Diagnosis and epidemiology of canine leishmaniasis in southeastern Bahia, Brazil.

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.

Canine hepatozoonosis in southeastern Bahia, Brazil.

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.

Leishmania (Viannia) braziliensis in dogs in Brazil: epidemiology, co-infection, and clinical aspects.

Leishmaniasis is an endemic disease present in 98 countries. In Brazil, the northeast region accounts for approximately half of the cases in humans, and has experienced an increased number of positive cases in dogs. In this study, we investigated the epidemiology of canine leishmaniasis in the city of Ilhéus, Bahia, using serological and molecular techniques and evaluated the possible environmental risk factors and associated clinical signs. Blood samples were collected from 560 dogs in urban and peri-urban areas in Ilhéus, northeastern Brazil. Genomic DNA was extracted from the selected animals and subjected to molecular analysis using Leishmania species-specific primers and diagnosis of Trypanosoma cruzi. A total of 54.72% of dogs were positive for Leishmania braziliensis, and animals positive for both Leishmania infantum and T. cruzi were not identified. Hematologic variables were not statistically associated with cases of L. braziliensis. However, the positive animal group showed lower red blood cell and platelet counts and higher levels of urea and serum creatinine. Few dogs presented clinical signs compatible with the presence of Leishmania. Age of more than 2 years and specific hair colors were associated with positive results for L. braziliensis. The geoclimatic characteristics of the region may improve parasite survival, reproduction, and vectors. This may explain the higher rate of dogs identified as positive in this study.

Detection of Toxoplasma gondii DNA in naturally infected sheep's milk.

The objective of this study was to verify whether Toxoplasma gondii is excreted in the milk of naturally infected sheep. In order to accomplish this, 275 lactating ewes were used; these were bred extensively in 17 estates distributed across nine cities. Polymerase chain reaction amplification was used to detect T. gondii DNA in milk samples, and the indirect immunofluorescence test was employed for the detection of anti-T. gondii IgG antibodies in the sera, with a cut-off value of 1:64. It was possible to verify the presence of the parasite DNA in 6.5% (18/275) of the studied animals. Anti-T. gondii antibodies were present in 41.5% of the animals studied (114/275). There was no correlation between parasite excretion in milk and the presence of IgG in 38.9% of the studied animals (7/18). The high seropositivity and the presence of parasite DNA in the milk led to the conclusion that T. gondii infection is present in the sheep population in southern and southwestern Bahia, and that there is a risk of the human population becoming infected due to the consumption of raw, in natura milk.

Detection of Toxoplasma gondii DNA in Brazilian oysters (Crassostrea rhizophorae).

The aim of this study was to detect evidence of Toxoplasma gondii using polymerase chain reaction (PCR)-based techniques in oysters (Crassostrea rhizophorae) obtained from the southern coastal region of Bahia, Brazil. A total of 624 oysters were collected, and the gills and digestive glands were dissected. Each tissue sample was separated into pools containing tissues (of the same type) from three animals, leading to a total of 416 experimental samples for analysis (208 samples each from the gills and digestive glands). Molecular analysis using PCR-based detection of the T. gondii AF 146527 repetitive fragment yielded negative results for all samples. However, when nested-PCR was used for detection of the T. gondii SAG-1 gene, 17 samples were positive, with the gills being the tissue with maximal detection of the parasite. These positive results were confirmed by sample sequencing. It is therefore suggested that C. rhizophorae oysters are capable of filtering and retaining T. gondii oocysts in their tissue. This represents a risk to public health because they are traditionally ingested in natura.

Identification and genetic characterization of a new Brazilian genotype of Toxoplasma gondii from sheep intended for human consumption.

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.

Genetic characterization of Toxoplasma gondii isolates from pigs intended for human consumption 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.

Seroprevalence of Toxoplasma gondii in free-living Amazon River dolphins (Inia geoffrensis) from central Amazon, Brazil.

Toxoplasma gondii is an important pathogen in aquatic mammals and its presence in these animals may indicate the water contamination of aquatic environment by oocysts. Serum samples from 95 free-living Amazon River dolphins (Inia geoffrensis) from the Mamirauá Sustainable Development Reserve (RDSM), Tefé, Amazonas, Central Amazon, Brazil were tested for T. gondii antibodies using the modified agglutination test (MAT). Antibodies (MAT ≥ 25) to T. gondii were found in 82 (86.3%) dolphins with titers of 1:25 in 24, 1:50 in 56, and 1:500 in 2. Results suggest a high level contamination of the aquatic environment of the home range of these animals.

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus.

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.

Risk factors associated with Toxoplasma gondii infection in dairy cattle, state of Rio de Janeiro / Fatores de risco associados à infecção por Toxoplasma gondii em bovinos leiteiros no estado do Rio de Janeiro

Toxoplasmosis is one of the most common parasitic zoonoses throughout the world. Infection in man and animals varies in different geographical areas influenced by many environmental conditions. Seroprevalence of Toxoplasma gondii infection in cattle in Brazil ranges from 1.03 to 71 percent. A cross-sectional survey was carried out in 58 out of 453 farms in the South Fluminense Paraiba Valley, State of Rio de Janeiro, Brazil. Over 3-year-old cattle (n=589) from dairy herds were selected for blood collection and detection of anti-T. gondii antibodies by indirect immunofluorescence reaction (IFA) with initial titration of 1:16; titers > 64 were considered positive. Univariate analysis of risk factors showed that cats in contact with cattle, cats in contact with drinking water, and number of cats were associated with T. gondii seroprevalence. Logistic regression revealed a two-fold increased risk for infection of cattle (p=0.0138) through larger number of cats (>3) compared with low numbers of cats (1-2) on the farm. In contrast, the presence of chickens was considered a protective factor (p=0.025).

Toxoplasmose é uma das mais comuns zoonoses parasitárias do mundo. Infecções em seres humanos e em animais variam nas diferentes áreas geográficas influenciadas pelas condições ambientais. A soroprevalência da infecção por Toxoplasma gondii em bovinos no Brasil varia de 1,03 a 71,0 por cento. O estudo transversal foi realizado em 58 de um total de 453 propriedades na região Sul Fluminense do estado do Rio de Janeiro. Vacas leiteiras acima de 3 anos de idade (n=589) foram selecionadas para coleta de sangue e a detecção de anticorpos anti-T. gondii foi feita pelo teste de imunofluorescência indireta (IFI) com titulação inicial de 1:16 e títulos > 64 foram considerados positivos. Após análise univariada dos fatores de risco, gatos em contato com bovinos, em contato com a água de beber dos animais e o número de gatos foram associados com a soroprevalência de T. gondii. A regressão logística demonstrou que o número maior de gatos (>3) teve um risco duas vezes maior (p=0,0138) que propriedades que tinham um número menor de gatos (1-2). Em contraste, a presença de galinhas foi considerada um fator de proteção (p=0,025).

Epidemiological and molecular study of Ehrlichia canis in dogs in Bahia, Brazil.

The objective of the present article was an epidemiological and molecular study of Ehrlichia canis in dogs of Ilhéus 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, using 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 Ilhéus-Itabuna microregion. The associated risk factors were exposure to tick-infested habitats and the fact that the dogs lived in the countryside.