Development of a Dual PCR Method for Detection of Pasteurella Multocida and Haemophilus Parasuis

Yu. Zhang; Halyna Rebenko; Jianhe Hu; Shouping Zhang

doi:10.32845/bsnau.vet.2021.1.2

Yu. Zhang Henan Institute of Science and Technology https://orcid.org/0000-0002-4848-8063
Halyna Rebenko Sumy National Agrarian University https://orcid.org/0000-0002-1884-4901
Jianhe Hu Henan Institute of Science and Technology https://orcid.org/0000-0002-6768-0851
Shouping Zhang Henan Institute of Science and Technology https://orcid.org/0000-0003-1774-1924

DOI: https://doi.org/10.32845/bsnau.vet.2021.1.2

Keywords: Pasteurella multocida; Haemophilus parasuis; Double PCR; Detection

Abstract

Pasteurella multocida and Haemophilus parasuis are the main pathogens of porcine respiratory disease syndrome, and often are the common pathogens of porcine pulmonary disease in collectivized pig farms. It has brought great influence on the pig industry and seriously hampered the healthy development of pigs. Therefore, the rapid detection method for Pasteurella multocida and Haemophilus parasuis is the key to its successful prevention and control. In the process of diagnosis, we need to conduct laboratory tests for confirming of preliminary diagnosis, that can be made based on the history, clinical symptoms, pathological changes of necropsy and epidemiological characteristics of the disease. Traditional diagnostic techniques, such as bacterial isolation and immunological test, are time-consuming and laborious, and are not suitable for rapid clinical diagnosis, nor for large-scale epidemiological investigation. Therefore, it is necessary to establish an accurate and rapid method to identify the two common respiratory pathogens in pigs.

In this paper, Pasteurella multocida and Haemophilus parasuis were used as research objects to establish a dual PCR detection method. The main research contents are as follows: A dual PCR assay was established to detect Pasteurella multocida and Haemophilus parasuis simultaneously. By using Pasteurella multocida and Haemophilus parasuis specific primers, and adjusting primer concentration and annealing temperature, a dual PCR method for detecting Pasteurella multocida and Haemophilus parasuis was established.

The rapid detection method for Pasteurella multocida and Haemophilus parasuis established in this study has high specificity and sensitivity, and can realize rapid and accurate identification of pathogenic bacteria in a relatively short period of time, providing a new technical means for rapid detection in clinical and grassroots laboratories

References

Cai X W. (2006). Isolation and characterization of Haemophilus parasuis and development of its diagnostic method and inactivated bacterin [D]. Wuhan: Huazhong Agricultural University,
Cardoso-Toset F, Gómez-Laguna J, Callejo M, et al. (2013). Septicaemic pasteurellosis in free-range pigs associated with an unusual biovar 13 of Pasteurella multocida. Vet Microbioogyl, 167(3-4): 690-694.
Gao PC, Chu YF, Zhao P, et al. (2008). Screening of culture conditions for Haemophilus parasuis and its pathogenicity test to guinea pigs. Advances in Veterinary Medicine, (10):10-13.
Harper M, Boyce J D. (2017). The Myriad Properties of Pasteurella multocida Lipopolysaccharide. Toxins, 21, 9(8): 254-275.
Li J Y, Zhang J M, Xu C G, et al. (2010). Research progress on main virulence factors and pathogenic mechanism of Haemophilus parasuis.China Animal Husbandry and Veterinary Medicine, 37 (12) : 173-176.
Li J, Xie Y Z, Xuan Xiongbiao, et al. (2011). Real time fluorescence PCR of Haemophilus parasuis Establishment and application of rapid detection method. Anhui Agricultural Sciences, 39(6) : 3607-3609.
Liang Q, Yang B, Yong S L, et al. (2018). PCR method for detection of Haemophilus parasuis. Modern Animal Husbandry and Veterinary Medicine, 358 (09) : 10-13. (in Chinese)
Lin, Wei-Hao, Shih, Hsing-Chun, Lin, Chuen-Fu, Yang, Cheng-Yao, Lin, Chao-Nan & Chiou, Ming-Tang. (2019). Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan. PeerJ. 7. e6960. https://doi.org/10.7717/peerj.6960.
Ma, Lina, Wang, Liyan, Chu, Yuefeng, Li, Xuerui, Cui, Yujun, Chen, Shengli, Zhou, Jianhua, Li, Chunling, Lu, Zhongxin, Liu, Jixing & Liu, Yongsheng. (2016). Characterization of Chinese Haemophilus parasuis Isolates by Traditional Serotyping and Molecular Serotyping Methods. PLoS ONE. 11. https://doi.org/10.1371/journal.pone.0168903.
Malorny B，Tassios P T，Radstrom P，et al． (2003). Standardization of diagnostic PCＲ for the detection of foodborne pathogens．Int J Food Microbiol，83( 1) : 39－48．
Moriy，Nagamine K，Tomita N，et al．(2010). Development of multiplex － PCＲ for identification of Pasteurella multocida，Haemophilus parasuis and Actinbacillus pleuropneumoniae． Anim Feed Sci Tech， ( Z2) : 25－27．
Nedbalcova, Katerina, Satran, P., Jaglic, Zoran, Ondriasova, R. & Kucerova, Z. (2018). Haemophilus parasuis and Glässer’s disease in pigs: a review. Veterinární Medicína. 51. 168-179. https://doi.org/10.17221/5537-VETMED.
Ni, Hong-Bo, Gong, Qing-Long, Zhao, Quan, Li, Xiao-Yue, Zhang, Xiao-Xuan. (2020). Prevalence of Haemophilus parasuis“Glaesserella parasuis” in pigs in China: A systematic review and meta-analysis. Preventive Veterinary Medicine. 182. 105083. https://doi.org/10.1016/j.prevetmed.2020.105083.
Rebenko H. I. (2014). Etiolohiia ta epizootolohiia infektsiinykh khvorob respiratornoho traktu svynei (ohliad literatury) [Etiology and epizootology of infectious diseases of the respiratory tract of pigs (literature review)] Visnyk SNAU seriia «Vet med». [Bulletin of the SNAU series "Vet Med"], № 1 (34), 114-121.
Si Z S, Wang G Y. (2011). Research progress of hemophilus parasuis . China Animal Husbandry and Veterinary Medicine, 38 (06): 179-182. (in Chinese with English abstract)
Starkey, S.R. & Donnelly, T.M.. (2012). Pasteurella multocida. Clinical Veterinary Advisor: Birds and Exotic Pets. 717-719. https://doi.org/10.1016/B978-1-4160-3969-3.00418-2.
Xue G C, Ren T. (2009). Epidemiology and pathogenic factors of Haemophilus parasuis. Chinese Journal of Animal Husbandry and Veterinary Medicine, 36 (05): 168-171. (in Chinese)
Zhang Bin, Tang Chen, Liao Ming, et al. (2014). Update on the pathogenesis of Haemophilus parasuis infection and virulence factors. Vet Microbiology, 168(1): 1-7.
Zhang Y, Li J, Li W J, et al. (2017). PCR method for detection of Bacillus multicidal bars Chinese Journal of Veterinary Medicine, 51 (05) : 16-21. (in Chinese with English abstract)
Zhang Yuan, Wei Caiwen, Li Jian, et al. (2014). Application of multiplex PCR in capsular sequencing of Pasteurella multocida. Chinese Journal of Veterinary Science, 44 (12): 1286-1291.