Davor Ojkic

Triple Reassortant H3N2 Influenza A Viruses, Canada, 2005

Since January 2005, H3N2 influenza viruses have been isolated from pigs and turkeys throughout Canada and from a swine farmer and pigs on the same farm in Ontario. These are human/classical swine/avian reassortants similar to viruses that emerged in US pigs in 1998 but with a distinct human-lineage neuraminidase gene.

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

Summary 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.

Genotyping of Canadian isolates of fowl adenoviruses

Five hundred and seventy-three clinical submissions with fowl adenovirus (FAdV) involvement were examined to investigate the association of different types of FAdV with clinical problems related to FAdV infection. Samples were received from 2000 to 2006 and originated from seven Canadian provinces. Four hundred and eighty-seven submissions were inclusion body hepatitis (IBH) related, while 86 were not IBH related. Viruses isolated from 287 samples were further analysed by hexon gene loop 1 sequencing. Twenty-seven genotyped FAdVs were from Alberta, 20 from British Columbia, 16 from Manitoba, one from Nova Scotia, 82 from Ontario, 64 from Quebec and 77 from Saskatchewan. Two hundred and forty-six analysed FAdVs were from IBH cases, confirmed by liver histopathology, by FAdV isolation from the liver, or both. Based on hexon gene loop 1 sequencing analysis, FAdVs associated with IBH outbreaks were genetically related to FAdV02 (nine isolates, 99.4%), FAdV08a (100 isolates, 99.4% to 100%) and FAdV11 (98 isolates, 99.4% to 100%). Thirty-nine viruses were 93.7% to 94.3% identical to FAdV07 strain x11a, but the genetic and immunogenic properties of this strain require further investigation. In IBH cases, the co-infection rates for infectious bursal disease virus, infectious bronchitis virus, reoviruses and Newcastle disease virus were 3.47%, 1.04%, 6.25% and 0.69%, respectively. Forty-one genotyped FAdVs were from “non-IBH” cases. Viruses isolated from non-IBH cases consisted of 22 FAdV01, 15 FAdV11, two FAdV08a and one each of FAdV02 and FAdV04 viruses. Co-infection rates in non-IBH submissions were 50.00% for infectious bursal disease virus, 40.70% for infectious bronchitis virus, 27.91% for reoviruses and 1.16% for Newcastle disease virus.

Wild bird influenza survey, Canada, 2005.

Of 4,268 wild ducks sampled in Canada in 2005, real-time reverse transcriptase–PCR detected influenza A matrix protein (M1) gene sequence in 37% and H5 gene sequence in 5%. Mallards accounted for 61% of samples, 73% of M1-positive ducks, and 90% of H5-positive ducks. Ducks hatched in 2005 accounted for 80% of the sample.

INCLUSION BODY HEPATITIS AS A PRIMARY DISEASE IN BROILERS IN SASKATCHEWAN, CANADA

Abstract In recent years inclusion body hepatitis (IBH) has emerged as an economically important disease in Western Canada. Historically, infections with infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) have been known to suppress the immune system of broilers and make them more susceptible to a secondary disease such as IBH. Recently it has been reported that virulent adenoviruses are able to cause IBH as a primary disease in broilers without apparent involvement of IBDV and CAV. The objectives of this study were to examine the possible association of IBH with IBDV and CAV infections in Western Canada and to identify adenoviruses involved in outbreaks. Serum samples from 17 broiler-breeder flocks and their progeny were collected when broilers were hatched and then again from broilers at the time of slaughter, and these samples were tested for IBDV and CAV antibodies by enzyme-linked immunosorbent assay (ELISA). Based on the ELISA titers the antibody response to vaccination against IBDV and CAV was at an expected level in all broiler flocks. Therefore, IBH outbreaks in these flocks were not due to inadequate levels of antibodies against IBDV and CAV. Moreover, there was no correlation found between occurrences of IBH outbreaks in broilers and their IBDV or CAV titers at the time of processing. Viruses that were isolated from livers of birds suffering from IBH could be classified into four different genotypes. Their hexon gene loop 1 sequences showed high percentages of identity to FAdV-7, FAdV-8a, FAdV-8b, and FAdV-11. The results of this study could not demonstrate an association of IBH with IBDV and CAV infections, but they supported the hypothesis that IBH in broilers in Western Canada is a primary disease with no apparent immunosuppressive involvement.

New genotype of avian bornavirus in wild geese and trumpeter swans in Canada

AVIAN bornavirus (ABV), a newly discovered agent, has been identified as the causal agent of proventricular dilation disease (PDD) in psittacine birds (Honkavuori and others 2008, Kistler and others 2008). Subsequent research, including bird inoculation studies (Gancz and others 2009, Gray and others 2010) and outbreak investigations (Kistler and others 2010) have provided strong supporting evidence. PDD is a significant pathological syndrome, with high mortality affecting primarily psittacine birds, that has been reported worldwide since the late 1970s. Characteristic pathological findings of PDD …

Avian bornavirus is present in many tissues of psittacine birds with histopathologic evidence of proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a neurologic disease of psittacine birds suspected to be caused by a recently identified Avian bornavirus (ABV). In the current report, data supporting the causal association of ABV with PDD are presented. Immunohistochemistry (IHC) with rabbit polyclonal antiserum raised against ABV nucleocapsid protein was used to identify cell and organ distribution of viral antigen. The ABV antigen was most consistently detected in brain, spinal cord, adrenal gland, pancreas, and kidney. Histopathologic evaluation was correlated with ABV-specific polymerase chain reaction (PCR) and immunohistochemical tests in multiple tissues from 16 psittacine birds with and without PDD. Using histopathologic diagnosis as the gold standard, the sensitivity and specificity of IHC for ABV antigens were found to be 100% and 100%, respectively. Many more tissues were positive for ABV RNA by reverse transcription PCR than were positive for pathologic changes or viral antigens by IHC, indicating the presence of subclinical or asymptomatic infection at many sites.

Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys.

Abstract Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/InDRE/4487/2009 was determined by inoculating groups of 8–10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 × 105 and 1 × 106 TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.

Multiplex PCR tests sentinel the appearance of pandemic influenza viruses including H1N1 swine influenza.

Abstract Background Since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. Both SARS Coronavirus which quickly spread to 29 countries in February 2003 and H1N1 swine influenza that recently spread from Mexico to 30 countries in three weeks represent major pandemic threats for mankind. Diagnostic assays are required to detect novel influenza strains with pandemic potential. Objective In this report we evaluate the ability of a multiplex PCR test (xTAG™ RVP) to detect new, “non-seasonal” influenza viruses including the H1N1 swine influenza A/swine/California/04/2009. Study design Laboratory based study using retrospective and prospective specimens. Results This multiplex PCR test detected the present of non-seasonal (non-H1, non-H3) influenza in 20 of 20 patients infected with H1N1 swine flu virus. In addition to detecting the current swine flu the xTAG™ RVP test detected the H5N1 A/Vietnam/1203/2004 high pathogenicity avian influenza virus that circulated in South East Asia in 2003 as well as 17 out of 17 influenza A viruses representing 11 HA subtypes isolated from birds, swine and horses not yet seen in the human population. Conclusion Based on these results we believe that this molecular test can perform an important role as a sentinel test to detect novel non-seasonal influenza A viruses in patients presenting with influenza-like illness (ILI) and therefore act as an early warning system for the detection of future pandemic influenza threats.

Complete genomic sequence of turkey coronavirus.

Abstract 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.