Influenza surveillance in pigs: balancing act between broad diagnostic coverage and specific virus characterization.

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.

Viral and Bacterial Profiles in Endemic Influenza A Virus Infected Swine Herds Using Nanopore Metagenomic Sequencing on Tracheobronchial Swabs.

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.

Implementation of piglet castration under inhalation anaesthesia on farrowing farms.

BACKGROUND: Since 01.01.2021, suckling piglets may no longer be castrated without anaesthesia in Germany. Previous studies showed castration using isoflurane anaesthesia in combination with a suitable analgesic, meet the requirements of the German Animal Welfare Act. It can be carried out independently by farmers and other qualified persons with an automated and certified isoflurane device. Therefore, the aim of the present field study was to implement the use of three different anaesthetic devices for surgical castration of male piglets under automated isoflurane anaesthesia on 15 conventional pig farms in southern Germany. In addition, the depth of anaesthesia based on defensive movements, the labour time required in contrast to anaesthetic-free castration, castration-related anaesthetic incidents and the piglet mortality rate as well as occupational safety were investigated. For this purpose, farrowing batches of 11,574 piglets castrated under isoflurane anaesthesia (IA) were compared with the results of the 1568 piglets of anaesthetic-free farrowing batches (AF). RESULTS: In total, 80.1% of the castrated piglets showed sufficient depth of anaesthesia, although this varied significantly between devices. 1.7% of the piglets suffered an anaesthetic incident, of which 0.1% died during or within 24 h after anaesthesia. The required time for the complete working process differed significantly between AF (1.7 ± 0.8 min/piglet) and IA batches (2.2 ± 0.8 min/piglet) but not for castration itself. The mean isoflurane consumption was 0.57 ± 0.27 ml/piglet and differed significantly between the devices (p < 0.001). The isoflurane concentration in the ambient air of the person-related workplace safety measurements was below the internationally lowest value of 15 mg/m3 from Ontario and Israel. CONCLUSION: In conclusion, 2 of the 3 types of devices used, a sufficient depth of anaesthesia during castration under isoflurane was achieved in 85% of castrated piglets. Anaesthetic incidents occurred in 1.7% of the animals, of which 0.1% died. Castration under isoflurane is more time-consuming than anaesthetic-free castration, but the castration time itself did not differ significantly. The occupational exposure limits were below the internationally lowest limit value of 15 mg/m3 for the persons involved. Even though castration under isoflurane is more time consuming than anaesthetic-free castration, it is a well-establishable method for practice and a dear improvement for animal welfare.

[Investigations on suckling piglets castrated under automated isoflurane anesthesia]. / Untersuchungen zur automatisierten Isoflurannarkose bei der Saugferkelkastration.

OBJECTIVE: Investigation of defensive reactions, piglet losses, post-bleeding and recovery time of suckling piglets castrated under automated isoflurane anesthesia as well as measurements of isoflurane concentrations in ambient air and estimation of the cleaning and disinfection success of anesthesia masks. MATERIAL AND METHODS: A total of 955 suckling piglets (age: 4.0 ± 1.2 days; weight: 2.0 ± 0.5 kg) were castrated under automated isoflurane anesthesia (PorcAnest 3000®) after the administration of a non-steroidal anti-inflammatory drug. Reactions during the procedure were assessed before castration using the interdigital claw reflex and during castration by defensive movements and vocal response. The piglets' recovery time was evaluated in the stable corridor (pass 1) and the farrowing pen (passes 4-5). In 73 animals, the spermatic cord was cut with a scalpel and the post-operative bleeding visually evaluated. In addition, exposure measurements of isoflurane were performed and anesthetic masks were examined for total bacterial contamination and indicator bacteria before and after the anesthetic procedure and following their disinfection. RESULTS: Following insufflation of isoflurane for 90 seconds 94.3 % of the piglets and after prior testing of the interdigital claw reflex and possible extension of the anesthetic supply 95.3 % of the piglets showed no or minimal defensive movements during castration. An anesthetic incident occurred in 0.9 % of the piglets, but no animal died. The recovery time lasted 7.3 ± 4.7 minutes (pass 1) and 6.2 ± 3.3 minutes (passes 4-5). Cutting the spermatic cord with a scalpel led to a higher post-bleeding score (p < 0.001) compared to use of the emasculator. Isoflurane concentrations in ambient air ranged between 4.5 und 28.1 mg/m3. Masks disinfection reduced the total germ count by 99.8 %. Contaminations with Escherichia coli and MRSA were no longer detectable in 4 of 6 cases after disinfection. CONCLUSION: Isoflurane anesthesia led in over 94 % of the piglets to no or minimal defensive reactions during castration. Anesthetic incidents occurred rarely and no piglet losses were recorded. Therefore, automated isoflurane anesthesia is associated with a low risk for suckling piglets. Measurements of isoflurane concentrations on persons involved were below the internationally lowest limit value. Disinfection of the anesthesia masks may prevent germ transmission between animal groups via this potential vector.

Reproductive performance of pandemic influenza A virus infected sow herds before and after implementation of a vaccine against the influenza A (H1N1)pdm09 virus.

BACKGROUND: Reproductive failure in sow herds due to infection with influenza A viruses has been described in the literature, but only a few studies have focused on the pathogenesis and the clinical signs of the infection. Case reports indicate an association between infections with influenza A viruses and reduced reproductive performance, although it has been difficult to experimentally reproduce the clinical outcome of poor reproductive performance. The aim of the present longitudinal field study was to compare the reproductive performance parameters before and after the implementation of vaccination against the influenza A (H1N1)pdm09 virus in sow herds infected with pandemic influenza A virus. Therefore, farm-specific data of 137 sow herds in Germany, including 60,153 sows, as well as the clinical presentation of the infection were surveyed via questionnaire. Furthermore, average performance parameters (return to oestrus rate, abortion rate, stillbirth rate, number of piglets born alive per litter, preweaning mortality rate and number of piglets weaned per sow per year) were recorded for 6 months before vaccination and 6 months after completion of primary vaccination. RESULTS: In 79.8% of the farms, the clinical presentation of the infection was characterised by a reduced reproductive performance. These findings were confirmed by analysis of the performance parameters, which revealed a significant decline in the return to oestrus rate (p < 0.001), abortion rate (p < 0.001) and preweaning mortality rate (p = 0.023) and a significant increase of the number in piglets born alive (p = 0.001) and piglets weaned per sow per year (p < 0.001) after immunisation. The stillbirth rate did not change significantly. CONCLUSION: The present study represents the first attempt to demonstrate the association of influenza A virus infection, vaccination and the alteration in reproductive performance parameters, investigating a large number of cases. The results show that by vaccinating against the influenza A (H1N1)pdm09 virus, an improvement in reproductive performance can be achieved in sow herds infected with pandemic influenza A virus. Additionally, the large number of herds that were affected by poor reproductive performance after infection with the aforementioned virus confirms the assumption of an association between pandemic influenza A virus and reproductive losses.

The Munich MIDY Pig Biobank - A unique resource for studying organ crosstalk in diabetes.

OBJECTIVE: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. METHODS: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. RESULTS: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. CONCLUSIONS: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.