Species-specific differences in Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti seroprevalence in Namibian wildlife.

BACKGROUND: Knowledge about parasitic infections is crucial information for animal health, particularly of free-ranging species that might come into contact with livestock and humans. METHODS: We investigated the seroprevalence of three tissue-cyst-forming apicomplexan parasites (Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti) in 506 individuals of 12 wildlife species in Namibia using in-house enzyme linked immunosorbent assays (indirect ELISAs applying purified antigens) for screening and immunoblots as confirmatory tests. We included six species of the suborder Feliformia, four species of the suborder Caniformia and two species of the suborder Ruminantia. For the two species for which we had most samples and life-history information, i.e. cheetahs (Acinonyx jubatus, n = 250) and leopards (Panthera pardus, n = 58), we investigated T. gondii seroprevalence in relation to age class, sex, sociality (solitary, mother-offspring group, independent sibling group, coalition group) and site (natural habitat vs farmland). RESULTS: All but one carnivore species (bat-eared fox Otocyon megalotis, n = 4) were seropositive to T. gondii, with a seroprevalence ranging from 52.4% (131/250) in cheetahs to 93.2% (55/59) in African lions (Panthera leo). We also detected antibodies to T. gondii in 10.0% (2/20) of blue wildebeest (Connochaetes taurinus). Adult cheetahs and leopards were more likely to be seropositive to T. gondii than subadult conspecifics, whereas seroprevalence did not vary with sex, sociality and site. Furthermore, we measured antibodies to N. caninum in 15.4% (2/13) of brown hyenas (Hyaena brunnea) and 2.6% (1/39) of black-backed jackals (Canis mesomelas). Antibodies to B. besnoiti were detected in 3.4% (2/59) of African lions and 20.0% (4/20) of blue wildebeest. CONCLUSIONS: Our results demonstrate that Namibian wildlife species were exposed to apicomplexan parasites at different prevalences, depending on parasite and host species. In addition to serological work, molecular work is also needed to better understand the sylvatic cycle and the clear role of wildlife in the epidemiology of these parasites in southern Africa.

BLOOD LEAD AND ZINC LEVELS AND THEIR IMPACT ON HEALTH OF FREE-LIVING SMALL CARNIVORES IN TAIWAN, REPUBLIC OF CHINA.

Lead and zinc are recognized as the most widespread trace metals in nature and can, at high levels, compromise the health of wildlife and their habitat. Because of their position in a higher trophic level, wild carnivores can be valuable biological indicator species of trace-metal contamination in the environment. We assessed blood lead and zinc concentrations of four small carnivore species native to Taiwan, the small Indian civet (Viverricula indica), the masked palm civet (Paguma larvata), the ferret badger (Melogale moschata), and the crab-eating mongoose (Herpestes urva), from urban and rural areas (Yangmingshan National Park, Xiuguluan River bank, and Da-an River bank). Blood samples were acquired from the anterior vena cava under general anesthesia, and lead and zinc concentrations, hematology, and serum biochemistry results were then obtained. Blood lead levels were significantly higher in ferret badgers in the Yangmingshan area. Although lead concentrations were comparable with those in humans and cats with lead toxicosis, there was no hematological or biochemical evidence that animal health was compromised. Blood zinc levels were within an acceptable range in all four species tested. Overall, we found significant differences in blood lead and zinc levels among four species of carnivores living in areas with different levels of land development in Taiwan. Anthropogenic pollution, mining history, and volcanic activities in Yangmingshan National Park may contribute to significantly high blood lead levels in ferret badgers in this area. Our results provided information about the potential impact of land development on wildlife and may be beneficial to wildlife conservation, public health, and environmental health in Taiwan.

Epizootological study on Toxoplasma gondii in zoo animals in the Czech Republic.

Toxoplasma gondii is protozoan parasite with ability of causing disease in wide-spectrum of animals; many species of animals in captivity died of clinical toxoplasmosis. The monitoring of T. gondii antibodies in zoo animals can be an important indicator of T. gondii circulation in zoo. The aim of this study was to examine sera of animals from eight Czech zoos by latex agglutination test with statistical evaluation and detect T. gondii DNA in stray cats and rodents captured in the zoos. T. gondii antibodies were detected in 33% of 1043 zoo animals without statistical difference between birds (27%, n = 74) and mammals (33%, n = 969). In birds, the chance to be infected with T. gondii was higher in Accipitriformes (71%) compared to Pelecaniformes (6%) (p < 0.0001). In mammals, the chance to be infected with T. gondii was higher in Carnivora (63%) compared to Cetarodactyla (30%), Perissodactyla (26%), Primates (28%) and Rodentia (13%) (p < 0.0001) and higher in Felidae (70%) compared to Bovidae (28%) and Equidae (28%) (p < 0.0001). Mammals with carnivore/scavenger way of feeding were in a higher risk of T. gondii infection compared to herbivores and omnivores (p < 0.0001). T. gondii DNA was detected in tissue of one stray cat while in none of 77 rodents caught in zoo. This study is the first report on toxoplasmosis in zoos from the Czech Republic including seroepidemiology and molecular detection.

Hematocrit of mammals (Artiodactyla, Carnivora, Primates) at 1500m and 2100m altitudes.

The rise in hematocrit (Hct) is one of the hallmarks of human acclimatization to high altitude and, in chronic conditions, reflects the hypoxia-induced polycythemia. However, it is not a uniform response among domestic species and it is not found in Andean camelids, species long adapted to high altitudes. Hence, we asked to what extent the polycythemia of humans is common among mammals. Hct data were collected from captive mammals of three orders (Primates, Artiodactyla, Carnivora), 70 specimens of 33 species at ∼1500m altitude (barometric pressure Pb=635mmHg) and 296 specimens of 64 species at ∼2100m (Pb=596mmHg), long-term residents at those altitudes. Sea level values and data in men and women at the corresponding altitudes were from a compilation of literature sources. At either altitude Hct was significantly higher than at sea level both in men and women; the increase (ΔHct) for genders combined averaged 3.4% (±0.7 SEM) at 1500m and 5.4% (±0.3) at 2100m. Differently, among the three mammalian orders studied a significant increase in Hct occurred only in females of Carnivora (at 1500m) and in males of Primates (at 2100m). The average ΔHct of all species combined was 0.8% (±0.7) at 1500m and 1.5% (±0.4) at 2100m, both significantly less than in humans (P<0.001). At 2100m the average ΔHct of nine species long adapted to high altitude was 0.4% (±1), significantly less than in non-adapted species (P<0.001). A polycythemic response like that of men and women at 2100m occurred in less than 10% of the mammals examined. We conclude that, at least for the altitudes studied, a minimal polycythemia is a general feature of both high-altitude adapted and non-adapted species, and the magnitude of the human response is exceptional among mammals.

Serologic survey for canine distemper virus and canine parvovirus in free-ranging wild carnivores from Portugal.

A serologic survey for Canine distemper virus (CDV) and canine parvovirus (CPV) was performed on serum and lung extract from an opportunistic sample of 120 free-ranging wild carnivores (13 species) from Portugal, collected from 1995 to 2006. Antibodies to CDV were detected in wolf (Canis lupus; 3/27) and red fox (Vulpes vulpes; 2/22). Antibodies to CPV were detected in wolf (9/28), red fox (2/14), wildcat (Felis silvestris;1/8), genet (Genetta genetta; 17/18), and stone marten (Martes foina; 3/17). Antibodies to CPV were detected throughout the study, whereas for CDV antibodies were detected in 3 of 10 yr and only during winter. The extremely high CPV antibody prevalence in genets is unprecedented. Although based on a limited sample, these data suggest widespread exposure of free-ranging Iberian carnivores to CDV and CPV.

Seroprevalence of Toxoplasma gondii antibodies in wild carnivores from Spain.

Serum samples from 282 wild carnivores from different regions of Spain were tested for antibodies to Toxoplasma gondii by the modified agglutination test using a cut-off value of 1:25. Antibodies to T. gondii were found in 22 of 27 (81.5%) of Iberian lynx (Lynx pardinus), 3 of 6 European wildcats (Felis silvestris), 66 of 102 (64.7%) red foxes (Vulpes vulpes), 15 of 32 (46.9%) wolves (Canis lupus), 26 of 37 (70.3%) Eurasian badgers (Meles meles), 17 of 20 (85.0%) stone martens (Martes foina), 4 of 4 pine martens (Martes martes), 6 of 6 Eurasian otters (Lutra lutra), 4 of 4 polecats (Mustela putorius), 1 of 1 ferret (Mustela putorius furo), 13 of 21 (61.9%) European genets (Genetta genetta), and 13 of 22 (59.1%) Egyptian mongooses (Herpestes ichneumon). Serological results indicated a widespread exposure to T. gondii among wild carnivores in Spain. The high T. gondii seroprevalence in Iberian lynx and the European wildcat reported here may be of epidemiologic significance because seropositive cats might have shed oocysts.

Tools for rabies serology to monitor the effectiveness of rabies vaccination in domestic and wild carnivores.

Serology remains the only way to monitor the effectiveness of vaccination of humans and animals against rabies. Many techniques for determining the level of rabies antibodies have been described, including seroneutralisation techniques such as tests for fluorescent antibody virus neutralisation (FAVN) and rapid fluorescent focus inhibition (RFFIT), enzyme-linked immunosorbent assay (ELISA), and in-vivo tests (the mouse neutralisation test, MNT). The need to verify the effectiveness of rabies vaccination has become widespread, particularly in the context of international trading of domestic carnivores from infected to rabies-free territories. The standardisation of serological techniques, approval of laboratories and proficiency tests are key concepts to ensure the practicability of such systems. Serological tests for rabies are also often used by laboratories in infected territories to assess the efficacy of campaigns aimed at the eradication of the disease via oral vaccination of wildlife. The adaptation of these methods should provide the means to titrate specific antibodies in dogs during mass parenteral vaccination in countries infected by canine rabies. However, in most cases these serological tests are carried without any standardised procedure. On the basis of our experience in rabies serology and its harmonisation throughout laboratories worldwide, we propose here an adapted standard technique for the serological monitoring for rabies in wildlife at the European level. Such harmonisation would allow the monitoring of vaccination campaigns to be enhanced by increasing the exchange of epidemiological data, with the ultimate goal being the eradication of rabies in Europe.

Collaborative study to evaluate a new ELISA test to monitor the effectiveness of rabies vaccination in domestic carnivores.

To prevent any introduction of rabies, many rabies-free countries have adopted a scheme requiring the rabies vaccination of pets associated with a serological test. FAVN test and RFFIT are the current OIE prescribed techniques to perform this assay. A qualitative indirect ELISA (Serelisa) test has been recently described as a screening test to monitor the effectiveness of rabies vaccination of pets. A lack of sensitivity requires ELISA negative samples to be retested using an OIE confirmatory test. This raised the question whether this new test could be reasonably proposed as an alternative tool in the context of international trades of pets. The Community Reference Institute of Nancy organized a short trial to answer this question. In this study, 16 laboratories tested a panel of their own samples with FAVN test/RFFIT and the Serelisa. The comparison of results revealed that the performance of the Serelisa is highly heterogeneous. A lack of sensitivity was detected in 50% of participants, when 25% of laboratories obtained a significant rate of false positive results. This last point questions the pertinence of using the Serelisa in the context of international trades by preventing any movements of insufficiently or non-protected animals.

Cortisol levels are positively associated with pup-feeding rates in male meerkats.

In societies of cooperative vertebrates, individual differences in contributions to offspring care are commonly substantial. Recent attempts to explain the causes of this variation have focused on correlations between contributions to care and the protein hormone prolactin, or the steroid hormone testosterone. However, such studies have seldom considered the importance of other hormones or controlled for non-hormonal factors that are correlative with both individual hormone levels and contributions to care. Using multivariate statistics, we show that hormone levels explain significant variation in contributions to pup-feeding by male meerkats, even after controlling for non-hormonal effects. However, long-term contributions to pup provisioning were significantly and positively correlated with plasma levels of cortisol rather than prolactin, while plasma levels of testosterone were not related to individual patterns of pup-feeding. Furthermore, a playback experiment that used pup begging calls to increase the feeding rates of male helpers gave rise to parallel increases in plasma cortisol levels, whilst prolactin and testosterone levels remained unchanged. Our findings confirm that hormones can explain significant amounts of variation in contributions to offspring feeding, and that cortisol, not prolactin, is the hormone most strongly associated with pup-feeding in cooperative male meerkats.

Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan.

Raccoons (Procyon lotor), which have recently become feral in Japan, were examined for the presence of Babesia microti-like parasites. Out of 372 raccoons captured in the west-central part of Hokkaido, 24 animals with splenomegaly were selected and tested by nested PCR targeting the babesial 18S rRNA gene. B. microti-like parasites were detected in two of the 24 individuals, and their DNA sequences were identical to that of the B. microti-like parasite reported from raccoons in the United States, suggesting that the parasites were probably imported into Japan and that the life cycle of the parasite has already been established in the country. The potential risk of this B. microti-like parasite spreading among dogs and foxes in Japan will need to be carefully monitored, as parasitization by phylogenetically very close parasites has been reported from such animals.

Neutralizing antibodies against feline herpesvirus type 1 in captive wild felids of Brazil.

Feline herpesvirus type 1 infection affects domestic cats, causing mainly upper respiratory tract diseases. Although this infection has been described in captive and free-ranging wild felids from Europe, Asia, North America, and Africa, no information is available on its occurrence among wild felids of Brazil. In this study, 250 serum samples of six species of Brazilian captive wild felids (Leopardus tigrinus, Leopardus wiedii, Herpailurus yaguarondi, Puma concolor, Leopardus pardalis, and Panthera onca) were examined for neutralizing antibodies to feline herpesvirus type 1. Positive sera were found in 72% of L. tigrinus samples, 15% of L. wiedii, 6% of L. pardalis, 8% of H. yaguarondi, 18% of P. concolor, and 14% of P. onca. The relatively low percentages of seropositivity and low antibody titers found among the last five species suggest that feline herpesvirus type 1 does not circulate extensively among these animals. Nevertheless, quarantine, serologic screening, and vaccination of newly introduced felids is recommended in zoos in order to prevent virus transmission and outbreaks of the disease among wild felids kept in captivity.

Cloning and sequencing of badger (Meles meles) interferon gamma and its detection in badger lymphocytes.

The European badger (Meles meles) has been identified as a reservoir for Mycobacterium bovis and is implicated in the maintenance and transmission of tuberculosis in cattle. There is a need for a sensitive test of M. bovis infection in badgers and the current serodiagnostic test used for this purpose has low sensitivity. As observed for other species, assay of interferon-gamma (IFNgamma) produced in response to M. bovis antigens is a more sensitive test of tuberculosis. With this objective in sight, we report the first step in the development of an ELISA for badger IFNgamma. The badger IFNgamma gene was cloned and sequenced and used to generate a specific polyclonal antibody to the cytokine. The gene sequence demonstrated regions that were conserved within the IFNgamma genes of other mammals. The badger sequence was most similar to the canine, showing similar structural organisation of the gene and 88% amino acid identity. Rabbits were immunised with DNA encoding badger IFNgamma and the resulting polyclonal antiserum demonstrated specificity for canine IFNgamma by immunoblot of a commercial recombinant canine IFNgamma. The antiserum was used to detect intracellular badger IFNgamma by flow cytometry analysis of badger lymphocytes stimulated with mitogen.

Serum lipid concentrations in six canid and four ursid species in four zoos.

Serum lipid levels were measured in healthy captive wild canids and ursids, and the values were compared with previously published data. Serum lipid levels were evaluated in blood samples collected from eight African wild dogs (Lycaon pictus), three arctic foxes (Alopex lagopus), nine gray wolves (Canis lupus), four maned wolves (Chrysocyon brachyurus), two Mexican wolves (Canis lupus baleiyi), nine red wolves (Canis rufus), two brown bears (Ursus arctos), six polar bears (Ursus maritimus), six spectacled bears (Tremarctos ornatus), and five sun bears (Ursus malayanus). Samples were analyzed for total cholesterol, triacylglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol. Although the results showed a great variation among species, circulating lipids appeared especially high, sometimes extremely so, in the spectacled bears, polar bears, sun bears, and maned wolves compared with all other species sampled. The study provides a substantial basis for comparing lipid levels in presumed healthy animals and indicates a need for controlled study of the effects of diet on circulating lipid levels.

Seasonal physiology of the wild raccoon dog (Nyctereutes procyonoides).

The raccoon dog (Nyctereutes procyonoides) is a canid omnivore with autumnal fattening and winter sleep. Farmraised raccoon dogs have elevated plasma leptin and growth hormone levels in the winter and depressed plasma cortisol and insulin concentrations during wintertime food deprivation. However, these parameters were not previously tested in the wild population. In the present study 37 wild raccoon dogs were sampled at different seasons and diverse biochemical variables were determined. The results mostly confirmed previous observations on farmraised raccoon dogs. The liver glycogen stores increased during the autumnal fattening period but were low in the winter. The liver glycogen phosphorylase activity decreased but lipase activity increased in the winter indicating the use of fat as the principal metabolic fuel. The plasma insulin concentrations were low in the winter allowing the release of fatty acids from adipose tissue. Low wintertime cortisol and thyroid hormone levels could contribute to protein sparing. Unlike on farms, wild raccoon dogs did not show seasonal fluctuations in their plasma ghrelin or growth hormone levels. The observed physiological phenomena emphasise the adaptation of the species to long periods of food scarcity in the winter.

Defense of females, but not social status, predicts plasma androgen levels in male spotted hyenas.

A positive correlation between male social status and testosterone levels is expected and often found in social species with high rates of agonistic interactions or when social relationships among males are unstable. In contrast, in species with low rates of agonistic interactions or when social relationships are stable, testosterone levels should not correlate with social status. The "challenge hypothesis" predicts that androgen levels should rise during periods of courtship or mate guarding. We addressed these questions in free-ranging spotted hyenas, a species with low rates and low intensities of aggression among males but where males spend extensive effort to court females. In males, we measured testosterone, its precursor androstenedione, and its metabolite 5alpha-dihydrotestosterone. As predicted, testosterone levels were significantly higher and androstenedione levels tended to be higher in males that, at the time of sampling, defended a female, compared with males that did not defend a female. Also, as predicted, there was no correlation between social status and androgen levels in male spotted hyenas.

Seasonal reproductive endocrine profile of the raccoon dog (Nyctereutes procyonoides)-effects of melatonin and food deprivation.

The raccoon dog (Nyctereutes procyonoides, Canidae, Carnivora) is a middle-sized omnivore with excessive autumnal fattening and winter sleep. We studied adaptations of the species to boreal climate and photoperiod by following the plasma reproductive and thyroid hormone concentrations of farm-bred raccoon dogs (n=32) for 12 months. On August 16, 2000, and February 8, 2001, half of the raccoon dogs received continuous-release melatonin implants (the MEL group). The other half was sham-operated (the SHAM group). Between November 27, 2000, and January 25, 2001, half of the animals of both groups were fasted. The plasma testosterone concentrations of the MEL males peaked in February, a month earlier than in the SHAM males. Autumnal melatonin treatment also advanced the gestation period reflected by the plasma progesterone concentrations by seven weeks. Food deprivation in winter seems to accentuate the sex steroid response during the mating as the fasted males had higher testosterone concentrations than the fed males in February and March.

A comparative study of white blood cell counts and disease risk in carnivores.

In primates, baseline levels of white blood cell (WBC) counts are related to mating promiscuity. It was hypothesized that differences in the primate immune system reflect pathogen risks from sexually transmitted diseases (STDs). Here, we test for the generality of this result by examining hypotheses involving behavioural, ecological and life-history factors in carnivores. Again, we find a significant correlation in carnivores between mating promiscuity and elevated levels of WBC counts. In addition, we find relationships with measures of sociality, substrate use and life-history parameters. These comparative results across independent taxonomic orders indicate that the evolution of the immune system, as represented by phylogenetic differences in basal levels of blood cell counts, is closely linked to disease risk involved with promiscuous mating and associated variables. We found only limited support for an association between the percentage of meat in the diet and WBC counts, which is consistent with the behavioural and physiological mechanisms that carnivores use to avoid parasite transmission from their prey. We discuss additional comparative questions related to taxonomic differences in disease risk, modes of parasite transmission and implications for conservation biology.

Experimental Babesia gibsoni infection in coyotes (Canis latrans).

Four 5 mo old captive raised coyotes (Canis latrans) were experimentally inoculated with approximately 1 x 10(6) Babesia gibsoni organisms. Parasites were detected 1 wk post-inoculation in all coyotes with maximum parasitemia of 8-11% occurring at 34 wk. Parasitemias remained at or above 1% for at least 12 wk and were still detectable 20 wk post-inoculation. All experimentally infected coyotes developed pale mucous membranes, splenomegaly, and a positive heme reaction in urine while one coyote exhibited mild depression and inappetence. Infected coyotes also developed a regenerative anemia, thrombocytopenia, and neutropenia. The mild clinical signs coupled with the high level and long duration of parasitemia indicate that coyotes could serve as reservoirs for B. gibsoni. Entrance of this foreign parasite into the United States suggests the need for strict quarantines and thorough health and blood film examinations for imported animals.

Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles).

A field study was carried out on Little Island (County Waterford, Ireland) in June 2000 to evaluate the potential of a bait-marking system for use in European badgers (Meles meles). Two oral biomarkers, sulfadimethoxine (SDM) and rhodamine B, were incorporated into fishmeal baits and distributed by hand at main sets in five test territories for 3 consecutive days. In parallel, non-biomarked baits were distributed at a single control territory. The objectives of the study were to: (1) assess the effects of SDM and rhodamine B on palatability and thus bait acceptance, and (2) investigate the marking capacity of SDM and rhodamine B in serum and hair samples taken from badgers. Trapping was carried out in each territory for 5 consecutive days immediately after bait distribution. Analysis of data revealed that 90-100% of baits were removed in four of the test territories and from the control territory. In the fifth test territory, 61% of baits were removed. Of the badgers (n = 26) trapped in the test territories, 18 (69%) were positive when tested for both biomarkers. In contrast, the remaining eight animals and those captured in the control territory (n = 6 badgers) were negative. In the marked animals, the highest levels of SDM were recorded in serum samples taken soon after bait distribution. Thereafter, the levels declined in each badger over the course of the study. In contrast, rhodamine B was readily detectable by fluorescence microscopy of hair samples throughout the period of study. The results indicate that SDM and rhodamine B act as systemic markers in badgers and have potential future applications for monitoring of oral vaccine uptake.

Detection of Mycobacterium bovis infection and production of interleukin-2 by in vitro stimulation of badger lymphocytes.

The Eurasian badger (Meles meles) is considered to be an important wildlife reservoir for Mycobacterium bovis infection of cattle in Ireland and in GB. However, rapid diagnosis of tuberculosis in live badgers has been constrained through a lack of suitable immuno-diagnostic reagents for detection of M. bovis-infected animals. To date, there have been no reports of cytokine activity in badgers that might be associated with specific immune responses to M. bovis infection. In this study, nine badgers were removed from an area with a persistent tuberculosis problem in cattle herds and tuberculosis was confirmed in four of the animals by "post-mortem" examination and M. bovis culture. In preliminary investigations of interleukin-2 (IL-2) activity, we were able to demonstrate that lymphoblasts prepared from badger peripheral blood mononuclear cells (PBMCs) proliferated when cultured in the presence of human recombinant IL-2 (HrIL-2). Supernatants derived from purified protein derivative of tuberculin (PPD-bovine) stimulated PBMC cultures also induced blastogenesis of badger-derived lymphoblasts. The results demonstrate that badger lymphocytes are responsive to HrIL-2 and that PPD-bovine stimulation of badger PBMC results in production of bio-active IL-2.