Serosurvey for antibodies against Brucella abortus and Leptospira interrogans in pampas deer from Brazil

A survey for antibodies against Brucella abortus, and Leptospira interrogans was conducted on 17 pampas deer (Ozotocerus bezoarticus) from Pantanal Matogrossense (State of Mato Grosso do Sul, Brazil) and on 24 pampas deer from Parque Nacional de Emas (State of Goiás, Brazil). Antibodies against B. abortus were detected by plate agglutination, rose Bengal, and complement fixation tests; antibodies against Leptospira interrogans were detected by the microscopic agglutination test. All sera were negative for B. abortus antibodies and all deer sera from Parque Nacional de Emas were negative for L. interrogans antibodies. Four (24%) of 17 sera from Pantanal Matogrossense were positive for L. interrogans serovar (n = 2) hardjo, wolffi (n = 1) and mini (n = 1). While these diseases do not appear to be of major importance to the health status of Pampas deer, it appears that deer are reservoir for leptospirosis in one of the study areas.     

Health evaluation of pampas deer (Ozotoceros bezoarticus celer) at Campos del Tuyú Wildlife Reserve, Argentina

Samples from 14 free-ranging pampas deer (Ozotoceros bezoarticus celer) were collected in 1995 and 1998, at Campos del Tuyú Wildlife Reserve, Buenos Aires, Argentina. Hematology, serum chemistries, minerals and metals, and fecal parasites were analyzed. In addition, fecal ova and parasites were evaluated seasonally during 1998-2000. Serology for infectious diseases included blue-tongue, brucellosis, bovine respiratory syncytial virus infection, bovine viral diarrhea/mucosal disease, infectious bovine rhinotracheitis, Johne's disease (paratuberculosis), foot and mouth disease (FMD), leptospirosis (eight serovars), epizootic hemorrhagic disease, and parainfluenza-3 (PI-3). Three (21%) pampas deer had antibodies to Leptospira spp. and six (43%) to PI-3 virus. Serologic results for all other infectious agents were negative. Domestic cattle (n = 27) included in this study for comparison had antibodies to Leptospira, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, and PI-3 virus (74-100% of tested animals) and one animal (4%) to Brucella sp. All cattle had antibodies to FMD virus attributable to vaccination. This study provides the first data on the health status of the southernmost sub-species of pampas deer.     

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.     

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples.     

Recombinant ligA for leptospirosis diagnosis and ligA among the Leptospira spp. clinical isolates

Rapid diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality is imperative for proper treatment. During infection, the pathogenic Leptospira spp. express virulence factors which induce antibody responses in the infected host. In this study, 50 referenced Leptospira spp. belonging to six genomospecies and 10 L. interrogans clinical isolates were studied for the presence of a gene encoding an in vivo expressed, surface exposed, immunoglobulin-like protein, LigA, by using PCR and southern hybridization specific to the 5' terminus sequence of the DNA. LigA was also detected in the Leptospira spp. whole cell homogenates by a direct ELISA using a mouse antiserum to the C-terminal portion of recombinant LigA (cLigA) as a detection reagent. All pathogenic Leptospira spp. except one of the two strains of L. santorasai were positive for the gene and its phenotype while all of the L. borgpetersenii and L. biflexa strains were negative. Recombinant cLigA was used as an antigen in ELISAs for detecting IgM and IgG in the sera of leptospirosis patients and in the sera of patients with other febrile illnesses and healthy subjects. When acute phase sera were tested by the cLigA IgM- and IgG-ELISAs, 92% and 100% of the MAT-positive sera were positive, respectively. The diagnostic sensitivity was 100% when both IgM- and IgG-ELISAs were performed on the same acute phase sera and the results were combined. Acute and convalescence sera of patients who were Leptospira culture positive but MAT/IgM-dipstick negative gave 88% and 100% positives by combined cLigA IgM/IgG ELISAs. The diagnostic specificities for the cLigA IgM- and IgG-ELISAs were 98% and 100%, respectively. Our cLigA based-serology has a high potential for early diagnosis of leptospirosis especially when the culture and MAT results are not yet available.     

Home range and habitat selection of pampas deer

The southernmost subspecies of pampas deer Ozotocerus bezoarticus celer is an endemic and endangered cervid of the Argentine Pampas. The aim of our study was to describe the habitat use of this deer on the coast of Samborombón Bay. Twelve adult pampas deer (seven female and five male) were radiotracked and their home-range sizes and habitat selection studied from 1995 to 2001. The mean home-range size was 898±181 ha, and the core area was concentrated in 22% of their range. The home-range size of males was three times larger than that of females (1422 vs. 523 ha). Deer home ranges overlapped extensively. No sex differences were found regarding habitat selection. Celtis tala forests and Spartina densiflora grasslands were used more than expected by their availability, while wetlands, coastal grasslands and Salicornia ambigua beaches were avoided. Their habitat selection was affected by cattle presence, suggesting avoidance: they tended to use areas free of cattle, and their home ranges were larger when cattle were absent. An action plan for this endangered population of pampas deer should include initiatives involving private landowners in pampas deer conservation, the use of fire and cattle grazing management tools to improve deer habitat, and studies to provide biological and health data related to pampas deer coexistence with cattle.     

Cross-amplification tests of ungulate primers in the endangered Neotropical pampas deer (Ozotoceros bezoarticus)

In cross-species amplification tests of 15 ungulate primers in pampas deer, five were retained to form a small panel of highly polymorphic loci that could be used to efficiently screen populations of this endangered species. The polymerase chain reactions were performed incorporating the universal fluorescent labeled M13 (-21) primer. In 69 pampas deer, average allelic diversity was 15, expected heterozygosity was 0.869 and the mean polymorphic information content value was 0.847. Paternity exclusion probabilities over loci were NE-1P = 0.01336 and NE-2P = 0.00135, and combined non-exclusion probability of identity was P(ID) = 3 x 10(-8).     

Exposure to Leptospira in stray dogs in the city of Cali

In Colombia, little information is available concerning the epidemiology of leptospirosis in urban environments. Furthermore, the role of dogs in the transmission cycle of leptospirosis in the urban setting is unclear. To explore the potential role of canines in the transmission of leptospirosis in Cali, a serological study was conducted with 197 serum samples collected from stray dogs during 2001 and 2003. Serum specimens were screened with the Microscopic Agglutination Test (MAT) and 7 serovars--Icterohaemorrhagiae, Canicola, Gryppotyphosa, Hardjo strain Hardjobovis, Pomona, Hardjo strain Hardjoprajitno and Bratislava. All serovars were provided by the Instituto Colombiano Agropecuario (ICA), Tuluá, Colombia. The MAT was considered positive when 50% or more leptospiras were agglutinated with one or more serovars in a serum dilution of 1:100. At least one serovar showed evidence of infection in 41.1% of the dogs. The most prevalent serovar was Icterohaemorrhagiae, found in 55.6% of the seropositive dogs. 48.1% were co-agglutinations. No reactions against the serovars Pomona, Hardjo strain Hardjoprajitno and Bratislava were observed. These findings suggested that stray dogs are potential reservoirs of Leptospira in Cali and underscored the need to study the epidemiology of this disease in Colombia.     

Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California

One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona.     

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in S?o Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.     

A serological survey of Australian wildlife for antibodies to Leptospires of the Hebdomadis serogroup.

A serological survey for antibodies to Leptospira interrograns serovar hardjo was conducted on 574 serum samples from 10 native and 4 introduced wildlife species in south-eastern Australia. The microscopic agglutination (MA) test was used, and titres to hardjo antigen were detected in 33.5% of 352 brushtailed possums (Trichosurus vulpecula) sampled in several areas of Victoria. Prevalence of reactors ranged from 14 to 66% in 4 populations examined intensively. Serovar balcanica was isolated from possums with hardjo antibodies from two different areas. Of 20 wombats Vombatus ursinus) examined in Victoria, antibodies to hardjo were found in sera from 4 and titres to Pyrogenes and Pomona serogroups were detected in another. Hardjo antibodies were demonstrated in sera from 13 of 19 rusa deer (Cervus timorensis). Negative MA test results to hardjo antigens were recorded in 55 mountain possums (T. caninus), 63 macropods (Macropus spp.), 17 water rats (Hydrmys chrysogaster), 39 fallow deer (Dama dama), 2 hog deer (Axis porcinus) and 2 water buffalo (Bubalus bubalus). No MA antibodies to any of 16 leptospiral serogroups were detected in 17 water rats tested. Kidneys were examined from 330 of these animals and focal interstitial nephritis suggestive of leptospirosis was found in kidneys of 63 of 169 T. vulpecula, 3 of 55 T. caninus, 12 of 18 V. ursinus, 6 of 22 Macropus spp., 9 of 16 H. chrysogaster, 5 of 11 C. timorensis and 3 of 39 D. dama. A statistical association between focal interstitial nephritis and MA antibodies to hardjo was found in T. vulpecula.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Discovery of an undescribed protostrongylid nematode from the endangered pampas deer (Ozotoceros bezoarticus celer) in Argentina

Dorsal-spined protostrongylid nematode larvae (Metastrongyloidea: Protostrongylidae) were recovered from the feces of the endangered pampas deer (Ozotocerus bezoarticus celer) in Campos del Tuyú Wildlife Reserve, Bahia Samborombón, Argentina. Partial DNA sequences from the large subunit ribosomal RNA (LSU rRNA) gene and from the second internal transcribed spacer region (ITS2) were amplified, cloned, sequenced, and compared to those of other nematodes. Nucleotide alignment and phylogenetic analysis of the sequences indicate that this protostrongylid nematode is most closely related to Parelaphostrongylus spp. as inferred from the LSU rRNA sequence analysis. Analysis of the ITS2 spacer indicated that the pampas deer protostrongylid is nested in a clade containing Parelaphostrongylus and Elaphostrongylus spp. These sequences differed considerably from those of other protostrongylid nematodes, and were most similar to those of Parelaphostrongylus spp. and Elaphostrongylus spp. in spite of clear variability from both genera. These results suggest that the protostrongylid from pampas deer is an undescribed nematode that likely belongs in the subfamily Elaphostrongylinae.     

Selenium status and antibodies to selected pathogens in white-tailed deer (Odocoileus virginianus) in Southern Minnesota

To determine exposure to a variety of infectious diseases potentially important for native ungulates, livestock, and humans, serum samples from 114 (94 adults, 20 fawns) female white-tailed deer (Odocoileus virginianus) were collected during January 2000-03 from multiple locations in southeast (SE) and southwest (SW) Minnesota. Antibody prevalence was determined for the following pathogens: Mycobacterium avium subsp. paratuberculosis, Leptospira interrogans (six serovars), Anaplasma marginale, Borrelia burgdorferi, Brucella abortus, epizootic hemorrhagic disease virus, and bovine viral diarrhea virus (BVDV) types 1 and 2. Samples collected in 2001 were screened for antibodies against Anaplasma phagocytophilum, and whole blood was submitted for polymerase chain reaction (PCR) testing for A. phagocytophilum and B. burgdorferi. In addition, serum selenium concentrations were evaluated for samples collected during 2001-03. Antibody prevalence and selenium concentration were compared by age-class and geographic region. Antibodies to all of the infectious agents except A. marginale and B. abortus were detected; when detected, antibody prevalence was highest in adults. Deer collected from SE Minnesota had a higher antibody prevalence to B. burgdorferi than SW deer. Blood culture and PCR results for A. phagocytophilum and B. burgdorferi were negative. Antibodies against BVDV (combined types 1 and 2) were more prevalent (chi(2) = 3.617, P< or = 0.029) in deer collected in SW (41%) than in SE (25%) Minnesota. No statistically significant differences in serum selenium concentrations were detected when data were analyzed by age-class or by geographic location.     

The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka,Japan

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi‐solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira‐positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi‐solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi‐solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.     

Prevalence of agglutinating antibodies to Toxoplasma gondii in adult and fetal mule deer (Odocoileus hemionus) from Nebraska

Toxoplasma gondii is an apicomplexan parasite of mammals and birds. Herbivores acquire postnatal infection by ingesting oocysts from contaminated food or water. Toxoplasma gondii infection is common in white-tailed deer, Odocoileus virginianus, but little is known about the prevalence of infection in mule deer, O. hemionus. We examined sera from 89 mule deer from Nebraska for agglutinating antibodies to T. gondii using the modified direct agglutination test (MAT) with formalin-fixed tachyzoites as antigen. Thirty-one (35%) of the samples were positive at dilutions of > or = 1:25. Samples were examined from 29 fetuses from these mule deer and none were positive in the MAT. Sera from 14 white-tailed deer from Nebraska were also examined and 6 (43%) were positive for T. gondii. Samples were examined from 5 fetuses from these white-tailed deer and none was positive in the MAT. Our results in both deer species from Nebraska are similar to studies conducted in white-tailed deer from other regions of the United States. Our findings indicate that mule deer are frequently infected with T. gondii and that mule-deer meat may be a source of human infection.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

Estimating the true prevalence of Mycobacterium bovis in hunter-harvested white-tailed deer in Michigan

Apparent prevalence, although useful as a consistent index, may underestimate the true prevalence of disease. In Michigan, the ability to estimate the true prevalence of bovine tuberculosis (TB; caused by Mycobacterium bovis) in free-ranging white-tailed deer (Odocoileus virginianus) will become increasingly important to accurately assess progress towards eradication. Our objectives were threefold: to estimate the true prevalence of M. bovis in free-ranging deer in Michigan, to evaluate the effectiveness of existing TB surveillance methods, and to indirectly assess whether TB epidemiologic data from captive cervid herds can be meaningfully extrapolated to free-ranging populations. The study population consisted of all free-ranging deer submitted for TB testing in 2001 from six townships in northeastern Lower Michigan. Tissue samples of tonsil and cranial lymph nodes were collected bilaterally from all deer eligible for the study that did not have gross lesions suggestive of TB (n = 701). Samples were subjected to histopathologic, acid-fast (AF) staining, mycobacterial culture, and polymerase chain reaction (PCR) testing. Seven deer cultured positive for M. bovis that would not have been detected by current surveillance, yielding apparent and true prevalence estimates (95% confidence limits) of 2.7% (1.6, 3.8) and 3.6% (2.3, 4.9), respectively. The sensitivity, specificity, and positive and negative predictive values of the current surveillance protocol were 75, 100, 100, and 99%, respectively. Histologic lesions were present only in tonsils, and ranged from simple necrosis to caseation, suppuration, and granuloma formation. Acid-fast staining and PCR detected M. bovis in only one of the seven culture-positive deer. Our study provides the first estimate of the true prevalence of M. bovis in Michigan's free-ranging deer population and suggests modest underestimation of that prevalence by current surveillance. This study also suggests that caution is warranted when extrapolating epidemiologic data on TB in captive cervids to free-ranging populations and confirms the pivotal role of the tonsil in early infections.     

Molecular detection of coccidian Apicomplexa Parasites isolated from wild crab-eating and pampas foxes through novel TaqMan™ probes: a contribution to their molecular epidemiology

Neospora caninum, Toxoplasma gondii and Hammondia spp. are coccidian parasites similar in morphology. Molecular techniques are necessary to detect parasite DNA isolated from stool samples in wild canids because they were reported as definitive hosts of N. caninum life cycle. The objective of this study was to develop a highly sensitive and accurate molecular method for the identification of coccidian Apicomplexa parasites in crab-eating fox (Cerdocyon thous) and pampas fox (Lycalopex gymnocercus). Tissue samples from road-killed animals (pampas fox?=?46, crab-eating fox?=?55) and feces (pampas fox?=?84, crab-eating fox?=?2) were collected, and species were diagnosed through molecular assay. PCR was used for the amplification of a fragment of the coccidian Apicomplexa nss-rRNA gene. Additionally, we developed a novel real-time PCR TaqMan? probe approach to detect T. gondii- Hammondia spp. and N. caninum. This is the first report of N. caninum DNA in pampas fox feces (n?=?1), thus it was also detected from pampas fox tissues (n?=?1). Meanwhile, T. gondii was found in tissues of pampas (n?=?1) and crab-eating (n?=?1) foxes and H. triffittae in one crab-eating fox tissue. Despite the low percentage (2.5%) of positive samples, the molecular method developed in this study proved to be highly sensitive and accurate allowing to conduct an extensive monitoring analysis for these parasites in wildlife.

     

Antibody responses to Four Corners hantavirus infections in the deer mouse (Peromyscus maniculatus): identification of an immunodominant region of the viral nucleocapsid protein.

Antibody responses to Four Corners hantavirus (FCV) infections in the deer mouse (Peromyscus maniculatus) were characterized by using FCV nucleocapsid protein (N), glycoprotein 1 (G1), and glycoprotein 2 (G2) recombinant polypeptides in Western immunoblot assays. Strong immunoglobulin G reactivities to FCV N were observed among FCV-infected wild P. maniculatus mice (n = 34) and in laboratory-infected P. maniculatus mice (n = 11). No immunoglobulin G antibody reactivities to FCV G1 or G2 linear determinants were detected. The strongest N responses were mapped to an amino-proximal segment between amino acids 17 and 59 (QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG). FCV N antibodies cross-reacted with recombinant N proteins encoded by Puumala, Seoul, and Hantaan viruses.