Diversity of influenza A viruses retrieved from respiratory disease outbreaks and subclinically infected herds in Spain (2017-2019).

The present study was aimed to assess the diversity of influenza A viruses (IAV) circulating in pig farms in the Iberian Peninsula. The study included two different situations: farms suffering respiratory disease outbreaks compatible with IAV (n = 211) and randomly selected farms without overt respiratory disease (n = 19). Initially, the presence of IAV and lineage determination was assessed by qRT-PCR using nasal swabs. IAV was confirmed in 145 outbreaks (68.7%), mostly in nurseries (53/145; 36.5%). Subtyping by RT-qPCR was possible in 94 of those cases being H1avN2hu (33.6%), H1avN1av (24.3%) and H1huN2hu (18.7%), the most common lineages. H3huN2hu and H1pdmN1pdm represented 7.5% and 6.5% of the cases, respectively. As for the randomly selected farms, 15/19 (78.9%) were positive for IAV. Again, the virus was mostly found in nurseries and H1avN2hu was the predominant lineage. Virus isolation in MDCK cells was attempted from positive cases. Sixty of the isolates were fully sequenced with Illumina MiSeq®. Within those 60 isolates, the most frequent genotypes had internal genes of avian origin, and these were D (19/60; 31.7%) and A (11/60; 18.3%), H1avN2hu and H1avN1av, respectively. In addition, seven previously unreported genotypes were identified. In two samples, more than one H or N were found and it was not possible to precisely establish their genotypes. A great diversity was observed in the phylogenetic analysis. Notably, four H3 sequences clustered with human isolates from 2004-05 (Malaysia and Denmark) that were considered uncommon in pigs. Overall, this study indicates that IAV is a very common agent in respiratory disease outbreaks in Spanish pig farms. The genetic diversity of this virus is continuously expanding with clear changes in the predominant subtypes and lineages in relatively short periods of time. The current genotyping scheme has to be enlarged to include the new genotypes that could be found in the future.

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.

Substantial Antigenic Drift in the Hemagglutinin Protein of Swine Influenza A Viruses.

The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level of genetic and antigenic differences even within the same swIAV lineage. The objective of this study was to investigate the level of genetic drift in one enzootically infected swine herd over one year. Nasal swabs were collected monthly from sows (n = 4) and piglets (n = 40) in the farrowing unit, and from weaners (n = 20) in the nursery. Virus from 1-4 animals were sequenced per month. Analyses of the sequences revealed that the hemagglutinin (HA) gene was the main target for genetic drift with a substitution rate of 7.6 × 10-3 substitutions/site/year and evidence of positive selection. The majority of the mutations occurred in the globular head of the HA protein and in antigenic sites. The phylogenetic tree of the HA sequences displayed a pectinate typology, where only a single lineage persists and forms the ancestor for subsequent lineages. This was most likely caused by repeated selection of a single immune-escape variant, which subsequently became the founder of the next wave of infections.

Longitudinal field studies reveal early infection and persistence of influenza A virus in piglets despite the presence of maternally derived antibodies.

A longitudinal study was performed in three Danish farrow to grower (30 kilos) herds over a 4-month period to investigate the dynamics and clinical impacts of influenza A virus (IAV) infections. In each herd, four batches consisting of four sows each with five ear-tagged piglets were included. Nasal swabs and/or blood were sampled from the sows and/or the piglets prior to farrowing and at weeks 1, 3, and 5 and at the end of the nursery period. Clinical examinations were performed at each sampling time. The sows and piglets were tested for IAV and IAV antibodies in nasal swabs and blood samples, respectively. The results revealed three enzootically infected herds, where the majority of the pigs were infected during the first 5 weeks after birth. Infected piglets of only 3 days of age were detected in the farrowing unit, where the sows were also shedding virus. In all herds, low to moderate numbers of infected pigs (ranging from 3.6 to 20.7%) were found to be virus positive in nasal swabs at two consecutive sampling times. Furthermore, clinical signs of respiratory disease were associated with IAV detection. The findings of this study documented that IAV can persist in herds and that piglets as young as 3 days can be infected despite the presence of maternally derived antibodies.

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.

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

BACKGROUND: A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. OBJECTIVES: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. METHODS: New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples. RESULTS: A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping. CONCLUSIONS: These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

New route for fast detection of antibodies against zoonotic pathogens in sera of slaughtered pigs by means of flow-through chemiluminescence immunochips.

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes.

Detection of IgM and IgG against hepatitis E virus in serum and meat juice samples from pigs at slaughter in Bavaria, Germany.

Hepatitis E virus (HEV) is an emerging foodborne pathogen with domestic and wild pigs (and likely other species such as deer or rabbits) recognized as reservoir. Pathogenesis in pigs usually leads to an asymptomatic course of disease. Since there is no enzyme-linked immunosorbent assay (ELISA) kit for the detection of anti-HEV antibodies in pigs commercially available, the objective of this study was to assess the seroprevalence in fattening pigs at slaughter and at herd level using a newly developed ELISA based on genotype (GT) 1 and GT 3 in Bavaria, Germany. Based on 516 serum and 198 meat juice samples collected from different herds at four different Bavarian slaughterhouses, the overall seroprevalence of anti-HEV IgG in serum and meat juice samples was 68.6% and 67.6%, respectively. Analyzing the serum for the presence of anti-HEV IgM, 36/516 (7%) were positive for anti-HEV IgM. At herd level, most of the herds were seropositive for anti-HEV antibodies. The present study shows that HEV is widespread among the Bavarian pig population and that some pigs might test positive for anti-HEV IgM even at the age of slaughter. Also, meat juice serves as an equivalent matrix to serum to test for anti-HEV antibodies in pigs.

Antimicrobial susceptibility and distribution of ß-lactamase A (blaA) and ß-lactamase B (blaB) genes in enteropathogenic Yersinia species.

One hundred eighty-six strains of enteropathogenic Yersinia (Y.) enterocolitica of bioserotypes 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 and 12 strains of Yersinia pseudotuberculosis of bioserotypes 1/O:1, 1/O:2, and 2/O:1 from different human (feces) and nonhuman (pig, pork, wild boar, monkey, chinchilla, mara, capybara, salad) sources collected in the years 1995-2009 were examined. Antimicrobial resistance patterns for 12 antimicrobial agents were generated using broth microdilution. The presence and characterization of the ß-lactamase genes blaA and blaB were studied using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP), respectively. The expression of ß-lactamase BlaA and BlaB was detected using double-disc diffusion. Y. enterocolitica strains showed resistance to ampicillin (92%), streptomycin (13%), and sulfamethoxazole (2%). Intermediate susceptibility to tetracycline was shown by two Y. enterocolitica strains. All Y. pseudotuberculosis strains were susceptible to all tested antimicrobial agents. Most (99%) of the Y. enterocolitica strains carried both ß-lactamase genes. One strain of bioserotype 3/O:3 lacked both genes. In contrast, all Y. pseudotuberculosis strains carried neither of the ß-lactamase genes. Homogeneity was detected in all blaA and blaB genes of Y. enterocolitica using PCR-RFLP. The majority (89%) of Y. enterocolitica strains expressed both ß-lactamase enzymes, whereas none of the Y. pseudotuberculosis strains showed expression of either enzyme. Also, it seems that the resistance of Y. enterocolitica has not changed during the last years.

Different enteropathogenic Yersinia strains found in wild boars and domestic pigs.

Yersinia enterocolitica and Yersinia pseudotuberculosis strains isolated from wild boars and fattening pigs were characterized and compared with each other. In wild boars, ail-positive Y. enterocolitica strains belonged to bioserotypes 4/O:3 (36%, 5/14), 2/O:9 (29%, 4/14), and 2/O:5,27 (21%, 3/14). Additionally, two ail-positive strains were untypable. Among fattening pigs, the bioserotype 4/O:3 was dominating (91%, 71/78), and bioserotypes 2/O:5,27 (8%, 6/78) and 2/O:9 (1%, 1/78) were rare. inv-positive Y. pseudotuberculosis strains of serotypes O:1 and O:2 were isolated only from wild boars. Antimicrobial resistance patterns between wild boar and fattening pig strains differed. Most of the ail-positive Y. enterocolitica strains carried yst, hreP, and virF genes. Several genotypes of Y. enterocolitica strains were obtained by PFGE using NotI, ApaI, XhoI, and SpeI enzymes. All genotypes of wild boar strains differed from fattening pig strains. Especially strains of bioserotype 4/O:3 were clearly different with all four enzymes. These results show that wild boar strains differed from domestic pig strains. More wild boar strains should be isolated to show that wild boars and domestic pigs are reservoirs for different Y. enterocolitica and Y. pseudotuberculosis strains.

Wild boars as an important reservoir for foodborne pathogens.

One hundred fifty-three wild boars shot in the canton of Geneva, Switzerland, were studied for the occurrence of foodborne pathogens. Tonsils and fecal samples of the animals were examined using real-time polymerase chain reaction, enzyme-linked fluorescent immunoassay, and cultural methods. The detection rate of Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, stx-positive Escherichia coli, and Listeria monocytogenes was 12%, 35%, 20%, 9%, and 17%, respectively, when tonsil samples were studied. Only Y. enterocolitica (5%) and L. monocytogenes (1%) were detected in fecal samples. None of the samples was positive for Campylobacter spp. Females (71%) and young animals (61%) carried more frequently one or more pathogens than males (53%) and older ones (44%). In total, 8 Salmonella spp., 14 Y. enterocolitica, 4 Y. pseudotuberculosis, and 26 L. monocytogenes strains were further characterized. Most of the Salmonella spp. strains were of serotype Salmonella Enteritidis (75%) followed by serotypes Salmonella Stourbridge (13%) and Salmonella Veneziana (13%). L. monocytogenes strains belonged to serotypes 1/2a (42%), 1/2b (19%), and 4b (38%). Serotypes O:3 (36%), O:5,27 (21%), and O:9 (29%) were identified among Y. enterocolitica strains and serotypes O:1 (75%) and O:2 (25%) among Y. pseudotuberculosis strains. This study shows that wild boars are frequent carriers of foodborne pathogens. High wild boar densities and increasing popularity of outdoor ranging of pigs may intensify the risk of transmission of these pathogens to fattening pigs.

Epidemiological data on pathogenic Yersinia enterocolitica in Southern Germany during 2000-2006.

Yersinia enterocolitica is the most common species causing enteric yersiniosis, which is still the third most frequently reported foodborne gastroenteritis in Europe. Y. enterocolitica generally causes sporadic human infections, and outbreaks are rare. The most important infection source of yersiniosis is believed to be contaminated pork and pork products. Data on the prevalence of pathogenic Y. enterocolitica in animals and foodstuffs are very limited and old; thus, more information on the extent and range of the prevalence of this enteropathogen in nonhuman sources is needed. In this work, prevalence of pathogenic Y. enterocolitica in different sources in Bavaria is presented. Further, the antimicrobial resistance of human and nonhuman strains is reported. The highest isolation rate of pathogenic Y. enterocolitica (67%) was found in tonsils of slaughter pigs. No pathogenic strains were isolated from cattle, sheep, turkey, and horses. ail-Positive Y. enterocolitica was detected in dogs (5%), cats (3%), and rodents (3%) by real-time PCR. Pathogenic Y. enterocolitica was isolated only from raw pork, especially from edible offal (51%). Surprisingly, 38% of game was contaminated with this pathogen when the samples were studied with PCR. Additionally, some raw pork sausages and one poultry sample were PCR positive. All pathogenic Y. enterocolitica isolates from nonhuman sources were belonging to bioserotype 4/O:3. Antimicrobial resistance of 60 human and 140 porcine strains of bioserotype 4/O:3 was tested by the agar disc diffusion method to 15 different antimicrobial agents. All Y. enterocolitica 4/O:3 strains were susceptible to most of the tested antibacterial agents.