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Leishmaniosis is a zoonosis caused by protozoa of the genus Leishmania. American cutaneous leishmaniosis (ACL) is mainly caused by the species L. amazonensis and L. braziliensis, and American visceral leishmaniosis (AVL) is caused by L. infantum chagasi. In addition to their proven roles as reservoirs of AVL, dogs are also suspected by researchers to be reservoirs of ACL due to reports of this infection in domestic environments and of infected dogs in endemic areas. The aim of this study was to detect Leishmania sp. infection in dogs from Vila Operária, Buerarema, Bahia, using parasitological tests, indirect immunofluorescent assay (IFA) and polymerase chain reaction (PCR). Furthermore, this study also aimed to identify risk factors associated with illness in dogs in this locality by conducting an epidemiological survey. For this purpose, 292 dogs were clinically evaluated for the presence of skin lesions, and the dogs that showed these changes were submitted to scarification injury to enable preparation of slides for microscopic study of amastigotes. Subsequently, the dogs underwent blood sampling for serological (IFA) and molecular (PCR) tests. Additionally, the owners of the dogs answered an epidemiological questionnaire to facilitate the identification of risk factors for exposure of dogs to pathogens of ACL. Of the 292 dogs studied, 13 (4.5%) had lesions suggestive of ACL, but with a negative parasitological examination and 147 (50.3%) were seropositive according to the IFA. Of the 273 dogs studied using PCR test, 10 (3.66%) were positive for L. braziliensis, and all samples were negative for L. infantum chagasi. Wastelands in the peridomicile and the presence of light in the household were risk factors associated with ACL. The results show that Vila Operária has asymptomatic dogs with ACL and that the detection sensitivity of the IFA was higher than that of PCR for the infected dogs.

Strains of Toxoplasma gondii in Brazil are highly genetically diverse compared to strains from North America and Europe. Dogs are epidemiologically important because they act as sentinels for T. gondii infections in humans and are good indicators of environmental contamination. The aim of this study was to isolate and genetically characterize T. gondii strains from tissues of naturally infected Brazilian dogs. For this study, 21 blood samples were collected from dogs at the Zoonosis Control Centers of Ilhéus and Itabuna cities, Bahia, Brazil. The sera were examined for T. gondii antibodies using the indirect hemagglutination test. Brains and hearts of seropositive dogs were bioassayed in mice to isolate and characterize T. gondii parasites by PCR-RFLP using 10 genetic markers (SAG1, newSAG2, SAG3, BTUB, c22-8, c29-2, GRA6, PK1, APICO, and L358). However, T. gondii was isolated from only 4 (57.1%) dogs, designated TgDgBr6, 13, 17, and 21. All strains were virulent, causing clinical changes (rough hair coat, lethargy, and abdominal distention) and the death of all mice within 8–20 days after inoculation. Genetic analysis of these 4 T. gondii isolates revealed 4 distinct genotypes with different clonal lineage combinations (types I, II, and III) and 2 atypical alleles. Using PCR-RFLP with several markers, this study contributes to evaluations of the genetic diversity of strains circulating in Brazil.

This study genetically Toxoplasma gondii isolates obtained from pigs intended for human consumption in northeastern Brazil; multilocus PCR-RFLP and sequencing techniques were utilized. Bioassays were conducted using the brain and tongue of 20 pig heads purchased at butcher shops in the city of Ilheus, Bahia, Brazil. Overall, 11 T. gondii isolates designated TgPgBr06-16 were identified. Application of multilocus PCR-RFLP with seven molecular markers (SAG1, SAG2, SAG3, BTUB, C22-8, PK1 and Apico) identified six different genotypes. Isolates TgPgBr 06, 08, 11, 12, 14 and 15 were indistinguishable by this technique, forming a single genotype; the remaining isolates were characterized as distinct genotypes. However, when five genetic markers (SAG1, SAG2, SAG3, BTUB and c22-8) were employed in multilocus PCR-sequencing, all eleven strains of T. gondii were shown to be different. All isolates differed from Type I, II and III clonal genotypes using both genotyping techniques. These results demonstrate that the multilocus PCR-RFLP assay underestimated the true diversity of the T. gondii population in this study. Thus, DNA sequencing is the preferred technique to infer the genetic diversity and population structure of T. gondii strains from Brazil. Moreover, it is necessary to develop new molecular markers to group and characterize atypical T. gondii isolates from South America.

Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi, is an anthropozoonosis of great importance for public health. Several species of mammals are reservoirs for this parasite, including dogs. The objective of this work was to verify the natural infection by T. cruzi in the population of dogs of the municipality of Ituberá, Bahia. A total of 392 domiciled dogs from all districts of the city were evaluated; five milliliters of blood was collected from the dogs for molecular diagnosis. T. cruzi DNA was amplified through the polymerase chain reaction (PCR); the primers P35 and P36, which amplify a fragment of 330 bp, were used. The PCR products were subjected to 2% agarose gel electrophoresis containing Sybr (Invitrogen). Of the 392 dogs evaluated, only 2 (0.51%) animals, one male and one female, tested positive for T. cruzi. This study concluded that there are dogs naturally infected by T. cruzi in the municipality of Ituberá-Bahia and that this finding is an alert to veterinarians, health professionals, and local health authorities that their own dogs can act as reservoirs of the disease. A Doença de Chagas, causada pelo protozoário flagelado Trypanosoma cruzi, é uma antropozoonose de grande importância para a saúde pública. Diversas espécies de mamíferos são reservatórios do parasita, incluindo o cão. O objetivo deste trabalho foi verificar a infecção natural por T. cruzi na população de cães do município de Ituberá, Bahia. Foram avaliados 392 cães domiciliados, em todos os bairros do município, dos quais foram coletados 5mL de sangue para realização do diagnóstico molecular. A amplificação do DNA de T. cruzi foi através da Reação em Cadeia da Polimerase (PCR), com os primers P35 e P36, que amplificam um fragmento de 330 pb. Os produtos das PCRs foram submetidos à eletroforese em gel de agarose a 2% contendo Sybr (Invitrogen®). Dos 392 cães avaliados, apenas 2 (0,51%) animais, um macho e uma fêmea, foram positivos no diagnóstico molecular de T. cruzi. Conclui-se com esse estudo que há cães naturalmente infectados pelo T. cruzi no município de Ituberá-Bahia e que este achado constitui um alerta aos veterinários, profissionais da saúde e autoridades sanitárias locais, cujos cães podem atuar como reservatórios da doença.

The objective of the present article was an epidemio- logical and molecular study of Ehrlichia canis in dogs of Ilheus and Itabuna in Bahia, as well as an evaluation of associated risk factors. Blood samples were collected from 153 dogs and DNA was extracted and analyzed by the nested-polymerase chain reaction, us- ing one pair of primers to detect Ehrlichia bacteria and another pair to detect the presence of E. canis. Of the 153 animals, 12 (7.8%) were polymerase chain reaction-positive for E. canis, indicating the presence of the parasite in dogs of the Ilheus-Itabuna microregion. The associated risk factors were exposure to tick-infested habitats and the fact that the dogs lived in the countryside.

In Brazil, canine hepatozoonosis is a tick-borne subclinical hemoparasitosis caused by a protozoa Hepatozoon canis and is highly prevalent in dogs in rural areas. An epizootiological study was conducted to investigate the prevalence of H. canis in the canine population of Ituberá, Bahia, and to analyze any associated risk factors. Blood samples were collected from 380 dogs and determined the presence of the protozoan by performing capillary blood smear and polymerase chain reaction (PCR). Epizootiological data were collected by asking dog owners to answer a structured questionnaire. H. canis gamonts were not detected in the blood smears. However, PCR detected H. canis in 163/380 (42.9%) dogs examined. Physical examination and anamnesis indicated 105 (64.4%) positive asymptomatic dogs. Hematological alterations were observed in 115 (70.5%) infected dogs. No clinical, hematological, or epizootiological variable was found to be significantly associated to the infection. In conclusion, the high prevalence of H. canis infection in local dogs may be because of the peri-urban features of this municipality. Moreover, to the best of our knowledge, this study the first study to report H. canis infection in the State of Bahia.

Leishmaniasis is a disease caused by protozoa of the genus Leishmania. Two distinct forms are recognized: visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). In the Americas, the causative agent of VL is L. infantum chagasi, whereas L. braziliensis is principally responsible for CL. Domestic dogs constitute the main source of VL in urban environments, and have also been implicated in CL epidemiology. We carried out molecular and serological surveys to detect Leishmania infection in dogs from the municipality of Ituberá in Bahia, Brazil. Furthermore, we identified risk factors associated with illness in dogs from this locality. Blood samples were collected from 399 dogs and tested using an indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR) to detect Leishmania spp antibodies and L. infantum chagasi and L. braziliensis DNA, respectively. Dogs were clinically evaluated and tissue samples from those exhibiting skin lesions were examined for parasites. In addition, the dog owners completed an epidemiological questionnaire to identify factors associated with infection. Skin lesions consistent with CL were found on 37 (9.3%) of the evaluated animals, but parasitological examination was negative for all samples. The IFA returned positive results for 60 (15%) dogs. PCR identified DNA from L. braziliensis in 86 (21.6%) animals, where as all samples proved negative for L. infantum chagasi. The 134 dogs (33.6%) testing positive using IFA and/ or PCR were considered infected, and of these, only 13 demonstrated skin lesions. Animals from rural areas were 3.39-times more likely to be infected compared to those in urban environments.

Ehrlichiosis is a zoonotic disease that is caused by bacteria of the genus Ehrlichia. The aims of this study were to detect the presence of Ehrlichia spp. in the blood of dogs in Ituberá, Bahia, and to compare the sensitivities and specificities of blood smear, serological, and molecular examinations. Furthermore, this study identified factors associated with exposure to the agent in dogs in this locality. Blood samples were collected from 379 dogs and submitted for indirect immunofluorescent assay and polymerase chain reaction testing for the detection of Ehrlichia spp. antibodies and DNA, respectively. Additionally, a peripheral blood smear was obtained from the ear tip for parasite identification. Of the 379 animals, 12.4%, 32.7%, and 25.6% were identified as positive on the blood smear, serological, and molecular tests, respectively. The dogs positive in one of the three techniques were considered exposed (46.9%). Younger dogs and rural habitat were protective factors and presence of ticks and contact with other dogs were the risk factors associated with exposure to the agent. It was concluded that dogs of Ituberá have high positivity for Ehrlichia spp. and that the diagnostic methods used for detection are complementary. Erliquiose é uma doença zoonótica causada por bactérias do gênero Ehrlichia. O objetivo desse estudo foi detectar a presença de Ehrlichia spp. no sangue de cães em Ituberá-BA, e comparar as sensibilidades e especificidades do esfregaço sanguíneo, e testes sorológico e molecular. Além disso, esse estudo identificou fatores associados com a exposição ao agente em cães desta localidade. Amostras de sangue foram coletadas de 379 cães e submetidas à Reação de Imunofluorescência Indireta e Reação em Cadeia de Polimerase para detecção de anticorpos e DNA de Ehrlichia spp., respectivamente. Adicionalmente, sangue periférico de ponta de orelha foi coletado para identificação do parasita. Dos 379 animais, 12,4%, 32,7% e 25,6% foram identificados como positivos no esfregaço sanguíneo, teste sorológico e molecular, respectivamente. Cães positivos em uma das três técnicas foram considerados expostos (46,9%). Cães mais novos e hábitat rural foram fatores de proteção e presença de carrapatos e contato com outros cães foram os fatores de risco associados à exposição ao agente. Foi concluído que, os cães do município de Ituberá têm alta positividade para Ehrlichia spp. e que os diferentes métodos diagnósticos utilizados para sua detecção são complementares.

Recent studies have demonstrated that strains of Toxoplasma gondii in Brazil are frequently different from those detected in other countries, thus making an accurate phylogenetic analysis difficult. The aim of this study was to genetically characterize T. gondii samples from sheep raised in southern Bahia and intended for human consumption, by means of PCR–RFLP and sequencing techniques. Experimental samples were obtained from 200 sheep brains purchased at butcher's shops in Itabuna, Bahia, Brazil. In total, three samples (#54, #124 and #127) were T. gondii-positive. The application of multilocus PCR–RFLP using ten molecular markers (SAG1, SAG2, SAG3, BTUB, c22-8, PK1, GRA6, L358, c-29-2 and Apico) revealed a single genotype common to all samples of this study, which differed from any other published T. gondii genotypes. An atypical allele was detected in the L358 genetic marker; this has not previously been shown in any other South American T. gondii isolates. Phylogenetic analysis on the sequences from multilocus PCR sequencing revealed that these three samples were classified into the same lineage. Extensive indel regions were detected in the Apico genetic marker. Together, our findings revealed a new Brazilian T. gondii genotype. Further research should be conducted to enrich the database of Brazilian T. gondii genotypes from different regions. This will make it possible to understand the phylogenetic relationship between isolates.

In order to verify the Trypanosoma cruzi infection in domestic domiciled dogs in a rural endemic area from the south region of the State of Bahia, Polymerase Chain Reaction (PCR) were performed using S35 and S36 primers in 272 dogs living in the district of Vila Operaria, in the municipality of Buerarema. All animals were clinically evaluated; 2.5 mL of blood were collected through venipuncture for the performance of molecular tests. None of these animals showed clinical signs of the illness and only two were identified with the DNA parasite. This result is the first report of natural infection by T. cruzi in domestic dogs in southern Bahia. Com o objetivo de verificar a infecção porTrypanosoma cruzi em cães domésticos domiciliados em área rural e endêmica do sul da Bahia, foi realizada a Reação em Cadeia da Polimerase (PCR), utilizando-se os iniciadores S35 e S36 em 272 cães domiciliados no distrito da Vila Operária, cidade de Buerarema. Todos os animais foram avaliados clinicamente e, posteriormente, foram coletados 2,5 mL de sangue por punção venosa para realização do diagnóstico molecular. Nenhum dos animais apresentou manifestação clínica da doença e, em apenas dois foram identificados DNA do parasito. Esse resultado é o primeiro relato de infecção natural por T. cruzi em cães domésticos no sul baiano.

This study aimed to compare the sensitivity of five diagnostic methods commonly used for the detection of Toxoplasma gondii in tissues of naturally infected pigs. We purchased 20 heads of pigs in butcher shops in the city of Ilheus, Bahia. Brain and tongue fragments were taken from each animal for the performance of PCR against T. gondii. The rest of these two tissues were processed and inoculated into three mice. These rodents were observed for 42 days and euthanized. We prepared slides with brain and lungs of each mouse for the visualization of T. gondii. From the tissues of mice, we carried out polymerase chain reaction (PCR), histopathology, and immunohistochemistry in an attempt to identify the parasite. The PCR direct from the tissue of pigs showed 10% (2/20) of positive samples, all from the brain. PCR in tissue from mice found that 55% (11/20) of pigs were positive: 55% (11/20) and 45% (9/20) for brains and tongues, respectively. Mice were inoculated with material obtained from the samples and examined by various methods for resulting Toxoplasma infection (bioassay). Cyst detection in bioassay mice identified 25% (5/20) and immunohistochemistry 30% (6/20) of the samples pigs as positive for T. gondii. Histopathology of mice tissue could not detect parasite; only suggestive pathological changes such as inflammation with foci of necrosis were seen. The results indicated PCR of mice tissue as the most sensitive among those tested.

We used PCR to test various protocols and define a technique for DNA extraction directly from chicken-shed stool samples for the identification of Eimeria species that parasitize birds. It was possible to extract and amplify DNA of seven Eimeria species from field stool samples, using both protocols tested; extractions made with phenol/chloroform protocols gave the best results. The primers were specific and sensitive, allowing amplification of samples containing as few as 20 oocysts, both in individual and in a multiplex PCR. Individualized PCR with the phenol/chloroform DNA extraction protocol detected a larger number of Eimeria species. Molecular diagnosis was found to be practical and precise, and can be used for monitoring and epidemiological studies of Eimeria. Made available in DSpace on 2018-01-26T23:36:59Z (GMT). No. of bitstreams: 1 APIMoleculardiagnosis.pdf: 661266 bytes, checksum: 8327ce0f55733a20b1b1bc705ecdbde1 (MD5) Previous issue date: 2011-12-02

The aim of this work was to study the clinical disorders and risk factors of canine ehrlichiosis in Ilhéus and Itabuna, Bahia, and compare different diagnostic methods. Blood samples were collected from 200 dogs. Each dog was clinically examined. A questionnaire was used to evaluate the risk factors. The blood samples were analyzed using the Dot-ELISA test; hematometry, platelet counts and searches for morulae on blood smears were performed. Nested PCR was carried out on 50 serologically positive samples and 50 negative samples. Three positive PCRs were sequenced. Thirty-six percent were serologically positivity and 5.5% from blood smears. The animals were anemic and thrombocytopenic. Presence of ticks and living in areas on the urban periphery were considered to be risk factors (p < 0.05). Nested PCR identified 11 positive dogs of which nine were serologically positive and two were negative. The DNA sequencing was consistent with the presence of Ehrlichia canis. Objetivou-se com este trabalho estudar as alterações clínicas, fatores de risco da ehrlichiose canina nos municípios de Ilhéus e Itabuna, Bahia, e comparar diferentes métodos de diagnóstico. Amostras de sangue foram coletadas de 200 cães e cada animal foi examinado clinicamente. Foi preenchido um questionário para avaliar os fatores de risco. As amostras de sangue foram analisadas pelo teste Dot-ELISA e foram realizadas hematimetria, contagem de plaquetas e procura de mórulas em esfregaço de sangue. Nested-PCR foi realizada em 50 amostras positivas e 50 negativas na sorologia. Três amostras PCRs positivas foram seqüenciadas. Foi encontrado 36,0% de positividade na sorologia e 5,5% nos esfregaços sanguíneos. Os animais apresentavam anemia e trombocitopenia. Ter carrapatos e residir em áreas suburbanas foram considerados fatores de risco (p < 0,05). A Nested-PCR identificou 11 cães positivos, sendo 9 com sorologia positiva e 2 negativos. O sequenciamento de DNA foi compatível com a presença de Ehrlichia canis.