Avian metapneumovirus subtype C in Wild Waterfowl in Ontario, Canada.

Avian metapneumovirus (aMPV) is an emerging poultry pathogen that has a significant economic impact on poultry production worldwide. The geographic range of the virus continues to expand, and wild birds have been implicated as reservoirs of aMPV that have the potential to spread the virus over long distances. Our objective was to determine the apparent prevalence of aMPV subtype C in wild waterfowl in Ontario, Canada. Wild waterfowl were captured in August and September, 2016 as part of routine migratory waterfowl population monitoring by the Ontario Ministry of Natural Resources and Forestry. Oropharyngeal and cloacal swabs were collected from each bird and placed together for aMPV testing using real-time RT-PCR. A total of 374 live wild birds from 23 lakes were sampled and tested for aMPV. Among all ducks tested, 84 (22%) were positive for aMPV. The proportion of samples that tested positive ranged from 0% in ring-necked ducks (Aythya collaris) and green-winged teal (Anas carolinensis) to 44% (8 of 18) in American black ducks (A. rubripes). Waterfowl positive for aMPV were found at 14 of 23 lakes in the study area and the percent positive at these 14 lakes ranged between 5% and 84%. Although subtype C aMPV has been detected in a variety of wild birds in North America, this is the first report of aMPV in wild ducks in Ontario, Canada. The high apparent prevalence, particularly in mallards and American black ducks (37 and 44%, respectively), suggests that these species may be important reservoirs of aMPV. Given the potential impact of aMPV on domestic poultry and the potential role of wild birds as reservoirs of the virus, further investigation of the geographic distribution, risk factors associated with aMPV carriage in wild waterfowl and potential role of other birds in the epidemiology of aMPV in Canada is warranted.

Investigation of the Occurrence of Porcine Reproductive and Respiratory Virus in Swine Herds Participating in an Area Regional Control and Elimination Project in Ontario, Canada.

The main goal of this study was to investigate the occurrence of porcine reproductive and respiratory syndrome virus (PRRSV)-specific genotypes in swine sites in Ontario (Canada) using molecular, spatial and network data from a porcine reproductive and respiratory syndrome (PRRS) regional control project. For each site, location, animal movement service provider (truck companies), PRRSV status and sequencing data of the open reading frame 5 (ORF5) were obtained. Three-kilometre buffers were created to evaluate neighbourhood characteristics for each site. Social network analysis was conducted on swine sites and trucking companies to assemble the network and define network components. Three different PRRSV genotypes were used as outcomes for statistical analysis based on the region's phylogenetic tree of the ORF5. Multivariable exact logistic regression was conducted to investigate the association between being positive for a specific genotype and two main exposures of interest: (i) having at least one neighbour within three km also positive for the same genotype outside the production system and (ii) having at least one positive site for the same genotype in the same truck network component outside the production system. Results showed that the importance of area spread and truck network on PRRSV occurrence differed according to genotype. Additionally, the Ontario PRRS database appears suitable for conducting regional disease investigations. Finally, the use of relatively new tools available for network, spatial and molecular analysis could be useful in investigation, control and prevention of endemic infectious diseases in animal populations.

Reoccurrence of Suspected Human-to-Turkey Transmission of H1N1 Pandemic 2009 Virus in Turkey Breeder Flocks in Ontario and Manitoba, 2016.

Soon after the emergence of 2009 pandemic H1N1, the first outbreaks in breeder turkey operations were reported that implicated human-to-turkey transmission. In the spring of 2016, the reoccurrence of 2009 pandemic H1N1 lineage viruses infecting breeder turkey flocks in Ontario and Manitoba, Canada, also implicated human-to-turkey transmission. In addition to raising concerns over biosecurity and vaccine failures, these cases once again raise the issue of whether turkeys have the potential to act as a bridge species to generate novel influenza A virus reassortants with public health implications.

Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada.

In January 2014, approximately 9 months following the initial detection of porcine epidemic diarrhea (PED) in the USA, the first case of PED was confirmed in a swine herd in south-western Ontario. A follow-up epidemiological investigation carried out on the initial and 10 subsequent Ontario PED cases pointed to feed as a common risk factor. As a result, several lots of feed and spray-dried porcine plasma (SDPP) used as a feed supplement were tested for the presence of PEDV genome by real-time RT-PCR assay. Several of these tested positive, supporting the notion that contaminated feed may have been responsible for the introduction of PEDV into Canada. These findings led us to conduct a bioassay experiment in which three PEDV-positive SDPP samples (from a single lot) and two PEDV-positive feed samples supplemented with this SDPP were used to orally inoculate 3-week-old piglets. Although the feed-inoculated piglets did not show any significant excretion of PEDV, the SDPP-inoculated piglets shed PEDV at a relatively high level for ≥9 days. Despite the fact that the tested PEDV genome positive feed did not result in obvious piglet infection in our bioassay experiment, contaminated feed cannot be ruled out as a likely source of this introduction in the field where many other variables may play a contributing role.

Survey of influenza A viruses circulating in wild birds in Canada 2005 to 2007.

A multi-agency, Canada-wide survey of influenza A viruses circulating in wild birds, coordinated by the Canadian Cooperative Wildlife Health Centre, was begun in the summer of 2005. Cloacal swab specimens collected from young-of-year ducks were screened for the presence of influenza A nucleic acids by quantitative, real-time reverse transcription-polymerase chain reaction (RRT-PCR). Specimens that produced positive results underwent further testing for H5 and H7 gene sequences and virus isolation. In addition to live bird sampling, dead bird surveillance based on RRT-PCR was also carried out in 2006 and 2007. The prevalence of influenza A viruses varied depending on species, region of the country, and the year of sampling, but generally ranged from 20% to 50%. All HA subtypes, with the exception of H14 and H15, and all NA subtypes were identified. The three most common HA subtypes were H3, H4, and H5, while N2, N6, and N8 were the three most common NA subtypes. H4N6, H3N2, and H3N8 were the three most common HA-NA combinations. The prevalence of H5 and H7 subtype viruses appears to have a cyclical nature.

Infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in Canada.

Turkey coronavirus (TCoV) is an important viral pathogen causing diarrhoea of young turkey poults that is associated with sizeable economic losses for the turkey industry. Using a field isolate that was found to be free from turkey astrovirus and avian reovirus we were able to reproduce the clinical disease associated with TCoV. Clinical signs and weight gain of poults during experimental infections were compared with age-matched, uninfected controls. Poults infected at 2 days of age had 100% morbidity and 10% mortality, and birds infected at 28 days of age showed 75% morbidity and no mortality. Diarrhoea was consistently seen in infected poults at 2 to 3 days post infection (d.p.i.) with a duration of about 3 to 5 days. Mean body weights of birds infected at 2 or 28 days of age were significantly reduced compared with uninfected birds by 7 d.p.i. and remained significantly lower for the duration of the study. At 44 days of age, poults infected at 2 or 28 days of age weighed only 68.1% or 77.7%, respectively, compared with uninfected turkeys of the same age on the same diet, a mean difference in body weights of 683 or 477g, respectively. Infected birds had profound villus atrophy with some compensatory crypt hyperplasia at 5 to 7 d.p.i. Villus heights in the duodenum were significantly reduced at 7 d.p.i. We were able to reproduce enteric disease using only a pathogenic field isolate (MG10) of TCoV that negatively affected growth performance and intestinal morphology of young turkey poults.

Virus shedding and serum antibody responses during experimental turkey coronavirus infections in young turkey poults.

The course of turkey coronavirus (TCoV) infection in young turkey poults was examined using a field isolate (TCoV-MG10) from a diarrhoeal disease outbreak on a commercial turkey farm in Ontario, Canada. Two-day-old and 28-day-old poults were inoculated orally with TCoV-MG10 to examine the effect of age on viral shedding and serum antibody responses to the virus. The presence of coronavirus particles measuring 105.8+/-21.8 nm in the cloacal contents was confirmed using transmission electron microscopy. The pattern of cloacal TCoV shedding was examined by reverse-transcription polymerase chain reaction amplification of the nucleocapsid gene fragment. TCoV serum antibody responses were assessed with two recently developed TCoV enzyme-linked immunosorbent assays that used TCoV nucleocapsid and S1 polypeptides as coating antigens. Poults were found equally susceptible to TCoV infection at 2 days of age and at 4 weeks of age, and turkeys of either age shed virus in their faeces starting as early as 1 day post-inoculation and up to 17 days post-inoculation. Poults infected at 2 days of age were immunologically protected against subsequent challenge at 20 days post-inoculation. The protection was associated with measurable serum antibody responses to both the nucleocapsid and S1 structural proteins of TCoV that were detectable as early as 1 week post-infection.

Complete genomic sequence of turkey coronavirus.

Turkey coronavirus (TCoV), one of the least characterized of all known coronaviruses, was isolated from an outbreak of acute enteritis in young turkeys in Ontario, Canada, and the full-length genomic sequence was determined. The full-length genome was 27,632 nucleotides plus the 3' poly(A) tail. Two open reading frames, ORFs 1a and 1b, resided in the first two thirds of the genome, and nine additional downstream ORFs were identified. A gene for hemagglutinin-esterase was absent in TCoV. The region between the membrane (M) and nucleocapsid (N) protein genes contained three potential small ORFs: ORF-X, a previously uncharacterized ORF with an associated putative TRS within the M gene (apparently shared among all group III coronaviruses), and previously described ORFs 5a and 5b. The TCoV genome is organized as follows: 5' UTR--replicase (ORFs 1a, 1b)--spike (S) protein--ORF3 (ORFs 3a, 3b)--small envelop (E or 3c) protein--membrane (M) protein--ORF5 (ORFs X, 5a, 5b)--nucleocapsid (N) protein--3' UTR--poly(A). TCoV genome structure and sequence was most similar, but distinct from, avian infectious bronchitis virus (IBV). This is the first complete genome sequence for a TCoV and confirms that TCoV belongs to group III coronaviruses.

The long repeat region is dispensable for fowl adenovirus replication in vitro.

Two regions containing tandemly repeated sequences are present in the fowl adenovirus 9 (FAdV-9) genome. The longer repeat region (TR-2) is composed of 13 contiguous 135-bp-long direct repeats, the function of which is unknown. An infectious FAdV-9 genomic clone, constructed by homologous recombination in Escherichia coli, was used for engineering of recombinant viruses. The enhanced green fluorescence protein (EGFP) coding sequence was cloned in both rightward and leftward orientations so as to replace TR-2. Replication-competent recombinant FAdVs were recovered, demonstrating that TR-2 was dispensable for FAdV-9 propagation in vitro. The expression of EGFP in infected cells was demonstrated by fluorescence microscopy, immunoprecipitation, and RT-PCR.

The complete nucleotide sequence of fowl adenovirus type 8.

The fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.

Application of the polymerase chain reaction to detect fowl adenoviruses.

The possibility of using the polymerase chain reaction (PCR) for the detection of fowl adenoviruses (FAdV) was tested. The optimal reaction parameters were evaluated and defined for purified genomic DNA of type 8 fowl adenovirus (FAdV-8), and then the same conditions were applied for nucleic acid extracted from infected cells. One hundred picograms of purified viral DNA, or 250 FAdV-8-infected cells, were detected by ethidium bromide staining of the PCR products in agarose gels. The sensitivity was increased to 10 pg purified viral DNA, or 25 infected cells, when the PCR products were hybridized with a specific labeled probe. Several field isolates of FAdV and the CELO virus (FAdV serotype 1) could be amplified by the same primers and conditions, but the size of the amplicons was smaller than that for the FAdV-8 PCR product. Other avian viruses and uninfected cell cultures tested negative.

Sequencing analysis of the region encoding the DNA polymerase of bovine adenovirus serotypes 2 and 3.

Bovine adenoviruses (BAVs) are important pathogens causing significant economic losses to the cattle industry. We have been interested in the differences among serotypes of these viruses, particularly in their pathogenicity and host range. As part of our efforts to better understand these viruses, we have determined the nucleotide sequences for serotype 3 (BAV3) at map coordinates beween 11.7 and 23.7% and for serotype 2 (BAV2) between 13.1 and 24.0%. Analyses of these sequences revealed large open reading frames (ORFs) encoded within the leftward-reading strand of the viral DNA. The coding capacity of the ORF in BAV3 is 1,167 amino acid residues and 1,138 in BAV2. A search in the GenEMBL protein sequence databank for homology to the predicted polypeptide products of these ORFs established their identity as that for the adenovirus (Ad) DNA polymerase (DNA pol). The deduced polypeptide sequences were aligned with each other and with other known Ad DNA pols to reveal regions of homology and similarity. The comparison at the amino acid sequence level not only showed that the bovine Ad DNA pols from the two serotypes are quite distinct from each other, but also revealed that Ad DNA pols contain multiple domains that are highly conserved among human, canine and bovine Ads. These conserved domains are likely important for the multiple functions attributed to Ad DNA pol, which include catalysis of its own initiation complex, elongation of nascent DNA strand, as well as correction of DNA replication errors.

Sequence analysis of the terminal protein precursor coding regions from bovine adenovirus serotypes 2 and 3.

As part of our efforts to develop bovine adenoviruses into a vector system, we sequenced the region predicted to encode the terminal protein precursor of either bovine adenoviruses type 2 and bovine adenoviruses type 3. We examined the regions containing the terminal protein precursors and showed that the bovine adenovirus early region 2B had an identical organization to all adenoviruses so far examined. The bovine adenovirus terminal protein precursors and those of other adenoviruses were also compared in a sequence alignment analysis and several highly conserved structural domains were identified. Finally, we showed how the various terminal protein precursors were related in a sequence pair similarity analysis and concluded that the terminal protein precursors of bovine adenoviruses types 2 and 3 are significantly different from each other at the protein sequence level.