Targeted genomic sequencing of avian influenza viruses in wetland sediment from wild bird habitats.

Diverse influenza A viruses (IAVs) circulate in wild birds, including highly pathogenic strains that infect poultry and humans. Consequently, surveillance of IAVs in wild birds is a cornerstone of agricultural biosecurity and pandemic preparedness. Surveillance is traditionally done by testing wild birds directly, but obtaining these specimens is labor intensive, detection rates can be low, and sampling is often biased toward certain avian species. As a result, local incursions of dangerous IAVs are rarely detected before outbreaks begin. Testing environmental specimens from wild bird habitats has been proposed as an alternative surveillance strategy. These specimens are thought to contain diverse IAVs deposited by a broad range of avian hosts, including species that are not typically sampled by surveillance programs. To enable this surveillance strategy, we developed a targeted genomic sequencing method for characterizing IAVs in these challenging environmental specimens. It combines custom hybridization probes, unique molecular index-based library construction, and purpose-built bioinformatic tools, allowing IAV genomic material to be enriched and analyzed with single-fragment resolution. We demonstrated our method on 90 sediment specimens from wetlands around Vancouver, Canada. We recovered 2,312 IAV genome fragments originating from all eight IAV genome segments. Eleven hemagglutinin subtypes and nine neuraminidase subtypes were detected, including H5, the current global surveillance priority. Our results demonstrate that targeted genomic sequencing of environmental specimens from wild bird habitats could become a valuable complement to avian influenza surveillance programs.IMPORTANCEIn this study, we developed genome sequencing tools for characterizing avian influenza viruses in sediment from wild bird habitats. These tools enable an environment-based approach to avian influenza surveillance. This could improve early detection of dangerous strains in local wild birds, allowing poultry producers to better protect their flocks and prevent human exposures to potential pandemic threats. Furthermore, we purposefully developed these methods to contend with viral genomic material that is diluted, fragmented, incomplete, and derived from multiple strains and hosts. These challenges are common to many environmental specimens, making these methods broadly applicable for genomic pathogen surveillance in diverse contexts.

Rat Hepatitis E Virus in Norway Rats, Ontario, Canada, 2018-2021.

We tested liver samples from 372 Norway rats (Rattus norvegicus) from southern Ontario, Canada, during 2018-2021 to investigate presence of hepatitis E virus infection. Overall, 21 (5.6%) rats tested positive for the virus. Sequence analysis demonstrated all infections to be rat hepatitis E virus (Rocahepevirus ratti genotype C1).

Culling of Urban Norway Rats and Carriage of Bartonella spp. Bacteria, Vancouver, British Columbia, Canada.

We investigated the effects of culling on Bartonella spp. bacteria carriage among urban rats in Canada. We found that the odds of Bartonella spp. carriage increased across city blocks except those in which culling occurred. Removing rats may have prevented an increase in Bartonella spp. prevalence, potentially lowering human health risks.

"They're always there": resident experiences of living with rats in a disadvantaged urban neighbourhood.

BACKGROUND: The presence of urban rats in the neighbourhood environment may negatively impact the physical and mental health of residents. Our study sought to describe the experiences with, perceptions of, and feelings towards rats and rat control efforts among a group of disadvantaged urban residents in Vancouver, Canada. METHODS: Semi-structured interviews were held with 20 members of the Vancouver Area Network of Drug Users (VANDU) recruited by VANDU staff. Interviews were audio recorded, transcribed, and analyzed using thematic analysis. RESULTS: Participants reported daily sightings of rats and close contact during encounters. Participants generally disliked encountering rats, raising issues of health and safety for themselves and the community due to the belief that rats carry disease. Fear of rats was common, and in some cases resulted in avoidance of rats. Effects of rats on participants were particularly pronounced for those living with rats in the home or for homeless participants who described impacts on sleep due to the sounds made by rats. Although rats were viewed as more problematic in their neighbourhood than elsewhere in Vancouver, participants believed there to be a lack of neighbourhood-level control initiatives that angered and disheartened participants. In combination with other community-level concerns (e.g., housing quality and availability), the presence of rats was viewed by some to align with a general disregard for the community and its residents. CONCLUSIONS: This study suggests that the presence of rats in urban centres may have several consequences on the physical and mental health of residents living in close contact with them. These effects may be exacerbated with continued contact with rats and when residents perceive a lack of initiative to control rats in their neighbourhood. As such, research and policies aimed at mitigating the health risks posed by rats should extend beyond disease-related risk and incorporate diverse health outcomes.

Effects of Culling on Leptospira interrogans Carriage by Rats.

We found that lethal, urban rat control is associated with a significant increase in the odds that surviving rats carry Leptospira interrogans. Our results suggest that human interventions have the potential to affect and even increase the prevalence of zoonotic pathogens within rat populations.

Urban rat races: spatial population genomics of brown rats (Rattus norvegicus) compared across multiple cities.

Urbanization often substantially influences animal movement and gene flow. However, few studies to date have examined gene flow of the same species across multiple cities. In this study, we examine brown rats (Rattus norvegicus) to test hypotheses about the repeatability of neutral evolution across four cities: Salvador, Brazil; New Orleans, USA; Vancouver, Canada; and New York City, USA. At least 150 rats were sampled from each city and genotyped for a minimum of 15 000 genome-wide single nucleotide polymorphisms. Levels of genome-wide diversity were similar across cities, but varied across neighbourhoods within cities. All four populations exhibited high spatial autocorrelation at the shortest distance classes (less than 500 m) owing to limited dispersal. Coancestry and evolutionary clustering analyses identified genetic discontinuities within each city that coincided with a resource desert in New York City, major waterways in New Orleans, and roads in Salvador and Vancouver. Such replicated studies are crucial to assessing the generality of predictions from urban evolution, and have practical applications for pest management and public health. Future studies should include a range of global cities in different biomes, incorporate multiple species, and examine the impact of specific characteristics of the built environment and human socioeconomics on gene flow.

Global population divergence and admixture of the brown rat (Rattus norvegicus).

Native to China and Mongolia, the brown rat (Rattus norvegicus) now enjoys a worldwide distribution. While black rats and the house mouse tracked the regional development of human agricultural settlements, brown rats did not appear in Europe until the 1500s, suggesting their range expansion was a response to relatively recent increases in global trade. We inferred the global phylogeography of brown rats using 32 k SNPs, and detected 13 evolutionary clusters within five expansion routes. One cluster arose following a southward expansion into Southeast Asia. Three additional clusters arose from two independent eastward expansions: one expansion from Russia to the Aleutian Archipelago, and a second to western North America. Westward expansion resulted in the colonization of Europe from which subsequent rapid colonization of Africa, the Americas and Australasia occurred, and multiple evolutionary clusters were detected. An astonishing degree of fine-grained clustering between and within sampling sites underscored the extent to which urban heterogeneity shaped genetic structure of commensal rodents. Surprisingly, few individuals were recent migrants, suggesting that recruitment into established populations is limited. Understanding the global population structure of R. norvegicus offers novel perspectives on the forces driving the spread of zoonotic disease, and aids in development of rat eradication programmes.

INFECTION OF A GOELDI'S MONKEY (CALLIMICO GOELDII) WITH A EUROPEAN STRAIN OF ECHINOCOCCUS MULTILOCULARIS IN A CANADIAN INSTITUTION.

A 12-yr-old female Goeldi's monkey (Callimico goeldii) in British Columbia, Canada was diagnosed with alveolar echinococcosis (AE) on postmortem examination. Echinococcus multilocularis has been identified in several species of nonhuman primates, most frequently Old World primates, in zoos and research facilities in Europe and Asia. The strain affecting this monkey was identified as a European haplotype, indistinguishable from E. multilocularis recently identified in several canids in British Columbia. The animal is suspected to have been exposed while living in a zoological institution in Alberta, where E. multilocularis has also been reported in urban coyotes. Alveolar echinococcosis is a zoonotic disease of increasing concern in the United States and Canada, and this disease should be considered on the differential list of any nonhuman primate exhibiting signs of abdominal pain or distension, along with diagnostic imaging consistent with cystic structures of the liver or other organs.

Carriage of Clostridium difficile by wild urban Norway rats (Rattus norvegicus) and black rats (Rattus rattus).

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 "novel" ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.

Urban rats are the 'fall-guy': Resident motivations for municipal rat complaints.

Rats are an important issue in cities globally. Despite their ubiquity, perceptions and concerns about rats vary with circumstance and the context in which a person interacts with them. Municipal rat management programs are a service to communities and therefore must be responsive to the varied concerns of their residents. Understanding why communities are concerned about rats can help inform rat management programs to meet the specific needs of their residents. The objective of this study was to identify why the residents of Vancouver, Canada care about rats and what they want done to address them. To do this, we qualitatively analyzed 6,158 resident complaints about rats made to the city's municipal government between January 2014 and May 2020. Using a qualitative descriptive coding process, we found that rats were a priority in a minority of cases. In general, people were more concerned about broader community issues, such as neighborhood disorder, of which rats were one part. Complaints tended to be made when problems were highly visible, nearby, and when the complainant wanted the city to take action to alleviate this issue, particularly when they were in and around their living spaces. The rates of complaints were highest in the most economically and socially deprived neighborhoods and lowest in the most privileged neighbourhoods. We synthesize this information with a view towards understanding how to develop objectives and actions for municipal management strategies that are grounded in community concerns.

SARS-CoV-2 Surveillance of Wild Mice and Rats in North American Cities.

From July 2020 to June 2021, 248 wild house mice (Mus musculus), deer mice (Peromyscus maniculatus), brown rats (Rattus norvegicus), and black rats (Rattus rattus) from Texas and Washington, USA, and British Columbia, Canada, were tested for SARS-CoV-2 exposure and infection. Two brown rats and 11 house mice were positive for neutralizing antibodies using a surrogate virus neutralization test, but negative or indeterminate with the Multiplexed Fluorometric ImmunoAssay COVID-Plex, which targets full-length spike and nuclear proteins. Oro-nasopharyngeal swabs and fecal samples tested negative by RT-qPCR, with an indeterminate fecal sample in one house mouse. Continued surveillance of SARS-CoV-2 in wild rodents is warranted.

Environmental and sociodemographic factors associated with zoonotic pathogen occurrence in Norway rats (Rattus norvegicus) from Windsor, Ontario.

AIMS: Rat-associated zoonotic pathogen transmission at the human-wildlife interface is a public health concern in urban environments where Norway rats (Rattus norvegicus) thrive on abundant anthropogenic resources and live in close contact with humans and other animal species. To identify potential factors influencing zoonotic pathogen occurrence in rats, we investigated associations between environmental and sociodemographic factors and Leptospira interrogans and Bartonella spp. infections in rats from Windsor, Ontario, Canada, while controlling for the potential confounding effects of animal characteristics (i.e., sexual maturity and body condition). METHODS AND RESULTS: Between November 2018 and June 2021, 252 rats were submitted by collaborating pest control professionals. Kidney and spleen samples were collected for L. interrogans and Bartonella spp. PCR and sequencing, respectively. Of the rats tested by PCR, 12.7% (32/252) were positive for L. interrogans and 16.3% (37/227) were positive for Bartonella species. Associations between infection status and environmental and sociodemographic variables of interest were assessed via mixed multivariable logistic regression models with a random intercept for social group and fixed effects to control for sexual maturity and body condition in each model. The odds of L. interrogans infection were significantly higher in rats from areas with high building density (odds ratio [OR]: 3.76; 95% CI: 1.31-10.79; p = 0.014), high human population density (OR: 3.31; 95% CI: 1.20-9.11; p = 0.021), high proportion of buildings built in 1960 or before (OR: 11.21; 95% CI: 2.06-60.89; p = 0.005), and a moderate number of reports of uncollected garbage compared to a low number of reports (OR: 4.88; 95% CI: 1.01-23.63; p = 0.049). A negative association was observed between median household income and Bartonella spp. infection in rats (OR: 0.26; 95% CI: 0.08-0.89; p = 0.031). CONCLUSIONS: Due to the complexity of the ecology of rat-associated zoonoses, consideration of environmental and sociodemographic factors is of critical importance to better understand the nuances of host-pathogen systems and inform how urban rat surveillance and intervention efforts should be distributed within cities.

A comparison of sampling and testing approaches for the surveillance of SARS-CoV-2 in farmed American mink.

Surveillance for SARS-CoV-2 in American mink (Neovison vison) is a global priority because outbreaks on mink farms have potential consequences for animal and public health. Surveillance programs often focus on screening natural mortalities; however, significant knowledge gaps remain regarding sampling and testing approaches. Using 76 mink from 3 naturally infected farms in British Columbia, Canada, we compared the performance of 2 reverse-transcription real-time PCR (RT-rtPCR) targets (the envelope [E] and RNA-dependent RNA polymerase [RdRp] genes) as well as serology. We also compared RT-rtPCR and sequencing results from nasopharyngeal, oropharyngeal, skin, and rectal swabs, as well as nasopharyngeal samples collected using swabs and interdental brushes. We found that infected mink were generally RT-rtPCR-positive on all samples; however, Ct values differed significantly among sample types (nasopharyngeal < oropharyngeal < skin < rectal). There was no difference in the results of nasopharyngeal samples collected using swabs or interdental brushes. For most mink (89.4%), qualitative (i.e., positive vs. negative) serology and RT-rtPCR results were concordant. However, mink were positive on RT-rtPCR and negative on serology and vice versa, and there was no significant correlation between Ct values on RT-rtPCR and percent inhibition on serology. Both the E and RdRp targets were detectable in all sample types, albeit with a small difference in Ct values. Although SARS-CoV-2 RNA can be detected in multiple sample types, passive surveillance programs in mink should focus on multiple target RT-rtPCR testing of nasopharyngeal samples in combination with serology.

Detection of multiple human enteropathogens in Norway rats (Rattus norvegicus) from an under-resourced neighborhood of Vancouver, British Columbia.

Urban Norway rats (Rattus norvegicus) can carry various human pathogens, and may be involved in pathogen propagation and transmission to humans. From January 31-August 14, 2021, a community outbreak of Shigella flexneri serotype 2a occurred among unhoused or poorly housed people in the Downtown Eastside neighborhood of Vancouver, British Columbia, Canada. The source could not be identified; however, patients reported contact with rats, and previous studies indicated transmission of rat-associated zoonotic pathogens among the unhoused or poorly housed residents of this neighborhood. The study objective was to determine if rats trapped in the outbreak area were carriers of Shigella spp. and other zoonotic enteric pathogens. From March 23-April 9, 2021, 22 rats were lethally trapped within the outbreak area. Colonic content was analyzed using the BioFire FilmArray Gastrointestinal (multiplex PCR) panel for human enteropathogens, which detected: Campylobacter spp. (9/22), Clostridioides difficile (3/22), Yersinia enterocolitica (5/22), Cryptosporidium spp. (8/22), Giardia duodenalis (5/22), Rotavirus A (1/22), enteroaggressive Escherichia coli (2/22), enteropathogenic E. coli (10/22), and Shigella spp. or enteroinvasive E. coli (EIEC) (3/22). An ipaH PCR assay was used for targeted detection of Shigella spp./EIEC, with five rats positive. Two samples contained insertion sites unique to S. flexneri isolated from the human outbreak. This study highlights the potential for rats to carry a broad range of human pathogens, and their possible role in pathogen maintenance and/or transmission.

No Isolate, No Problem: Using a Novel Insertion Sequence PCR to Link Rats to Human Shigellosis Cases in an Underserved Urban Community.

During an investigation into a cluster of Shigella flexneri serotype 2a cases in an underserved community, we assessed the relatedness of human and rat S. flexneri isolates utilizing a novel PCR targeting insertion sites (IS-PCR) of mobile elements in the Shigella genome characteristic of the cluster strain. Whole-genome sequences of S. flexneri (n = 50) associated with the cluster were analyzed. De novo genome assemblies were analyzed by a Geneious V10.2.6 motif search, and two unique IS were identified in all human Shigella sequences of the local cluster. Hydrolysis probe PCR assays were designed to detect these sequences consisting of forward and reverse primers to amplify across each insertion site and a hydrolysis probe spanning the insertion site. IS-PCR was performed for three Shigella PCR-positive culture-negative rat intestine specimens from this community. Both insertion sites were detected in the de novo genome assemblies of all clinical S. flexneri isolates (n = 50). Two of the three PCR-positive culture-negative rat samples were positive for both unique ISs identified in the human S. flexneri isolates, suggesting that the rat Shigella species strains were closely related to the human strains in the cluster. The cycle threshold (Ct) values were >35, indicating that the bacterial load was very low in the rat samples. Two unique IS were identified in clinical isolates from a community S. flexneri cluster. Both IS targets were identified in PCR-positive (Shigella spp.), culture-negative rat tissue and clinical isolates from humans, indicating relatedness. IMPORTANCE This article describes a novel molecular method to show relatedness between bacterial infections, which may not be able to grow in the laboratory due to treatment with antibiotics or for bacteria requiring unique conditions to grow well. Uniquely, we applied this technique to Shigella isolates from human cases associated with a local cluster in an underserved community, as well as rat samples from the same community. We believe that this novel approach can serve as a complementary method to support outbreak/cluster investigation for Shigella spp.

A comparison of assays for the detection of Cryptosporidium parvum in the feces of scouring calves.

Cryptosporidium parvum is a zoonotic, protozoan parasite that causes potentially life-threatening diarrhea in the host and can be transmitted via the fecal-oral route. C. parvum can infect cattle and may be detected in their feces using a variety of tests. We compared the level of agreement, ease of procedure, and cost among PCR, lateral flow immunoassay, fluorescent antibody, and Kinyoun acid-fast stain direct smear tests. Over the course of 9 mo, 74 calf fecal samples were submitted and tested for C. parvum using all 4 tests. A Fleiss kappa value of 0.813 was obtained, indicating an excellent level of agreement among tests. Overall, the best test based on cost and ease of procedure was the Kinyoun acid-fast stain direct smear.

A threat from both sides: Multiple introductions of genetically distinct H5 HPAI viruses into Canada via both East Asia-Australasia/Pacific and Atlantic flyways.

From 2016 to 2020, high pathogenicity avian influenza (HPAI) H5 viruses circulated in Asia, Europe, and Africa, causing waves of infections and the deaths of millions of wild and domestic birds and presenting a zoonotic risk. In late 2021, H5N1 HPAI viruses were isolated from poultry in Canada and also retrospectively from a great black-backed gull (Larus marinus), raising concerns that the spread of these viruses to North America was mediated by migratory wild bird populations. In February and April 2022, H5N1 HPAI viruses were isolated from a bald eagle (Haliaeetus leucocephalus) and broiler chickens in British Columbia, Canada. Phylogenetic analysis showed that the virus from bald eagle was genetically related to H5N1 HPAI virus isolated in Hokkaido, Japan, in January 2022. The virus identified from broiler chickens was a reassortant H5N1 HPAI virus with unique constellation genome segments containing PB2 and NP from North American lineage LPAI viruses, and the remaining gene segments were genetically related to the original Newfoundland-like H5N1 HPAI viruses detected in November and December 2021 in Canada. This is the first report of H5 HPAI viruses' introduction to North America from the Pacific and the North Atlantic-linked flyways and highlights the expanding risk of genetically distinct virus introductions from different geographical locations and the potential for local reassortment with both the American lineage LPAI viruses in wild birds and with both Asian-like and European-like H5 HPAI viruses. We also report the presence of some amino acid substitutions across each segment that might contribute to the replicative efficiency of these viruses in mammalian host, evade adaptive immunity, and pose a potential zoonotic risk.

Evaluating the utility of pest control sourced rats for zoonotic pathogen surveillance.

Norway rats (Rattus norvegicus) inhabit cities worldwide and live in close association with humans. Studies of urban rat zoonoses often rely on live-trapping, with fewer studies using rats sourced through lethal pest control interventions. Our objectives were to evaluate the utility of rats collected by pest control professionals for zoonotic pathogen surveillance and determine whether we could detect Leptospira interrogans and Streptobacillus moniliformis in pest control sourced rats. Rat carcasses were submitted from Windsor, Canada by pest control professionals between November 2018 and March 2020. Submissions were categorized by season and land use. Necropsies were performed to classify carcass quality, collect tissue samples, and record demographic data. The association between carcass quality and the ability to collect tissue samples for pathogen surveillance was assessed via an exact logistic regression model. Using PCR, a subset of kidney and spleen samples were tested for L. interrogans and S. moniliformis, respectively. Our sample of pest control sourced rats had similar sex and age distributions to those of live-trapping studies. Rats were primarily submitted from residential and industrial locations during fall, winter, and spring, which may reflect pest control service areas and peak business periods, rather than rat distribution. Of 124 submissions, 98 (79.0%) of rats showed only mild decomposition. The odds of collecting all tissue samples were reduced for fair compared to good-quality carcasses (OR: 0.029; 95% CI: 0-0.25; p = .0009) and for poor compared to fair-quality carcasses (OR: 0.048; 95% CI: 0.00085-0.53; p = .0065). Leptospira interrogans and S. moniliformis were detected in 9.1% (4/44) and 27.3% (15/55) of a subset of rats tested, respectively. Our results suggest that pest control sourced rats are suitable for surveillance for multiple zoonotic pathogens in urban environments. This method of rat collection may provide preliminary information to guide more detailed ecological studies.

Is Carriage of Leptospira interrogans by Rats Influenced by the Urban Environment or Population Density?

Leptospira interrogans is one of the most important zoonotic pathogens globally. In urban settings, Norway rats (Rattus norvegicus) are important reservoirs of L. interrogans, but it is unclear how this bacterium is transmitted among rats. Both environmental features and rat population density may determine the prevalence of this pathogen in rat populations as well as the spillover risk to people. While these factors could play an important role in transmission between rats, it is unknown whether such factors influence prevalence among rats at a fine scale. Our objective was to determine if carriage of L. interrogans by rats could be explained by variation in the environment or in rat population density. Rats were live-trapped in a single neighborhood of Vancouver, Canada during two study periods (2011-12; 2016-17) and were tested for L. interrogans. The physical environment of each city block was recorded using a comprehensive, in-person environmental survey. Using generalized linear mixed modelling, we found no evidence of an association between carriage of L. interrogans and environmental features or rat population density, suggesting that these were not the primary drivers of its distribution among rats within this neighborhood. Understanding factors that promote L. interrogans transmission can be used to inform management approaches to minimize public health risks.

Flea Presence and Abundance Are not Predictors of Bartonella tribocorum Carriage in Norway Rats (Rattus norvegicus) from an Underserved Neighborhood of Vancouver, Canada.

Urban Norway rats (Rattus norvegicus) carry pathogenic Bartonella spp. that are transmitted among rats and from rats to people through arthropod vectors, particularly fleas. There is marked temporospatial variation in Bartonella spp. carriage among Norway rats in Vancouver, Canada, and we investigated whether this variation is associated with flea presence or abundance. Bartonella triborocum was isolated from 96/370 (35%) rats and 211 (57%) rats had fleas with an average of one flea per rat. All fleas were identified as Nosopsyllus fasciatus. There was no significant relationship between B. tribocorum carriage and flea presence or abundance, suggesting that, in contrast to other rat-associated zoonoses transmitted by fleas (e.g., Yersinia pestis) flea indices may not be informative for understanding the ecology of Bartonella spp. in rats, particularly for N. fasciatus.