Newcastle disease (ND) still remains one of the most important diseases affecting domestic poultry in Colombia. Here, for the first time, we report on the molecular characterization of 12 virulent and 12 avirulent or lentogenic avian paramyxovirus type 1 (APMV-1) strains that were isolated from commercial, backyard, and game poultry in Colombia from 2008 to 2010. The 12 virulent isolates had a fusion (F) protein cleavage site with basic amino acids at positions 113, 115, and 116 and a phenylalanine at position 117 (112RRQKR*F117), characteristic of virulent strains. The remaining 12 isolates had the F protein cleavage sites 112GKQGR*L117 or 112GRQGR*L117 typical of avirulent or lentogenic APMV-1 strains. Phylogenetic analysis of full-length F genes of all isolates was performed, and based on the recently proposed criteria for classification of APMV-1 strains, the 24 Colombian isolates were found to belong to class II viruses and clustered into four different genotypes. Ten virulent isolates clustered with genotype VII (sub-genotype VIId), seven lentogenic strains within genotype II, five lentogenic strains with genotype I (sub-genotype Ia), and two virulent isolates within genotype XII. Our data provide essential information on the genetic diversity of AMPV-1 isolates circulating in Colombia.

In November 2010, an outbreak of avian influenza (AI) due to the H5N2 subtype virus occurred in a turkey breeder farm in northern Manitoba, Canada. The only clinical signs observed were depression, decrease in food consumption, and loss of egg production. The hemagglutinin (HA) cleavage (HA(0)) site of the isolated H5N2 virus was PQRETR/GLF, consistent with low pathogenic AI viruses. The intravenous pathogenicity index of this virus was zero. Whole-genome sequencing of two isolates that originated from two different barns was performed, and both isolates had 100% identical protein sequence in PB2, HA, NP, M1, M2, NS1, and NS2. The remaining gene segments (PB1, PA, and NA) had a single amino-acid difference when compared with each other. The nucleotide and protein sequences of eight gene segments from both isolates showed 99 or greater identity with other AI viruses that have been circulating in free-living aquatic birds in Canada and the United States within the last 10 yr. Phylogenetic analysis of the HA and neuraminidase (NA) gene segments showed that these viruses are closely related to other H5 strains that have been isolated from Manitoba and other parts of Canada. Serologic testing of archived serum samples collected from these turkeys a week before the outbreak showed no evidence of AI infection. In addition, other farms that were located within 3 km radius from the infected farm and farms that had epidemiologic connection with the farm also tested negative for the presence of H5N2 AI virus or antibody. This indicates that the virus might have been introduced to the farm from wild aquatic birds only a short time before detection. Results of this study highlight the importance of early detection and the significance of ongoing Canada-wide surveillance of AI in domestic poultry as well as in wild aquatic birds/ducks.

Highly pathogenic avian influenza (HPAI) H5N1 virus infections have caused unprecedented morbidity and mortality in different species of domestic and wild birds in Asia, Europe, and Africa. In our previous study, we demonstrated the susceptibility and potential epidemiologic importance of H5N1 HPAI virus infections in Canada geese. In this study, we investigated the potential of preexposure with North American lineage H3N8, H4N6, and H5N2 low pathogenicity avian influenza (LPAI) viruses to cross-protect Canada geese against a lethal H5N1 HPAI virus challenge. Based on our results, birds that were primed and boosted with an H5N2 LPAI virus survived a lethal H5N1 challenge. In contrast, only two of five birds from the H3N8 group and none of the birds preexposed to H4N6 survived a lethal H5N1 challenge. In vitro cell proliferation assays demonstrated that peripheral blood mononuclear cells collected from each group were no better stimulated by homologous vs. heterologous antigens.

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.

In September 2007, an H7N3 highly pathogenic avian influenza outbreak (HPAI) occurred on a multiple-age broiler breeder operation near Regina Beach, Saskatchewan, Canada. Mortality was initially observed in a barn that housed 24-wk-old roosters, with later involvement of 32-wk-old breeders. All birds on the affected premises were destroyed, and surveillance of surrounding farms demonstrated no further spread. The use of water from a dugout pond during periods of high demand, and the proximity of the farm to Last Mountain Lake, the northern end of which is a bird sanctuary, implicated wild aquatic birds as a possible source of the virus. Of particular note, the H7-specific real-time reverse transcription polymerase chain reaction assay that was in use at the time did not detect the virus associated with this outbreak. A Canadian national influenza A virus survey of wild aquatic birds detected no H7 subtype viruses in 2005 and 2006; however, H7 subtype viruses were detected in the fall of 2007. Phylogenetic analysis of a number of these H7 isolates demonstrated an evolutionary relationship with each other, as well as with the H7N3 HPAI virus that was isolated from the Saskatchewan broiler breeder farm.

In February 2004 a highly pathogenic avian influenza (HPAI) outbreak erupted in British Columbia. Investigations indicated that the responsible HPAI H7N3 virus emerged suddenly from a low pathogenic precursor. Analysis of the haemagglutinin (HA) genes of the low and high pathogenic viruses isolated from the index farm revealed the only difference to be a 21 nt insert at the HA cleavage site of the highly pathogenic avian influenza virus. It was deduced that this insert most probably arose as a result of non-homologous recombination between the HA and matrix genes of the same virus. Over the course of the outbreak, a total of 37 isolates with, and 3 isolates without inserts were characterized. The events described here appear very similar to those which occurred in Chile in 2002 where the virulence shift of another H7N3 virus was attributed to non-homologous recombination between the HA and nucleoprotein genes.

Pigs were experimentally inoculated with Glentorf, Lelystad/97, and Alfort/187: representative low, moderate, and high virulent strains of classical swine fever virus (CSFV). Animals were tested for viremia using virus isolation and reverse transcriptase–polymerase chain reaction (RT-PCR) assays run under routine diagnostic conditions. The virus was detected in the peripheral blood by virus isolation and RT-PCR assays of all Glentorf- and Lelystad/97-infected pigs beginning at 3 days postinoculation (dpi) and in all Alfort/187-infected pigs beginning at 2 dpi. Viremia, as determined by virus isolation, remained detectable in Lelystad/97- and Alfort/187-infected pigs until the last animal within each cohort was euthanized on days 12 and 7 postinoculation, respectively. In contrast, the virus could be isolated from the blood of all Glentorf-infected pigs between 3 and 7 dpi but not from 10 to 21 dpi when the experiment was concluded. Viremia, as determined by RT-PCR, became apparent in Alfort/187-infected pigs at 2 dpi and in Glentorf- and Lelystad/97-infected pigs at 3 dpi. All pigs, regardless of the CSFV strain used, remained RT-PCR positive until they were euthanized. Tonsils were harvested from all the pigs and frozen sections tested for the presence of the CSFV antigen using polyclonal pestivirus and monoclonal CSFV horseradish peroxidase (HRPO) conjugates. Immunostaining reactions were positive for all the Alfort/187- and Lelystad/97-infected pigs. By contrast, tonsils from the Glentorf-infected pigs gave negative to equivocal results. These data suggest that an RT-PCR assay performed on blood may be the best test when dealing with pigs infected with low virulent strains of CSFV.

The National Centre for Foreign Animal Disease (NCFAD) in Winnipeg, Manitoba, is the Canadian Food Inspection Agency's (CFIA) newest high biocontainment laboratory. One of the functions of the NCFAD is to serve as a national reference laboratory for avian influenza. Between 1997 and 2001, 15 avian influenza virus isolates were characterized. These isolates originated from domestic poultry, imported caged birds held in quarantine, and wild birds. Diagnostic specimens were submitted to the NCFAD by CFIA field veterinarians, provincial veterinary diagnostic laboratories, and veterinary colleges. Characterization of isolates included the determination of H and N subtypes: H1, H6, H7, and H10 subtypes were isolated from domestic poultry; H3, H4, and three H13 viruses were isolated from water fowl, and six H3 viruses were isolated from caged birds being held in import quarantine. Selected isolates were characterized with respect to their pathogenic potential by intravenous inoculation of 4-to-6-wk-old chickens. A molecular-based protocol was used to assess the pathogenicity of one H7 isolate. During this period, work was also carried out toward validating our molecular pathotyping protocol for avian influenza viruses with H5 and H7 hemagglutinin subtypes.