Assessment of exposure to influenza A viruses in pigs between weaning and market age.

Influenza A viruses (IAVs) are common causes of respiratory infection in pigs. The objective of this study was to characterize the circulation of IAVs between weaning and market age on the basis of development of antibody response and molecular epidemiology of detected viruses. Two batches of weaned pigs were followed in the nursery and finisher barns with a sample of 81 and 75 pigs. Nasal swabs and blood samples were collected from individual pigs for virological and serological analyses. A H3N2 subtype virus, of cluster IV, was detected in Study 1, with a maximum of 97.9% identity to HA gene of viruses previously isolated in Ontario. In Study 2, a H1N1 subtype virus, of 2009 H1N1 pandemic lineage, was detected, with a maximum of 97.8% identity to HA gene of viruses previously isolated in Ontario. On the basis of HA gene, it was observed that pigs were being detected with the same virus over time. The existence of antibody titers for IAV other than the isolated one confirmed that more than one subtype can circulate in the same population. In Study 1, pigs with higher numbers of IAV detection had lower serological titers for the same virus that was confirmed to circulate in the nursery (P < 0.01). Thorough knowledge of all endemic viral strains is fundamental for development of infection and disease control, particularly in complex production systems. This may include consideration of sampling and testing strategies which could detect circulation of all IAV variants, even if they have low prevalence.

Longitudinal study of influenza A virus circulation in a nursery swine barn.

Commercial production of swine often involves raising animals in large groups through the use of multi-stage production systems. In such systems, pigs can experience different degrees of contact with animals of the same or different ages. Population size and degree of contact can greatly influence transmission of endemic pathogens, including influenza A virus (IAV). IAV can display high genetic variability, which can further complicate population-level patterns. Yet, the IAV transmission in large multi-site swine production systems has not been well studied. The objectives of this study were to describe the IAV circulation in a multi-source nursery facility and identify factors associated with infection in nursery pigs. Pigs from five sow herds were mixed in one all-in/all-out nursery barn, with 81 and 75 pigs included in two longitudinal studies. Virus isolation was performed in Madin-Darby canine kidney cells and serology was performed using hemagglutination inhibition assays. Risk factor analysis for virological positivity was conducted using logistic regression and stratified Cox's regression for recurrent events. In Study 1, at ≈30 days post-weaning, 100% of pigs were positive, with 43.2% of pigs being positive recurrently over the entire study period. In study 2, 48% of pigs were positive at the peak of the outbreak, and 10.7% were positive recurrently over the entire study period. The results suggest that IAV can circulate during the nursery phase in an endemic pattern and that the likelihood of recurrent infections was associated in a non-linear way with the level of heterologous (within-subtype) maternal immunity (p < 0.05). High within-pen intracluster correlation coefficients (> 0.75) were also observed for the majority of sampling times suggesting that pen-level factors played a role in infection dynamics in this study.

Virological Surveillance of Influenza A Subtypes Isolated in 2014 from Clinical Outbreaks in Canadian Swine.

Influenza A viruses (IAVs) are respiratory pathogens associated with an acute respiratory disease that occurs year-round in swine production. It is currently one of the most important pathogens in swine populations, with the potential to infect other host species including humans. Ongoing research indicates that the three major subtypes of IAV-H1N1, H1N2, and H3N2-continue to expand in their genetic and antigenic diversity. In this study, we conducted a comprehensive genomic analysis of 16 IAVs isolated from different clinical outbreaks in Alberta, Manitoba, Ontario, and Saskatchewan in 2014. We also examined the genetic basis for probable antigenic differences among sequenced viruses. On the basis of phylogenetic analysis, all 13 Canadian H3N2 viruses belonged to cluster IV, eight H3N2 viruses were part of the IV-C cluster, and one virus belonged to the IV-B and one to the IV-D cluster. Based on standards used in this study, three H3N2 viruses could not be clearly classified into any currently established group within cluster IV (A to F). Three H1N2 viruses were part of the H1α cluster.

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012.

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.

Molecular characterization of H3N2 influenza A viruses isolated from Ontario swine in 2011 and 2012.

BACKGROUND: Data about molecular diversity of commonly circulating type A influenza viruses in Ontario swine are scarce. Yet, this information is essential for surveillance of animal and public health, vaccine updates, and for understanding virus evolution and its large-scale spread. METHODS: The study population consisted of 21 swine herds with clinical problems due to respiratory disease. Nasal swabs from individual pigs were collected and tested by virus isolation in MDCK cells and by rtRT-PCR. All eight segments of 10 H3N2 viruses were sequenced using high-throughput sequencing and molecularly characterized. RESULTS: Within-herd prevalence ranged between 2 and 100%. Structurally, Ontario H3N2 viruses could be classified into three different groups. Group 1 was the most similar to the original trH3N2 virus from 2005. Group 2 was the most similar to the Ontario turkey H3N2 isolates with PB1 and NS genes originating from trH3N2 virus and M, PB2, PA and NP genes originating from the A(H1N1)pdm09 virus. All Group 3 internal genes were genetically related to A(H1N1)pdm09. Analysis of antigenic sites of HA1 showed that Group 1 had 8 aa changes within 4 antigenic sites, A(1), B(3), C(2) and E(2). The Group 2 viruses had 8 aa changes within 3 antigenic sites A(3), B(3) and C(2), while Group 3 viruses had 4 aa changes within 3 antigenic sites, B(1), D(1) and E(2), when compared to the cluster IV H3N2 virus [A/swine/Ontario/33853/2005/(H3N2)]. CONCLUSIONS: The characterization of the Ontario H3N2 viruses clearly indicates reassortment of gene segments between the North American swine trH3N2 from cluster IV and the A(H1N1)pdm09 virus.

Comparison of fiber gene sequences of inclusion body hepatitis (IBH) and non-IBH strains of serotype 8 and 11 fowl adenoviruses.

Fowl adenoviruses (FAdVs) are common in broiler operations, and the most frequently isolated FAdVs belong to serotypes 1, 8, and 11. Serotype 1 viruses are considered nonpathogenic. While some serotype 8 and 11 viruses cause inclusion body hepatitis (IBH), these virus serotypes can also be isolated from non-IBH cases. The fiber protein is one of the major constituents of the adenoviral capsid, involved in virus entry, and it has been implicated in the variation of virulence of FAdVs. The fiber gene sequences of four FAdV-8 and four FAdV-11 isolates from both IBH and non-IBH cases were determined and analyzed for a possible association of the fiber gene sequence in virulence. The fiber protein can be divided into tail, shaft, and head domains comprising some specific features. The conserved "RKRP" sequence motif (aa 17-aa 20) fit the consensus sequence predicted for the nuclear localization signal, while the "VYPF" motif (aa 53-aa 56), involved in the penton base interaction, was also found. Similar to mammalian adenoviruses, 17 pseudo-repeats with an average length of 16 aa were detected in the FAdV-8 fiber shaft region, while 20 pseudo-repeats with an average length of 18 aa were found in FAdV-11 fibers. There was a 144-147 nt difference between the fiber genes of the two FAdV serotypes. In the shaft region, the TLWT motif that marks the beginning of the fiber head domain of the mastadenovirus was not evident among examined FAdVs. The FAdV-11 isolates had 99.1 % aa sequence identity and 99.3 % similarity to each other, and there was no conserved aa substitution within the fibers. The FAdV-8 fiber proteins showed an overall lower, 89 % aa sequence identity and 93.4 % similarity, to each other and 22 nonsynonymous mutations were detected. Virulence markers were not detected in the analyzed fiber gene sequences of the different pathotypes of the two FAdV serotypes.

Pathogenicity and cytokine gene expression pattern of a serotype 4 fowl adenovirus isolate.

Hydropericardium-hepatitis syndrome (HHS), a recently emerged disease of chickens, is caused by some strains of fowl adenovirus serotype 4 (FAdV-4). In this study, a Canadian FAdV-4 isolate, designated as FAdV-4 ON1, was evaluated for pathogenicity after oral and intramuscular (im) infection of specific pathogen free (SPF) chickens. Pathogenicity was evaluated by observation of clinical signs and gross and histological lesions. The highest viral DNA copy numbers, irrespective of the inoculation route, were detected in the cecal tonsils. Virus titers in cloacal swabs collected over the entire study period were compared between the orally and im inoculated chickens, and the difference in titers between the two groups was significant (P<0.001), the oral group had a higher rank. The antibody response of infected chickens tested by an adenovirus-specific ELISA showed a statistically significant (P<0.001) difference between the orally and im inoculated chickens. The im inoculated chickens had higher values than birds inoculated orally (P<0.001). Serum samples from both groups collected at 14 days post-infection completely neutralized FAdV-4 ON1. In addition, the effects of FAdV-4 ON1 infection on transcription of a number of avian cytokines were studied in vivo. The expression of interferon (IFN)-γ and interleukin (IL)-10 in the liver was induced at early times after infection. This FAdV-4 ON1 potentially could be used as a live vaccine against HHS and developed as vaccine vector. The GenBank/EMBL/DDBJ accession number for the FAdV-4 ON1 sequence is GU188428.

Cytokine patterns associated with a serotype 8 fowl adenovirus infection.

This study examined cytokine gene expression patterns associated with fowl adenovirus (FAdV) infection. The selected cytokine mRNA was quantified by quantitative real-time reverse transcription-PCR in spleen, liver, and cecal tonsil during the course of infection of chickens with a serotype 8 FAdV (FAdV-8). Compared to uninfected chickens, infected birds had higher mRNA expression of interleukin (IL)-18 and IL-10 in spleen and liver, respectively. Interferon gamma (IFN-γ) mRNA expressed in spleen and liver of infected chickens was significantly upregulated, while the expression of IL-8 mRNA in spleen and liver of infected chickens was significantly downregulated. There was no significant difference between infected and uninfected groups in terms of cytokine gene expression in cecal tonsil. These results indicate that these four cytokines might play an important role in driving the immune responses following FAdV-8 infection.

Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate.

In this study we determined and analyzed the complete nucleotide sequence of the genome of a fowl adenovirus serotype 8 (FAdV-8) isolate and examined its pathogenicity in chickens. The full genome of FAdV-8 was 44,055 nucleotides in length with a similar organization to that of FAdV-1 and FAdV-9 genomes. No regions homologous to early regions E1, E3 and E4 of mastadenoviruses were recognized. Along with FAdV-9, FAdV-8 has only one fiber gene and with regard to sequence composition and genome organization, FAdV-8 is closer to FAdV-9 than to FAdV-1. Moreover, our findings suggest that FAdV-1 of species Fowl adenovirus A as the current type species despite its historical priority is not representative of the genus Aviadenovirus, and that FAdV-8 or FAdV-9 in species Fowl adenovirus E and Fowl adenovirus D, respectively, would be more suitable for that designation. Additionally, pathogenicity of FAdV-8 was studied in specific pathogen free chickens following oral and intramuscular inoculations. Despite lack of clinical signs and pathological changes virus was found in tissues and cloacal swabs of all birds with the highest viral copy numbers present in the cecal tonsils. The highest virus titers in the feces for orally and intramuscularly inoculated chickens were recorded at days 10 and 3 post-infection, respectively.

Vaccine efficacy against Ontario isolates of infectious bronchitis virus.

Infectious bronchitis (IB) is an economically important viral disease with worldwide distribution. Every country with an intensive poultry industry has infectious bronchitis virus (IBV). The virus rapidly spreads from bird to bird through horizontal transmission by aerosol or ingestion. Sentinel bird studies were carried out in southern Ontario and IBV has been isolated from layer flocks. Genetic analysis of the S1 region of the strains showed that they were not vaccine related. The pathogenicity of selected Ontario variants of IBV isolates was studied and the subsequent work was to determine the degree of protection against field isolates provided by a commonly used vaccine MILDVAC-Ma5 in Ontario. The protection was evaluated by challenging immunized chickens with the respiratory (IBV-ON1) and nephropathogenic (IBV-ON4) viruses. The mean vaccine efficacy for IBV-ON1 was 66.7% indicating that a Massachusetts serotype vaccine would provide some protection against IBV field isolates.

Pathogenicity of infectious bronchitis virus isolates from Ontario chickens.

Infectious bronchitis (IB) is one of the important viral diseases of chickens, and in spite of regular vaccination, IB is a continuous problem in Canadian poultry operations. In an earlier study using sentinel chickens we determined the incidence of infectious bronchitis virus (IBV) in Ontario commercial layer flocks. The objective of this study was to determine the pathogenicity of 5 nonvaccine-related IBV isolates recovered from the sentinel birds. The clinical signs, gross, and histological lesions in specific pathogen-free chickens indicated that all 5 isolates caused mild lesions in the respiratory tract. An important finding of this study was the significantly lower average daily weight gain among virus-inoculated groups of chickens during the acute phase of infection. Based on sequences of part of the S1 gene IBV-ON2, IBV-ON3, and IBV-ON5 formed a cluster and they were closely related to strain CU-82792. IBV-ON4 had 98.7% identity with the strain PA/1220/9, a nephropathogenic variant.

Serologic monitoring of a broiler breeder flock previously affected by inclusion body hepatitis and testing of the progeny for vertical transmission of fowl adenoviruses.

The increasing number of clinical cases of inclusion body hepatitis (IBH) associated with fowl adenoviruses (FAdVs) is a growing concern in different parts of the world, including Canada. After an outbreak of IBH in a 10-d-old pullet broiler breeder flock, we serologically monitored the flock from 8 to 46 wk of age, using the agar gel precipitation test (AGPT) offered by diagnostic laboratories and an FAdV group-specific enzyme-linked immunosorbemt assay (ELISA) developed earlier. In addition, we tested 1-d-old progeny for possible vertical transmission of FAdV when the breeder flock approached the peak of egg production by performing virus isolation and polymerase chain reaction (PCR) procedures on target organs. As in previous studies comparing the 2 tests, ELISA was more sensitive than AGPT. With ELISA, a few birds had weakly positive results at 8 wk of age, and all the birds had strongly positive results from 12 wk of age until the end of the study. This group-specific ELISA is therefore a sensitive and practical way to monitor FAdV antibodies in commercial flocks. None of the 1-d-old chicks tested were positive by PCR, nor was FAdV isolated from the same tissues, indicating an absence of transmission of infectious virus to the progeny. The lack of virus production and transmission could be due to the presence of high antibody titers in the layers.

Study of vertical transmission of fowl adenoviruses.

The vertical transmission of fowl adenoviruses (FAdVs) was studied by polymerase chain reaction (PCR) and virus isolation. Liver, spleen, kidney, and bursa of Fabricius were collected from 60 chicks 1 d old representing progenies hatched to 6 broiler breeder flocks in 6 geographically different premises in Ontario, Canada. The presence of FAdV DNA sequences was detected by PCR with the use of primers specific for the conserved pVI gene of FAdV-9 in 58 (24%) of the 240 samples tested. All samples from 1 flock were negative for FAdV sequences, and only a few samples were positive in 3 flocks, whereas 32% and 72% of the samples from the other 2 flocks were positive. Testing of 1 sample with primers designed to amplify the L1 region of the hexon protein gene and amino acid sequence analysis of the PCR product indicated that the sequences were similar to serotype-8a FAdV sequences. No fowl adenoviruses were isolated in chicken hepatoma cells from any of the 30 samples inoculated. These findings imply that vertical transmission and establishment of latent infection with FAdVs can occur in chickens.