Comparative metal analysis in a species assemblage of mammals from the Southeastern United States.

Metal pollution commonly occurs in many terrestrial environments and may pose a threat for the animals inhabiting such areas. Here, we present concentrations of six metals (cadmium [Cd], copper [Cu], nickel [Ni], lead [Pb], selenium [Se], and zinc [Zn]) in the liver tissues of seven species of mammals obtained from a study that examined the impact of mesopredator removal on northern bobwhite (Colinus virginianus) populations. A total of 1326 samples were collected from 2003 to 2006 at four sites in southwest Georgia and north Florida from nine-banded armadillos (Dasypus novemcinctus), bobcats (Lynx rufus), feral cats (Felis catus), coyotes (Canis latrans), grey foxes (Urocyon cinereoargenteus), opossums (Didelphis virginiana), and raccoons (Procyon lotor). Data from armadillos, bobcats, opossums, and raccoons were published previously to examine age, sex, spatial (between sites), and temporal (between years) variation. In this paper, we present similar comparisons for the remaining three species as well as comparisons of metal concentrations among all seven species. Concentrations of Cu and Pb exhibited strong negative relationships with body weight in coyotes, while Ni was positively correlated with weight in feral cats. Concentrations of these metals, as well as the other two tested (Cd and Zn), were not significantly correlated with one another in any of the three species. The only sex difference in liver metal concentrations was observed in female feral cats, which had higher levels of Pb than did males. Coyotes exhibited significant differences in Cu concentrations between sites and between years (2005 versus 2006). We also found significant differences between sites in Pb concentrations for both feral cats and grey foxes. There were significant differences in metal concentrations among all seven species for all metals except Cd. With the exception of Cd and Se (tested only in bobcats and opossums), a three-way ANOVA with species, year, and site as the three factors revealed significant differences among species for every metal but only a single main effect of year for Cu, and no main effects of site. In sum, our results provide an extensive survey of metal concentrations in a diverse assemblage of mammals and suggest that metal accumulation may be heavily influenced by species identity, which in turn may reflect ecological lifestyle.

Metal accumulation in bobcats in the Southeastern USA.

Bobcats (Lynx rufus) are wide-ranging mammals found throughout the continental USA. As carnivores near the top of their food chain, bobcats would seem to be a useful bioindicator of metal pollution in terrestrial environments. However, there is very limited research on bobcats in toxicology studies. Here, we offer the first analysis of metal (copper, selenium, silver, and zinc) contaminants in the livers of wild bobcats. Liver tissues from 120 adult bobcats (i.e., estimated to be ≥1 year old) were collected from 2003 to 2006 at four sites in Georgia and Florida, USA that experienced relatively similar levels of human disturbance. We found no differences in metal concentrations between males and females. At two of the sites sampled over three consecutive years, there was substantial year-to-year variation in the concentrations of Cu, Se, and Zn. We also documented some variation between sites, but only between sites sampled in different years, which may reflect additional temporal, rather than spatial, variation. Concentrations of Cu and Ag were significantly positively correlated with one another, as were concentrations of Se and Zn. Contrary to expectation, there were no significant relationships between body weight and metal concentrations. Finally, comparison with results from previous metal toxicology studies of nine-banded armadillos (Dasypus novemcinctus) and Virginia opossums (Didelphis virgianus), collected from the same sites during the same years, showed differential patterns of accumulation across species, suggesting that ecological lifestyle is an important influence on metal accumulation. This study provides reference levels of metal contaminants in the liver of bobcats as well as insight into metal accumulation in a top level carnivore.

Metal accumulation in wild-caught opossum.

The Virginia opossum (Didelphis virginiana) is widespread in the USA, ranging south through Latin America. The ecology of opossums is such that they are in frequent contact with soils, suggesting that they may function as a valuable bioindicator for chemical contamination in terrestrial environments. Surprisingly, there have been virtually no toxicology studies on opossums. Here, we provide the first analysis of metal contaminants in opossum liver tissues. Liver samples were obtained from 471 opossums, collected from 2003 to 2006, at four sites in North Florida and South Georgia, USA, and concentrations of copper, lead, nickel, selenium, and zinc were measured. We found little evidence of age differences in the concentration of any of the metals. However, there were at least some significant differences between years, males and females, and between sites for each metal, although the pattern of these differences was not always consistent across metals. Concentrations of metals in liver tissue were positively correlated with one another, primarily of each metal (except Pb) with zinc. Reference levels of metal contaminants are not available for opossums, but concentrations of Cu, Ni, Pb, and Zn in our samples were for the most part significantly higher than those reported from liver tissues of nine-banded armadillos (Dasypus novemcinctus) collected at the same sites and in the same years. Data from other small mammals studied elsewhere further indicate that metal concentrations in opossums were high, but at this time, it is not possible to determine if these elevated levels generated toxicity. The substantial temporal and spatial variation we found in metal concentrations suggests that determination of baseline levels for opossums may not be straightforward. Nonetheless, this is the first study quantifying metal accumulation in the livers of Didelphis virginiana and, as such, provides an important starting point for future research.

Zoonotic Leprosy in the Southeastern United States.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and have been implicated in zoonotic transmission of leprosy. Early studies found this disease mainly in Texas and Louisiana, but armadillos in the southeastern United States appeared to be free of infection. We screened 645 armadillos from 8 locations in the southeastern United States not known to harbor enzootic leprosy for M. leprae DNA and antibodies. We found M. leprae-infected armadillos at each location, and 106 (16.4%) animals had serologic/PCR evidence of infection. Using single-nucleotide polymorphism variable number tandem repeat genotyping/genome sequencing, we detected M. leprae genotype 3I-2-v1 among 35 armadillos. Seven armadillos harbored a newly identified genotype (3I-2-v15). In comparison, 52 human patients from the same region were infected with 31 M. leprae types. However, 42.3% (22/52) of patients were infected with 1 of the 2 M. leprae genotype strains associated with armadillos. The geographic range and complexity of zoonotic leprosy is expanding.

Serologic survey of wild turkeys (Meleagris gallopavo) and evidence of exposure to avian encephalomyelitis virus in Georgia and Florida, USA.

Wild Turkeys (Meleagris gallopavo) are susceptible to many of the same diseases as domestic turkeys. Before 2005, most Wild Turkeys in southern Georgia, US, had little or no exposure to commercial poultry operations. As part of a pathogen survey examining the effects of commercial poultry on Wild Turkeys, samples were collected from Wild Turkeys from March 2005 through May 2008. The turkeys were collected from 13 counties in southern Georgia and Madison County, Florida, and tested for antibodies to various pathogens of poultry. Three (13%) of the turkeys were positive for antibodies to Salmonella. Thirteen turkeys (54%) were positive for Newcastle disease virus antibodies, and 15 turkeys (63%) were positive for antibodies to reticuloendotheliosis virus. One turkey (4%) from Madison County was positive for avian encephalomyelitis virus antibodies.

Surveillance for upper respiratory tract disease and Mycoplasma in free-ranging gopher tortoises (Gopherus polyphemus) in Georgia, USA.

Abstract Upper respiratory tract disease (URTD) in the gopher tortoise (Gopherus polyphemus) is highly contagious and has been implicated in the reduction of populations throughout the range. With the exception of a few limited studies, the prevalence of URTD in Georgia, USA tortoise populations is poorly known. We found that exposure to Mycoplasma agassizii and Mycoplasma testudineum, associated with URTD, varied geographically among 11 Georgia tortoise populations. The prevalence of antibodies to M. agassizii in individual populations was either very low (0-3%, n=7 populations) or very high (96-100%, n=4 populations), whereas there was variation in the prevalence of antibodies to M. testudineum among populations (20-61%, n=10) with only one site being negative. Five sites had tortoises with antibodies to both pathogens, and these were the only sites where we observed tortoises with clinical signs consistent with URTD. We did not find tortoises with clinical signs of URTD at sites with tortoises with antibodies only to M. testudineum, which provides evidence that this organism may be of limited pathogenicity for gopher tortoises. Collectively, these data indicate that both M. agassizii and M. testudineum are present in Georgia populations of gopher tortoises and that clinical disease is apparent in populations where both pathogens are present. Additional research is needed to better understand the role of these two pathogens, and other potential pathogens, in the overall health of tortoise populations, especially if future conservation efforts involve translocation of tortoises.

Metal accumulation in wild nine-banded armadillos.

Nine-banded armadillos (Dasypus novemcinctus) are widespread and abundant New World mammals with a lifestyle that entails prolonged, intimate contact with soils. Thus, armadillos would seem a promising candidate as a sentinel species to monitor chemical contamination in terrestrial ecosystems. Surprisingly, there have been virtually no toxicology studies on armadillos. Here, we provide the first analysis of metal contaminants for wild armadillos. Liver tissues were obtained from 302 armadillos collected at 6 sites in Georgia and Florida, USA that varied in their extent of human disturbance, from rural pine plantations to highly modified military/space installations. Data were stratified by age (juvenile and adult), sex, and site. Temporal (yearly) variation was examined at two of the sites that were sampled over three consecutive years. Concentrations of aluminum, cadmium, copper, nickel, lead, and zinc were measured in liver samples from each site. Although reference levels are not available for armadillos, accumulated metal concentrations were comparable to those reported for other mammals. We found no evidence of sex or age differences in the concentrations of any metal, except for Cd (age) and Pb (sex and age). However, concentrations of most metals varied substantially across sites and over time. Finally, concentrations of many metals were positively correlated with one another, suggesting that they likely co-occurred in some areas. Collectively, this study indicates the utility of armadillos as a sentinel species for studies of metal contamination in terrestrial systems, and highlights the need for further studies of other toxicants in these animals.

Variation in the ITS-1 and ITS-2 rRNA genomic regions of Cytauxzoon felis from bobcats and pumas in the eastern United States and comparison with sequences from domestic cats.

Cytauxzoon felis, a tick-borne protozoan parasite, is the causative agent of cytauxzoonosis in domestic cats in the United States. The natural reservoir for this parasite is the bobcat (Lynx rufus), which typically does not develop clinical signs. Although not likely important reservoirs, C. felis has also been detected in pumas (Puma concolor) in Florida and Louisiana. Recent studies suggest that specific genotypes of C. felis that circulate in domestic cats may be associated with variable clinical outcomes and specific spatial locations. In the current study, we investigated the intraspecific variation of the C. felis internal transcribed spacer (ITS)-1 and ITS-2 rRNA regions from 145 wild felids (139 bobcats and six pumas) from 11 states (Florida, Georgia, Kansas, Kentucky, Louisiana, Missouri, North Carolina, North Dakota, South Carolina, Oklahoma, and Pennsylvania). Unambiguous ITS-1 and ITS-2 data were obtained for 144 and 112 samples, respectively, and both ITS-1 and ITS-2 sequences were obtained for 111 (77%) samples. For the ITS-1 region, sequences from 65 samples collected from wild felids were identical to those previously reported in domestic cats, while the other 79 sequences were unique. C. felis from 45 bobcats and one puma had ITS-1 sequences identical to the most common sequence reported from domestic cats. Within the ITS-2 region, sequences from 49 bobcats were identical to those previously reported in domestic cats and 63 sequences were unique (with some occurring in more than one bobcat). The most common ITS-2 sequence from domestic cats was also common in wild felids (31 bobcats and a puma). Samples from three pumas from Florida and two bobcats from Missouri had a 40- or 41-bp insert in the ITS-2 similar to one described previously in a domestic cat from Arkansas. Additionally, a previously undescribed 198- or 199-bp insert was detected in the ITS-2 sequence from four bobcats. Collectively, based on combined ITS-1 and ITS-2 sequences, five different genotypes were detected in the wild felids. Genotype ITSa was the most common genotype (11 bobcats and one puma) and fewer numbers of ITSb, ITSe, ITSg, and ITSi were detected in bobcats. These data indicate that, based on ITS-1 and ITS-2 sequences, numerous C. felis strains may circulate in wild felids.

Distribution and prevalence of Cytauxzoon felis in bobcats (Lynx rufus), the natural reservoir, and other wild felids in thirteen states.

Cytauxzoon felis, a protozoan parasite of wild and domestic felids, is the causative agent of cytauxzoonosis in domestic and some exotic felids in the United States. The bobcat (Lynx rufus) is the natural reservoir for this parasite, but other felids such as Florida panthers (Puma concolor coryii) and domestic cats may maintain long-term parasitemias and serve as reservoirs. Experimentally, two tick species, Dermacentor variabilis and Amblyomma americanum, have demonstrated the ability to transmit C. felis. These two tick species have overlapping distributions throughout much of the southeastern United States. The objective of the current study was to determine the distribution and prevalence of C. felis in free-ranging bobcat populations from 13 states including California, Colorado, Florida, Georgia, Kansas, Kentucky, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, South Carolina, and West Virginia. These states were selected because of differential vector presence; D. variabilis is present in each of these states except for the region of Colorado sampled and A. americanum is currently known to be present only in a subset of these states. Blood or spleen samples from 696 bobcats were tested for C. felis infection by a polymerase chain reaction (PCR) assay which targeted the first ribosomal internal transcribed spacer region (ITS-1). Significantly higher prevalences of C. felis were detected from Missouri (79%, n=39), North Carolina (63%, n=8), Oklahoma (60%, n=20), South Carolina (57%, n=7), Kentucky (55%, n=74), Florida (44%, n=45), and Kansas (27%, n=41) compared with Georgia (9%, n=159), North Dakota (2.4%, n=124), Ohio (0%, n=19), West Virginia (0%, n=37), California (0%, n=26), and Colorado (0%, n=67). In addition to bobcats, seven cougars (Puma concolor) from Georgia, Louisiana, and North Dakota and one serval (Leptailurus serval) from Louisiana were tested for C. felis. Only one cougar from Louisiana was PCR positive, which represents the first report of an infected cougar outside of the Florida panther population. These data also indicate that C. felis is present in North Dakota where infection has not been reported in domestic cats. Based on a nonparametric analysis, prevalence rates were significantly higher in states where there are established populations of A. americanum, which supports recent data on the experimental transmission of C. felis by A. americanum and the fact that domestic cat clinical cases are temporally associated with A. americanum activity. Collectively, these data confirm that bobcats are a common reservoir for C. felis and that A. americanum is likely an epidemiologically important vector.

Identification and genetic characterization of Cytauxzoon felis in asymptomatic domestic cats and bobcats.

The objectives of the current study were to assess the prevalence of Cytauxzoon felis infection among a population of domestic cats that were clinically healthy but at higher risk for parasite exposure and to determine if the strains present in these asymptomatically infected cats were genetically unique as compared to those present both in domestic cats that were fatally infected and in the natural reservoir host, the bobcat. Using real-time PCR analysis targeting a portion of the parasite 18S rRNA gene specific for C. felis, 27/89 (30.3%) high-risk asymptomatic domestic cats from Arkansas and Georgia, and 34/133 (25.6%) bobcats from Arkansas, Georgia and Florida, were identified as positive for C. felis infection. Conventional PCR analysis was performed on all positive samples, targeting the C. felis ribosomal internal transcribed spacer regions 1 and 2 (ITS1, ITS2) in order to utilize the ITS sequences as markers to assess the genotype variability of the parasite population. Within the asymptomatically infected domestic cat samples, 3 genetically distinct parasite populations were identified. The C. felis ITS sequences from asymptomatic cats were identical to those previously reported from clinically ill infected cats, and 2 of the 3 sequence types were also present in infected bobcat samples. While sequence diversity exists, evaluation of the ITS region does not appear to be useful to verify pathogenicity of C. felis strains within host species. However, the presence of asymptomatic C. felis infections in clinical healthy domestic cats warrants further investigation to determine if these cats can serve as a new reservoir for C. felis transmission.

Salmonella from gopher tortoises (Gopherus polyphemus) in south Georgia.

From 2002 to 2006, gopher tortoises (Gopherus polyphemus) were collected at Moody Air Force Base, Lowndes/Lanier counties, Georgia, USA, and opportunistically surveyed for the presence of Salmonella species. Four of 155 (2.6%) cloacal swabs collected from 80 tortoises were positive for the presence of Salmonella enterica, and the following serovars were identified: Give, Hartford, Javiana, and Luciana. Female tortoises (5%) were infected at a rate similar to male tortoises (5%). All isolates were obtained from adult tortoises (n = 73); subadults (n = 7) were all negative. Each isolated serovar is a potential human pathogen, suggesting appropriate precautions should be emphasized when handling these animals.

Gnathostoma procyonis from south Georgia and north Florida raccoons.

From 2004 to 2006, 511 raccoons collected by the United States Department of Agriculture Wildlife Services as part of a study to evaluate the effects of mesomammalian predator removal on bobwhite-quail reproduction, were examined for the presence of Gnathostoma procyonis, a nematode that parasitizes the stomach of its definitive host, the raccoon. One hundred forty-one raccoons (27.6%) contained G. procyonis, with males being equally infected (27.5%) as females (27.8%). Mean intensity did not differ significantly between sexes (males-mean = 2.88, range 1-17; females-mean = 2.78, range 1-10) or between collection sites. A significant seasonal prevalence of G. procyonis was noted with a peak in March (43.3%, n = 120) and a minimum in September (5.4%, n = 37). Both collection sites exhibited significant seasonal decreases in G. procyonis, but were not significantly different from each other. There was no cumulative seasonal pattern in mean intensity of G. procyonis, and no seasonal pattern at either collection site. Host weight was not related to either G. procyonis prevalence or mean intensity. Host removal did not have an apparent effect on prevalence or mean intensity of G. procyonis.

Evaluation of a prototype Ehrlichia chaffeensis surveillance system using white-tailed deer (Odocoileus virginianus) as natural sentinels.

The natural history of Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, includes the lone star tick (LST, Amblyomma americanum) as a vector and white-tailed deer (WTD; Odocoileus virginianus) as both a natural reservoir of E. chaffeensis and a major host of LST. The goal of the current study was to implement and evaluate a prototype surveillance system to delineate the geographic distribution of E. chaffeensis using WTD as natural sentinels. To accomplish this goal, serologic testing using the indirect immunofluorescent antibody (IFA) test was performed on WTD serum samples, and to confirm serologic results, polymerase chain reaction (PCR) assays and culture isolation were conducted. Considerations relevant to the applicability of a surveillance system utilizing WTD were analyzed (e.g., age and gender relationships to serologic status, adequacy of sample sizes needed to distinguish between uninfected and infected populations, presence of LST, and ability to detect stability and spread of E. chaffeensis in WTD populations). Of 3275 WTD serologically tested, 549 (47%) from 17 of 18 states had antibodies reactive to E. chaffeensis (IFA titer > or = 1:128). No difference between age groups or gender was noted with serologic testing, thus these variables would not be a concern for a surveillance system using WTD. Significantly more deer in younger age groups (< or = 1.5 yr) were PCR and culture positive, and 46% of 122 seropositive WTD populations were confirmed positive by PCR or culture isolation. A significant association between LST infestation and E. chaffeensis seroreactivity was noted. Furthermore, the surveillance system was able to detect stability of E. chaffeensis within WTD populations and also spread to new populations, both of which were associated with LST status. These data clearly demonstrate that WTD are useful as natural sentinels for this emerging human pathogen, and establish a prototypical framework for a WTD surveillance system.