Pathogenicity of Nipah henipavirus Bangladesh in a swine host.

In 1998 an outbreak of fatal encephalitis among pig farm workers in Malaysia and Singapore led to the discovery of Nipah henipavirus (NiV), a novel paramyxovirus closely related to Hendra henipavirus with case fatality rates of nearly 40%. Following its initial emergence nearly annual outbreaks of NiV have occurred in Bangladesh with a different, NiV Bangladesh, genotype, where the role of pigs in its transmission remains unknown. The present study provides the first report on susceptibility of domestic pigs to NiV Bangladesh following experimental infection, characterizing acute and long-term phases of disease and pathogenesis. All pigs were successfully infected with NiV Bangladesh following oronasal inoculation, with viral shedding confirmed by a novel genotype-specific qRT-PCR in oral, nasal and rectal excretions and dissemination from the upper respiratory tract to the brain, lungs, and associated lymphatic tissues. Unlike previous NiV Malaysia findings in pigs, clinical signs were absent, viremia was undetectable throughout the study, and only low level neutralizing antibody titers were measured by 28/29 days post-NiV-B infection. Results obtained highlight the need for continued and enhanced NiV surveillance in pigs in endemic and at-risk regions, and raise questions regarding applicability of current serological assays to detect animals with previous NiV-B exposure.

Foot-and-Mouth Disease in Red Deer - Experimental Infection and Test Methods Performance.

The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

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.

Characterization of a low pathogenic avian influenza H5N2 virus isolated from a turkey breeder flock in Manitoba, Canada.

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.

Experimental foot-and-mouth disease virus infection in white tailed deer.

White tailed deer (Odocoileus virginianus) were inoculated with foot-and-mouth disease virus (FMDV) O UKG 11/2001 and monitored for the development of clinical signs, histopathological changes and levels of virus replication. All FMDV-infected deer developed clinical signs starting at 2 days post inoculation and characterized by an increase in body temperature, increased salivation and lesions in the mouth and on the feet. Virus spread to various tissues was determined by quantifying the amount of FMDV RNA using quantitative reverse transcriptase polymerase chain reaction. Virus or viral antigen was also detected in tissues using traditional isolation techniques, enzyme linked immunosorbent assay and immunohistochemistry. Deer-to-cattle transmission of the virus was observed in this experimental setting; however, inoculated deer were not found to become carriers of FMDV.

Pathology and viral antigen distribution following experimental infection of sheep and goats with capripoxvirus.

Current understanding of capripoxvirus pathogenesis is limited since there have been no detailed studies examining cell tropism at well-defined intervals following infection. We undertook time-course studies in sheep and goats following inoculation of sheeppox or goatpox viruses in their respective homologous hosts, and examined tissues by light microscopy. A monoclonal antibody generated to a sheeppox virus core protein was used for immunohistochemical detection of viral antigen in tissue sections. Lesions and virus antigen were observed consistently in the skin, lung and lymph nodes. Antigen was detected at 6 and 8 days post inoculation for skin and lung, respectively, within cells which appeared to be of monocyte/macrophage lineage. In sheep skin capripoxvirus immunoreactivity was detected within previously unreported large multinucleated cells. In the lung, double immunolabelling detected the simultaneous expression of capripoxvirus antigen and cytokeratin indicating the presence of virus within pneumocytes. Lung double immunolabelling also detected the expression of capripoxvirus antigen in CD68(+) cells, confirming the presence of viral antigen within macrophages. Based on early detection of infected macrophages, dissemination of virus within the host and localization to tissues likely occurred through cells of the monocyte/macrophage lineage. Histological findings revealed similarities with both monkeypox and smallpox, thus capripoxvirus infection in sheep and goats may represent useful models with which to study strategies for poxvirus-specific virus vaccine concepts and therapeutics.

Studying possible cross-protection of Canada geese preexposed to North American low pathogenicity avian influenza virus strains (H3N8, H4N6, and H5N2) against an H5N1 highly pathogenic avian influenza challenge.

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.

Pathology of highly pathogenic avian influenza virus (H5N1) infection in Canada geese (Branta canadensis): preliminary studies.

Susceptibility of Canada geese (Branta canadensis) to highly pathogenic avian influenza (HPAI) virus (H5N1) infection was studied by inoculating 10 naïve (antibody-negative) animals (5 adults and 5 juveniles) with A/chicken/Vietnam/14/05 (H5N1) virus. In the adults, 1 of 5 became infected, and 4 of 5 remained normal; in the juvenile group, 5 of 5 became infected. The pathology observed in the affected animals was similar to that reported in natural occurrences. Peripheral and parasympathetic nervous systems were examined and found infected, as well as cerebrospinal fluid-contacting neurons. In some locations with significant virus infection in cells, the expected inflammatory reaction was absent or very mild. Immunohistochemistry was used to locate influenza A virus nucleoprotein in brain, spinal cord, respiratory and digestive systems, pancreas, heart, and peripheral and parasympathetic nervous systems. Further studies are needed to explain age-related differences in susceptibility.

Quantification of lumpy skin disease virus following experimental infection in cattle.

Lumpy skin disease along with sheep pox and goatpox are the most serious poxvirus diseases of livestock, and are caused by viruses that belong to the genus Capripoxvirus within the subfamily Chordopoxvirinae, family Poxviridae. To facilitate the study of lumpy skin disease pathogenesis, we inoculated eight 4- to 6-month-old Holstein calves intravenously with lumpy skin disease virus (LSDV) and collected samples over a period of 42 days for analysis by virus isolation, real-time PCR and light microscopy. Following inoculation, cattle developed fever and skin nodules, with the extent of infection varying between animals. Skin nodules remained visible until the end of the experiment on day post-inoculation (DPI) 42. Viremia measured by real-time PCR and virus isolation was not observed in all animals but was detectable between 6 and 15 DPI. Low levels of viral shedding were observed in oral and nasal secretions between 12 and 18 DPI. Several tissues were assessed for the presence of virus at DPI 3, 6, 9, 12, 15, 18 and 42 by virus isolation and real-time PCR. Virus was consistently detected by real-time PCR and virus isolation at high levels in skin nodules indicating LSDV has a tropism for skin. In contrast, relatively few lesions were observed systemically. Viral DNA was detected by real-time PCR in skin lesions collected on DPI 42. Cattle developing anti-capripoxvirus antibodies starting at DPI 21 was detected by serum neutralization. The disease in this study varied from mild with few secondary skin nodules to generalized infection of varying severity, and was characterized by morbidity with no mortality.

Bacterial infections in pigs experimentally infected with Nipah virus.

Nipah virus (NiV; Paramyxoviridae) caused fatal encephalitis in humans during an outbreak in Malaysia in 1998/1999 after transmission from infected pigs. Our previous study demonstrated that the respiratory, lymphatic and central nervous systems are targets for virus replication in experimentally infected pigs. To continue the studies on pathogenesis of NiV in swine, six piglets were inoculated oronasally with 2.5 x 10(5) PFU per animal. Four pigs developed mild clinical signs, one exudative epidermitis, and one neurologic signs due to suppurative meningoencephalitis, and was euthanized at 11 days post-inoculation (dpi). Neutralizing antibodies reached in surviving animals titers around 1280 at 16 dpi. Nasal and oro-pharyngeal shedding of the NiV was detected between 2 and 17 dpi. Virus appeared to be cleared from the tissues of the infected animals by 23 dpi, with low amount of RNA detected in submandibular and bronchial lymph nodes of three pigs, and olfactory bulb of one animal. Despite the presence of neutralizing antibodies, virus was isolated from serum at 24 dpi, and the viral RNA was still detected in serum at 29 dpi. Our results indicate slower clearance of NiV from some of the infected pigs. Bacteria were detected in the cerebrospinal fluid of five NiV inoculated animals, with isolation of Streptococcus suis and Enterococcus faecalis. Staphylococcus hyicus was isolated from the skin lesions of the animal with exudative epidermitis. Along with the observed lymphoid depletion in the lymph nodes of all NiV-infected animals, and the demonstrated ability of NiV to infect porcine peripheral blood mononuclear cells in vitro, this finding warrants further investigation into a possible NiV-induced immunosuppression of the swine host.