Co-quantification of crAssphage increases confidence in wastewater-based epidemiology for SARS-CoV-2 in low prevalence areas.

Wastewater surveillance of SARS-CoV-2 RNA is increasingly being incorporated into public health efforts to respond to the COVID-19 pandemic. In order to obtain the maximum benefit from these efforts, approaches to wastewater monitoring need to be rapid, sensitive, and relatable to relevant epidemiological parameters. In this study, we present an ultracentrifugation-based method for the concentration of SARS-CoV-2 wastewater RNA and use crAssphage, a bacteriophage specific to the human gut, to help account for RNA loss during transit in the wastewater system and sample processing. With these methods, we were able to detect, and sometimes quantify, SARS-CoV-2 RNA from 20 mL wastewater samples within as little as 4.5 hours. Using known concentrations of bovine coronavirus RNA and deactivated SARS-CoV-2, we estimate recovery rates of approximately 7-12% of viral RNA using our method. Results from 24 sewersheds across Upstate New York during the spring and summer of 2020 suggested that stronger signals of SARS-CoV-2 RNA from wastewater may be indicative of greater COVID-19 incidence in the represented service area approximately one week in advance. SARS-CoV-2 wastewater RNA was quantifiable in some service areas with daily positives tests of less than 1 per 10,000 people or when weekly positive test rates within a sewershed were as low as 1.7%. crAssphage DNA concentrations were significantly lower during periods of high flow in almost all areas studied. After accounting for flow rate and population served, crAssphage levels per capita were estimated to be about 1.35 × 1011 and 2.42 × 108 genome copies per day for DNA and RNA, respectively. A negative relationship between per capita crAssphage RNA and service area size was also observed likely reflecting degradation of RNA over long transit times. Our results reinforce the potential for wastewater surveillance to be used as a tool to supplement understanding of infectious disease transmission obtained by traditional testing and highlight the potential for crAssphage co-detection to improve interpretations of wastewater surveillance data.

High-throughput wastewater analysis for substance use assessment in central New York during the COVID-19 pandemic.

Wastewater entering sewer networks represents a unique source of pooled epidemiological information. In this study, we coupled online solid-phase extraction with liquid chromatography-high resolution mass spectrometry to achieve high-throughput analysis of health and lifestyle-related substances in untreated municipal wastewater during the coronavirus disease 2019 (COVID-19) pandemic. Twenty-six substances were identified and quantified in influent samples collected from six wastewater treatment plants during the COVID-19 pandemic in central New York. Over a 12 week sampling period, the mean summed consumption rate of six major substance groups (i.e., antidepressants, antiepileptics, antihistamines, antihypertensives, synthetic opioids, and central nervous system stimulants) correlated with disparities in household income, marital status, and age of the contributing populations as well as the detection frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater and the COVID-19 test positivity in the studied sewersheds. Nontarget screening revealed the covariation of piperine, a nontarget substance, with SARS-CoV-2 RNA in wastewater collected from one of the sewersheds. Overall, this proof-of-the-concept study demonstrated the utility of high-throughput wastewater analysis for assessing the population-level substance use patterns during a public health crisis such as COVID-19.

Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii).

Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle's wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.

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.

Identification of Specialists and Abundance-Occupancy Relationships among Intestinal Bacteria of Aves, Mammalia, and Actinopterygii.

The coalescence of next-generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineation of host-microbe associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macroorganisms, but it remains unclear how the specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA genes amplified from 106 fecal samples spanning Aves, Mammalia, and Actinopterygii (ray-finned fish). We investigated the interspecific abundance-occupancy relationship, where widespread taxa tend to be more abundant than narrowly distributed taxa, among operational taxonomic units (OTUs) within and among host species. In a separate analysis, we identified specialist OTUs that were highly abundant in a single host and rare in all other hosts by using a multinomial model without excluding undersampled OTUs a priori. We show that intestinal microbes in humans and other vertebrates display abundance-occupancy relationships, but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest that intestinal bacteria can be shared among diverse hosts in ways that resemble the distribution of "free-living" bacteria in the extraintestinal environment.

Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups.

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.

Development of rapid canine fecal source identification PCR-based assays.

The extent to which dogs contribute to aquatic fecal contamination is unknown despite the potential for zoonotic transfer of harmful human pathogens. We used genome fragment enrichment (GFE) to identify novel nonribosomal microbial genetic markers potentially useful for detecting dog fecal contamination with PCR-based methods in environmental samples. Of the 679 sequences obtained from GFE, we used 84 for the development of PCR assays targeting putative canine-associated genetic markers. Twelve genetic markers were shown to be prevalent among dog fecal samples and were rarely found in other animals. Three assays, DG3, DG37, and DG72, performed best in terms of specificity and sensitivity and were used for the development of SYBR Green and TaqMan quantitative PCR (qPCR) assays. qPCR analysis of 244 fecal samples collected from a wide geographic range indicated that marker concentrations were below limits of detection in noncanine hosts. As a proof-of-concept, these markers were detected in urban stormwater samples, suggesting a future application of newly developed methods for water quality monitoring.

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.

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples.

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers--is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.

Sensitive detection of sample interference in environmental qPCR.

Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR.

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.

Genetic markers for rapid PCR-based identification of gull, Canada goose, duck, and chicken fecal contamination in water.

Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well.

Differential decay of human faecal Bacteroides in marine and freshwater.

Genetic markers from Bacteroides and other faecal bacteria are being tested for inclusion in regulations to quantify aquatic faecal contamination and estimate public health risk. For the method to be used quantitatively across environments, persistence and decay of markers must be understood. We measured concentrations of contaminant molecular markers targeting Enterococcus and Bacteroides spp. in marine and freshwater microcosms spiked with human sewage and exposed to either sunlight or dark treatments. We used Bayesian statistics with a delayed Chick-Watson model to estimate kinetic parameters for target decay. DNA- and RNA-based targets decayed at approximately the same rate. Molecular markers persisted (could be detected) longer in marine water. Sunlight increased the decay rates of cultured indicators more than those of molecular markers; sunlight also limited persistence of molecular markers. Within each treatment, Bacteroides markers had similar decay profiles, but some Bacteroides markers significantly differed in decay rates. The role of extracellular DNA in persistence appeared unimportant in the microcosms. Because conditions were controlled, microcosms allowed the effects of specific environmental variables on marker persistence and decay to be measured. While marker decay profiles in more complex environments would be expected to vary from those observed here, the differences we measured suggest that water matrix is an important factor affecting quantitative source tracking and microbial risk assessment applications.

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.