RESUMO
BACKGROUND: Monitoring of infectious diseases on swine farms requires a high diagnostic sensitivity and specificity of the test system. Moreover, particularly in cases of swine influenza A virus (swIAV) it is desirable to include characterization of the virus as precisely as possible. This is indispensable for strategies concerning prophylaxis of swIAV and furthermore, to meet the requirements of a purposeful monitoring of newly emerging swIAV strains in terms of vaccine design and public health. Within the present cross-sectional study, we compared the diagnostic value of group samples (wipes of surfaces with direct contact to mouth/nose, dust wipes, udder skin wipes, oral fluids) to individual samples (nasal swabs, tracheobronchial swabs) for both swIAV identification and characterization. Sampling included different stages of pig production on 25 sow farms with attached nursery considered as enzootically infected with swIAV. Firstly, samples were analyzed for IAV genome and subsequently samples with Ct-values < 32 were subtyped by multiplex RT-qPCR. RESULTS: Nasal swabs of suckling piglets and nursery pigs resulted in a higher odds to detect swIAV (p < 0.001) and to identify swIAV subtypes by RT-qPCR (p < 0.05) compared to nasal swabs of sows. In suckling piglets, significant higher rates of swIAV detection could be observed for nasal swabs (p = 0.007) and sow udder skin wipes (p = 0.036) compared to contact wipes. In the nursery, group sampling specimens were significantly more often swIAV positive compared to individual samples (p < 0.01), with exception of the comparison between contact wipes and nasal swabs (p = 0.181). However, in general nasal swabs were more likely to have Ct-value < 32 and thus, to be suitable for subtyping by RT-qPCR compared to dust wipes, contact wipes, udder skin wipes and tracheobronchial swabs (p < 0.05). Interestingly, different subtypes were found in different age groups as well as in different specimens in the same holding. CONCLUSION: Although population-based specimens are highly effective for swIAV monitoring, nasal swabs are still the preferable sampling material for the surveillance of on-farm circulating strains due to significantly higher virus loads. Remarkably, sampling strategies should incorporate suckling piglets and different age groups within the nursery to cover as many as possible of the on-farm circulating strains.
RESUMO
Swine influenza A virus (swIAV) plays an important role in porcine respiratory infections. In addition to its ability to cause severe disease by itself, it is important in the multietiological porcine respiratory disease complex. Still, to date, no comprehensive diagnostics with which to study polymicrobial infections in detail have been offered. Hence, veterinary practitioners rely on monospecific and costly diagnostics, such as Reverse Transcription quantitative PCR (RT-qPCR), antigen detection, and serology. This prevents the proper understanding of the entire disease context, thereby hampering effective preventive and therapeutic actions. A new, nanopore-based, metagenomic diagnostic platform was applied to study viral and bacterial profiles across 4 age groups on 25 endemic swIAV-infected German farms with respiratory distress in the nursery. Farms were screened for swIAV using RT-qPCR on nasal and tracheobronchial swabs (TBS). TBS samples were pooled per age, prior to metagenomic characterization. The resulting data showed a correlation between the swIAV loads and the normalized reads, supporting a (semi-)quantitative interpretation of the metagenomic data. Interestingly, an in-depth characterization using beta diversity and PERMANOVA analyses allowed for the observation of an age-dependent interplay of known microbial agents. Also, lesser-known microbes, such as porcine polyoma, parainfluenza, and hemagglutinating encephalomyelitis viruses, were observed. Analyses of swIAV incidence and clinical signs showed differing microbial communities, highlighting age-specific observations of various microbes in porcine respiratory disease. In conclusion, nanopore metagenomics were shown to enable a panoramic view on viral and bacterial profiles as well as putative pathogen dynamics in endemic swIAV-infected herds. The results also highlighted the need for better insights into lesser studied agents that are potentially associated with porcine respiratory disease. IMPORTANCE To date, no comprehensive diagnostics for the study of polymicrobial infections that are associated with porcine respiratory disease have been offered. This precludes the proper understanding of the entire disease landscape, thereby hampering effective preventive and therapeutic actions. Compared to the often-costly diagnostic procedures that are applied for the diagnostics of porcine respiratory disease nowadays, a third-generation nanopore sequencing diagnostics workflow presents a cost-efficient and informative tool. This approach offers a panoramic view of microbial agents and contributes to the in-depth observation and characterization of viral and bacterial profiles within the respiratory disease context. While these data allow for the study of age-associated, swIAV-associated, and clinical symptom-associated observations, it also suggests that more effort should be put toward the investigation of coinfections and lesser-known pathogens (e.g., PHEV and PPIV), along with their potential roles in porcine respiratory disease. Overall, this approach will allow veterinary practitioners to tailor treatment and/or management changes on farms in a quicker, more complete, and cost-efficient way.
