Disease outbreaks select for mate choice and coat color in wolves.

We know much about pathogen evolution and the emergence of new disease strains, but less about host resistance and how it is signaled to other individuals and subsequently maintained. The cline in frequency of black-coated wolves (Canis lupus) across North America is hypothesized to result from a relationship with canine distemper virus (CDV) outbreaks. We tested this hypothesis using cross-sectional data from wolf populations across North America that vary in the prevalence of CDV and the allele that makes coats black, longitudinal data from Yellowstone National Park, and modeling. We found that the frequency of CDV outbreaks generates fluctuating selection that results in heterozygote advantage that in turn affects the frequency of the black allele, optimal mating behavior, and black wolf cline across the continent.

Evaluation of the Presence of ASFV in Wolf Feces Collected from Areas in Poland with ASFV Persistence.

African swine fever (ASF), caused by a DNA virus (ASFV) belonging to genus Asfivirus of the Asfarviridae family, is one of the most threatening diseases of suids. During last few years, it has spread among populations of wild boars and pigs in countries of Eastern and Central Europe, causing huge economical losses. While local ASF occurrence is positively correlated with wild boar density, ecology of this species (social structure, movement behavior) constrains long-range disease transmission. Thus, it has been speculated that carnivores known for high daily movement and long-range dispersal ability, such as the wolf (Canis lupus), may be indirect ASFV vectors. To test this, we analyzed 62 wolf fecal samples for the presence of ASFV DNA, collected mostly in parts of Poland declared as ASF zones. This dataset included 20 samples confirmed to contain wild boar remains, 13 of which were collected near places where GPS-collared wolves fed on dead wild boars. All analyzed fecal samples were ASFV-negative. On the other hand, eight out of nine wild boar carcasses that were fed on by telemetrically studied wolves were positive. Thus, our results suggest that when wolves consume meat of ASFV-positive wild boars, the virus does not survive the passage through intestinal tract. Additionally, wolves may limit ASFV transmission by removing infectious carrion. We speculate that in areas where telemetric studies on large carnivores are performed, data from GPS collars could be used to enhance efficiency of carcass search, which is one of the main preventive measures to constrain ASF spread.

Patterns and processes of pathogen exposure in gray wolves across North America.

The presence of many pathogens varies in a predictable manner with latitude, with infections decreasing from the equator towards the poles. We investigated the geographic trends of pathogens infecting a widely distributed carnivore: the gray wolf (Canis lupus). Specifically, we investigated which variables best explain and predict geographic trends in seroprevalence across North American wolf populations and the implications of the underlying mechanisms. We compiled a large serological dataset of nearly 2000 wolves from 17 study areas, spanning 80° longitude and 50° latitude. Generalized linear mixed models were constructed to predict the probability of seropositivity of four important pathogens: canine adenovirus, herpesvirus, parvovirus, and distemper virus-and two parasites: Neospora caninum and Toxoplasma gondii. Canine adenovirus and herpesvirus were the most widely distributed pathogens, whereas N. caninum was relatively uncommon. Canine parvovirus and distemper had high annual variation, with western populations experiencing more frequent outbreaks than eastern populations. Seroprevalence of all infections increased as wolves aged, and denser wolf populations had a greater risk of exposure. Probability of exposure was positively correlated with human density, suggesting that dogs and synanthropic animals may be important pathogen reservoirs. Pathogen exposure did not appear to follow a latitudinal gradient, with the exception of N. caninum. Instead, clustered study areas were more similar: wolves from the Great Lakes region had lower odds of exposure to the viruses, but higher odds of exposure to N. caninum and T. gondii; the opposite was true for wolves from the central Rocky Mountains. Overall, mechanistic predictors were more informative of seroprevalence trends than latitude and longitude. Individual host characteristics as well as inherent features of ecosystems determined pathogen exposure risk on a large scale. This work emphasizes the importance of biogeographic wildlife surveillance, and we expound upon avenues of future research of cross-species transmission, spillover, and spatial variation in pathogen infection.

Follicle-sinus complexes in muzzle skin of domestic and wild animals as diagnostic material for detection of rabies.

We previously reported a novel diagnostic method using follicle-sinus complexes (FSCs) in the muzzle skin for postmortem diagnosis of rabies in dogs. However, whether this method works in other animal species remains unclear. Here, FSCs were collected from a wolf, a red fox, 2 bats, and a cat, and examined for the presence of viral antigen, viral mRNA, and viral particles. Viral antigen and viral mRNA were confirmed in Merkel cells (MCs) in FSCs of all species. Electron microscopy performed using only samples from wolf and cat confirmed viral particles in MCs of FSCs. These results suggested that this novel diagnostic method using FSCs might be useful for detection of rabies not only in domestic but also wild animals.

First report of a pseudorabies-virus-infected wolf (Canis lupus) in China.

We provide the first report of a wolf infected with pseudorabies virus (PRV) in China. We observed the clinical symptoms and also dissected tissue samples from the wolf. The samples were ground under sterile conditions and injected subcutaneously into the necks of rabbits, which subsequently developed intense pruritus symptoms and died. The PRV strain from the wolf was isolated in porcine kidney (PK)-15 cells and was specifically recognized by pig PRV antibody-positive serum, as shown by indirect immunofluorescence. Tissues from the dead wolf and rabbits were examined by polymerase chain reaction (PCR), and the PCR-amplified partial glycoprotein E gene was sequenced, which confirmed that the wolf had died as a result of PRV infection.

Molecular detection of canine bufaviruses in wild canids.

Novel protoparvoviruses genetically related to human and non-human primate bufaviruses (BuVs) have been detected recently in respiratory and enteric specimens collected from dogs and cats. In this study, by molecular screening of archival collections of faecal samples from wolves and foxes, we detected BuVs with a rate of 17.1% (7/41) and 10.5% (9/86), respectively. Sequence analysis of a portion of the ORF2 gene region of nine positive samples showed that the viruses in these samples were closely related to BuVs (97.5-99.0% nucleotide sequence identity) found in domestic carnivores.

Dual infection with an emergent strain of canine distemper virus and canine parvovirus in an Arctic wolf under managed care.

A 6-wk-old managed male Arctic wolf with lethargy, drooling, dehydration, elevated temperature, and acute onset of seizures was submitted for autopsy. The wolf had been vaccinated with a multivalent vaccine exactly 2 wk prior to presentation. Grossly, long bones were brittle and easily fractured under pressure; the intestinal contents were mucoid and yellow. Histologically, there was widespread lymphoid and hematopoietic necrosis, failure of endochondral ossification within long bones, as well as intranuclear and intracytoplasmic inclusions in various tissues and cell types. Canine distemper virus was detected in numerous tissues by IHC and confirmed by RT-rtPCR and sequencing as an American-4 strain, an emerging strain in domestic dogs and wildlife species in the southeastern United States. The clinical and pathologic findings associated with this emergent CDV strain have not been reported previously in wolves, to our knowledge. Canine parvovirus 2 (CPV-2b) was also detected in the spleen by IHC and confirmed by conventional PCR as a wild-type strain. The exact impact of CPV-2b on the clinical course is unknown. Early vaccination in this case may have predisposed this Artic wolf to developing clinical disease.

Genetic Characterization of Canine Adenovirus Type 1 Detected by Real-Time Polymerase Chain Reaction in an Oral Sample of an Italian Wolf (Canis Lupus).

We report the detection of canine adenovirus type 1 DNA by real-time PCR technique in an oral sample of an Italian wolf (Canis lupus italicus). Genetic characterization of the virus revealed a strict relationship with viruses detected in dogs (Canis lupus familiaris), wolves, and red foxes (Vulpes vulpes), suggesting that transmission between wild animals and dogs had occurred.

Effect of Canine Parvovirus and Canine Distemper Virus on the Mexican Wolf (Canis lupus baileyi) Population in the USA.

Mexican wolves (Canis lupus baileyi), classified as probably extinct in the wild in Mexico and endangered in the US, were reintroduced into Arizona in 1998. We combined annual serologic testing results from samples collected between 2003 and 2016 from 108 wolves and known survival data from 118 wolves born in the recovery area from 2003 to 2014 to evaluate whether exposure to canine distemper virus (CDV) or canine parvovirus (CPV) was associated with a greater risk of mortality before 2 yr of age. We used mixed-effects logistic regression to estimate the effect of CDV and CPV on the probability of mortality. Annual seroprevalence rates for CDV and CPV ranged from 0% to 62% and from 33% to 100%, respectively (median, 14.2% and 90.3%, respectively). The covariate, age at testing, had a negative effect on mortality, indicating that younger animals had lower survival, whereas sex had little effect on mortality. The best-supported model excluded any effect of CPV or CDV on death before 2 yr old at both the pack and individual level. Although our analysis did not detect an effect of these viruses on mortality before 2 yr old, CDV was later identified as the cause of mortality in two individuals in 2017. Additional information is needed to assess the impact of these diseases on Mexican wolves.

Identification of Pantropic Canine Coronavirus in a Wolf ( Canis lupus italicus) in Italy.

We report a case in an Italian wolf ( Canis lupus italicus) of pantropic canine coronavirus infection, which has previously been detected only in dogs. The wolf was coinfected by canine parvovirus type 2b and canine adenovirus type 2, which highlighted the crucial role of epidemiologic surveys in European wild carnivores.

First molecular identification of kobuviruses in wolves (Canis lupus) in Italy.

Canine kobuviruses (CaKoVs) were first identified in diarrhoeic and asymptomatic dogs in 2011 in the USA. Subsequent studies have demonstrated a worldwide distribution of these viruses, but it is not clear if CaKoVs play a role as enteric pathogens of dogs. More recently, CaKoV RNA has been detected in wild carnivores, including red fox, golden jackal, side-striped jackal and spotted hyena. In this study, we addressed the hypothesis that wolves are susceptible to CaKoV infections. A total of 185 wolf stool samples were collected from necropsied animals and from transects in the Liguria, Piemonte and Valle D'Aosta regions of Italy, and CaKoV RNA was identified in two of these specimens. Both samples were obtained from necropsied wolves, with a prevalence rate of 4.9% (2/41). Sequence analysis of the full-length VP1 region showed that these strains displayed the highest nucleotide (nt) sequence identity (86.3-98.5%) to canine strains identified in the UK and Africa, and to kobuviruses that were previously detected in other African wild carnivores. This suggests that genetically related CaKoV strains circulate in domestic and wild carnivores, with interspecies transmission being not uncommon among carnivores of different ecosystems.

Potential role of wolf (Canis lupus) as passive carrier of European brown hare syndrome virus (EBHSV).

European brown hare syndrome virus (EBHSV) was detected in a faecal swab collected from a wolf carcass in Northern Italy. The full-length genome of the EBHSV WOLF/17/2016/ITA strain was determined. In the VP60 capsid gene, the wolf strain displayed the highest genetic identity (99.2-99.1% nucleotide and 99.6-99.7% amino acid) with two EBHSV strains recently found in the intestinal content of a red fox and in the spleen and liver of a hare in Northern Italy. This finding poses interrogatives on the potential role of carnivores as EBHSV passive carriers, favoring the introduction and spread of the virus among different hare populations.

Rabies and Distemper Outbreaks in Smallest Ethiopian Wolf Population.

Widespread deaths recently devastated the smallest known population of Ethiopian wolves. Of 7 carcasses found, all 3 tested were positive for rabies. Two wolves were subsequently vaccinated for rabies; 1 of these later died from canine distemper. Only 2 of a known population of 13 wolves survived.

GENETIC CHARACTERIZATION OF CANINE PARVOVIRUS IN SYMPATRIC FREE-RANGING WILD CARNIVORES IN PORTUGAL.

Since its emergence in the 1970s, canine parvovirus (CPV) has been reported in domestic and nondomestic carnivores worldwide with severe implications on their health and survival. Here, we aim to better understand CPV circulation in multihost-pathogens systems by characterizing CPV DNA or viruses in 227 free-ranging wild carnivores of 12 species from Portugal. Collected samples during 1995-2011 were analyzed by PCR and sequence analysis. The canine parvovirus DNA was detected in 4 (2%) animals of two species, namely in wolves (Canis lupus; 3/63, 5%, 95% confidence interval=1.6-3.15) and in a stone marten (Martes foina; 1/36, 3%, 95% confidence interval=0.5-14.2). Viruses in two wolves had VP2 residue 426 as aspartic acid (so-called CPV-2b) and the third had VP2 residue 426 as asparagine (CPV-2a), while the virus in the stone marten uniquely had VP2 residue 426 as glutamic acid (CPV-2c). The comparative analysis of the full-length VP2 gene of our isolates showed other nonsynonymous mutations. The phylogenetic analysis demonstrated that the sequences from wolves clustered together, showing a close relationship with European domestic dogs (Canis lupus familiaris) and wolf strains while the viral sequence from the stone marten grouped with other viruses contained the glutamic acid VP2 426 along with raccoon (Procyon lotor), bobcat (Lynx rufus), and domestic dog strains. This study confirmed that wild carnivores in Portugal are infected by CPV variants, strongly suggesting viral transmission between the wild and domestic populations and suggesting a need for a better understanding of the epidemiology of the disease and its management in wild populations.

A SEROSURVEY OF DISEASES OF FREE-RANGING GRAY WOLVES (CANIS LUPUS) IN MINNESOTA, USA.

We tested serum samples from 387 free-ranging wolves ( Canis lupus ) from 2007 to 2013 for exposure to eight canid pathogens to establish baseline data on disease prevalence and spatial distribution in Minnesota's wolf population. We found high exposure to canine adenoviruses 1 and 2 (88% adults, 45% pups), canine parvovirus (82% adults, 24% pups), and Lyme disease (76% adults, 39% pups). Sixty-six percent of adults and 36% of pups exhibited exposure to the protozoan parasite Neospora caninum . Exposure to arboviruses was confirmed, including West Nile virus (37% adults, 18% pups) and eastern equine encephalitis (3% adults). Exposure rates were lower for canine distemper (19% adults, 5% pups) and heartworm (7% adults, 3% pups). Significant spatial trends were observed in wolves exposed to canine parvovirus and Lyme disease. Serologic data do not confirm clinical disease, but better understanding of disease ecology of wolves can provide valuable insight into wildlife population dynamics and improve management of these species.

Lethal distemper in badgers (Meles meles) following epidemic in dogs and wolves.

Canine distemper virus (CDV) represents an important conservation threat to many wild carnivores. A large distemper epidemic sustained by an Arctic-lineage strain occurred in Italy in 2013, mainly in the Abruzzi region, causing overt disease in domestic and shepherd dogs, Apennine wolves (Canis lupus) and other wild carnivores. Two badgers were collected by the end of September 2015 in a rural area of the Abruzzi region and were demonstrated to be CDV-positive by real time RT-PCR and IHC in several tissues. The genome of CDV isolates from badgers showed Y549H substitution in the mature H protein. By employing all publicly available Arctic-lineage H protein encoding gene sequences, six amino acid changes in recent Italian strains with respect to Italian strains of dogs from 2000 to 2008, were observed. A CDV strain belonging to the European-wildlife lineage was also identified in a fox found dead in the same region in 2016, proving co-circulation of an additional CDV lineage.

Reassortment among picobirnaviruses found in wolves.

We conducted a viral metagenomics study in diarrheic free-ranging wolves in Portugal, revealing for the first time the presence of reassortant picobirnaviruses. These viruses shared identical capsid segments together with diverse RNA-dependent RNA polymerase segments. Even though causality between these picobirnaviruses and diarrhea could not be established, the study nonetheless confirms for the first time that wolves are a potential reservoir for picobirnaviruses, which might play a role as enteric pathogens.

PREVALENCE OF ANTIBODIES FOR SELECTED CANINE PATHOGENS AMONG WOLVES (CANIS LUPUS) FROM THE ALASKA PENINSULA, USA.

We collected blood samples from wolves ( Canis lupus ) on the Alaska Peninsula, southwest Alaska, US, 2006-11 and tested sera for antibodies to canine adenovirus (CAV), canine coronavirus (CCV), canine distemper virus (CDV), canine herpesvirus (CHV), canine parainfluenza (CPI), canine parvovirus (CPV), Neospora caninum , and Toxoplasma gondii . Detected antibody prevalence was 90% for CAV, 28% for CCV, 12% for CDV, 93% for CHV, 0% for CPI, 20% for CPV, 0% for N. caninum, and 86% for T. gondii . Prevalence of CCV antibodies suggested a seasonal pattern with higher prevalence during spring (43%) than in fall (11%). Prevalence of CCV antibodies also declined during the 6-yr study with high prevalence during spring 2006-08 (80%, n=24) and low prevalence during spring 2009-11 (4%, n=24). Prevalence of N. caninum and T. gondii antibodies were highly variable in the study area during 2006-11. Results suggested that some pathogens might be enzootic on the Alaska Peninsula (e.g., CAV and CHV) while others may be epizootic (e.g., CCV, N. caninum , T. gondii ).

Patterns of Exposure of Iberian Wolves (Canis lupus) to Canine Viruses in Human-Dominated Landscapes.

Wildlife inhabiting human-dominated landscapes is at risk of pathogen spill-over from domestic species. With the aim of gaining knowledge in the dynamics of viral infections in Iberian wolves (Canis lupus) living in anthropized landscapes of northern Spain, we analysed between 2010 and 2013 the samples of 54 wolves by serology and polymerase chain reaction (PCR) for exposure to four pathogenic canine viruses: canine distemper virus (CDV), canine parvovirus-2 (CPV), canine adenovirus 1 and 2 (CAV-1 and CAV-2) and canine herpesvirus. Overall, 76% of the studied wolves presented evidence of exposure to CPV (96% by HI, 66% by PCR) and 75% to CAV (75% by virus neutralization (VN), 76% by PCR, of which 70% CAV-1 and 6% CAV-2). This represents the first detection of CAV-2 infection in a wild carnivore. CPV/CAV-1 co-infection occurred in 51% of the wolves. The probability of wolf exposure to CPV was positively and significantly correlated with farm density in a buffer zone around the place where the wolf was found, indicating that rural dogs might be the origin of CPV infecting wolves. CPV and CAV-1 appear to be enzootic in the Iberian wolf population, which is supported by the absence of seasonal and inter-annual variations in the proportion of positive samples detected. However, while CPV may depend on periodical introductions by dogs, CAV-1 may be maintained within the wolf population. All wolves were negative for exposure to CDV (by VN and PCR) and CHV (by PCR). The absence of acquired immunity against CDV in this population may predispose it to an elevated rate of mortality in the event of a distemper spill-over via dogs.

Presence of anti-Toxoplasma gondii, -Neospora caninum, - Leishmania spp. and -Ehrlichia canis antibodies in free-ranging maned wolves (Chrysocyon brachyurus) in the northeastern region of the state of São Paulo, Brazil / Presença de anticorpos anti-Toxoplasma gondii, -Neospora caninum, -Leishmania spp. e - Ehrlichia canis em lobos-guará (Chrysocyon brachyurus) de vida livre na Região Nordeste do Estado de São Paulo, Brasil

The maned wolf (Chrysocyon brachyurus), an inhabitant of the Brazilian savanna, is considered the largest canid of South America and is classified as a near-threatened species by the International Union for Conservation of Nature (IUNC). The purpose of this study was to investigate the presence of antibodies against Toxoplasma gondii, -Neospora caninum, - Leishmania spp., and -Ehrlichia canis in free-ranging maned wolves in the northeastern region of the state of São Paulo, Brazil. Of the 17 serum samples tested by the indirect fluorescent antibody test (IFAT), 88.2% (15/17), 17.6% (3/17) and 52.9% (9/17) showed anti-T. gondii, -Leishmania spp., and -E. canis antibodies, respectively. None of the studied maned wolves tested positive for N. caninum antibodies. Our results indicated the exposure of free-ranging maned wolves to the agents in question. The presence of industrial complexes, extensive agriculture and habitat fragmentation in the northeastern region of the state of São Paulo puts these wild animals in proximity to urban areas, possibly contributing to the transmission of diseases between wild and domestic animals and human beings.(AU)

O lobo-guará (Chrysocyon brachyurus) habita o ecossistema de Cerrado e é considerado o maior canídeo da América do Sul e uma espécie ameaçada de extinção pela "International Union for Conservation of Nature" (IUNC). O objetivo desse estudo foi investigar a presença de anticorpos anti-Toxoplasma gondii, -Neospora caninum, -Leishmania spp. e -Ehrlichia canis em lobosguará da região nordeste do estado de São Paulo, Brasil. Das 17 amostras de soro testadas por meio da reação de imunofluorescência indireta (RIFI), 88,2% (15/17), 17,6% (3/17) e 52,9% (9/17) apresentaram anticorpos anti-T. gondii, - Leishmania spp. e -E. canis, respectivamente. Todos os animais testados foram soronegativos para N. caninum. Esses resultados indicam a exposição dos lobos-guará dessa região aos agentes pesquisados. A presença de um complexo industrial, agricultura extensiva e fragmentação de habitat na região nordeste do estado de São Paulo, favorece a proximidade desses animais silvestres a ambientes urbanos o que pode contribuir para a transmissão de doenças entre os animais silvestres, domésticos e o homem.(AU)