Design of a High-Throughput Real-Time PCR System for Detection of Bovine Respiratory and Enteric Pathogens

Abstract

Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves, Pasteurella multocida (38.4%), rotavirus A (27.4%), Mycoplasma spp. (26.2%), and Trueperella pyogenes (25.5%) were the most prevalent pathogens, while P. multocida (71.4%), Mycoplasma spp. (58.9%), Mannheimia haemolytica (53.6%), and Mycoplasma bovis (42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.

Characterization of Influenza D Virus in Danish Calves

“…No prior cleaning of the nostril was performed. Further information about the handling and storage of the samples is described elsewhere [36].…”

“…A previous study investigated the occurrence of 11 respiratory and enteric pathogens in the 100 Danish calf herds at the first sampling time using a high-throughput real-time PCR platform, BioMark HD (Fluidigm, South San Francisco, CA, USA), and the 192.24 (samples.assays) dynamic array integrated fluidic circuit nanofluidic chip (Fluidigm) [36]. The herds that were sampled at multiple time points were also tested on the high-throughput real-time PCR (data not shown).…”

Lesions and pathogens found in pigs that died during the nursery period in five Danish farms

“…The pre-amplified cDNA and DNA was stored at − 20 °C until high-throughput real-time PCR amplification, using the BioMark HD (Fluidigm, South San Franscisco, USA) and 192.24 dynamic array (DA) integrated fluidic circuit (IFC) system (Fluidigm) as previously described [ 45 , 46 ].…”

In silico analysis to develop PCR assays for identification of bacterial pathogens in animals: what can we improve?

“…We found polymorphic positions in 50% of the cases, which was common among bacterial species with the highest numbers of genomes in wgs database. However, the sodA ( 15 ) and lktD ( 17 ) genes were found to be very conservative with single-variant sequences among 1,613 Mannheimia haemolytica genomes in the wgs database. The frequency of sodA and lktD sequences with a polymorphic nucleotide in the primer was only 0.06%.…”

“…It should also be noted that a BLAST search in the wgs database allows detection if a target PCR sequence is not ubiquitous for the genome sequenced of a given bacterial species. For instance, the wgs database comprises 1,613 genomes of Mannheimia haemolytica and the target sequence for PCR in the sodA gene ( 15 ) is present in 1,611 cases.…”