[Challenges in Influenza diagnostics in a swine herd - a case report]. / Herausforderungen bei der Influenzadiagnostik in einem Schweinebetrieb ­ ein Fallbericht.

In a gilt producing farm in Lower Austria, respiratory diseases occurred over the previous years in self-reared gilts after being introduced into the sow herd. In addition, fertility disorders in terms of late abortions and re-breeders were observed in the fall of 2019. Nasal swabs of 3 gilts with respiratory signs and fever were tested positive for influenza A virus (IAV) subtype H1avN1 by PCR. However, examination of serum samples from these animals at 2 different time points did not detect antibodies using the standard hemagglutination inhibition (HI) test of the laboratory. Examination of additional age groups likewise failed to detect H1avN1 antibody titers. In consequence to the extension of the diagnostic panel of the HI test by 7 additional H1avN1 test antigens, a clear seroconversion of the PCR positive sows against 2 different H1avN1 isolates could be measured. In addition, high antibody titers against these 2 H1avN1 strains were also detectable in the majority of the remaining age groups tested. Following the administration of the trivalent influenza vaccine, which has been approved throughout Europe, a significant improvement of the clinical presentation in the herd was achieved. The present case report illustrates that direct and indirect pathogen detection should be used in combination for targeted influenza diagnostics. In addition, it was shown that the continuous adaptation of test antigens to the isolates circulating in the field would be extremely crucial for the significance of the HI test.

Surveillance of European Domestic Pig Populations Identifies an Emerging Reservoir of Potentially Zoonotic Swine Influenza A Viruses.

Swine influenza A viruses (swIAVs) can play a crucial role in the generation of new human pandemic viruses. In this study, in-depth passive surveillance comprising nearly 2,500 European swine holdings and more than 18,000 individual samples identified a year-round presence of up to four major swIAV lineages on more than 50% of farms surveilled. Phylogenetic analyses show that intensive reassortment with human pandemic A(H1N1)/2009 (H1pdm) virus produced an expanding and novel repertoire of at least 31 distinct swIAV genotypes and 12 distinct hemagglutinin/neuraminidase combinations with largely unknown consequences for virulence and host tropism. Several viral isolates were resistant to the human antiviral MxA protein, a prerequisite for zoonotic transmission and stable introduction into human populations. A pronounced antigenic variation was noted in swIAV, and several H1pdm lineages antigenically distinct from current seasonal human H1pdm co-circulate in swine. Thus, European swine populations represent reservoirs for emerging IAV strains with zoonotic and, possibly, pre-pandemic potential.

A newly developed tetraplex real-time RT-PCR for simultaneous screening of influenza virus types A, B, C and D.

BACKGROUND: Human- or avian-to-swine transmissions have founded several autonomously circulating influenza A virus (IAV) lineages in swine populations that cause economically important respiratory disease. Little is known on other human influenza virus types, like B (IBV) and C (ICV) in European swine, and of the recently detected novel animal influenza virus type D (IDV). OBJECTIVES: Development of a cost-effective diagnostic tool for large-scale surveillance programmes targeting all four influenza virus types. METHODS: An influenza ABCD tetraplex real-time RT-PCR (RT-qPCR) was developed in the frame of this study. A selection of reference virus strains and more than 4000 porcine samples from a passive IAV surveillance programme in European swine with acute respiratory disease were examined. RESULTS: Two IBV, a single IDV but no ICV infections were identified by tetraplex RT-qPCR. IBV and IDV results were confirmed by conventional RT-PCR and partial sequence analysis. CONCLUSIONS: The tetraplex RT-qPCR proved fit for purpose as a sensitive, specific and high-throughput tool to study influenza virus transmission at the human-animal interface. Complementing close-meshed active virological and serological surveillance is required to better understand the true incidence and prevalence of influenza virus type B, C and D infections in swine.

Expanded cocirculation of stable subtypes, emerging lineages, and new sporadic reassortants of porcine influenza viruses in swine populations in Northwest Germany.

The emergence of the human 2009 pandemic H1N1 (H1N1pdm) virus from swine populations refocused public and scientific attention on swine as an important source of influenza A viruses bearing zoonotic potential. Widespread and year-round circulation of at least four stable lineages of porcine influenza viruses between 2009 and 2012 in a region of Germany with a high-density swine population is documented here. European avian influenza virus-derived H1N1 (H1N1av) viruses dominated the epidemiology, followed by human-derived subtypes H1N2 and H3N2. H1N1pdm viruses and, in particular, recently emerging reassortants between H1N1pdm and porcine HxN2 viruses (H1pdmN2) were detected in about 8% of cases. Further reassortants between these main lineages were diagnosed sporadically. Ongoing diversification both at the phylogenetic and at the antigenic level was evident for the H1N1av lineage and for some of its reassortants. The H1avN2 reassortant R1931/11 displayed conspicuously distinct genetic and antigenic features and was easily transmitted from pig to pig in an experimental infection. Continuing diverging evolution was also observed in the H1pdmN2 lineage. These viruses carry seven genome segments of the H1N1pdm virus, including a hemagglutinin gene that encodes a markedly antigenically altered protein. The zoonotic potential of this lineage remains to be determined. The results highlight the relevance of surveillance and control of porcine influenza virus infections. This is important for the health status of swine herds. In addition, a more exhaustive tracing of the formation, transmission, and spread of new reassortant influenza A viruses with unknown zoonotic potential is urgently required.

Porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infections in wild boar (Sus scrofa) in southwestern Germany.

Samples were collected from 203 wild boars (Sus scrofa) hunted in Baden-Wurtemburg, Germany from November-January 2008 and 2009. Samples from the lung and tonsil were analyzed by quantitative polymerase chain reaction (qPCR) for porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European type) and type 2 (American type). A qPCR to detect porcine circovirus type 2 (PCV2)-specific genome was performed on tissue homogenates including lung, tonsils, and inguinal lymph nodes. Serum samples were tested for antibodies against PRRSV and PCV2 by enzyme-linked immunosorbent assay (ELISA). No PRRSV was detected in any of the 203 samples and one sample had detectable antibodies against PRRSV. We detected PCV2 in organ materials from 103 wild boars with a prevalence of 50.7%. The number of wild boars positive for PCV2 by PCR varied according to the population density of wild boars among woodlands. More positive samples were detected in woodlands with a high density of wild boars. We found no correlation between the number of PCV2-positive wild boars and the density of domestic pigs in the surrounding area. The number of wild boars positive for antibodies against PCV2 by the INGEZIM Circovirus IgG/IgM test kit was low (53 sera positive for IgG- and three sera positive for IgM-antibodies) in comparison to the higher positive results from the INGEZIM CIRCO IgG test kit (102 positive and 12 inconclusive results).

Respiratory function and pulmonary lesions in pigs infected with porcine reproductive and respiratory syndrome virus.

Pulmonary dysfunction was evaluated in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV, isolate VR-2332) and compared to clinical and pathological findings. Infected pigs developed fever, reduced appetite, respiratory distress and dullness at 9 days post-inoculation (dpi). Non-invasive pulmonary function tests using impulse oscillometry and rebreathing of test gases (He, CO) revealed peripheral airway obstruction, reduced lung compliance and reduced lung CO-transfer factor. PRRSV-induced pulmonary dysfunction was most marked at 9-18 dpi and was accompanied by a significantly increased respiratory rate and decreased tidal volume. Expiration was affected more than inspiration. On histopathological examination, multifocal areas of interstitial pneumonia (more severe and extensive at 10 dpi than 21 dpi) were identified as a possible structural basis for reduced lung compliance and gas exchange disturbances.

Porcine Torque teno virus: determination of viral genomic loads by genogroup-specific multiplex rt-PCR, detection of frequent multiple infections with genogroups 1 or 2, and establishment of viral full-length sequences.

Torque teno virus (TTV) is a non-enveloped virus with a circular, single-stranded DNA genome. TTV is currently classified in the unassigned genus Anellovirus, and distinct TTVs of tentative species-status infect a wide range of vertebrates. In domestic pigs and wild boars, porcine TTV occurs in two genogroups, TTV1 and TTV2, which are currently detected using only conventional PCR assays. To allow high-throughput testing, the present study describes development of a multiplex real-time (rt)-PCR assay for efficient simultaneous detection of TTV1 and TTV2. To demonstrate usefulness of this rt-PCR assay for large-scale testing, 203 serum samples from domestic pigs were screened for TTV infection. The detected rates of single TTV1, single TTV2, and double TTV1/TTV2 infections were 32, 17, and 32% and represent the first report on the occurrence of porcine TTV in Germany. In addition, 100 wild boar lung samples were tested that confirmed high prevalences of TTV infection. Moreover, establishment of genogroup-specific rt-PCR standards allowed the determination of mean viral genomic loads in sera from TTV-infected swine to about 10(4.5)/ml, respectively. To verify the specificity of the rt-PCR assay, conventional PCR assays that amplify genogroup-specific, size-distinguishable products from the TTV untranslated regions were designed. In total, 50 clones derived from 24 PCR products obtained from 19 TTV1 and TTV2 single- or double-infected animals were sequenced. Phylogenetic analyses of these sequences demonstrated the frequent occurrence of multiple infections with distinct porcine TTVs of the same genogroup. Moreover, two porcine TTV full-length sequences were established, one for each genogroup.