First report of Angiostrongylus vasorum (French heartworm) in red foxes (Vulpes vulpes) on Prince Edward Island

Objective To identify first-stage nematode larvae (L1) recovered from a red fox scat sample and adult female worms recovered from 2 red fox lungs at necropsy, using published molecular methods to confirm a morphological diagnosis of Angiostrongylus vasorum (French heartworm).Animal Red fox (Vulpes vulpes).Procedure Nematode larvae recovered from a Baermann examination survey of wild canid scats (n = 101) conducted from January 2017 to August 2020, were identified by size and morphology and subjected to PCR and DNA sequencing of the small subunit (SSU) rRNA gene, the large subunit (LSU) rRNA gene, or the second internal transcribed spacer (ITS2). In addition, these techniques were applied to adult female worms recovered from the heart/lungs of 2 red foxes (obtained from PEI trappers and stored frozen at −20°C since December of 2018 and 2020).Results Size and morphology of L1 recovered by Baermann examination from a wild canid scat sample (presumed to be red fox) collected near Montague, PEI and adult female worms recovered at necropsy from 2 red fox carcasses were identified as A. vasorum. Molecular analysis confirmed the larvae and adult worms were A. vasorum.Conclusion These findings indicated that A. vasorum has become endemic in the red fox population on PEI.Clinical relevance Angiostrongylus vasorum infection is potentially fatal in dogs. Veterinarians and regional diagnostic laboratories in the Maritime provinces should consider the possibility of A. vasorum infection in dogs with clinical signs of cardiopulmonary and/or central nervous system disease or bleeding disorders.     

Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum

The present study was undertaken to determine if red foxes are natural intermediate and/or definitive host for Neospora caninum and to study the importance of infection of N. caninum in this species in North-eastern Spain. Faecal samples and brain tissues were obtained from 122 foxes from 21 rural areas of Catalonia. Faeces collected were examined for parasite eggs and coccidian oocysts using sucrose flotation. For PCR-based diagnosis of N. caninum in brain tissues, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. To control for PCR failure and facilitate identification of truly negative samples, the competitor pNc5C molecule was added to all negative samples in a second round of PCR reactions. Of the 122 foxes analysed, 13 (10.7%) were positive by PCR for N. caninum. Signal intensities of all positive samples were relatively weak with the exception of one sample from a 3-month male animal, that also showed the highest repeatability. No differences were observed by sex, age or area of sampling analysis. Detection of stages of N. caninum in brain from naturally infected red foxes demonstrated that red foxes are a natural intermediate host for N. caninum. Faecal samples were analysed for the presence of N. caninum oocysts, however, no oocysts compatible with N. caninum were found. A widespread latent infection of red foxes in North-eastern Spain found in the present study indicates that red foxes could have a very important role in the epidemiology of neosporosis in our area.     

Seroprevalences of Toxoplasma gondii and Neospora caninum in Swedish red foxes (Vulpes vulpes).

The prevalences of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and ELISA, respectively, in 221 red foxes (Vulpes vulpes) from different parts of Sweden. A total of 84 (38%) of the analysed sera had antibodies to T. gondii, but none of the foxes had antibodies to N. caninum. The results indicate that T. gondii infection is fairly common in Swedish red foxes and that the infection is present in most parts of the country. They also show that N. caninum is not widespread as a latent infection among red foxes in Sweden.     

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in Hungarian red foxes (Vulpes vulpes)

In the present study we have investigated the seroprevalence to the protozoan parasites Toxoplasma gondii and Neospora caninum in 337 red foxes (Vulpes vulpes) from 16 out of 19 counties in Hungary. The foxes were originally collected within a National vaccination program against rabies. Antibodies to T. gondii were detected in as many as 228 (68%) of the foxes using a commercial direct agglutination test (DAT). In an indirect iscom ELISA, five foxes (1.5%) were positive for antibodies against N. caninum. The high prevalence of foxes positive for T. gondii might be explained by the widespread occurrence of the parasite in the diet of foxes. As a contrast, latent infections of N. caninum among red foxes in Hungary are much less common.     

High seroprevalence of Neospora caninum in the red fox (Vulpes vulpes) in the Pyrenees (NE Spain)

Antibodies to Neospora caninum were determined in the red fox (Vulpes vulpes) in the Pyrenees, north-eastern Spain. Sera from 53 red foxes (29 male and 24 female) were tested using a Neospora agglutination test (NAT). Seroprevalence at dilutions of 1/40, 1/80 and 1/160 was 69.8%, 47.2% and 7.5%, respectively. Sex differences were significant only at a dilution of 1:40. The high seroprevalence observed in red fox suggests that this species is highly exposed to N. caninum in this area.     

Use of an enzyme-linked immunosorbent assay in bulk milk to estimate the prevalence of Neospora caninum on dairy farms in Prince Edward Island, Canada

This study evaluated the use of bulk milk as a diagnostic tool for estimation of herd-level Neospora caninum exposure in Atlantic Canada; it was used to estimate the prevalence of dairy farms with a within-herd N. caninum-seroprevalence > or = 15% in Prince Edward Island (PEI). The variation over time of N. caninum antibodies in bulk milk is also reported. Skimmed bulk milk and individual serum samples were analyzed for N. caninum antibodies by using an enzyme-linked immunosorbent assay (ELISA). Bulk milk samples were collected in May 2004 (n = 235), May 2005 (n = 189), and June 2005 (n = 235). The prevalence of dairy farms with a within-herd seroprevalence > or = 15% on PEI was 6.4% in May 2004. In May and June 2005, respectively, 10.1% and 10.2% of farms had a > or = 15% within-herd seroprevalence. In 11 farms that were considered positive based on bulk milk samples, blood samples were collected from all adult cows in September 2005, in conjunction with a 4th bulk milk sample on the same day. The correlation coefficient between serology and bulk milk ELISA was 0.87. The results of this study demonstrate that the prevalence of N. caninum in dairy farms can be estimated by using a bulk milk ELISA.     

Evaluation of a serological test (indirect ELISA) for the diagnosis of sarcoptic mange in red foxes (Vulpes vulpes)

Sarcoptic mange occurs in many parts of the world and is common in populations of domestic and wild canids, including red foxes (Vulpes vulpes). In recent years, an indirect antibody enzyme-linked immunosorbent assay (ELISA), with higher sensitivity and specificity than traditional diagnostic methods, has been successfully applied in the diagnosis of sarcoptic mange in dogs. The same ELISA has also demonstrated specific antibodies to Sarcoptes scabiei in experimentally infected red foxes. The aim of this study was to evaluate the indirect ELISA when used to detect antibodies to S. scabiei in field sera from Swedish red foxes. One cohort of both infected and non-infected red foxes (cohort 1; n = 88), and one cohort of apparently non-infected foxes (cohort 2; n = 67) were examined for skin lesions and presence of S. scabiei by thorough visual examination at autopsy and skin scrapings. Samples of blood-tinted body liquid from the abdomen or thorax cavity were collected and analysed by the indirect ELISA. The relative sensitivity and specificity of the ELISA at different cut-offs (OD values) were estimated by comparing the test results to the infection status as determined by examination and skin scrapings. The highest combination of relative sensitivity and specificity, calculated based on cohort 1, was 95.4 and 100.0%, respectively. These estimates were constant for cut-offs 0.150-0.225, which included the cut-off based on the mean plus three standard deviations of test results from cohort 2 (0.165). It is concluded that this test can be useful in diagnosis and epidemiological studies of S. scabiei infection in red foxes.     

Spatial analysis of Yersinia pestis and Bartonella vinsonii subsp. berkhoffii seroprevalence in California coyotes (Canis latrans)

Zoonotic transmission of sylvatic plague caused by Yersinia pestis occurs in California, USA. Human infections with various Bartonella species have been reported recently. Coyotes (Canis latrans) are ubiquitous throughout California and can become infected with both bacterial agents, making the species useful for surveillance purposes. This study examined the geographic distribution of 863 coyotes tested for Y. pestis and Bartonella vinsonii subsp. berkhoffii serologic status to gain insight into the natural history of B. vinsonii subsp. berkhoffii and to characterize the spatial distribution of the two agents.

We found 11.7% of specimens positive to Y. pestis and 35.5% positive to B. vinsonii subsp. berkhoffii. The two pathogens had distinct spatial clusters: Y. pestis was more prevalent in eastern portions of the state and B. vinsonii subsp. berkhoffii in coastal regions. Prevalence of Y. pestis increased with increasing elevation, whereas prevalence of B. vinsonii subsp. berkhoffii decreased with increasing elevation. There were differences in the proportions of positive animals on a yearly basis to both pathogens.     

Studies on the role of the red fox (Vulpes vulpes) as a potential definitive host of Neospora caninum

Neospora (N.) caninum is a protozoan parasite which is regarded as a major cause of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum which may shed environmentally resistant stages, oocysts, in their feces. Epidemiological studies in Germany showed that the presence of dogs increased the risk of a bovine herd to be N. caninum-positive in a bulk-milk ELISA test. However, there were also N. caninum-positive herds where dogs were not kept together with cattle.This leads to the question whether canids other than dogs, e.g., foxes, might be involved in the horizontal transmission of N. caninum. Therefore, the aim of our examinations in wild animals was to find out whether there are indications for a sylvatic cycle with foxes as definitive hosts and deer, roe deer and wild mice samples contained structures which resembled those of coccidian oocysts. In 13 of these 65 samples coccidian DNA was detected using a 18S rRNA gene based polymerase chain reaction (PCR).The examination of the 65 samples in a N. caninum-specific PCR revealed no positive result. Hammondia (H.) heydorni-DNA was detected in two samples. In addition, brain samples from 528 foxes, 224 wild mice, 16 deer and roe deer as well as from 1 wild boar were examined for the presence of N. caninum DNA by real time PCR. All samples tested negative by PCR. In conclusion, our study yielded no evidence indicating that the examined animals were part of a sylvatic cycle for N. caninum.     

Active use of coyotes (Canis latrans) to detect Bovine Tuberculosis in northeastern Michigan, USA

Seroconversion after early vaccination at four weeks against canine parvovirus (CPV) using a high antigen titre vaccine was evaluated in 121 puppies from three breeds of dogs housed in kennels representative of the private practitioner's environment. The trial included 52 German shepherd pups, 25 Rottweiler pups and 44 Boerboel pups. From each group 11, 4, and 18 puppies acted as control dogs, respectively. Depending on the different groups, puppies were vaccinated at 4, 6, 9 and 12 weeks. The experimental group differed from the control group in that they received the high titre vaccine at 4 weeks of age, whereas the control group was not vaccinated at 4 weeks. Blood was collected from all pups prior to vaccination to measure maternally derived colostral antibody. The results indicated that vaccination at 4 weeks of age in pups with high maternally derived antibody levels, results in seroconversion rates that may lead to a reduction in the window of susceptibility with respect to CPV infection. The implications of the findings with respect to dogs in heavily contaminated environments are discussed.     

Evaluation of 6 ONRAB Ultralite bait flavor matrices delivered to coyotes (Canis latrans): Implications for oral rabies vaccination

The spread of rabies in terrestrial wildlife throughout the United States is primarily controlled through oral rabies vaccination. Relatively low bait acceptance and seroconversion rates by some target species have prompted investigation into an alternative to the RABORAL V-RG bait currently used. In Canada, ONRAB Ultralite baits are used to vaccinate raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). Comparative studies between RABORAL V-RG and ONRAB found higher seroconversion rates among raccoons that ingested ONRAB, suggesting that it may be a suitable alternative. However, ONRAB has not been evaluated in many rabies reservoir species, including coyotes (Canis latrans). Vaccination of coyotes is a critical element in preventing reemergence of canine strain of rabies in the United States. We evaluated flavor preference of ONRAB Ultralite oral rabies vaccine baits by coyotes. Preferences among bait types differed (Friedman χ² = 13.28; df = 5; P = 0.02). Of the 6 bait flavors evaluated, cheese ranked the highest, followed by fish, chicken, sugar-vanilla, egg, and bacon flavors. Pairwise trials among the top 3 flavors (cheese, fish, and chicken) showed no difference (Friedman χ² = 3.00; df = 2; P = 0.22). Our research suggests that among the bait flavors we evaluated, cheese, fish, or chicken-flavored baits may be an appropriate flavor for delivery of ONRAB Ultralite baits to coyotes.     

Study on the prevalence of Toxoplasma gondii and Neospora caninum and molecular evidence of Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis infections in red foxes (Vulpes vulpes) in rural Ireland

Thoracic fluid (pleural fluid and clotted blood) from 206 foxes were examined for antibodies to Toxoplasma gondii and 220 thoracic fluid samples were tested for Neospora caninum antibodies using indirect immunofluorescent antibody tests (IFAT). A total of 115 (56%) and six (3%) foxes had antibodies to T. gondii and N. caninum, respectively. The brains from 148 foxes were examined for histological lesions and pathological changes suggestive of parasitic encephalitis were observed in 33 (22%). Two thirds of these foxes had antibodies to T. gondii and one fox had antibodies to both T. gondii and N. caninum. PCR assays carried out on DNA extracted from the 33 brains with histological lesions were negative for N. caninum but one of the brains was positive for T. gondii. Microsporidian DNA was also amplified from the brains of two of these foxes. Sequencing these amplicons revealed 100% homology with Encephalitozoon (Septata) intestinalis in one fox and Encephalitozoon cuniculi in the second fox. This is the first report of Encephalitozoon infections in wildlife in Ireland.     

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8 months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II.All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.     

A parasitological survey of wild red foxes (Vulpes vulpes) from the province of Guadalajara, Spain

An epizootiological survey of leishmaniosis, coccidiosis and parasitic helminths in 67 foxes (Vulpes vulpes) was conducted in Guadalajara (central Spain). Examination for parasitic protozoa revealed prevalences of 74% Leishmania (determined by molecular methods) and 2.9% coccidia oocysts (fecal flotation). Survey of parasitic helminths (fecal flotation/necropsy) demonstrated the presence of nine species, including six nematodes, two cestodes and one trematode. Nematodes were the most common parasites of foxes, followed by cestodes and trematodes. Greater levels of nematodes like Uncinaria, with a free-living stage in its life-cycle, were found in foxes in areas where moist soils were likely to exist, in contrast to areas of semiarid characteristics, where Toxascaris leonina or Trichuris vulpis were predominant. With regard to helminths of importance as human pathogens, trichinoscopy revealed the presence of a relatively high number of foxes (8.9%) infected with Trichinella spiralis. Finally, Toxocara canis infection was less frequent (4.4%) than trichinellosis.     

Spongy degeneration of white matter in the central nervous system of silver foxes (Vulpes vulpes)

A disorder of central nervous white matter in Norwegian-bred silver foxes is described from the case histories of 21 clinically affected foxes. The main presenting sign of this disorder was caudal limb ataxia, which appeared between 2 1/2 and 4 months of age and progressed over the next 4-8 weeks. Only four affected foxes were allowed to live beyond this period, but they showed moderate to marked improvement. Light microscopic examination of specimens from 16 affected foxes necropsied between 3 1/2 and 6 1/2 months of age revealed lesions that were restricted to the white matter of brain and spinal cord. The lesions were characterized by a symmetrical spongy change with vacuoles of varying sizes and included significant myelin deficiency. There was a relative preservation of axons and nerve cells and no significant inflammation or vascular reaction. An astrocytic hypertrophy was usually associated with the spongy change. Ultrastructural examination of central nervous tissue from two, perfusion-fixed, 6-month-old foxes showed intramyelin vacuoles resulting from splitting of the myelin lamellae at the intraperiod line and was interpreted as indicating myelin edema. Expanded extracellular spaces and watery astrocytic processes also contributed to the vacuolar appearance. Astrocytic processes in affected areas were hypertrophic and contained abundant filaments. Although the 16 silver foxes had severe clinical signs, their lesions had features in common with the juvenile form of Canavan's disease in children and a spongy degeneration reported in Labrador Retrievers; however, the clinical course in the foxes was not uniformly progressive.     

Potential application of serological tests on fluids from carcasses: detection of antibodies against Toxoplasma gondii and Sarcoptes scabiei in red foxes (Vulpes vulpes)

Background

Serological surveys for disease investigation of wild animal populations require obtaining blood samples for analysis, which has logistic, ethic and economic difficulties. Applying serological test to fluids collected from dead animals is an alternative. The aim of this study was to assess if antibodies could be detected in two types of fluids collected from 56 carcasses of red foxes (Vulpes vulpes): pleural fluid and lung extract.

Findings

In 22 (39%) foxes antibodies against Sarcoptes scabiei were detected in both fluid types by ELISA and Western blot. In 46 (82%) foxes, antibodies against Toxoplasma gondii were detected in pleural fluid and in 41 (73%) in lung extract applying a Toxo-screen test (DAT). Antibodies were still detectable in the same fluids kept at room temperature for 28 days, although in fewer foxes (16 and 14 foxes tested for T. gondii in lung extract and pleural fluid respectively; and 1 and 4 tested for S. scabiei in lung extract and pleural fluid respectively.

Conclusions

These results indicate the potential utility of using fluids from carcasses for antibody screening of wild animals at the population level.     

Cost-efficient vaccination of foxes (Vulpes vulpes) against rabies and the need for a new baiting strategy

In this study, ecological models, optimisation algorithms and threshold analysis were linked to develop oral-vaccination strategies against rabies in fox populations. It is important that such strategies are cost-efficient and resistant to environmental conditions which would lessen their success.The model validation shows that the ecological models used are suited to predict the proportion of tetracycline- (TC) marked foxes in the course of time. This figure indicates the proportion of foxes which had at least one contact to vaccine baits, and is based on the design of the vaccination strategy (i.e. the number and timing of vaccination campaigns and the number of baits used per square kilometre and campaign). The design of a vaccination strategy also determines the costs.It is the combination of ecological models and optimisation algorithms that helped us to design a vaccination strategy which is capable of achieving a continuous rate of >70% of TC-marked foxes within an analytical horizon of 3 years at low costs. Compared to the standard strategy (baseline comparator), the improved strategy incurs just over half of the cost while almost doubling the number of weeks during which the proportion of TC-marked foxes is >70%.In the improved strategy, June is recommended as the time for bait distribution. The standard strategy, however, avoids summer months (because high temperatures reduce the durability of the baits) which again leads to a reduction of the bait intake by the foxes. Using threshold analysis, we examined the effect of a reduced durability of the baits on the design of the improved vaccination strategy. We concluded that distribution of baits in June was optimal given that the durability of baits is above a threshold of 7 days.     

Evaluation of selenium status and its distribution in organs of free living foxes (Vulpes vulpes) from an Se deficient area

The objective of the study was to determine selenium status and its distribution in the organs of free living foxes from selenium deficient areas of north-western Poland. Samples of organs harvested from 40 foxes shot during the 2008-2009 hunting seasons served as experimental material. Selenium concentration in the organs was determined spectrofluorometrically. Selenium distribution in tissues depends largely on its dietary content. Our study indicated that concentrations of selenium in the examined organs followed the order: kidney>liver>spleen>lung>heart and kidneys were the organ with the highest retention of this element. Mean selenium concentration in fox kidneys was 0.60 +/- 0.15 microg/g wet weight. Several times less selenium on average was found in the liver (0.27 +/- 0.09 microg/g w.w.), lungs (0.17 +/- 0.06 microg/g w.w.), spleen (0.19 +/- 0.06 microg/g w.w.) and heart (0.13 +/- 0.05 microg/g w.w.). All the animals studied were deficient in selenium.