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Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens
Preventive Veterinary Medicine 91 (2009) 274–279
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Short communication
Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens Jun-xing Li, Ping Jiang *, Yan Wang, Yu-feng Li, Wen Chen, Xian-wei Wang, Peng Li Key Laboratory of Animal Diseases Diagnostic and Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Ministry of Agriculture, Nanjing 210095, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2008 Received in revised form 10 May 2009 Accepted 3 June 2009
From December 2003 to July 2006, a total of 131 (28.4%) Haemophilus parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. These strains were isolated from clinically diseased pigs, and 50 of them along with 15 reference strains of all known serovars were subjected to PCR–FRLP (restriction fragment length polymorphism) analysis by tbpA gene. The analysis of the 1.9-kb tbpA amplicon using TaqI, AvaI and RsaI endonucleases produced 9 RFLP patterns for the15 reference strains and 13 patterns for the 50 field isolates. And the first three prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). Meanwhile, co-infection of H. parasuis, PRRSV and PCV2 was examined in the 462 pig herds. It is indicated that 11.5% cases (53), 27.9% cases (129) and 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens. It is confirmed that H. parasuis existed widely in southeast China with numerous genotypes. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Haemophilus parasuis PCR–RFLP Genotype Prevalence Co-infection
1. Introduction Haemophilus parasuis, a member of the family Pasteurellaceae, is a non-motile, nicotinamide adenine dinucleotide (NAD)-dependent, Gram-negative bacteria (Biberstein and White, 1969). It is the causative agent of Gla¨sser’s disease, porcine polyserositis and arthritis, which is a significant disease in the pork industry. Recently in farms with high sanitary standards, a significant increase in the incidence, morbidity and mortality has occurred, which leads to increasing economic losses in the swine industry worldwide (Rapp-Gabrielson et al., 2006). Classification of H. parasuis has been traditionally based on serotyping. So far 15 serovars have been recognized (Kielstein and Rapp-Gabrielson, 1992). But serotyping does not provide enough discrimination of isolates for epidemiological studies and between 15% and
* Corresponding author. Tel.: +86 25 84395504; fax: +86 25 84396640. E-mail address: [email protected] (P. Jiang). 0167-5877/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2009.06.004
41% of isolates are non-typeable (Oliveira and Pijoan, 2004). Analysis of nucleic acid amplification by PCR could be used in rapid identification and typing of bacteria. Several genes, including the transferrin binding protein A (tbpA) (De la Puente Redondo et al., 2003), the fragment of the 16s rRNA gene (Lin, 2003) and the 5-enolpyruvylshikimate-3-phosphate synthase (aroA) (Del Rio et al., 2006) of H. parasuis, have been used for genotype of H. parasuis by PCR–RFLP. The PCR–RFLP based on tbpA gene was proved to be a quick and effective method for typing clinical H. parasuis because of its sensitivity and high degree of discrimination (De la Puente Redondo et al., 2003). In this paper, we report the genotypes of H. parasuis isolated from clinically ill pigs based on RFLP analysis of tbpA gene in order to understand the molecular epidemiology of H. parasuis in China. The prevalence of coinfection with H. parasuis and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) or PCV2 (Porcine Circovirus type 2) was also studied from December 2003 to July 2006.
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2. Materials and methods
2.3. PCR–RFLP
2.1. Bacteria isolation and virus detection
2.3.1. DNA extraction and PCR amplification Genomic DNA was isolated using the method previously described (De la Puente Redondo et al., 2003), except that the preheating and boiling time was extended to 40 min and 20 min, respectively. The PCR assay was performed in a 50 mL reaction-mixture containing 8 mL of template DNA, 2 mL of each primer (10 pM), 4 mL of mixture of each dNTP (2.5 mM) and 5.0 units Taq DNA polymerase (Promega). The amplification reaction protocol was as follows: denaturation at 94 8C for 5 min; 35 cycles (94 8C for 45 s, annealing at 40 8C for 45 s, 72 8C for 2 min); and final extension at 72 8C for 10 min. PCR products were screened by electrophoresis on 1% (w/v) agarose gels.
From December 2003 to July 2006, a total of 131 strains of (28.4%) H. parasuis was isolated from 462 cases examined in our diagnostic laboratory. These H. parasuis strains were isolated from clinically ill pigs (6–11 weeks old) from different pig herds in 6 provinces (or municipality) in south-east of China, which were sent to our veterinary diagnostic laboratory. The clinical signs of the pigs included high fever (>40.0 8C), weakness, abdominal breathing, diarrhea, swollen joints and/or central nervous system signs. The complete necropsy of each animal was performed. Diseased pigs show fibrinous polyserositis, arthritis or meningitis which are typical lesions of Gla¨sser’s disease. Samples of lung were used for isolation of H. parasuis. Tryptic Soy Agar (TSA, Difco Laboratories, Detroit, MI) containing10 mg/ mL nicotinamide adenine dinucleotide (NAD) and 5% bovine serum was used as growth medium. Plates were incubated at 37 8C for 24–48 h, and the suspected bacteria clones which were small and translucent were selected for further identification. A group of 15 H. parasuis reference strains of all known serovars (kindly provided by Dr. Li in Zhangjiang University in China) were also studied. To examine the co-infection of H. parasuis with other pathogens, a sample mixture of lung, spleen and inguinal lymph nodes was taken from each case for RNA or DNA extraction. PRRSV and PCV2 were detected by specific RTPCR (targeted gene of nucleocapsid protein of PRRSV) and PCR (targeted gene of capsid protein of PCV2) described previously (Li et al., 2003; Wang et al., 2006). Briefly, the sample mixture was homogenized in PBS with ratio of 1:1. Viral RNA was extracted from the homogenates using TRIzol REAGENT (Invitrogen) as protocol; viral DNA was extracted from the homogenates using DNAzol (Invitrogen) according to the manufacturer’s instruction; RT-PCR and PCR protocols were described in detail in previous report, and primers for the RT-PCR and PCR are listed in Table 1. 2.2. Bacteria identification with PCR and biochemical tests The suspected H. parasuis isolates were identified using a specific PCR as previously described and primers for the PCR are listed in Table 1 (Oliveira et al., 2001). And then the suspected isolates were examined using biochemical test as previously described (Kielstein et al., 2001).
2.3.2. RFLP analysis Of the 131 strains of H. parasuis isolated, 50 were used for genotyping. PCR amplification product for each sample subjected to RFLP analysis was as follows: 10 mL of each PCR product was digested in final volume of 20 mL reactions separately for each restriction endonuclease including TaqI, AvaI and RsaI according to manufacturers’ recommendations (TaKaRa Bio. Co. Ltd.). After incubation for 3 h, the digestion products were analyzed by electrophoresis on 1.5% (w/v) agarose gels. The RFLP patterns were analyzed using GelDoc-It imaging computer programme (UVP, USA). A same name was assigned to each RFLP pattern if it was already showed in the original paper by De la Puente Redondo et al. (2003). 3. Results From December 2003 to July 2006, a total of 131 (28.4%) H. parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. For those 462 cases, the presence of PRRSV and PCV2 was also examined by specific RT-PCR and PCR (Li et al., 2003; Wang et al., 2006). The presence of H. parasuis and/or PRRSV and/or PCV2 in those cases is summarized in Table 2. The pair of primers for tbpA gene used in this study successfully amplified an expected 1900-bp DNA fragment, being similar to that previously reported for 15 reference strains (De la Puente Redondo et al., 2000, 2003). Restriction analysis of tbpA gene from 15 H. parasuis reference strains with TaqI, AvaI and RsaI generates 4 (Fig. 1(a)), 3 (Fig. 1(b)), 7 (Fig. 1 (c)) RFLP patterns, respectively. Combining the results of the three endonucleases, RFLP analysis of the 15 H. parasuis reference strains
Table 1 Primer sequences for amplification of 16s rRNA gene from H. parasuis, nucleocapsid (N) protein gene of PRRSV and capsid (Cap) protein gene of PCV2. Name P1 P2 N1 N2 Cap1 Cap2
Sequence 0
Amplified gene 0
5 -GTGATGAGGAA GGGTGGTGT-3 50 -GGCTTCGTCACCCTCTGT-30 50 -CGAGGATCCAATATGCCAAATAACAACGG-30 50 -CCAGAATTCCATCATGCTGCTGAGGGTGATGC-30 50 -TTCGGTACCAGCTATGACGTATCCAAG-30 50 -GCCAAGCTTTCACTTCGTAATGGTTTT-30
16s rRNA of H. parasuis (822 bp) N protein gene of PRRSV (376 bp) Cap protein gene of PCV2 (751 bp)
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Table 2 Simultaneous presence of H. parasuis, PRRSV and PCV2 in 462 cases from which H. parasuis was isolated during December 2003 to July 2006.
Table 3 RFLP patterns of tbpA gene obtained after TaqI, AvaI and RsaI digestion from 15 reference strains of all known serovars.
No. of cases
H. parasuis
PRRSV
PCV2
Strain
53 51 13 14 25 129 22 155
+ + + +
+ + + +
+ + + +
(+) Present; () absent.
produced 9 different groups (Table 3), two (CCA, BBE) of which are not the same with any of them showed in previous report (De la Puente Redondo et al., 2003). Of the 131 H. parasuis isolates, 50 H. parasuis strains were subjected to PCR–RFLP. The PCR products of 50 H. parasuis strains were digested with TaqI, AvaI and RsaI endonucleases. The results showed that 5 (Fig. 2(a)), 2 (Fig. 2 (b)), 9 (Fig. 2 (c)) patterns were obtained when PCR products were subjected to TaqI, AvaI and RsaI, respectively, and the combination of the three endonucleases
Hs-DY01 Hs-DY02 Hs-DY03 Hs-DY04 Hs-DY05 Hs-DY06 Hs-DY07 Hs-DY08 Hs-DY09 Hs-DY10 Hs-DY11 Hs-DY12 Hs-DY13 Hs-DY14 Hs-DY15
Restriction terns with
(serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar
1 reference strain) 2 reference strain) 3 reference strain) 4 reference strain) 5 reference strain) 6 reference strain) 7 reference strain) 8 reference strain) 9 reference strain) 10 reference strain) 11 reference strain) 12 reference strain) 13 reference strain) 14 reference strain) 15 reference strain)
pat-
TaqI
AvaI
RsaI
C B B D D B B B E B E D B D D
C B B B B A B A A B A B B Ba B
A B J E E F B H I E F E J E E
RFLP group
CCA BBB BBJ DBE DBE BAF BBB BAH EAI BBE EAF DBE BBJ DBE DBE
allowed grouping all 50 strains of H. parasuis into 13 RFLP groups (Table 4). Of these genotypes, the first three prevalent genotypes were DBN (19/50), ABN (9/50) and DBP (6/50).
Fig. 1. RFLP analysis of tbpA gene from 15 reference strains of all known serovars. (a) PCR products digested with TaqI, letters C, B, B, etc., show results of digestion with PCR products from reference strains of serovars 1–15. (b) tbpA PCR product digested with AvaI, letters C, B, B, etc., represent each restriction pattern for reference strains of serovars 1–15. (c) PCR product digested with RsaI, letters A, B, J, etc., show results of digestion with tbpA PCR product from reference strains of serovars 1–15. Lanes M: molecular size marker.
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Fig. 2. RFLP analysis of tbpA gene amplification products from H. parasuis isolates. (a) Digestion with TaqI. Letters A–E show different restriction pattern. (b) Digestion with AvaI. Letters A and B show each different restriction pattern. (c) Digestion with RsaI. One type of each different restriction pattern is showed. Lanes M: molecular size marker. Lanes PCR: tbpA gene amplification product from H. parasuis.
Table 4 RFLP Pattern, gross lesions and number of different genotypic strains of H. parasuis isolated from clinically diseased pigs. RFLP pattern
Gross lesions
No. of isolates
Percentage (%)
19
38
JS060220
9
18
JS051230
6
12
1 1 1 2 2 1 1 2 1 4
2 2 2 4 4 2 2 4 2 8
TaqI
AvaI
RsaI
Polyserositis + arthritis
Polyserositis
Arthritis
D
B
N
B
N
D
B
P
B B A A A C A E D D
B B B B A B A A B B
N B D B I J F I O E
SH040414, ZJ040926 SH041216, JS041009 JX041015, JS041101 GX050108, GX060525 SH040415, GX041118 GX060115, ZJ060613 ZJ050908, GX041118 SH060706, JX060817 JS040602 SH050929 GX040610 ZJ040217, JS041222 GX050414
SH051127, SH051201 JS051205a
A
SH031203, SH040912, SH060422, SH050512, SH031222, GX050509 JS050324
a
JS060522
SH031206 JS041010 SH040316 GX060429 JS040921
JS060118 AH040922 SH050309
GX060115 GX060302 JS060211 JS050929, SH060406 JX060718
The first two letter: abbreviation of the province name, the numbers (051205): year/month/days.
4. Discussion To date, a lot of H. parasuis isolates were characterized by PCR–RFLP worldwide (De la Puente Redondo et al., 2003; Lin, 2003; Del Rio et al., 2006). There was evidence
that the tbpA PCR–RFLP was an effective method for typing clinical H. parasuis strains, and it could be used as an alternative method for molecular epidemiological study (De la Puente Redondo et al., 2003). Here, we first used this method in China to study the prevalence of H. parasuis.
278
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In this study, the tbpA RFLP–PCR was used to characterize 15 reference strains of all known serovars and 50 H. parasuis clinical strains from China. The results showed that reference strains used here and these clinical isolates could be divided into 9 and 13 (three patterns were same with those from the reference strains used in this article) RFLP groups, respectively. The RFLP profiles were similar to those previously described (De la Puente Redondo et al., 2003) in heterogeneity and typeability as shown in Figs. 1 and 2. In this article, digestion of the amplicon of tbpA gene with AvaI yielded three RFLP patterns, which were the same with all three patterns obtained in their study (De la Puente Redondo et al., 2003); all of the five different patterns in our study were the same with their report in which five RFLP patterns were produced when digested with TaqI; eight out of eleven RFLP patterns generated by RsaI were the same with previous report; the rest three RFLP patterns (N, O, P) could not be compared with that of the study (De la Puente Redondo et al., 2003), since all of the RFLP patterns by RsaI were not shown in that report. So we named these three patterns differently from the previous report. The results showed that DBN (38%), ABN (18%) and DBP (12%) were the first three genotypes of H. parasuis prevailing in China. Interestingly, in this study, the most prevalent H. parasuis genotype was DBN (38%), which is different from the previous report in which the most prevalent one is DBE (De la Puente Redondo et al., 2003). This suggests that the prevalent H. parasuis strains may vary geographically in genotype. In this study, H. parasuis strains of most genotypes were found with the presence of polyserositis and/or arthritis, no strict correlation between genotype and virulence was found. Differentiation of strains is of significant importance in diagnosis and control of H. parasuis infection, since it is necessary to differentiate between ‘colonising’ strains and ‘disease-causing’ strains. Moreover, more than one strain can colonize the same animal (Smart et al., 1988), it is possible that a particular clinical isolate is not the one responsible for the outbreak of disease. In our study, some of the H. parasuis strains that were isolated from lungs of diseased pigs show only arthritis or even no remarkable gross lesions. Thus it is hard to know whether the clinical signs and gross lesion we observed were caused by infection of H. parasuis alone or co-infection with other pathogens. So H. parasuis isolates belonging to some of the genotypes could be nonvirulent strains, and were just found causing disease when there is a co-infection. To address the pathogenic properties of H. parasuis strains of different genotype, it is necessary to do experimental infection with pigs free of those pathogens that could cause similar lesions with H. parasuis. The simultaneous presence of PRRSV and PCV2 with H. parasuis was studied. It shows that 11.5% cases (53), 27.9% cases (129), 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens (Table 2).
Clinically, co-infection of H. parasuis with other pathogens, such as porcine reproductive and respiratory syndrome virus (Solano et al., 1997), porcine circovirus type 2 (Kim et al., 2002), Streptococcus suis, Escherichia coli, Bordetella bronchiseptica and Pasteurella multocida (Cai et al., 2006), are frequently found in Glasser’s disease. Clinical and diagnostic evidence have suggested that epizootic out breaks caused by porcine reproductive and respiratory syndrome virus (PRRSV) in a naı¨ve herd mediate a sequence of clinical events that include a high prevalence of secondary viral and bacterial infections, mainly in nursery and fattening pigs (Pijoan et al., 1994). Frequency of H. parasuis infection is only slightly higher in PRRSV positive diseased pigs (29.7%) than that of PRRSV negative ones (27.2%) and vice versa (49.6% vs 46.5%). It is indicated that PRRSV or H. parasuis infection may not necessarily facilitate the secondary infection of H. parasuis or PRRSV, which clinically confirmed the result that there was no influence of the previous infection with PRRSV in the occurrence of H. parasuis infection (Segales et al., 1998, 1999). Of all the 74 PCV2 positive cases examined in our study, a frequency of 36.5% (27/74) of H. parasuis positive was observed, which is similar to a previous study with the frequency of 32.3% (43/133); but a much higher frequency (52.7%, 39/74) of PRRSV positive cases were found than that (29.3%, 39/133) of the previous study (Kim et al., 2002). The higher prevalence of PRRSV in PCV2 positive pigs may be due to the existence and transmission of highly pathogenic PRRSV strains which caused outbreak in many provinces in China recently (Li et al., 2007; Tian et al., 2007; Zhou et al., 2008). Our study showed that multi-infection of H. parasuis, PRRSV and PCV2 existed with high frequency in China. So attention should be paid to the multi-infection of H. parasuis, PRRSV and PCV2, for disease diagnostic and control, and this study indicated that H. parasuis existed widely in China with numerous genotypes. Further studies need to be done to know the mechanisms of co-infections of H. parasuis, PRRSV and PCV2. Acknowledgements This work was supported by grants from Jiangsu Science and Technique Foundation (BE2007342), Zhejiang Province Science and Technique Foundation (2007C22045), and partly National Key Technology R&D Program (2007BAD86B02-3). References Biberstein, E.L., White, D.C., 1969. A proposal for the establishment of two new Haemophilus species. Journal of Medical Microbiology 2, 75–78. Cai, X., Chen, H., Blackall, P.J., Yin, Z., Wang, L., Liu, Z., Jin, M., 2006. Serological characterization of Haemophilus parasuis isolates from China. Veterinary Microbiology 111, 231–236. De la Puente Redondo, V.A., Garcı´a del Blance, N., Gutie´rrez Martı´n, C.B., Navas Me´ndez, J., Rodrı´guez Ferri, E.F., 2000. Detection and subtyping of Actinobacillus pleuropneumoniae strains by PCR–RFLP analysis of the tbpA and tbpB genes. Research in Microbiology 151, 669–681. De la Puente Redondo, V.A., Me´ndez, J.N., Blanco, N.G., Boronat, N.L., Mart’n, C.B.G., Ferri, E.F.R., 2003. Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene. Veterinary Microbiology 2511, 1–10.
J.- Li et al. / Preventive Veterinary Medicine 91 (2009) 274–279 Del Rio, M.L., Martin, C.B., Navas, J., Gutierrez-Muniz, B., RodriguezBarbosa, J.I., Rodriguez Ferri, E.F., 2006. aroA gene PCR–RFLP diversity patterns in Haemophilus parasuis and Actinobacillus species. Research in Veterinary Science 80, 55–61. Kielstein, P., Rapp-Gabrielson, V.J., 1992. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heatstable antigen extracts. Journal of Clinical Microbiology 30, 862–865. Kielstein, P., Wuthe, H., Angen, O., Mutters, R., Ahrens, P., 2001. Phenotypic and genetic characterization of NAD-dependent Pasteurellaceae from the respiratory tract of pigs and their possible pathogenic importance. Veterinary Microbiology 81, 243–255. Kim, J., Chung, H.K., Jung, T., Cho, W.S., Choi, C., Chae, C., 2002. Postweaning multisystemic wasting syndrome of pigs in Korea: prevalence, microscopic lesions and coexisting microorganisms. Journal of Veterinary Medical Science 64, 57–62. Li, Y.F., Wang, X.L., Bo, K.T., Wang, X.W., Tang, B., Yang, B.S., et al., 2007. Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of China. The Veterinary Journal 174, 577–584. Li, Y., Wang, X., Jiang, P., 2003. Occurrence of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus in Shanghai area. Chinese Journal of Veterinary Science 23, 442–444. Lin, B., 2003. Identification and differentiation of Haemophilus parasuis sero-nontypeable strains using a species specific PCR and the digestion of PCR products with HindIII endonuclease. In: Proceedings of the American Association of Swine Veterinarians Annual Meeting, Orlando, FL, USA, p. 299. Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. Journal of Veterinary Diagnostic Investigation 13, 495–501. Oliveira, S., Pijoan, C., 2004. Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Veterinary Microbiology 99, 1–12.
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Pijoan, C., Solano, G.I., Segale´s, J., Drew, T., 1994.In: PRRS virus and secondary disease, Proceedings of the Allen D. Leman Swine Conference, vol. 21, pp. 225–226. Rapp-Gabrielson, V.J., Oliveira, S.R., Pijoan, C., 2006. Haemophilus parasuis. In: Straw, B., Zimmerman, J.J., D’Allaire, S., Taylor, D.J. (Eds.), Diseases of Swine. Blackwell Publishing, Williston, FL, USA, pp. 681–690. Segales, J., Domingo, M., Balasch, M., Solano, G.I., Pijoan, C., 1998. Ultrastructural study of porcine alveolar macrophages infected in vitro with porcine reproductive and respiratory syndrome (PRRS) virus, with and without Haemophilus parasuis. Journal of Comparative Pathology 118, 231–243. Segales, J., Domingo, M., Solano, G.I., Pijoan, C., 1999. Porcine reproductive and respiratory syndrome virus and Haemophilus parasuis antigen distribution in dually infected pigs. Veterinary Microbiology 64, 287–297. Smart, N.L., Miniats, O.P., MacInnes, J.I., 1988. Analysis of Haemophilus parasuis isolates from southern Ontario swine by restriction endonuclease fingerprinting. Canadian Journal of Veterinary Research 52, 319–324. Solano, 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, K., Yu, X., Zhao, T., Feng, Y., Cao, Z., et al., 2007. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE 2 (6), e526, doi:10.1371/journal.pone.0000526. Wang, X., Jiang, W., Jiang, P., Li, Y., Feng, Z., Xu, J., 2006. Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice. Vaccine 24, 3374–3380. Zhou, Y.J., Hao, X.F., Tian, Z.J., Tong, G.Z., Yoo, D., An, T.Q., Zhou, T., Li, G.X., Qiu, H.J., Wei, T.C., Yuan, X.F., 2008. Highly virulent porcine reproductive and respiratory syndrome virus emerged in China. Transboundary and Emerging Diseases 55 (3–4), 152–164.
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Short communication
Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens Jun-xing Li, Ping Jiang *, Yan Wang, Yu-feng Li, Wen Chen, Xian-wei Wang, Peng Li Key Laboratory of Animal Diseases Diagnostic and Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Ministry of Agriculture, Nanjing 210095, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2008 Received in revised form 10 May 2009 Accepted 3 June 2009
From December 2003 to July 2006, a total of 131 (28.4%) Haemophilus parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. These strains were isolated from clinically diseased pigs, and 50 of them along with 15 reference strains of all known serovars were subjected to PCR–FRLP (restriction fragment length polymorphism) analysis by tbpA gene. The analysis of the 1.9-kb tbpA amplicon using TaqI, AvaI and RsaI endonucleases produced 9 RFLP patterns for the15 reference strains and 13 patterns for the 50 field isolates. And the first three prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). Meanwhile, co-infection of H. parasuis, PRRSV and PCV2 was examined in the 462 pig herds. It is indicated that 11.5% cases (53), 27.9% cases (129) and 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens. It is confirmed that H. parasuis existed widely in southeast China with numerous genotypes. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Haemophilus parasuis PCR–RFLP Genotype Prevalence Co-infection
1. Introduction Haemophilus parasuis, a member of the family Pasteurellaceae, is a non-motile, nicotinamide adenine dinucleotide (NAD)-dependent, Gram-negative bacteria (Biberstein and White, 1969). It is the causative agent of Gla¨sser’s disease, porcine polyserositis and arthritis, which is a significant disease in the pork industry. Recently in farms with high sanitary standards, a significant increase in the incidence, morbidity and mortality has occurred, which leads to increasing economic losses in the swine industry worldwide (Rapp-Gabrielson et al., 2006). Classification of H. parasuis has been traditionally based on serotyping. So far 15 serovars have been recognized (Kielstein and Rapp-Gabrielson, 1992). But serotyping does not provide enough discrimination of isolates for epidemiological studies and between 15% and
* Corresponding author. Tel.: +86 25 84395504; fax: +86 25 84396640. E-mail address: [email protected] (P. Jiang). 0167-5877/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2009.06.004
41% of isolates are non-typeable (Oliveira and Pijoan, 2004). Analysis of nucleic acid amplification by PCR could be used in rapid identification and typing of bacteria. Several genes, including the transferrin binding protein A (tbpA) (De la Puente Redondo et al., 2003), the fragment of the 16s rRNA gene (Lin, 2003) and the 5-enolpyruvylshikimate-3-phosphate synthase (aroA) (Del Rio et al., 2006) of H. parasuis, have been used for genotype of H. parasuis by PCR–RFLP. The PCR–RFLP based on tbpA gene was proved to be a quick and effective method for typing clinical H. parasuis because of its sensitivity and high degree of discrimination (De la Puente Redondo et al., 2003). In this paper, we report the genotypes of H. parasuis isolated from clinically ill pigs based on RFLP analysis of tbpA gene in order to understand the molecular epidemiology of H. parasuis in China. The prevalence of coinfection with H. parasuis and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) or PCV2 (Porcine Circovirus type 2) was also studied from December 2003 to July 2006.
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2. Materials and methods
2.3. PCR–RFLP
2.1. Bacteria isolation and virus detection
2.3.1. DNA extraction and PCR amplification Genomic DNA was isolated using the method previously described (De la Puente Redondo et al., 2003), except that the preheating and boiling time was extended to 40 min and 20 min, respectively. The PCR assay was performed in a 50 mL reaction-mixture containing 8 mL of template DNA, 2 mL of each primer (10 pM), 4 mL of mixture of each dNTP (2.5 mM) and 5.0 units Taq DNA polymerase (Promega). The amplification reaction protocol was as follows: denaturation at 94 8C for 5 min; 35 cycles (94 8C for 45 s, annealing at 40 8C for 45 s, 72 8C for 2 min); and final extension at 72 8C for 10 min. PCR products were screened by electrophoresis on 1% (w/v) agarose gels.
From December 2003 to July 2006, a total of 131 strains of (28.4%) H. parasuis was isolated from 462 cases examined in our diagnostic laboratory. These H. parasuis strains were isolated from clinically ill pigs (6–11 weeks old) from different pig herds in 6 provinces (or municipality) in south-east of China, which were sent to our veterinary diagnostic laboratory. The clinical signs of the pigs included high fever (>40.0 8C), weakness, abdominal breathing, diarrhea, swollen joints and/or central nervous system signs. The complete necropsy of each animal was performed. Diseased pigs show fibrinous polyserositis, arthritis or meningitis which are typical lesions of Gla¨sser’s disease. Samples of lung were used for isolation of H. parasuis. Tryptic Soy Agar (TSA, Difco Laboratories, Detroit, MI) containing10 mg/ mL nicotinamide adenine dinucleotide (NAD) and 5% bovine serum was used as growth medium. Plates were incubated at 37 8C for 24–48 h, and the suspected bacteria clones which were small and translucent were selected for further identification. A group of 15 H. parasuis reference strains of all known serovars (kindly provided by Dr. Li in Zhangjiang University in China) were also studied. To examine the co-infection of H. parasuis with other pathogens, a sample mixture of lung, spleen and inguinal lymph nodes was taken from each case for RNA or DNA extraction. PRRSV and PCV2 were detected by specific RTPCR (targeted gene of nucleocapsid protein of PRRSV) and PCR (targeted gene of capsid protein of PCV2) described previously (Li et al., 2003; Wang et al., 2006). Briefly, the sample mixture was homogenized in PBS with ratio of 1:1. Viral RNA was extracted from the homogenates using TRIzol REAGENT (Invitrogen) as protocol; viral DNA was extracted from the homogenates using DNAzol (Invitrogen) according to the manufacturer’s instruction; RT-PCR and PCR protocols were described in detail in previous report, and primers for the RT-PCR and PCR are listed in Table 1. 2.2. Bacteria identification with PCR and biochemical tests The suspected H. parasuis isolates were identified using a specific PCR as previously described and primers for the PCR are listed in Table 1 (Oliveira et al., 2001). And then the suspected isolates were examined using biochemical test as previously described (Kielstein et al., 2001).
2.3.2. RFLP analysis Of the 131 strains of H. parasuis isolated, 50 were used for genotyping. PCR amplification product for each sample subjected to RFLP analysis was as follows: 10 mL of each PCR product was digested in final volume of 20 mL reactions separately for each restriction endonuclease including TaqI, AvaI and RsaI according to manufacturers’ recommendations (TaKaRa Bio. Co. Ltd.). After incubation for 3 h, the digestion products were analyzed by electrophoresis on 1.5% (w/v) agarose gels. The RFLP patterns were analyzed using GelDoc-It imaging computer programme (UVP, USA). A same name was assigned to each RFLP pattern if it was already showed in the original paper by De la Puente Redondo et al. (2003). 3. Results From December 2003 to July 2006, a total of 131 (28.4%) H. parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. For those 462 cases, the presence of PRRSV and PCV2 was also examined by specific RT-PCR and PCR (Li et al., 2003; Wang et al., 2006). The presence of H. parasuis and/or PRRSV and/or PCV2 in those cases is summarized in Table 2. The pair of primers for tbpA gene used in this study successfully amplified an expected 1900-bp DNA fragment, being similar to that previously reported for 15 reference strains (De la Puente Redondo et al., 2000, 2003). Restriction analysis of tbpA gene from 15 H. parasuis reference strains with TaqI, AvaI and RsaI generates 4 (Fig. 1(a)), 3 (Fig. 1(b)), 7 (Fig. 1 (c)) RFLP patterns, respectively. Combining the results of the three endonucleases, RFLP analysis of the 15 H. parasuis reference strains
Table 1 Primer sequences for amplification of 16s rRNA gene from H. parasuis, nucleocapsid (N) protein gene of PRRSV and capsid (Cap) protein gene of PCV2. Name P1 P2 N1 N2 Cap1 Cap2
Sequence 0
Amplified gene 0
5 -GTGATGAGGAA GGGTGGTGT-3 50 -GGCTTCGTCACCCTCTGT-30 50 -CGAGGATCCAATATGCCAAATAACAACGG-30 50 -CCAGAATTCCATCATGCTGCTGAGGGTGATGC-30 50 -TTCGGTACCAGCTATGACGTATCCAAG-30 50 -GCCAAGCTTTCACTTCGTAATGGTTTT-30
16s rRNA of H. parasuis (822 bp) N protein gene of PRRSV (376 bp) Cap protein gene of PCV2 (751 bp)
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Table 2 Simultaneous presence of H. parasuis, PRRSV and PCV2 in 462 cases from which H. parasuis was isolated during December 2003 to July 2006.
Table 3 RFLP patterns of tbpA gene obtained after TaqI, AvaI and RsaI digestion from 15 reference strains of all known serovars.
No. of cases
H. parasuis
PRRSV
PCV2
Strain
53 51 13 14 25 129 22 155
+ + + +
+ + + +
+ + + +
(+) Present; () absent.
produced 9 different groups (Table 3), two (CCA, BBE) of which are not the same with any of them showed in previous report (De la Puente Redondo et al., 2003). Of the 131 H. parasuis isolates, 50 H. parasuis strains were subjected to PCR–RFLP. The PCR products of 50 H. parasuis strains were digested with TaqI, AvaI and RsaI endonucleases. The results showed that 5 (Fig. 2(a)), 2 (Fig. 2 (b)), 9 (Fig. 2 (c)) patterns were obtained when PCR products were subjected to TaqI, AvaI and RsaI, respectively, and the combination of the three endonucleases
Hs-DY01 Hs-DY02 Hs-DY03 Hs-DY04 Hs-DY05 Hs-DY06 Hs-DY07 Hs-DY08 Hs-DY09 Hs-DY10 Hs-DY11 Hs-DY12 Hs-DY13 Hs-DY14 Hs-DY15
Restriction terns with
(serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar (serovar
1 reference strain) 2 reference strain) 3 reference strain) 4 reference strain) 5 reference strain) 6 reference strain) 7 reference strain) 8 reference strain) 9 reference strain) 10 reference strain) 11 reference strain) 12 reference strain) 13 reference strain) 14 reference strain) 15 reference strain)
pat-
TaqI
AvaI
RsaI
C B B D D B B B E B E D B D D
C B B B B A B A A B A B B Ba B
A B J E E F B H I E F E J E E
RFLP group
CCA BBB BBJ DBE DBE BAF BBB BAH EAI BBE EAF DBE BBJ DBE DBE
allowed grouping all 50 strains of H. parasuis into 13 RFLP groups (Table 4). Of these genotypes, the first three prevalent genotypes were DBN (19/50), ABN (9/50) and DBP (6/50).
Fig. 1. RFLP analysis of tbpA gene from 15 reference strains of all known serovars. (a) PCR products digested with TaqI, letters C, B, B, etc., show results of digestion with PCR products from reference strains of serovars 1–15. (b) tbpA PCR product digested with AvaI, letters C, B, B, etc., represent each restriction pattern for reference strains of serovars 1–15. (c) PCR product digested with RsaI, letters A, B, J, etc., show results of digestion with tbpA PCR product from reference strains of serovars 1–15. Lanes M: molecular size marker.
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277
Fig. 2. RFLP analysis of tbpA gene amplification products from H. parasuis isolates. (a) Digestion with TaqI. Letters A–E show different restriction pattern. (b) Digestion with AvaI. Letters A and B show each different restriction pattern. (c) Digestion with RsaI. One type of each different restriction pattern is showed. Lanes M: molecular size marker. Lanes PCR: tbpA gene amplification product from H. parasuis.
Table 4 RFLP Pattern, gross lesions and number of different genotypic strains of H. parasuis isolated from clinically diseased pigs. RFLP pattern
Gross lesions
No. of isolates
Percentage (%)
19
38
JS060220
9
18
JS051230
6
12
1 1 1 2 2 1 1 2 1 4
2 2 2 4 4 2 2 4 2 8
TaqI
AvaI
RsaI
Polyserositis + arthritis
Polyserositis
Arthritis
D
B
N
B
N
D
B
P
B B A A A C A E D D
B B B B A B A A B B
N B D B I J F I O E
SH040414, ZJ040926 SH041216, JS041009 JX041015, JS041101 GX050108, GX060525 SH040415, GX041118 GX060115, ZJ060613 ZJ050908, GX041118 SH060706, JX060817 JS040602 SH050929 GX040610 ZJ040217, JS041222 GX050414
SH051127, SH051201 JS051205a
A
SH031203, SH040912, SH060422, SH050512, SH031222, GX050509 JS050324
a
JS060522
SH031206 JS041010 SH040316 GX060429 JS040921
JS060118 AH040922 SH050309
GX060115 GX060302 JS060211 JS050929, SH060406 JX060718
The first two letter: abbreviation of the province name, the numbers (051205): year/month/days.
4. Discussion To date, a lot of H. parasuis isolates were characterized by PCR–RFLP worldwide (De la Puente Redondo et al., 2003; Lin, 2003; Del Rio et al., 2006). There was evidence
that the tbpA PCR–RFLP was an effective method for typing clinical H. parasuis strains, and it could be used as an alternative method for molecular epidemiological study (De la Puente Redondo et al., 2003). Here, we first used this method in China to study the prevalence of H. parasuis.
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In this study, the tbpA RFLP–PCR was used to characterize 15 reference strains of all known serovars and 50 H. parasuis clinical strains from China. The results showed that reference strains used here and these clinical isolates could be divided into 9 and 13 (three patterns were same with those from the reference strains used in this article) RFLP groups, respectively. The RFLP profiles were similar to those previously described (De la Puente Redondo et al., 2003) in heterogeneity and typeability as shown in Figs. 1 and 2. In this article, digestion of the amplicon of tbpA gene with AvaI yielded three RFLP patterns, which were the same with all three patterns obtained in their study (De la Puente Redondo et al., 2003); all of the five different patterns in our study were the same with their report in which five RFLP patterns were produced when digested with TaqI; eight out of eleven RFLP patterns generated by RsaI were the same with previous report; the rest three RFLP patterns (N, O, P) could not be compared with that of the study (De la Puente Redondo et al., 2003), since all of the RFLP patterns by RsaI were not shown in that report. So we named these three patterns differently from the previous report. The results showed that DBN (38%), ABN (18%) and DBP (12%) were the first three genotypes of H. parasuis prevailing in China. Interestingly, in this study, the most prevalent H. parasuis genotype was DBN (38%), which is different from the previous report in which the most prevalent one is DBE (De la Puente Redondo et al., 2003). This suggests that the prevalent H. parasuis strains may vary geographically in genotype. In this study, H. parasuis strains of most genotypes were found with the presence of polyserositis and/or arthritis, no strict correlation between genotype and virulence was found. Differentiation of strains is of significant importance in diagnosis and control of H. parasuis infection, since it is necessary to differentiate between ‘colonising’ strains and ‘disease-causing’ strains. Moreover, more than one strain can colonize the same animal (Smart et al., 1988), it is possible that a particular clinical isolate is not the one responsible for the outbreak of disease. In our study, some of the H. parasuis strains that were isolated from lungs of diseased pigs show only arthritis or even no remarkable gross lesions. Thus it is hard to know whether the clinical signs and gross lesion we observed were caused by infection of H. parasuis alone or co-infection with other pathogens. So H. parasuis isolates belonging to some of the genotypes could be nonvirulent strains, and were just found causing disease when there is a co-infection. To address the pathogenic properties of H. parasuis strains of different genotype, it is necessary to do experimental infection with pigs free of those pathogens that could cause similar lesions with H. parasuis. The simultaneous presence of PRRSV and PCV2 with H. parasuis was studied. It shows that 11.5% cases (53), 27.9% cases (129), 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens (Table 2).
Clinically, co-infection of H. parasuis with other pathogens, such as porcine reproductive and respiratory syndrome virus (Solano et al., 1997), porcine circovirus type 2 (Kim et al., 2002), Streptococcus suis, Escherichia coli, Bordetella bronchiseptica and Pasteurella multocida (Cai et al., 2006), are frequently found in Glasser’s disease. Clinical and diagnostic evidence have suggested that epizootic out breaks caused by porcine reproductive and respiratory syndrome virus (PRRSV) in a naı¨ve herd mediate a sequence of clinical events that include a high prevalence of secondary viral and bacterial infections, mainly in nursery and fattening pigs (Pijoan et al., 1994). Frequency of H. parasuis infection is only slightly higher in PRRSV positive diseased pigs (29.7%) than that of PRRSV negative ones (27.2%) and vice versa (49.6% vs 46.5%). It is indicated that PRRSV or H. parasuis infection may not necessarily facilitate the secondary infection of H. parasuis or PRRSV, which clinically confirmed the result that there was no influence of the previous infection with PRRSV in the occurrence of H. parasuis infection (Segales et al., 1998, 1999). Of all the 74 PCV2 positive cases examined in our study, a frequency of 36.5% (27/74) of H. parasuis positive was observed, which is similar to a previous study with the frequency of 32.3% (43/133); but a much higher frequency (52.7%, 39/74) of PRRSV positive cases were found than that (29.3%, 39/133) of the previous study (Kim et al., 2002). The higher prevalence of PRRSV in PCV2 positive pigs may be due to the existence and transmission of highly pathogenic PRRSV strains which caused outbreak in many provinces in China recently (Li et al., 2007; Tian et al., 2007; Zhou et al., 2008). Our study showed that multi-infection of H. parasuis, PRRSV and PCV2 existed with high frequency in China. So attention should be paid to the multi-infection of H. parasuis, PRRSV and PCV2, for disease diagnostic and control, and this study indicated that H. parasuis existed widely in China with numerous genotypes. Further studies need to be done to know the mechanisms of co-infections of H. parasuis, PRRSV and PCV2. Acknowledgements This work was supported by grants from Jiangsu Science and Technique Foundation (BE2007342), Zhejiang Province Science and Technique Foundation (2007C22045), and partly National Key Technology R&D Program (2007BAD86B02-3). References Biberstein, E.L., White, D.C., 1969. A proposal for the establishment of two new Haemophilus species. Journal of Medical Microbiology 2, 75–78. Cai, X., Chen, H., Blackall, P.J., Yin, Z., Wang, L., Liu, Z., Jin, M., 2006. Serological characterization of Haemophilus parasuis isolates from China. Veterinary Microbiology 111, 231–236. De la Puente Redondo, V.A., Garcı´a del Blance, N., Gutie´rrez Martı´n, C.B., Navas Me´ndez, J., Rodrı´guez Ferri, E.F., 2000. Detection and subtyping of Actinobacillus pleuropneumoniae strains by PCR–RFLP analysis of the tbpA and tbpB genes. Research in Microbiology 151, 669–681. De la Puente Redondo, V.A., Me´ndez, J.N., Blanco, N.G., Boronat, N.L., Mart’n, C.B.G., Ferri, E.F.R., 2003. Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene. Veterinary Microbiology 2511, 1–10.
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