-encoded Tet B tetracycline resistance in Haemophilus parasuis. Antimicrobial Agents and Chemotherapy 49, 1927–1931.

97

Li, J.X., Jiang, P., Wang, Y., Li, Y.F., Chen, W., Wang, X.W., Li, P., 2009. Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens. Preventive Veterinary Medicine 91, 274–279. Lin, L., Jiang, B., Wu, S.H., Zhang, S.Z., 2006. Serologic investigation of Haemophilus parasuis in some pig farms of Fujian Province in 2005. Fujian Journal of Animal Husbandry and Veterinary Medicine 5, 57 (in Chinese). Lu, Y., Wang, G.P., Cai, Q.G., Ma, L.Q., 2009a. Serological survey of Haemphilus parasuis in Qinghai Province. Acta Ecologiae Animalis Domastici 30, 139–140 (in Chinese). Lu, Z.X., Gao, P.C., He, Y., Chu, Y.F., Zhao, P., Zhao, H.Y., Ma, L.Q., Lu, Y., Cai, Q.G., 2009b. Epidemiological investigation of Haemophilus parasuis in pigs in Qinghai Province. Acta Agriculturae Boreali-Sinica 24, 236–239 (in Chinese). Luo, T.Y., Mu, Y.C., Wang, L.K., 2010. Serological investigation of Haemophilus parasuis in pigs in Qiqihar City. Technical Advisor for Animal Husbandry 10, 154–155 (in Chinese). Ma, L., Tan, Y.Y., Tan, S.M., Lu, Q.Z., 2010. Investigations on the molecular epidemiology of Haemophilus parasuis in Guangxi. Guangxi Agricultural Sciences 41, 1113–1116 (in Chinese). MacInnes, J.I., Desrosiers, R., 1999. Agent of the “Sui-side Diseases” of swine: Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis. Canadian Journal of Veterinary Research 63, 83–89. Oliveira, S., Pijoan, C., 2004. Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Veterinary Microbiology 99, 1–12. Olvera, A., Calsamiglia, M., Aragon, V., 2006. Genotypic diversity of Haemophilus parasuis field strains. Applied and Environmental Microbiology 72, 3984–3992. Olvera, A., Segalés, J., Aragón, V., 2007. Update on the diagnosis of Haemophilus parasuis infection in pigs and novel genotyping methods. Veterinary Journal 174, 522–529. Rapp-Gabrielson, V.J., Oliveira, S.R., Pijoan, C., 2006. Haemophilus parasuis. In: Straw, B., et, al. (Eds.), Diseases of Swine. , 8th ed. Blackwell, Williston, USA, pp. 681– 690. ˜ E., 1999. Prevalence of Rubies, X., Kielstein, P., Costa, L., Riera, P., Artigas, C., Espuna, Haemophilus parasuis serovars isolated in Spain from 1993 to 1997. Veterinary Microbiology 66, 245–248. San, M.A., Escudero, J.A., Catalan, A., Nieto, S., Farelo, F., Gibert, M., Moreno, M.A., Dominguez, L., Gonzalez-Zorn, B., 2007. ˇ-Lactam resistance in Haemophilus parasuis is mediated by plasmid pB1000 bearing blaROB-1. Antimicrobial Agents and Chemotherapy 51, 2260–2264. Solano-Aguilar, G.I., Pijoan, C., Rapp-Gabrielson, V., Collins, J., Carvalho, L.F., Winkelman, N., 1999. Protective role of maternal antibodies against Haemophilus parasuis infection. American Journal of Veterinary Research 60, 81–87. Solano-Aguilar, G.I., Segales, J., Collins, J.E., Molitor, T.W., Pijoan, C., 1997. Porcine reproductive and respiratory syndrome virus (PRRSv) interaction with Haemophilus parasuis. Veterinary Microbiology 55, 247–257. Tian, H., Fu, F., Li, X., Chen, X., Wang, W., Lang, Y., Cong, F., Liu, C., Tong, G., Li, X., 2011. Identification of the immunogenic outer membrane protein A antigen of Haemophilus parasuis by a proteomics approach and passive immunization with monoclonal antibodies in mice. Clinical and Vaccine Immunology 18, 1695–1701. Vengust, G., Valencak, Z., Bidovec, A., 2006. A serological survey of selected pathogens in wild boar in Slovenia. Journal of Veterinary Medicine Series B 53, 24–27. Wan, C.Y., Wang, W., Gong, D.C., 2010. Serologic investigation of Haemophilus parasuis in Jinzhou of Hubei province. Swine Production 5, 74 (in Chinese). Wu, S.M., Ciren, D., Huang, S.Y., Xu, M.J., Ga, G., Yan, C., Mahmoud, M.S., Zou, F.C., Zhu, X.Q., 2012. First report of Toxoplasma gondii prevalence in Tibetan pigs in Tibet, China. Vector-Borne and Zoonotic Diseases 12, 654–656. Xu, C., Zhang, J., Zhao, Z., Guo, L., Zhang, B., Feng, S., Zhang, L., Liao, M., 2011. Antimicrobial susceptibility and PFGE genotyping of Haemophilus parasuis isolates from pigs in South China (2008–2010). Journal of Veterinary Medical Science 73, 1061–1065. Xuan, X.B., Xie, Y.Z., Li, J., Peng, H., Chen, Z.X., Hu, S., Xie, Y.P., Ma, C.X., Xu, L.G., Yang, W., Pan, Y., 2011. Epidemiological investigation of Haemophilus parasuis in Guangxi. China Animal Husbandry and Veterinary Medicine 38, 196–200 (in Chinese). Zhao, P., He, Y., Chu, Y.F., Gao, P.C., Lu, Z.X., 2009. Serological investigation of Haemophilus parasuis in some pig farms of Jiangxi Province. Progress in Veterinary Medicine 30, 107–109 (in Chinese). Zheng, J., 2006. Serological investigation of Haemophilus parasuis in Fuzhou. Shanghai Journal of Animal Husbandry and Veterinary Medicine 4, 35 (in Chinese). Zhou, X., Xu, X., Zhao, Y., Chen, P., Zhang, X., Chen, H., Cai, X., 2010. Distribution of antimicrobial resistance among different serovars of Haemophilus parasuis isolates. Veterinary Microbiology 141, 168–173.