Development and validation of high-throughput real-time PCR systems for diagnosis and typing of pathogens in pigs

Abstract

Millions of pigs are produced in Denmark each year, making it one of the largest pig producing countries in the world. Production of pigs can be associated with various challenges such as environmental and management issues and with the presence of pathogens. Respiratory and intestinal diseases caused by pathogens are of major importance to the pig production as it can result in reduced productivity, impaired animal welfare, increased mortality or morbidity. Vaccination and medical treatment are often initiated based on clinical observations and lacks support from laboratory investigations. This can lead to an overuse of medication and suboptimal vaccination programs. A reason why laboratory diagnostic analyses are often neglected is related to the high costs associated with the diagnostics and difficulties in interpretation of the results of random cross-sectional samples. This PhD thesis consists of an introduction in which the project and relevant background is introduced. Here, the focus is on influenza A virus (IAV), especially in pigs, and on respiratory and enteric viral and bacterial pathogens, which all have an impact on the Danish pig production. Furthermore, the background contains a section describing the history of methodology used for diagnostic analyses with the greatest focus put on polymerase chain reaction (PCR). The results of the work performed during the PhD are presented in the form of three manuscripts. Finally, the three manuscripts and suggestions to further work are discussed in a broader context. Manuscript I focus on the design and development of a high-throughput system to improve surveillance of swine influenza A virus (swIAV) and lower the costs of virus subtyping. For this, the high-throughput real-time PCR (qPCR) BioMark platform (Fluidigm) was used and optimized. To validate the platform, 12 different qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six different assays specific for the internal genes of the 2009 human pandemic strain (A(H1N1)pdm09) were tested. To test the sensitivity and specificity of the established high-throughput system, virus isolates and field samples, which had earlier been subtyped by full genome sequencing or multiplex reverse transcription qPCR (RT-qPCR), respectively, were used. Based on our findings, it was concluded that the high-throughput system is sensitive and specific and, thereby, the system provides a cost-effective alternative for subtyping of swIAVs. In manuscript II, a high-throughput diagnostic system specific for 17 respiratory and enteric viral and bacterial pathogens was designed and established again using the high-throughput qPCR BioMark platform (Fluidigm). A total of 21 qPCR assays were validated and optimized to run under the same reaction conditions. Furthermore, the sensitivity and specificity were assessed based on testing of known positive samples. The results revealed a performance of the diagnostic system similar to that of the qPCR analyses on the current qPCR platform. In addition, by using the high-throughput diagnostic system it is possible to offer diagnostic services with reduced costs and turnover time. Manuscript III describes three monthly sampling of oral fluid and faecal sock samples together with observations of clinical signs in six nursery and four finisher herds in Denmark. The collected samples were analysed with the high-throughput diagnostic system described in manuscript II and the occurrence of significant correlation between presence of pathogen and clinical signs, such as coughing and diarrhoea, was investigated. The study revealed that swIAV, porcine circovirus type 2 (PCV2), porcine cytomegalovirus (PCMV), Brachyspira pilosicoli, Lawsonia intracellularis, Escherichia coli fimbria type F4 and F18 were prevalent in several of the herds. Furthermore, for some of the pathogens a significant correlation was observed between their detection and the presence of coughing and/or diarrhoeic events. The study further showed that the use of the high-throughput diagnostic system for continuous monitoring of pathogens provides a broader knowledge on the distribution of pathogens in a given herd. This information can be useful for veterinary consultancy as it creates a more objective basis for intervention, including treatment regimens and vaccination strategies. Based on the studies described in manuscripts I, II and III, it was concluded that the use of the high-throughput qPCR BioMark platform provides a new and innovative way of carrying out diagnostic analyses. Changing to this cheaper platform will favour the use of laboratory analyses in the pig industry and provide a more objective basis for the selection of preventive measures to the benefit of animal health, animal welfare, production economics and food safety. The high-throughput system is presently under establishment in the Diagnostic Centre at the National Veterinary Institute, Technical University of Denmark and it will soon be offered to costumers as a diagnostic service