The role of medium-sized mammals as reservoirs of Borrelia burgdorferi in southern New York

The ability of raccoons (Procyon lotor), striped skunks (Mephitis mephitis) and opossums (Didelphis virginiana) to serve as reservoirs of Borrelia burgdorferi, the spirochetal agent of Lyme disease, was compared with that of white-footed mice (Peromyscus leucopus). Twenty-eight (28) medium-sized mammals and 34 white-footed mice were captured in Westchester County, New York (USA) in summer 1986. Animals were caged over pans of water for 1 to 2 days to recover engorged tick larvae (Ixodes dammini) that detached from the hosts after feeding. With the exception of mice, numbers of engorged tick larvae recovered exceeded those counted during initial examinations of the hosts by 30% (opossums) to nearly 90% (raccoons). Newly-molted nymphal ticks derived from the engorged larvae were examined for the presence of spirochetes by darkfield microscopy. Percentage infection was 5% (n = 22) for ticks from skunks and 14% (n = 191) for ticks from raccoons. None of 24 nymphs from larvae that fed on opossums survived long enough for spirochete examination. By comparison, 40% (n = 72) of nymphs from larvae which fed on white-footed mice were infected. Of the individual hosts from which molted nymphs had fed as larvae, 67% of mice, 33% of skunks, and 55% of raccoons produced spirochete-positive ticks.     

Prevalence of agglutinating antibodies to Toxoplasma gondii in striped skunks (Mephitis mephitis), opossums (Didelphis virginiana), and raccoons (Procyon lotor) from Connecticut

The prevalence of agglutinating antibodies to Toxoplasma gondii was examined in striped skunks (Mephitis mephitis), opossums (Didelphis virginiana), and raccoons (Procyon lotor) from 8 cities in Connecticut. Ten (42%) of the 24 striped skunks, 2 of 7 (29%) opossums, and 12 of 12 (100%) raccoons were positive at dilutions of 1:50 or greater. These results suggest that T. gondii is prevalent in the environment, or prey items, or both, of these omnivores in Connecticut.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.     

Host association and seasonal activity of Amblyomma americanum (Acari: Ixodidae) in Missouri

From June 1993 through June 1996, 2,260 adult, 4,426 nymphal, and 2,178 larval lone star ticks Amblyomma americanum (L.) were collected in Missouri from vertebrate hosts and by dragging a cloth over vegetation. Prevalence, mean intensity, and relative abundance of each stage varied among hosts. The relative abundance of adult lone star ticks was highest on white-tailed deer, but this stage was also collected from raccoons, opossum, red fox, coyotes, and wild turkey. Nymphs were collected from gray squirrels, eastern cottontail rabbits, opossums, red fox, Carolina wren, and bobwhite quail, but the highest relative abundance occurred on wild turkey, white-tailed deer, and raccoons. Eastern cottontail rabbits, white-tailed deer, raccoons, and squirrels had the highest relative abundance of larval lone star ticks, but they were also found on opossums and wild turkey. The activity of adult lone star ticks was greatest from May through July. The activity for nymphs was highest from May through August, and for larvae, July through September.     

Characterization of Toxoplasma gondii from raccoons (Procyon lotor), coyotes (Canis latrans), and striped skunks (Mephitis mephitis) in Wisconsin identified several atypical genotypes

During 2005-2006, sera and tissues from raccoons (Procyon lotor), coyotes (Canis latrans), and skunks (Mephitis mephitis) from the state of Wisconsin were tested for Toxoplasma gondii infection. Antibodies to T. gondii were found in 32 of 54 (59.2%) raccoons, 18 of 35 (51.4%) coyotes, and 5 of 7 (71.4%) skunks using the modified agglutination test and a cut-off titer of 1:20. Pooled tissues (brains, hearts, and tongues) from 30 raccoons, 15 coyotes, and 1 skunk were bioassayed for T. gondii infection in mice or cats. Viable T. gondii was isolated from 5 of 30 (16.7%) raccoons, 6 of 15 (40.0%) coyotes, and the skunk. Genetic characterization of the 12 parasite isolates by multilocus PCR-RFLP markers revealed 6 different genotypes including 5 atypical and I archetypal II lineages. The results indicate the prevalence of T. gondii in wildlife mammals is high and that these animals may serve as an important reservoir for transmission of T. gondii.     

Intramuscular vaccination of skunks and raccoons against rabies

Live-captured striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) were immunized with inactivated rabies vaccine by intramuscular injection and released at the point of capture during a rabies control program in Metropolitan Toronto (Ontario, Canada). Serum samples collected prior to and following vaccination revealed that 100% of the skunks and 98% of the raccoons seroconverted. Rabies antibody was still detectable 314 to 757 days postvaccination. Five of six skunks vaccinated in the laboratory survived challenge with rabies virus 90 days postvaccination. To our knowledge, this is the first documentation of the successful seroconversion of skunks and raccoons vaccinated against rabies in the field.     

Oral efficacy of an attenuated rabies virus vaccine in skunks and raccoons

Raccoons and skunks are major rabies reservoirs in North America. Oral vaccination is one method to consider for disease control in these carnivores. Under field conditions in the USA, only one oral rabies vaccine has been used. It is efficacious in wildlife such as raccoons (Procyon lotor), coyotes (Canis latrans), and foxes (Vulpes vulpes) but not in skunks (Mephitis mephitis). The objectives of this study were to evaluate an attenuated SAG-2 rabies virus vaccine for safety, immunogenicity, and efficacy by the oral route in skunks and raccoons. Two of five skunks and three of five raccoons developed virus neutralizing antibodies (VNA) by day 14 following oral administration of SAG-2 vaccine. All animals remained healthy. Upon challenge, naive controls succumbed to rabies. Among vaccinated animals, four of five skunks and all five raccoons had VNA on day 7 post-challenge and all survived. Given these results, SAG-2 is a promising candidate vaccine that may satisfy both safety and efficacy concerns for oral rabies immunization of major North American rabies reservoirs.     

Frequency of deposition and location of Baylisascaris procyonis eggs in raccoon feces

Baylisascaris procyonis is a large ascarid nematode found in the small intestine of raccoons (Procyon lotor). Infection with larvae of B. procyonis can produce visceral, ocular, and neural larval migrans in humans. Infected raccoons can shed millions of eggs a day in their feces. However, it is unknown whether eggs are consistently shed or whether eggs occur at irregular intervals by the population of female nematodes within a host. We trapped, infected, and collected daily fecal samples from 11 raccoons maintained in captivity. Eggs from B. procyonis were obtained from anterior, central, and posterior sections of raccoon feces, isolated by flotation, and quantified under 100× magnification. Naturally infected raccoons were collected and used as a comparison with the experimentally infected group. All raccoons in the experimental group (n=11) became infected with B. procyonis after consuming one infected mouse. Additionally, differential egg deposition rates were observed among individual raccoons from the experimental and naturally infected groups. Mean number of eggs per gram of feces (means±SE) was 16,563±4,321, which was less than previously reported for the species. However, no differences (F(2,30)=0.84, P=0.45) were noted in mean number of eggs per gram of feces among fecal sections. Wildlife biologists, veterinarians, health officials, and researchers of B. procyonis should collect daily fecal samples for a minimum of 3 days before identifying a raccoon as negative for B. procyonis infection. However, it does not matter where within the fecal matter the sample is obtained.     

Lack of Sarcocystis neurona antibody response in Virginia opossums (Didelphis virginiana) fed Sarcocystis neurona-infected muscle tissue

Serum was collected from laboratory-reared Virginia opossums (Didelphis virginiana) to determine whether experimentally infected opossums shedding Sarcocystis neurona sporocysts develop serum antibodies to S. neurona merozoite antigens. Three opossums were fed muscles from nine-banded armadillos (Dasypus novemcinctus), and 5 were fed muscles from striped skunks (Mephitis mephitis). Serum was also collected from 26 automobile-killed opossums to determine whether antibodies to S. neurona were present in these opossums. Serum was analyzed using the S. neurona direct agglutination test (SAT). The SAT was modified for use with a filter paper collection system. Antibodies to S. neurona were not detected in any of the serum samples from opossums, indicating that infection in the opossum is localized in the small intestine. Antibodies to S. neurona were detected in filter-paper-processed serum samples from 2 armadillos naturally infected with S. neurona.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Comparing ONRAB® AND RABORAL V-RG® oral rabies vaccine field performance in raccoons and striped skunks, New Brunswick, Canada, and Maine, USA

Control of rabies in mesocarnivore reservoirs through oral rabies vaccination (ORV) requires an effective vaccine bait. Oral rabies vaccine performance in the field may be affected by a variety of factors, including vaccine bait density and distribution pattern, habitat, target species population density, and the availability of competing foods. A field study in which these covariates were restricted as much as possible was conducted along the international border of the state of Maine (ME), USA, and the province of New Brunswick (NB), Canada, to compare the performance of two oral rabies vaccines in raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). RABORAL V-RG(?) (vaccinia-rabies glycoprotein recombinant oral vaccine in fishmeal-coated sachet) or ONRAB(?) (adenovirus-rabies glycoprotein recombinant oral vaccine in Ultralite bait matrix) were distributed in ME and NB, respectively, by fixed-wing aircraft at a density of 75 baits/km(2) along parallel flight lines spaced 1.0 km apart. Sera were collected from live-trapped raccoons and skunks 5-7 wk post-ORV and assayed to determine antibody prevalence in each area. Duplicate serum samples were provided blind to two different laboratories for analyses by rabies virus serum neutralization assays (at both laboratories) and a competitive enzyme-linked immunosorbent assay (at one laboratory). There was no significant difference in the proportion of antibody-positive animals determined by the three serologic methods, nor was there a significant difference between ONRAB and RABORAL V-RG in the proportion of antibody-positive striped skunks observed post-ORV. In contrast, the proportion of antibody-positive raccoons was significantly higher in the ONRAB- versus the RABORAL V-RG-baited areas (74% vs. 30%; χ(2)=89.977, df=5, P<0.0001). These data support that ONRAB may serve as an effective tool for raccoon rabies control.     

Dietary breadth and overlap among five sympatric prairie carnivores

Ecological communities are shaped, in part, by the manner in which similar species consume and partition food resources. To better understand the structure of a mammalian carnivore community from a prairie habitat in central North America, we determined the dietary breadth and overlap of sympatric American badgers Taxidea taxus , coyotes Canis latrans , red foxes Vulpes vulpes , raccoons Procyon lotor and striped skunks Mephitis mephitis , via stomach content analysis of 411 carcasses salvaged as part of a predator control program in southern Saskatchewan, Canada (2000–2001). Carnivores consumed a total of 25 separate food items; for all species except raccoons, the majority of stomachs contained mammals, whereas other foods such as amphibians, bird remains and insects were also recovered. Raccoon stomachs tended to contain wheat seed, eggshells and birds. Dietary breadth varied among the five carnivore species, being narrowest for raccoons and widest for skunks. Overall, dietary overlap tended to be highest for species pairings associated with the highest level of presumed niche similarity, which included raccoon–skunk and coyote–fox dyads. Yet, levels of dietary overlap exhibited notable interannual variability for most species pairings. The assessment of a larger assemblage of carnivores within a North American prairie community shows that marked interspecific and temporal variation in dietary breadth and overlap may characterize a guild of sympatric species occupying similar habitat.     

Trichinella murrelli in scavenging mammals from south-central Wisconsin, USA

Tissues and serum from 59 raccoons (Procyon lotor), 42 coyotes (Canis latrans), and seven Striped Skunks (Mephitis mephitis) collected in Dane and Iowa Counties, Wisconsin, USA, between October 2005 and March 2006 were microscopically and serologically examined for the presence of Trichinella spp. Encapsulated larvae were found on compression slides prepared from tongue tissues from a few animals. Complete tissue digestion of tongues revealed that 19% of the raccoons, 26% of the coyotes, and none of the seven skunks tested were infected with Trichinella spp. Cats were subsequently experimentally infected by feeding them the raccoon tissues containing muscle larvae, and muscle larvae isolated from the collected tongues were experimentally transmitted to mice. Multiplex polymerase chain reaction analysis of the isolated muscle larvae demonstrated two distinct bands migrating at 127 base pairs (bp) and 316 bp in all samples, which together are diagnostic for Trichinella murrelli; the isolates were assigned Istituto Superiore di Sanita (ISS) codes ISS1656 through ISS1667, and ISS1708 through ISS1710 by the International Trichinella Reference Centre. These findings extend the geographic range of T. murrelli into Wisconsin, USA.     

Patterns of gastrointestinal parasitism among five sympatric prairie carnivores: are males reservoirs?

Male vertebrates are believed to be disproportionately vulnerable to parasites, but empirical support for this contention is mixed. We tested the hypothesis of higher levels of parasitism in males with the use of counts of gastrointestinal helminths in 5 sympatric mammalian carnivores (American badgers, coyotes, red foxes, raccoons, striped skunks) from central Saskatchewan. Parasite burdens for females and males of each host species were compared with the use of prevalence (percentage of hosts infected), intensity (parasites per infected host), and overdispersion (proportion of heavily infected hosts that were male). Of 30 comparisons (13 each for prevalence and intensity, 4 for overdispersion), male bias was detected 8 times (27%), whereas female bias was detected only once (3%), adding some support to the notion that male mammals are more susceptible to parasitism. However, most of the statistical comparisons we undertook revealed no sexual bias (n=21, 70%), suggesting that differential patterns of infection are not ubiquitous in mammals. Moreover, when detected, the magnitude and direction of bias varied among host species, helminth species, and metrics of infection. We conclude that sympatric and ecologically similar mammal species will not always share the tendency for males to be more susceptible to parasitism, and that studies incorporating multiple parasites and metrics of infection are more likely to detect sex bias.     

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r² = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Rabies and rabies control in striped skunks (Mephitis mephitis) in three prairie regions of western North America

The number and geographic distribution of rabies cases in striped skunks (Mephitis mephitis) from Saskatchewan (n = 2,506 cases), Montana (n = 1,142), and Alberta (n = 199) since 1963 were reviewed. In Saskatchewan the number of cases increased steadily for 5 yr and then fluctuated consistently in a 4 yr cyclic pattern. Similarly an initial sweep across the province was followed by a cyclic pattern of geographic expansion (3 to 4 yr) and reduction (1 to 2 yr). No organized control efforts were conducted in Saskatchewan. Similar cyclic pattern were not seen in data from Montana or Alberta. In the latter areas, the number and distribution of rabies cases in skunks appeared to reflect efforts to reduce the population of skunks. An integrated program of skunk removal using poison and live-traps in association with research and public education successfully contributed to limiting the spread and establishment of rabies in striped skunks within prairie habitats. Rabies did not persist in skunks in other habitats.     

Comparison of immune responses of brown-headed cowbird and related blackbirds to west Nile and other mosquito-borne encephalitis viruses

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.     

Seroprevalence of Toxoplasma gondii in mesocarnivores of the Canadian prairies

The protozoon Toxoplasma gondii has a worldwide distribution and affects many species of warm-blooded animals. In the Canadian prairies, mesocarnivores such as striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) have experienced an increase in density and distribution, and they are in close contact with human dwellings. However, there has been no systematic study on the seroprevalence of T. gondii in these mesocarnivore populations. The objectives of the current project were to determine the serum antibody prevalence of T. gondii in Canadian prairie mesocarnivores and to study the relationship between antibody prevalence and species, sex, age, location, and year of collection. Antibodies to T. gondii were found in 5 of 24 (20.8%) skunks from Saskatchewan trapped in 1999 and 5 of 40 (12.5%) in 2000. Seroprevalences for T. gondii in raccoons and skunks trapped in Manitoba were 2 of 10 (20%) raccoons trapped in 2002, 7 of 44 (15.9%) trapped in 2003, and 16 of 37 (43.2%) trapped in 2004; and in 13 of 99 (13.1%) skunks trapped in 2003, 29 of 131 (22.1%) trapped in 2004, 53 of 165 (32.1%) trapped in 2005, and 30 of 51 (58.8%) trapped in 2006. Age, location, and year, but not the host species, were important variables in the determining the seroprevalence of T. gondii in skunks and raccoons. Results confirm that T. gondii is endemic in the skunk and raccoon populations in the Canadian prairies.