DNA extraction from hair shafts of wild Brazilian felids and canids

Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the Sao Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol: chloroform: isoamyl alcohol general method, with proteinase K as digestive enzyme.

HEMOPLASMAS IN WILD CANIDS AND FEUDS IN BRAZIL

Hemotropic mycoplasmas, epicellular erythrocytic bacterial parasites lacking a cell wall, are the causative agents of infectious anemia in numerous mammalian species. The presence of hemotropic mycoplasmas in blood samples of neotropical and exotic wild canids and felids from Brazilian zoos were recorded using molecular techniques. Blood samples were collected from 146 Brazilian wild felids, 19 exotic felids, 3 European wolves (Canis lupus), and from 97 Brazilian wild canids from zoos in the Brazilian states of Sao Paulo and Mato Grosso and the Federal District. Using conventional polymerase chain reaction (PCR), this work found 22 (13%) wild felids positive to Candidatus Mycoplasma haemominutum [4 jaguars (Panthera onca); 3 pumas (Puma concolor); 10 ocelots (Leopardus pardalis); 2 jaguarondis (Puma yagouaroundi); and 3 little spotted cats (Leopardus tigrinus)]. Only one little spotted cat (Leopardus tigrinus) was positive to Mycoplasma haemofelis, and none was positive to Candidatus Mycoplasma turicensis. Two bush dogs (Speothos venaticus) were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haematoparvum, and two European wolves were positive for a Mycoplasma sp. closely related to candidatus Mycoplasma haemominutum. This is the first study regarding the molecular detection of hemotropic mycoplasmas in wild canids.

Wild canids, domestic dogs and their pathogens in Southeast Brazil: disease threats for canid conservation

Wild canids are under many pressures, including habitat loss, fragmentation and disease. The current lack of information on the status of wildlife health may hamper conservation efforts in Brazil. In this paper, we examined the prevalence of canine pathogens in 21 free-ranging wild canids, comprising 12 Cerdocyon thous (crab-eating fox), 7 Chrysocyon brachyurus (maned wolf), 2 Lycalopex vetulus (hoary fox), and 70 non-vaccinated domestic dogs from the Serra do Cip National Park area, Southeast Brazil. For wild canids, seroprevalence of antibodies to canine parvovirus, canine adenovirus, canine coronavirus and Toxoplasma gondii was 100 (21/21), 33 (7/21), 5 (1/19) and 68 (13/19) percent, respectively. Antibodies against canine distemper virus, Neospora caninum or Babesia spp. were not found. We tested domestic dogs for antibodies to canine parvovirus, canine distemper virus and Babesia spp., and seroprevalences were 59 (41/70), 66 (46/70), and 42 (40/70) percent, respectively, with significantly higher prevalence in domestic dogs for CDV (P < 0.001) and Babesia spp. (P = 0.002), and in wild canids for CPV (P < 0.001). We report for the first time evidence of exposure to canine coronavirus in wild hoary foxes, and Platynossomun sp. infection in wild maned wolves. Maned wolves are more exposed to helminths than crab-eating foxes, with a higher prevalence of Trichuridae and Ancylostomidae in the area. The most common ectoparasites were Amblyomma cajennense, A. tigrinum, and Pulex irritans. Such data is useful information on infectious diseases of Brazilian wild canids, revealing pathogens as a threat to wild canids in the area. Control measures are discussed.

Comparative analysis of rabies virus isolates from Brazilian canids and bats based on the G gene and G-L intergenic region

Rabies virus (RABV) isolates from two species of canids and three species of bats were analyzed by comparing the C-terminal region of the G gene and the G-L intergenic region of the virus genome. Intercluster identities for the genetic sequences of the isolates showed both regions to be poorly conserved. Phylogenetic trees were generated by the neighbor-joining and maximum parsimony methods, and the results were found to agree between the two methods for both regions. Putative amino acid sequences obtained from the G gene were also analyzed, and genetic markers were identified. Our results suggest that different genetic lineages of RABV are adapted to different animal species in Brazil.

Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Anticorpos anti Brucella abortus, anti Brucella canis e anti Leptospira spp. em raposas (Pseudalopex vetulus) do semiárido paraibano, Nordeste do Brasil

O objetivo do presente trabalho foi determinar a ocorrência de anticorpos anti Brucella abortus, anti Brucella canis e anti Leptospira spp. em raposas (Pseudalopex vetulus). Para tanto, foram utilizadas 60 raposas atropeladas em rodovias no semiárido da Paraíba, Nordeste do Brasil. Para a detecção de anticorpos anti Brucella abortus, o teste do antígeno acidificado tamponado (AAT) foi empregado como teste de triagem, e a prova do 2-mercaptoetanol foi empregada como método confirmatório. Para o diagnóstico sorológico das infecções por Brucella canis e Leptospira spp., foram utilizados os testes de imunodifusão em gel de agar (IDGA) e soroaglutinação microscópica, respectivamente. Todas as amostras foram negativas na pesquisa de anticorpos anti Brucella canis e anti Leptospira spp. Das 60 raposas testadas, 16 (26,6%) foram positivas para anticorpos anti Brucella abortus no teste de AAT, e quatro (6,7%) amostras foram confirmadas no teste de 2-mercaptoetanol, sendo duas amostras com título 100 e duas com título 50.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Differential diagnosis of oocysts of Hammondia-like organisms of dogs and cats by PCR-RFLP analysis of 70-kilodalton heat shock protein (HSP70) gene

Organisms of the genera Toxoplasma, Hammondia and Neospora, the Hammondia-like organisms, are closely related coccidian with similarly sized oocysts. Therefore, a diagnosis based on microscopy of oocysts in feces is not a method of choice for species identification of these important parasites. In this paper, we present a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method to differentially diagnose oocysts of Toxoplasma gondii from oocyst of Hammondia hammondi. Another PCR-RFLP was designed to differentiate oocysts of Hammondia heydorni from oocysts of Neospora spp. Both PCR-RFLP are based on nucleotide sequences of the Hsp70 coding gene. In conclusion, we presented two alternative molecular diagnostic assays that can be successfully applied for the differentiation of oocysts of Hammondia-like organisms shed by felids and canids.

Crab-eating fox (Cerdocyon thous), a South American canid, as a definitive host for Hammondia heydorni

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15 mu m, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70 kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. (C) 2009 Elsevier B.V. All rights reserved.