Molecular and serological detection of Anaplasma spp. in small ruminants in an area of Cerrado Biome in northeastern Brazil.

Anaplasmosis, caused by bacteria of the genus Anaplasma, is an important tick-borne disease that causes economic losses to livestock farms in many countries. Even though Anaplasma spp. have been detected in goats and sheep worldwide, few studies investigate the occurrence and genetic identity of these agents in small ruminants from Brazil. Thus, this work aimed to detect and determine the genetic identity of Anaplasma spp. in small ruminants from the Baixo Parnaíba region, state of Maranhão, northeastern Brazil. For this purpose, blood samples were collected from 161 animals (91 goats; 70 sheep) from 4 municipalities in the Baixo Parnaíba region. Sheep and goat serum samples were subjected to recombinant membrane surface protein (MSP5)-based iELISA. Whole blood samples were subject to DNA extraction and molecular diagnosis using PCR assays for Anaplasma spp. targeting msp1ß, msp1α, 16S rRNA and msp4 genes. Positive samples were sequenced and then subjected to Anaplasma marginale msp1α genetic diversity analysis and phylogenetic inferences based on the 16S rRNA and msp4 genes. The serological survey detected the presence of anti-A. marginale IgG antibodies in 18 animals (11.1%): 2.9% (2/70) sheep and 17.4% (16/91) goats. Anaplasma marginale DNA was detected in 2 goats (1.2%) using qPCR based on the msp1ß gene. Two distinct A. marginale msp1α strains, namely α ß and α ß ΓγΓγΓγΓγ were found in the infected goats, each one found in a different animal, both belonging to the H genotype. Phylogenetic analysis based on the 16S rRNA gene showed the sequences positioned in three different clades and grouped with sequences from 'Candidatus Anaplasma boleense', A. platys and A. marginale. Phylogenetic inferences based on the msp4 gene positioned the sequence variants in the A. marginale clade. The present work represents the first molecular detection of sequence variants phylogenetic associated to 'Candidatus Anaplasma boleense' and A. platys and α ß and α ß ΓγΓγΓγΓγ in goats from Brazil.

Molecular detection and characterization of vector-borne agents in common opossums (Didelphis marsupialis) from northeastern Brazil.

Opossums are synanthropic marsupials able to interchange among wild, peri­urban and urban environments, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of relevance in public health. The present study aimed to detect and molecularly characterize vector-borne agents in a population of common opossums (Didelphis marsupialis) from the Island of São Luís do Maranhão, northeastern Brazil. Of the 45 animals analyzed, one (2.22%) was positive in the nested PCR assay based on the 18S rRNA gene of piroplasmids. The obtained sequence was phylogenetically positioned in a clade containing sequences of Babesia sp. previously detected in Didelphis aurita, Didelphis albiventris and associated ticks from Brazil. Eight (17.77%) samples were positive in PCR for Ehrlichia spp. based on the dsb gene; four samples were sequenced and positioned into a new clade, sister to E. minasensis and Ehrlichia sp. clade detected in Superorder Xenarthra mammals. No samples tested positive in the screening PCR assays based on the 16S rRNA gene of Anaplasma spp. Two samples were positive in the qPCR for Bartonella spp. based on the nuoG gene. Seven animals (15.56%) were positive in the nPCR based on the 16S rRNA gene of hemoplasmas. Of these, three were positive in a PCR based on the 23S rRNA gene. The phylogenies based on both 16S rRNA and 23S rRNA genes corroborated to each other and positioned the sequences in the same clade of hemoplasmas previously detected in D. aurita and D. albiventris sampled in Brazil. Finally, three (6.66%) animals were positive in the PCR for Hepatozoon spp.; the obtained 18S rRNA sequence was positioned into the H. felis clade.The present study showed, for the first time, the circulation of piroplasmids, Hepatozoon spp., Ehrlichia spp., hemoplasmas and Bartonella spp. in D. marsupialis sampled in northeastern Brazil, with description of putative novel genotypes of Ehrlichia and Hepatozoon and copositivity by different vector-borne agents. The present work consolidates the "South American Marsupialia" piroplasmid clade, adding one more genotype of Babesia sp. to this clade.

Cryptosporidium sp. in cultivated oysters and the natural oyster stock of the state of Maranhão, Brazil / Detecção de Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão, Brasil

ABSTRACT: This study detected Cryptosporidium spp. in cultivated oysters and the natural oyster stock of the state of Maranhão and determine the elective tissue(s) to examine this protozoan. For this purpose, 200 cultivated oysters were purchased from the municipality of Raposa and another 100 from Paço do Lumiar. Additionally, 100 oysters were extracted from the natural stock of the municipality of Primeira Cruz, thus making up a total of 400 oysters. They were grouped into 80 pools consisting of 5 oysters each. From each pool, the gills and visceral mass were removed to obtain 160 pools, 80 pools for the gill group and another 80 for the visceral mass group. Then, DNA was extracted from each pool using a commercial kit with modifications. Subsequently, the protozoan DNA was detected using nested polymerase chain reaction. With this technique, the DNA of the protozoan under investigation was detected in 2.5% (n = 2/80) of the pools containing gills, with 1.25% of the pools (n = 1/80) belonging to the cultivation group of oysters and the other 1.25% (n = 1/80) to the natural stock. With the results obtained in this study, it was concluded that the analyzed oysters of the genus Crassostrea, from cultivation and natural stock groups, found in the state of Maranhão, were contaminated by Cryptosporidium spp. and may become potential sources of infection in humans and other animals. In addition, the gills are the elective tissue for the study of Cryptosporidium spp. in oysters.

RESUMO: Objetivou-se com o estudo detectar Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão e determinar o(s) tecido(s) eletivo(s) para pesquisa desse protozoário. Para a realização do estudo foram adquiridas 200 ostras de cultivo do município de Raposa e 100 de Paço do Lumiar, além de 100 ostras extraídas de estoque natural do município de Primeira Cruz, totalizando 400 ostras. Estas foram agrupadas em 80 pools constituídos por cinco animais. De cada pool, as brânquias e a massa visceral foram removidas totalizando 160 pools, sendo 80 para o grupo das brânquias e 80 para o grupo de massa visceral. Na sequência, procedeu-se à extração de DNA de cada pool com a utilização de kit comercial com modificações. Posteriormente, realizou-se a detecção do DNA do protozoário por meio da técnica de Nested-PCR. Com a técnica utilizada, foi detectado o DNA do protozoário pesquisado em 2,5% (n=2/80) pools apenas de brânquias, sendo 1,25% pools (n=1/80) oriundos de cultivo e os outros 1,25% (n=1/80) de estoque natural. Com os resultados obtidos nesse estudo, conclui-se que as ostras analisadas do gênero Crassostrea sp., oriundas de cultivo e estoque natural no estado do Maranhão, estavam contaminadas por Cryptosporidium sp. e podem se reverter em fontes potenciais para seres humanos e outros animais. Para a pesquisa de Cryptosporidium sp. em ostras, as brânquias são o tecido eletivo.

Cryptosporidium sp. in cultivated oysters and the natural oyster stock of the state of Maranhão, Brazil / Detecção de Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão, Brasil

This study detected Cryptosporidium spp. in cultivated oysters and the natural oyster stock of the state of Maranhão and determine the elective tissue(s) to examine this protozoan. For this purpose, 200 cultivated oysters were purchased from the municipality of Raposa and another 100 from Paço do Lumiar. Additionally, 100 oysters were extracted from the natural stock of the municipality of Primeira Cruz, thus making up a total of 400 oysters. They were grouped into 80 pools consisting of 5 oysters each. From each pool, the gills and visceral mass were removed to obtain 160 pools, 80 pools for the gill group and another 80 for the visceral mass group. Then, DNA was extracted from each pool using a commercial kit with modifications. Subsequently, the protozoan DNA was detected using nested polymerase chain reaction. With this technique, the DNA of the protozoan under investigation was detected in 2.5% (n = 2/80) of the pools containing gills, with 1.25% of the pools (n = 1/80) belonging to the cultivation group of oysters and the other 1.25% (n = 1/80) to the natural stock. With the results obtained in this study, it was concluded that the analyzed oysters of the genus Crassostrea, from cultivation and natural stock groups, found in the state of Maranhão, were contaminated by Cryptosporidium spp. and may become potential sources of infection in humans and other animals. In addition, the gills are the elective tissue for the study of Cryptosporidium spp. in oysters.

Objetivou-se com o estudo detectar Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão e determinar o(s) tecido(s) eletivo(s) para pesquisa desse protozoário. Para a realização do estudo foram adquiridas 200 ostras de cultivo do município de Raposa e 100 de Paço do Lumiar, além de 100 ostras extraídas de estoque natural do município de Primeira Cruz, totalizando 400 ostras. Estas foram agrupadas em 80 pools constituídos por cinco animais. De cada pool, as brânquias e a massa visceral foram removidas totalizando 160 pools, sendo 80 para o grupo das brânquias e 80 para o grupo de massa visceral. Na sequência, procedeu-se à extração de DNA de cada pool com a utilização de kit comercial com modificações. Posteriormente, realizou-se a detecção do DNA do protozoário por meio da técnica de Nested-PCR. Com a técnica utilizada, foi detectado o DNA do protozoário pesquisado em 2,5% (n=2/80) pools apenas de brânquias, sendo 1,25% pools (n=1/80) oriundos de cultivo e os outros 1,25% (n=1/80) de estoque natural. Com os resultados obtidos nesse estudo, conclui-se que as ostras analisadas do gênero Crassostrea sp., oriundas de cultivo e estoque natural no estado do Maranhão, estavam contaminadas por Cryptosporidium sp. e podem se reverter em fontes potenciais para seres humanos e outros animais. Para a pesquisa de Cryptosporidium sp. em ostras, as brânquias são o tecido eletivo.

Endoparasite diversity and liver alterations in Hoplerythrinus unitaeniatus and Cichlasoma bimaculatum in a quilombola area in Maranhão, Brazil.

Our aim was to assess endoparasite diversity and liver alterations in Hoplerythrinus unitaeniatus (jeju) and Cichlasoma bimaculatum (acará preto) in a quilombola area in Maranhão, Brazil. For this, 21 H. unitaeniatus and 21 C. bimaculatum were caught in a natural environment and transported to a laboratory. After these had been euthanized, endoparasites were collected and identified. Liver alterations were evaluated histological analysis based on the severity of each lesion: stage I, organ functioning not compromised; stage II, more severe lesions that impair normal functioning of the organs; and stage III, very severe and irreversible lesions. Among the fish evaluated, 71.43% H. unitaeniatus and 61.90% C. bimaculatum were parasitized. Contracaecum sp. was found in both species; while acanthocephalans, only in H. unitaeniatus. The alterations were vacuolization, nucleus in the cell periphery, deformation of the cell outline, melanomacrophage center, hyperemia, cytoplasmic degeneration and nuclear vacuolization. Through calculating a histological alteration index, it was found that 26.19% of the specimens presented lesions in stage I; 38.09% lesions in stage II and 9.52% lesions in stage III. It was concluded that there is high prevalence of Contracaecum sp. and that the liver lesions may be adaptive responses by the fish to endoparasitic infection.

Diagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil.

Enzootic bovine leukosis (EBL) is a chronic viral disease of wide distribution in cattle herds and may take several years for the first manifestation of clinical signs. Most animals do not present clinical signs. However, the economic losses are underestimated due to this disease. Thus, this work aimed to detect and characterize BLV in dairy cattle in the Maranhão state, northeastern Brazil. Blood samples were collected from 176 animals from 8 municipalities in the southeastern state of Maranhão. Bovine blood samples were subjected to DNA extraction and molecular diagnosis using nested PCR assays for BLV, targeting gp51 gene. Positive samples were then sequenced and then subjected to phylogenetic inferences. BLV DNA was detected in 16 cattle (16/176, 9.09%) in 4 municipalities. Phylogenetic analyzes showed that the sequence obtained clustered in a clade containing BLV sequences classified as genotype 6, with a high degree of support. Our data shows BLV occurrence in the Northeast of Brazil and the identification of genotype 6 in this region. These findings contribute to the molecular epidemiology of this agent in Brazil.

Endoparasite diversity and liver alterations in Hoplerythrinus unitaeniatus and Cichlasoma bimaculatum in a quilombola area in Maranhão, Brazil / Diversidade endoparasitária e alterações hepáticas em Hoplerythrinus unitaeniatus e Cichlasoma bimaculatum de área quilombola maranhense, Brasil

Our aim was to assess endoparasite diversity and liver alterations in Hoplerythrinus unitaeniatus (jeju) and Cichlasoma bimaculatum (acará preto) in a quilombola area in Maranhão, Brazil. For this, 21 H. unitaeniatus and 21 C. bimaculatum were caught in a natural environment and transported to a laboratory. After these had been euthanized, endoparasites were collected and identified. Liver alterations were evaluated histological analysis based on the severity of each lesion: stage I, organ functioning not compromised; stage II, more severe lesions that impair normal functioning of the organs; and stage III, very severe and irreversible lesions. Among the fish evaluated, 71.43% H. unitaeniatus and 61.90% C. bimaculatum were parasitized. Contracaecum sp. was found in both species; while acanthocephalans, only in H. unitaeniatus. The alterations were vacuolization, nucleus in the cell periphery, deformation of the cell outline, melanomacrophage center, hyperemia, cytoplasmic degeneration and nuclear vacuolization. Through calculating a histological alteration index, it was found that 26.19% of the specimens presented lesions in stage I; 38.09% lesions in stage II and 9.52% lesions in stage III. It was concluded that there is high prevalence of Contracaecum sp. and that the liver lesions may be adaptive responses by the fish to endoparasitic infection.(AU)

Objetivou-se avaliar a diversidade de endoparasitos e alterações hepáticas em Hoplerythrinus unitaeniatus (jeju) e Cichlasoma bimaculatum (acará preto) de área quilombola maranhense, Brasil. Assim, 21 H. unitaeniatus e 21 C. bimaculatum foram capturados de ambiente alagável e transportados vivos ao laboratório. Após a eutanásia, procedeu-se à coleta, identificação de endoparasitos e avaliação de alterações hepáticas por meio de análise histológica, baseada na severidade das lesões: (i) alterações de estágio I, não comprometem o funcionamento dos órgãos; (ii) estágio II, lesões mais severas que prejudicam o funcionamento normal dos órgãos; (iii) estágio III, lesões muito severas e irreversíveis. Dos peixes avaliados, 71,43% H. unitaeniatus e 61,90% C. bimaculatum estavam parasitados. Contracaecum sp. foi encontrado nas duas espécies e acantocéfalos apenas em H. unitaeniatus. As alterações hepáticas foram vacuolização, núcleo na periferia das células, deformação do contorno celular, centro de melanomacrófagos, hiperemia, degeneração citoplasmática e vacuolização nuclear. Com o cálculo do índice das alterações, constatou-se que 26,19% dos exemplares apresentaram alterações de estágio I; 38,09% de estágio II e 9,52% de estágio III com a constatação de larvas de Contracaecum sp. encistadas no fígado. Conclui-se que existe alta prevalência de Contracaecum sp., e lesões hepáticas podem ser respostas adaptativas dos peixes à infecção endoparasitária.(AU)

Cutaneous candidiasis caused by antifungal-resistant Candida sp. strain in canine individual / Candidíase cutânea causada por cepa de Candida sp. resistente a antifúngicos em indivíduo canino

The aim of the current report is to describe a cutaneous candidiasis case affecting a canine individual treated at the University Veterinary Hospital of State University of Maranhão (UEMA), in São Luís City. The patient had three-month history of skin diseases; it had been previously subjected to several treatments based on antibiotics, corticosteroids and antifungal drugs that have failed to show clinical improvements. Dermatological assessment has indicated generalized moist dermatitis, intense skin desquamation, alopecia, pruritus and meliceric crusts along the animal's body, mainly in its dorsal region. Complementary tests, such as skin cytology and microscopy, trichogram, qualitative PCR and serology for canine visceral leishmaniasis, as well as fungal culture and antifungigram were requested based on this scenario. Serology recorded inconclusive results for leishmaniasis, whereas PCR recorded negative results in the presence of the agent's DNA. Cytology, microscopy and trichogram results have evidenced fungal infection in the assessed samples. Moreover, mycological culture and antifungigram resulted in the growth of Candida sp. specimens capable of resisting antifungal agents such as amphotericin B, fluconazole, itraconazole and nystatin. The therapy adopted after candidiasis diagnosis confirmation comprised oral doses of manipulated ketoconazole, in combination to topical therapy with shampoo based on moisturizing formulas associated with Miconazole and Chlorhexidine (at 2%), for four weeks. After 30 days, when the adopted therapy was over, the aforementioned animal presented remission of the previously observed lesions and fully improved condition.

The aim of the current report is to describe a cutaneous candidiasis case affecting a canine individual treated at the University Veterinary Hospital of State University of Maranhão (UEMA), in São Luís City. The patient had three-month history of skin diseases; it had been previously subjected to several treatments based on antibiotics, corticosteroids and antifungal drugs that have failed to show clinical improvements. Dermatological assessment has indicated generalized moist dermatitis, intense skin desquamation, alopecia, pruritus and meliceric crusts along the animal's body, mainly in its dorsal region. Complementary tests, such as skin cytology and microscopy, trichogram, qualitative PCR and serology for canine visceral leishmaniasis, as well as fungal culture and antifungigram were requested based on this scenario. Serology recorded inconclusive results for leishmaniasis, whereas PCR recorded negative results in the presence of the agent's DNA. Cytology, microscopy and trichogram results have evidenced fungal infection in the assessed samples. Moreover, mycological culture and antifungigram resulted in the growth of Candida sp. specimens capable of resisting antifungal agents such as amphotericin B, fluconazole, itraconazole and nystatin. The therapy adopted after candidiasis diagnosis confirmation comprised oral doses of manipulated ketoconazole, in combination to topical therapy with shampoo based on moisturizing formulas associated with Miconazole and Chlorhexidine (at 2%), for four weeks. After 30 days, when the adopted therapy was over, the aforementioned animal presented remission of the previously observed lesions and fully improved condition.

First detection of diarrheagenic Escherichia coli pathotypes in the Mearim River Watershed, Maranhão State, Brazil / Primeira detecção de patótipos diarreiogênicos de Escherichia coli na Bacia Hidrográfica do Rio Mearim, Estado do Maranhão, Brasil

The Mearim River Watershed has multiple uses e.g. leisure, navigation, fishing and subsistence agriculture and constitutes the main source of supply for the populations of municipalities situated along its course. In addition to being a water supply source, the existence of the 'pororoca' (tidal bore) effect in a stretch of the lower course of the Mearim River attracts people from several Brazilian states and different countries, as it offers excellent conditions for surfing in fresh water. In this respect, given the importance of the watershed, this study was developed to report the detection of diarrheagenic Escherichia coli pathotypes in a stretch of the lower course of the Mearim River, located in the state of Maranhão, Brazil. Thirty water samples were collected from 10 sampling points. To quantify E. coli, the chromogenic enzymatic system was used and positive samples were isolated and biochemically identified. Pure cultures underwent DNA extraction by heating followed by polymerase chain reaction (PCR) characterization. At the time of the collections, an observation schedule was used to record information on the existence of rearing of livestock and domestic animals; businesses; residences; and fruit and vegetable farming on the riverbanks. The samples were analyzed for the mean populations of E. coli, which ranged from 444 to 2,585 MPN mL-1. Twenty bacterial isolates were identified and the diarrheal pathotypes ETEC, typical EPEC and atypical EPEC were detected. The detection of these pathotypes can represent an epidemiological risk and compromise several uses of this water resource, such as irrigation of fruits and vegetables consumed raw, fishing, animal watering and recreation. Structural investments in basic sanitation are essential to minimize environmental degradation resulting from anthropic activities and to act preventively in public health. In addition, the recovery of riparian forests along the watershed and the maintenance of vegetation in these areas are measures to reduce the transport of particles from the soil to the watercourses, improving the qualitative and quantitative characteristics of this water resource.(AU)

A Bacia Hidrográfica do Rio Mearim apresenta múltiplos usos - lazer, navegação, pesca e agricultura de subsistência - e constitui a principal fonte de abastecimento para as populações dos municípios inseridos em seu curso. Além de fonte hídrica de abastecimento, a existência do efeito pororoca em um trecho do baixo curso do Rio Mearim atrai pessoas de vários estados brasileiros e diferentes países, pois apresenta excelentes condições para a prática de surfe em água doce. Nesse sentido, considerando a importância da Bacia Hidrográfica, objetivou-se relatar a detecção de patótipos diarreiogênicos de Escherichia coli em um trecho do baixo curso do rio Mearim, localizado no estado do Maranhão. Para isso, foram realizadas coletas de 30 amostras de água em 10 pontos amostrais. Para a quantificação de E. coli utilizou-se o sistema cromogênico enzimático e das amostras positivas procedeu-se ao isolamento e identificação bioquímica dos isolados. A extração do DNA das culturas puras foi realizada por aquecimento seguido da caracterização por reação em cadeia da polimerase (PCR). No momento das coletas utilizou-se uma pauta de observação para anotação de informações sobre a existência de criação de animais de interesse pecuário e doméstico, empreendimentos comerciais, residências e cultivo de frutas e hortaliças nas margens do rio. Nas amostras analisadas, foram quantificadas populações médias de E. coli que variaram de 444 a 2.585 NMP.mL-1, identificados 20 isolados bacterianos e detectados os patótipos diarreiogênicos ETEC, EPEC-típica e EPEC-atípica. A detecção destes patótipos pode representar risco epidemiológico e compromete diversos usos desse recurso hídrico, como a irrigação de frutas e hortaliças ingeridas cruas, pesca, dessedentação animal e recreação. Investimentos estruturais em saneamento básico são fundamentais para minimizar a degradação ambiental resultante das atividades antrópicas e para atuar preventivamente na saúde pública. Adicionalmente, a recuperação das matas ciliares ao longo da Bacia Hidrográfica e manutenção da vegetação nestas áreas são medidas para a redução do transporte de partículas do solo para os cursos d'água, e em consequência, acarretará na melhoria das características quali-quantitativas desse recurso hídrico.(AU)