Hepatozoon spp. infection in wild canids in the eastern United States.

BACKGROUND: Hepatozoon spp. are apicomplexan parasites known to cause musculoskeletal disease in a variety of animals. Two species are known to infect wild and domestic canids in the US: Hepatozoon canis and H. americanum. METHODS: In this study, blood, heart, and/or spleen samples were collected from 278 wild canids (180 coyotes, 93 red foxes, and 5 gray foxes) in the eastern US and tested via PCR for Hepatozoon. Histology slides of heart and skeletal muscle were assessed for Hepatozoon cysts and associated inflammation when fresh tissue was available (n = 96). RESULTS: Hepatozoon spp. were found in 24.2% (59/278) of individuals, with Hepatozoon canis in 14.0% (34/278) and H. americanum in 10.7% (26/278). One coyote was positive for both H. canis and H. americanum. Foxes were more likely to be positive for H. canis than coyotes (23% and 7% respectively, P = 0.0008), while only coyotes were positive for H. americanum. Of the eight sampled states, H. canis was present in six (Louisiana, North Carolina, Pennsylvania, South Carolina, Tennessee, and Virginia) while H. americanum was found in two southern states (South Carolina and Louisiana). Infection status was positively correlated with myositis and myocarditis, and heart or muscle cysts were found in 83% (5/6) of H. americanum-positive coyotes. CONCLUSION: This survey showed a moderate prevalence of H. canis and H. americanum in states where the parasite was previously unrecorded including South Carolina and Pennsylvania.

Hepatozoon parasites (Apicomplexa: Hepatozoidae) in fish Hoplias aimara (Characiformes, Erythrinidae) from the Eastern Amazon, Brazil.

This study reports the occurrence of parasites belonging to the Hepatozoon genus in fish Hoplias aimara from the Eastern Amazon. Fish (n = 54) were sampled from the Falsino River, located in the Amapá National Forest (FLONA), in the state of Amapá, northern Brazil. Fresh liver preparations were examined in the field between a slide and a coverslip under a light microscope. Cysts containing Hepatozoon cystozoites were observed in the liver of 5 (9%) out of 54 H. aimara individuals. The cysts were ovoid (mean dimensions 10.28 × 9.8 µm), presenting up to four elongated cystozoites (mean dimensions 11.04 × 1.68 µm), containing 1 to 4 residual bodies of different sizes. A single liver sample containing cysts was submitted to DNA extraction and PCR analyses based on a fragment of the 18S rRNA gene. The sequencing revealed a 465 bp fragment exhibiting 99% query coverage, 0.0 E-value, and 98.7% identity with Hepatozoon caimani (MF322538 and MF322539), detected in caimans (Caiman yacare) from Brazil. This is the first report of the occurrence of cysts containing Hepatozoon cystozoites in free-living fishes.

Molecular detection of Hepatozoon species infections in domestic cats living in Germany.

OBJECTIVES: Three species of protozoal Hepatozoon species (H felis, H canis and H silvestris) are known to infect cats in Europe. The objective of this study was to determine the prevalence of Hepatozoon species in samples from cats living in Germany that were submitted to a veterinary laboratory. METHODS: The study included cats tested for Hepatozoon species by PCR between 2007 and 2020 by the Laboklin laboratory. Travel history and haematological results were documented for cats with positive test results. From 2018 onwards, a partial 18S rRNA Hepatozoon gene fragment was sequenced from cats with positive PCR results. RESULTS: Sixty-four of 931 cats (7%) tested positive for Hepatozoon species. Sex and age did not have a statistically significant impact. Sequencing was carried out for 16 samples and revealed H felis in all cases. All cats with positive test results and a relevant travel history had been imported from the Mediterranean or south-eastern Europe. There were no autochthonous infections with Hepatozoon species. Leukocytosis, haemoconcentration and anaemia were the most common haematological abnormalities. CONCLUSIONS AND RELEVANCE: Although infections with Hepatozoon species in cats are usually subclinical, it may be useful to screen cats imported from the Mediterranean and south-eastern Europe for these pathogens to prevent local transmission cycles. There was no evidence of autochthonous infections in Germany; however, further investigations regarding a possible transmission of Hepatozoon species from infected cats to blood-feeding arthropods in Germany may be of interest. To avoid potential spread of the pathogens, ectoparasite prophylaxis is advisable.

Ocorrência de Babesia sp, Ehrlichia canis e Hepatozoon canis em cães domiciliados, em dois municípios do estado do Espírito Santo - Brasil / Occurrence of Babesia sp, Ehrlichia canis and Hepatozoon canis in domiciled dogs in two municipalities of the state of Espírito Santo - Brazil / Ocurrencia de Babesia sp, Ehrlichia canis y Hepatozoon canis em perros domiciliados em dos municipios del estado de Espírito Santo - Brasil

As doenças transmitidas por carrapatos são afecções de grande importância na clínica médica de pequenos animais, devido à alta casuística e ampla distribuição vetorial no território brasileiro. Os principais agentes responsáveis pelas infecções em cães são Babesia sp., Ehrlichia canis e Hepatozoon canis. Os animais infectados são assintomáticos ou apresentam sinais clínicos inespecíficos, sendo necessário a utilização de testes diagnósticos para definição do agente etiológico, e diagnóstico seguro. O objetivo do presente estudo foi determinar a ocorrência desses micro-organismos em cães naturalmente infectados, domiciliados nos municípios de Vila Velha e Anchieta, Espírito Santo, utilizando diferentes testes de detecção: Reação em cadeia polimerase (PCR), sorologia para detecção de anticorpos anti Ehrlichia canis e pesquisa de hematozoários em esfregaço sanguíneo. Foram analisadas 65 amostras de sangue obtidas por venopunção de veia cefálica de cães. No teste de PCR, 4,62% dos animais foram positivos para Babesia vogeli e 1,54% para Ehrlichia canis sendo os resultados para Hepatozoon canis negativos. No teste sorológico para E. canis 90,77% dos animais foram positivos para a presença de anticorpos, e na pesquisa em lâminas de esfregaço sanguíneo 3,02% apresentavam outros hemoparasitas. Os resultados indicam a dispersão desses hemoparasitas na população canina da região de estudo, entretanto com baixa ocorrência. O teste de PCR demonstrou-se como o mais sensível no qual Babesia vogeli foi o agente mais observado.(AU)

Tick-borne diseases are diseases of great importance in the medical practice of small animals, due to the high casuistry and wide vectorial distribution in the Brazilian territory. The main agents responsible for infections in dogs are Babesia sp., Ehrlichia canis and Hepatozoon canis. Infected animals are asymptomatic or present nonspecific clinical signs, requiring the use of diagnostic tests to define the etiologic agent, and safe diagnosis. The objective of the present study was to determine the occurrence of these microorganisms in naturally infected dogs domiciled in the municipalities of Vila Velha and Anchieta, Espírito Santo, using different detection tests: polymerase chain reaction (PCR), serology to detect antibodies against Ehrlichia canis and research of hematozoa in blood smears. Sixty-five blood samples obtained by venipuncture of the cephalic vein of dogs were analyzed. In the PCR test, 4.62% of the animals were positive for Babesia vogeli and 1.54% for Ehrlichia canis, and the results for Hepatozoon canis were negative. In the serological test for E. canis, 90.77% of the animals were positive for the presence of antibodies, and in the research in blood smear slides, 3.02% presented other hemoparasites. The results indicate the dispersion of these hemoparasites in the canine population of the study region, however with low occurrence. The PCR test proved to be the most sensitive, in which Babesia vogeli was the most observed agent.(AU)

Las enfermedades transmitidas por garrapatas son enfermedades de gran importancia en la práctica médica de los pequeños animales, debido a la alta casuística y amplia distribución vectorial en el territorio brasileño. Los principales agentes responsables de las infecciones en los perros son Babesia sp., Ehrlichia canis y Hepatozoon canis. Los animales infectados son asintomáticos o presentan signos clínicos inespecíficos, siendo necesario el uso de pruebas diagnósticas para la definición del agente etiológico, y el diagnóstico seguro. El objetivo del presente estudio fue determinar la ocurrencia de estos microorganismos en perros infectados naturalmente, domiciliados en los municipios de Vila Velha y Anchieta, Espírito Santo, utilizando diferentes pruebas de detección: reacción en cadena de la polimerasa (PCR), serología para detectar anticuerpos anti Ehrlichia canis e investigación de hematozoos en frotis de sangre. Se analizaron sesenta y cinco muestras de sangre obtenidas por venopunción de la vena cefálica de los perros. En la prueba PCR, el 4,62% de los animales fueron positivos para Babesia vogeli y el 1,54% para Ehrlichia canis, y los resultados para Hepatozoon canis fueron negativos. En la prueba serológica para E. canis, el 90,77% de los animales fueron positivos a la presencia de anticuerpos, y en la investigación en láminas de frotis de sangre el 3,02% presentaron otros hemoparásitos. Los resultados indican la dispersión de estos hemoparásitos en la población canina de la región de estudio, aunque con una baja presencia. La prueba PCR resultó ser la más sensible, en la que Babesia vogeli fue el agente más observado.(AU)

Hepatic coccidiosis in Serrasalmus rhombeus Linnaeus, 1766 from the Amazon basin: morphological and histopathological aspects.

Calyptospora species are coccids that commonly cause liver infections in fish all around the world. This paper describes the morphology and histopathological characteristics of liver infection caused by Calyptospora sp. in black piranha Serrasalmus rhombeus, from the Capim River, in the municipality of Ipixuna do Pará, state of Pará (Brazil). Specimens were collected, analyzed and necropsied and tissue fragments containing parasites were prepared for histology and scanning electron microscopy. Parasitism was detected in 33.0% of the analyzed specimens, which had spherical oocysts in the liver, with four pyriform sporocysts presenting sporozoites internally. A histological examination revealed oocysts positioned close to blood vessels, causing necrosis and degeneration of hepatic parenchyma, while the presence of mononuclear cell infiltrate and melanomacrophages indicated the onset of an inflammatory process. This is the first record of the genus Calyptospora in fish from the Capim River.

Description of a New Species Hepatozoon quagliattus sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting the Sleep Snake, Dipsas mikanii (Squamata: Colubridae: Dipsadinae) from Goiás State, Brazil.

PURPOSE: Species of Hepatozoon Miller, 1908 (Hepatozoidae) are blood protozoans with a cosmopolitan distribution and are reported to parasitize a range of vertebrate hosts including mammals, birds, reptiles, and amphibians. The present study aimed to describe a new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) found infecting the sleep snake Dipsas mikanii (Schlegel, 1837) (Squamata: Colubridae: Dipsadinae). METHODS: The snake was collected in 2017 at the municipality of Britânia, Goiás State, Brazil. Blood smears were made in order to find blood gametocytes and PCR was performed targeting the 18S rRNA gene. RESULTS: Microscopy screening of blood smears revealed the presence of intraerythrocytic gamont stages of Hepatozoon sp. in the peripheral blood with a parasitemia of 0.25%. Furthermore, meronts and monozoic cysts were observed in histological sections of the liver from the infected individual. The interspecific divergence of 18S rRNA sequences fragments isolated from D. mikanii had differences (2.39-11.3%) as compared to other sequences of species of Hepatozoon from snakes. CONCLUSIONS: Based on morphological and molecular data, a new species of Hepatozoon infecting D. mikanii from Brazil is described.

Genetic diversity of Hepatozoon spp. in rodents from Chile / Diversidade genética de Hepatozoon spp. em roedores do Chile

Abstract This study aimed to investigate the genetic diversity of Hepatozoon spp. in rodents from Valdivia, Chile. A total of 74 rodents (synanthropic n=38; wild n=36) were trapped in Valdivia. We performed conventional PCR assays for Apicomplexa organisms targeting two overlapping 18S rDNA gene fragments (600 bp and 900 bp) followed by sequencing of selected amplicons. Hepatozoon spp. occurrence was 82.43% (61/74). Twelve sequences obtained from the 600 bp and ten from the 900 bp 18S rDNA fragments were identified as Hepatozoon sp. Six sequences obtained from 18S rDNA-based overlapping PCR protocols were used for concatenated (1,400 bp) phylogenetic, haplotype and distance analyses. Hepatozoon spp. 18S rDNA concatenated sequences from the present study were detected in Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus, and Abrothrix longipilis grouped with Hepatozoon species earlier described in rodents and reptiles from Chile and Brazil. Nucleotide polymorphism of the six 18S rDNA sequences (1,400 bp) from this study, and other Chilean sequences from rodents and rodent's ticks, showed high diversity with a total of nine Chilean haplotypes. Three haplotypes from Valdivia were identified for the first time in this study, suggesting the circulation of novel haplotypes in rodents from southern Chile.

Resumo Este estudo teve como objetivo investigar a diversidade genética de Hepatozoon spp. em roedores de Valdivia, Chile. Um total de 74 roedores (sinantrópicos n=38; selvagens n=36) foram capturados. PCR convencional foi realizada para organismos Apicomplexa, visando dois fragmentos sobrepostos do gene 18S rDNA (600 bp e 900 bp), seguida pelo sequenciamento de amplicons selecionados. A ocorrência de Hepatozoon spp. foi de 82,43% (61/74). Doze sequências obtidas dos fragmentos de 18S rDNA de 600 pb e dez dos fragmentos de 18S rDNA de 900 pb foram identificadas como Hepatozoon sp. Seis sequências obtidas, a partir de protocolos de PCR sobrepostos, foram usadas para análises filogenéticas (1.400 bp), de haplótipos e de distância. Sequências concatenadas 18S rDNA do presente estudo foram detectadas em Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus e Abrothrix longipilis e agrupadas com Hepatozoon descrito em roedores e répteis do Chile e do Brasil. A análise de polimorfismos das seis sequências deste estudo, junto com outras sequências chilenas de roedores e carrapatos de roedores, mostrou alta diversidade com um total de nove haplótipos no Chile. Três haplótipos detectados em Valdivia foram identificados pela primeira vez neste estudo, sugerindo que novos haplótipos circulam em roedores do sul do Chile.

Hepatic coccidiosis in Serrasalmus rhombeus Linnaeus, 1766 from the Amazon basin: morphological and histopathological aspects / Coccidiose hepática em Serrasalmus rhombeus Linnaeus, 1766 da Amazônia paraense: aspectos morfológicos e histopatológicos

Abstract Calyptospora species are coccids that commonly cause liver infections in fish all around the world. This paper describes the morphology and histopathological characteristics of liver infection caused by Calyptospora sp. in black piranha Serrasalmus rhombeus, from the Capim River, in the municipality of Ipixuna do Pará, state of Pará (Brazil). Specimens were collected, analyzed and necropsied and tissue fragments containing parasites were prepared for histology and scanning electron microscopy. Parasitism was detected in 33.0% of the analyzed specimens, which had spherical oocysts in the liver, with four pyriform sporocysts presenting sporozoites internally. A histological examination revealed oocysts positioned close to blood vessels, causing necrosis and degeneration of hepatic parenchyma, while the presence of mononuclear cell infiltrate and melanomacrophages indicated the onset of an inflammatory process. This is the first record of the genus Calyptospora in fish from the Capim River.

Resumo Calyptosporídeos são coccídeos constantemente encontrados causando infecções hepáticas em peixes de todo o planeta. Este estudo descreve a morfologia e características histopatológicas da infecção hepática causada por Calyptospora sp. encontrados em Serrasalmus rhombeus, a piranha negra, oriunda do rio Capim, em Ipixuna do Pará, Brasil. Os animais foram coletados, analisados e necropsiados; fragmentos teciduais com a presença dos parasitos foram processados para histologia e microscopia eletrônica de varredura. O parasitismo ocorreu em 33,0% dos espécimes analisados, onde foram encontrados oocistos esféricos no fígado, com esporocistos piriformes, apresentando internamente esporozoítos. No exame histológico, os oocistos estavam dispostos próximos aos vasos sanguíneos, causando necrose e degeneração do parênquima hepático; a presença de infiltrado de células mononucleares e melanomacrófagos indicaram o início de um processo inflamatório. Este é o primeiro registro do gênero Calyptospora em peixes do rio Capim.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Molecular detection of Cercopithifilaria bainae and other tick-borne pathogens in Rhipicephalus sanguineus s.l. isolated from dogs in Midwest Brazil / Detecção molecular de Cercopithifilaria bainae e outros patógenos transmitidos por carrapatos em Rhipicephalus sanguineus s.l. isolados de cães no Centro-Oeste do Brasil

Abstract The aim of the present study was to detect Cercopithifilaria bainae and other tick-borne pathogens and to perform molecular characterization of the tick Rhipicephalus sanguineus s.l. collected from dogs. Ticks (n = 432, including 8 larvae, 59 nymphs, and 365 adults) were sampled from domiciled dogs (n = 73) living in Campo Grande, Mato Grosso do Sul (Midwest Brazil). All ticks were morphologically identified as R. sanguineus. Genomic DNA was extracted in pools (three to five ticks per animal) and was used for definition of R. sanguineus haplotypes (based on 16S rRNA analysis) and pathogen identification (Cercopithifilaria sp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, Babesia vogeli and Rickettsia spp.). Rhipicephal us sanguineus specimens were identified as haplotypes A and B. DNA of Cercopithifilaria bainae (43.83%; 32/73), Ehrlichia canis (24.65%; 18/73), Anaplasma platys (19.17%; 14/73), and Hepatozoon canis (5.47%; 4/73) was detected. The identity of pathogens was confirmed by DNA sequence analysis. The present study confirms the presence of haplotypes A and B of R. sanguineus in the state of Mato Grosso do Sul and its importance as a vector of several pathogens of veterinary concern. Finally, this is the first report to identify C. bainae in ticks in the Midwestern region of Brazil.

Resumo O objetivo do presente estudo foi detectar Cercopithifilaria bainae e outros patógenos transmitidos por carrapatos e realizar a caracterização molecular do carrapato Rhipicephalus sanguineus s.l. coletado em cães. Carrapatos (n = 432, incluindo 8 larvas, 59 ninfas e 365 adultos) foram amostrados de cães domiciliados (n = 73) residentes no município de Campo Grande, Mato Grosso do Sul (centro-oeste do Brasil). Todos os carrapatos foram identificados morfologicamente como R. sanguineus. O DNA genômico foi extraído em pools (três a cinco carrapatos por animal), seguido pela definição de haplótipos (com base no gene 16S rRNA) e pela investigação de patógenos (Cercopithifilaria sp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, Babesia vogeli e Rickettsia spp.). Os espécimes coletados foram identificados como haplótipos A e B de R. sanguineus. Foram detectados DNA de Cercopithifilaria bainae (43,83%; 32/73), Ehrlichia canis (24,65%; 18/73), Anaplasma platys (19,17%; 14/73) e Hepatozoon canis (5,47%; 4/73). A identidade dos patógenos foi confirmada por análise de sequência de DNA. O presente estudo confirma a circulação dos haplótipos A e B de R. sanguineus no estado de Mato Grosso do Sul e sua importância como vetor de vários patógenos de interesse veterinário. Finalmente, este é o primeiro relato de C. bainae em carrapatos na região centro-oeste do Brasil.

First autochthonous case of clinical Hepatozoon felis infection in a domestic cat in Central Europe.

Three different Hepatozoon (Apicomplexa, Hepatozoidae) species have been described infecting domestic cats in Europe (i.e. H. felis, H. canis and H. silvestris), however, reports on clinical hepatozoonosis are uncommon and treatment protocols are not clearly defined. A six-year-old male European short-hair cat from Austria presented poor general condition, lethargy, anorexia, icterus, a painful abdomen, fever, ruffled hair and a tick infestation, and it had never left Austria. Laboratory tests revealed leukopenia, thrombocytopenia and increased serum levels of symmetric dimethylarginine (SDMA) and bilirubin. In May Grünwald-Giemsa-stained blood smears, structures resembling Hepatozoon gamonts were observed inside neutrophil granulocytes. A PCR targeting a fragment of the 18S rRNA gene of Hepatozoon spp. and DNA sequencing allowed the diagnosis of H. felis-DNA in blood samples. The cat was treated with imidocarb dipropionate (6 mg/kg body weight, repeated after 14 days) and doxycycline monohydrate (5 mg/kg body weight twice a day, p.o., for four weeks) and recovered completely. A broad haematological and biochemical laboratory control after six months showed all evaluated parameters under normal ranges. Coinfection with other feline pathogens (i.e. feline leukaemia virus, feline immunodeficiency virus, feline Coronavirus, Leishmania and Dirofilaria immitis) could not be detected. This study reveals the presence of H. felis in Austria and provides more evidence on the geographical distribution and pathogenicity of this parasite for domestic cats. To the authors' knowledge, this is the first autochthonous case of feline hepatozoonosis in Central Europe.

Occurrence of Ehrlichia canis and Hepatozoon canis and probable exposure to Rickettsia amblyommatis in dogs and cats in Natal, RN / Ocorrência de Ehrlichia canis, Hepatozoon canis e provável exposição a Rickettsia amblyommatis em cães e gatos de Natal, RN

Abstract Occurrence of infection or exposure to Ehrlichia canis, Hepatozoon canis and Rickettsia spp. was detected in feral cats living in two fragments from Atlantic rainforest, in Natal, RN, Brazil, and in dogs living around the parks. While serum samples were collected from 155 animals (53 cats living in the parks; 29 dogs living in human homes around the parks; and 73 dogs living at an animal control center - ACC), spleen samples were collected from 20 dogs that were euthanized at ACC. Serum samples were analyzed to Rickettsia spp. and E. canis antibodies using the indirect immunofluorescence assay. Seventeen of the 102 dogs (17%) had E. canis antibodies and 13% (20/155) of all dogs and cats (i.e. 3% (3/102) of the dogs and 32% (17/53) of the cats) were seropositive for Rickettsia spp. antigens. The animals were therefore been exposed to R. amblyommatis or by a very closely related genotype. Among the 20 dog spleen samples analyzed, eight were PCR positive for E. canis and two for H. canis (GenBank accession number MG772657 and MG772658, respectively). In none of the spleen samples were obtained amplicons for Babesia spp. through PCR. This study provided the first evidence that Rickettsia of the spotted fever group is circulating among dogs and cats in Natal.

Resumo A ocorrência de infecção ou exposição para Ehrlichia canis, Hepatozoon canis e Rickettsia spp. foi determinada em gatos ferais que viviam em dois fragmentos da Mata Atlântica, localizados em Natal, RN, Brasil e em cães que viviam em torno dos parques e em outras regiões da cidade. Enquanto amostras de soro foram coletadas de 155 animais (53 gatos que viviam nos parques, 29 cães com domicilio em torno dos parques e 73 cães do Centro de Controle de Animais -CCA), fragmentos de baço foram coletados de 20 cães eutanasiados no CCA. A detecção de anticorpos nas amostras de soros coletadas contra Rickettsia spp. e E. canis foi realizada pela Reação de Imunofluorescência Indireta. Dezessete dos 102 cães (17%) apresentaram anticorpos anti E. canis e 13% (20/155) de todos os cães e gatos (ou seja, 3% (3/102) dos cães e 32% (17/53) dos gatos) foram soropositivos para antígenos de Rickettsia spp. Os animais foram considerados expostos à R. amblyommatis ou a um genótipo muito relacionado. Entre as 20 amostras de baço de cães analisadas, oito foram positivas para E. canis e duas para Hepatozoon canis (números de acesso ao Genbank MG772657 e MG772658, respectivamente). Nenhuma das amostras de baço produziram amplicons de Babesia spp. na PCR. Observou-se, pela primeira vez, a circulação de Rickettsia do grupo da febre maculosa em cães e gatos em Natal, RN.

Molecular detection of vector borne pathogens in anemic and thrombocytopenic dogs in southern Brazil / Detecção molecular de patógenos transmitidos por vetores em cães anemicos e trombocitopenicos no Sul do Brasil

Abstract Arthropod-borne pathogens are medically important because of their ability to cause diseases in their hosts. The purpose of this study was to detect the occurrence of Ehrlichia spp., piroplasmids and Hepatozoon spp. in dogs with anemia and thrombocytopenia in southern Brazil. EDTA-whole blood was collected from 75 domestic dogs presenting anemia or/and thrombocytopenia from Guarapuava, state of Paraná, Brazil. DNA samples were subjected to conventional PCR assays for Ehrlichia spp. (dsb), piroplasmids (18S rRNA) and Hepatozoon spp. (18S rRNA), followed by sequencing and phylogenetic analyses. Among the 75 dogs, one (1.33%) was positive for Hepatozoon sp. and six (8%) were positive for piroplasmids in 18S rRNA cPCR assays. None of the dogs showed positive results in Ehrlichia spp.-cPCR targeting dsb gene. The phylogenetic analyses revealed that three piroplasm sequences were clustered with Rangellia vitalii, while one sequence was grouped with B. vogeli. The only sequence obtained from Hepatozoon spp.-PCR protocol was pooled with H. canis. Therefore, there is urgent need for differential molecular diagnosis of the two piroplasm species cited as etiological agents in clinical cases of canine hemoparasitic diseases, given the higher pathogenic potential of R. vitalii than of B. vogeli.

Resumo Agentes transmitidos por artrópodes têm grande importância na medicina veterinária devido à sua capacidade de causar doenças graves em seus hospedeiros. O presente estudo objetivou investigar a ocorrência de três patógenos transmitidos por vetores, Ehrlichia canis, Rangelia vitalii e Hepatozoon canis, em cães na região sul do Brasil. Foram coletadas amostras de sangue total de 75 cães domésticos que apresentavam anemia e/ou trombocitopenia, em Guarapuava, Paraná, Brasil. As amostras de DNA foram submetidas à técnica de PCR convencional para E. canis (dsb), piroplasmídeos (18S rRNA) e Hepatozoon spp. (18S rRNA), seguida de sequenciamento e análises filogenéticas. Das 75 amostras, uma (1,33%) foi positiva para Hepatozoon spp. e seis (8%) foram positivas para Babesia spp. Nenhuma amostra mostrou resultados positivos para Ehrlichia spp. utilizando a detecção pelo gene dsb. As análises filogenéticas revelaram que três sequências obtidas foram agrupadas no mesmo clado que R. vitalii , enquanto uma foi agrupada juntamente com B. vogeli. A única sequência obtida pelo protocolo de PCR para Hepatozoon spp. foi agrupada juntamente com H. canis. Assim, é justificada necessidade de diferenciação das espécies de piroplasmas, através do diagnóstico molecular, como agentes etiológicos nos casos clínicos de hemoparasitose canina, considerando o potencial patogênico de R. vitalii quando comparado à B. vogeli.

Rangelia vitalii and Hepatozoon canis coinfection in pampas fox Lycalopex gymnocercus from Santa Catarina State, Brazil / Infecção por Rangelia vitalii e Hepatozoon canis em Lycalopex gymnocercus proveniente do Estado de Santa Catarina, Brasil

Abstract Rangelia vitalii is a haemoparasite that infects erythrocytes, white blood cells and the cytoplasm of endothelial cells of blood capillaries of canids in South America, and has been detected in both domestic dogs and sylvatic canids. Hepatozoon canis is a parasite that infects neutrophils and monocytes of many mammalian hosts. This study reports the infection of Lycalopex gymnocercus from Santa Catarina, Brazil, with R. vitalii and H. canis. The piroplasm was observed on both blood smears and molecular tests. Many large piroplasms were detected inside the erythrocytes, with round, oval, or teardrop-shaped organism, that occurred singly or in pairs. They had an abundant, pale blue cytoplasm and decentral dark red small nucleus. The animal was also infected with H. canis that was detected only by molecular tests. The majority of haematological and biochemistry parameters were within the reference values for domestic dog and wild canids.

Resumo Rangelia vitalii é um hemoparasita que infecta eritrócitos, macrófagos e células endoteliais de canídeos na América do Sul, e vem sendo detectado tanto em cães domésticos quanto em canídeos silvestres. Hepatozoon canis é um parasita que infecta monócitos e neutrófilos de mamíferos. No presente estudo, é descrita a infecção de Lycalopex gymnocercus, proveniente de Santa Catarina, Brasil, por R. vitalii e H. canis. O piroplasma foi diagnosticado nos esfregaços sanguíneos e por técnicas moleculares. Nos eritrócitos foram observados vários merozoítos grandes, ovais, arredondados ou em forma de gota, ocorrendo isoladamente ou em pares. Estes piroplasmas apresentavam citoplasma abundante, corado em azul claro, com núcleo pequeno, avermelhado e descentralizado. O animal apresentou coinfecção com H. canis, que foi diagnosticado somente pelos testes moleculares. A maior parte dos parâmetros hematológicos e bioquímicos do animal estava dentro dos valores de referência para cães domésticos e canídeos silvestres.

Hepatozoon caimani Carini, 1909 (Adeleina: Hepatozoidae) in wild population of Caiman yacare Daudin, 1801 (Crocodylia: Alligatoridae), Pantanal, Brazil.

Previous studies showed infections of Hepatozoon caimani in wild populations of caimans in wide regions from Brazil; some of those demonstrated that trophic chain are linked to natural infections through paratenic hosts or by the direct ingestion of vectors. These studies life cycle of H. caimani contributed inestimably to the knowledge of transmission routes, yet but lack enhancement tools for better detail of parasite. This study reports the forms in the blood and tissues, and also partial molecular characterization of the H. caimani following part of the 18S rRNA region. In the southern Pantanal, there were sampling 39 adult caimans (Caiman yacare), where 31 (79.5%) were parasitized by H. caimani. Free gametocytes had an average intensity of 19.6% and intraerythrocytic forms 7.42%, in the blood smears. In stained smears of the liver and lungs of naturally infected caimans which were examined, monozoic and dizoic cysts were found in these tissues, generally next to the vessels. In the histopathology, meronts were observed in the wall of vessels from liver and kidney ducts. Blood samples were forwarded to PCR process and produced amplicons with about 600 and 900 bp, respectively, for the primers HEPF300/HEP900 and HEMO1/HEMO2. This was the first report of molecular confirmation of Hepatozoon in populations of naturally infected caimans of morphological detail of the gametocytes in scanning electron microscopy and histology of merogony in livers and kidneys of C. yacare.

Small rodents as paratenic or intermediate hosts of carnivore parasites in Berlin, Germany.

Rodents are important intermediate and paratenic hosts for carnivore parasites, including the important zoonotic agents Toxoplasma, Echinococcus and Toxocara. Monitoring of such parasites in rodents can be used to detect increasing risks for human and veterinary public health. Rodents were trapped at four sites in Berlin, two near the city center, two at the periphery. PCRs were conducted to detect Coccidia (target ITS-1) and specifically Toxoplasma gondii (repetitive element) in brain and ascarids (ITS-2) in muscle or brain tissue. During necropsies, metacestodes were collected and identified using ITS-2 and 12S rRNA PCRs. An ELISA to detect antibodies against Toxocara canis ES antigens was performed. Within the 257 examined rodents, the most frequently observed parasite was Frenkelia glareoli predominantly found in Myodes glareolus. T. gondii was only detected in 12 rodents and Microtus spp. (although strongly underrepresented) had a significantly increased chance of being positive. Neither Echinococcus nor typical Taenia parasites of dogs and cats were found but Mesocestoides litteratus and Taenia martis metacestodes were identified which can cause severe peritoneal or ocular cysticercosis in dogs, primates and humans. Using PCR, the ascarids T. canis (n = 8), Toxocara cati (4) and Parascaris sp. (1) were detected predominantly in muscles. Seroprevalence of T. canis was 14.2% and ELISA was thus more sensitive than PCR to detect infection with this parasite. Non-parametric multidimensional scaling and cluster analysis revealed that parasite communities could be grouped into an urban and a peri-urban cluster with high frequency of ascarid-positive rodents in urban and high frequency of F. glareoli in peri-urban sites. Prevalence rates of parasites in rodents with potential impact for human or veterinary public health are considerable and the monitoring of transmission cycles of carnivore parasites in intermediate rodent hosts is recommended to estimate the health risks arising from wild and domesticated carnivores.

Aspectos anatomopatológicos em cães naturalmente infectados por Hepatozoon canis / Anatomopathological findings in dogs naturally infected by Hepatozoon canis

A hepatozoonose canina é causada principalmente pelos protozoários Hepatozoon canis e H. americanum, transmitida por ingestão de carrapatos parasitados. Os sinais clínicos podem ser inespecíficos ou de difícil reconhecimento, pois geralmente ocorre associada a outras doenças. No Brasil, o parasito, e a doença, já foram identificados em vários Estados, no entanto pouco se sabe sobre as alterações clínicas e anátomo-patológicas decorrentes da infecção. O presente trabalho relata cinco casos de infecções naturais por Hepatozoon canis em cães do Estado de Minas Gerais e descreve pela primeira vez no Brasil os achados de necropsias e histopatológicos relacionados à infecção. Merontes de Hepatozoon sp., submetidos a avaliação morfométrica, foram observados em cortes histológicos de fígado, baço, medula óssea e rim.(AU)

Canine hepatozoonosis is mainly caused by protozoa Hepatozoon canis and H. americanum that are transmitted by ingestion of infected ticks. Clinical signs may be unspecific or difficult to identify, because usually hepatozoonosis occurs associated with other disease. In Brazil, the parasite and the disease, have been identified in several states, however little is known about the clinical and anatomopathological lesions resulting from the infection. This paper reports five cases of natural infection by Hepatozoon canis in dogs from Minas Gerais State and describes for the first time in Brazil the necropsy and histopathological findings related to infection. Meronts of Hepatozoon sp., submitted to morphometric evaluation, were observed in histological sections of liver, spleen, bone marrow and kidney.(AU)

Molecular diagnosis of Hepatozoon canis in symptomatic dogs in the city of Goiania, Goiás, Brazil / Diagnóstico molecular de Hepatozoon canis em cães sintomáticos do município de Goiânia, Goiás, Brasil

More than 300 species have been described in the genus Hepatozoon, occurring in different vertebrates. Among these, only Hepatozoon canis and Hepatozoon americanum are seen in dogs. Different methods may be used for laboratory diagnosis. The most common of these is direct parasitological examination of parasite stages in blood smears. The aim of this investigation was to conduct a phylogenetic study on Hepatozoon isolates from symptomatic dogs in the city of Goiânia, Goiás, Brazil. Blood samples were obtained from 40 symptomatic dogs that had been referred to the Veterinary Hospital of the Federal University of Goiás. Among these, only two samples were positive for Hepatozoon spp. using the direct parasitological method. These samples were then subjected to a DNA extraction process and amplification of a fragment of the 18S rRNA by means of PCR. Subsequently, the PCR products from each sample were purified and sequenced. The sequences obtained were then analyzed using the BLASTn algorithm, which identified both sequences of this study as Hepatozoon canis. By applying the Mega4 software, it was confirmed that these isolates of H. canis from dogs in Goiânia are similar to other reference isolates of the same species from other regions of Brazil and worldwide.(AU)

São descritas mais de 300 espécies do gênero Hepatozoon que acometem diferentes vertebrados. Entre estas, apenas Hepatozoon canis e Hepatozoon americanum são descritas em cães. Diferentes métodos podem ser utilizados para o diagnóstico laboratorial. O mais empregado é o exame parasitológico direto do parasito em esfregaços sanguíneos. O objetivo deste trabalho foi realizar um estudo filogenético em Hepatozoon isolados de cães sintomáticos de Goiânia, Goiás. As amostras de sangue foram obtidas de 40 cães sintomáticos encaminhados ao Hospital Veterinário da Universidade Federal de Goiás. Entre essas, duas únicas amostras foram positivas para Hepatozoon spp. pelo método parasitológico direto. Estas amostras foram, então, submetidas ao processo de extração de DNA e de amplificação de um fragmento de 18S rRNA por PCR. Ambas as amostras foram positivas na PCR. Posteriormente, os produtos de PCR de cada amostra foram purificados e sequenciados. As sequências obtidas foram analisadas pelo algoritmo BLASTn, sendo identificadas como Hepatozoon canis. Por meio do software Mega4 foi confirmado que estes isolados de H. canis de cães de Goiânia são semelhantes a outros isolados de referência da mesma espécie de outras regiões do Brasil e do mundo.(AU)

Hematological disorders detected in dogs infected by Hepatozoon canis in a municipality in Mato Grosso do Sul State, Brazil / Alterações hematológicas detectadas em cães infectados por Hepatozoon canis em um município do estado do Mato Grosso do Sul, Brasil

A retrospective review of hematological reports of nine dogs detected with Hepatozoon canis infection by microscopic examination of blood smears in a laboratory in the municipality of Dourados, Mato Grosso do Sul, Brazil was conducted. This study aimed to evaluate the hematological profile of these infected dogs, in addition to the occurrence of coinfections with other agents that infect blood cells, since studies concerning canine hepatozoonosis in Brazil are scarce and there are some divergences regarding H. canis infection that still require a resolution. The nine cases of H. canis infection were identified among all dogs examined at the studied laboratory in 2009 and 2010, with an occurrence of 7/1,192 (0.59%; 95% CI 0.15 - 1.02%) positive dogs in the first year and 2/1,313 (0.15%; 95% CI 0.02 - 0.55%) cases in 2010. The analysis of the hematological reports showed an occurrence of coinfection between H. canis and other agents in two (2/9; 22.22%; 95% CI 2.81 - 60.00%) dogs, one with E. canis and another with Babesia spp. (1/9; 11.11%; 95% CI 0.28 - 48.24%). Only the blood test of one dog had no alterations, based on reference values. Anemia was the most frequent hematological alteration (6/9; 66.67%; 95% CI 29.93 - 92.51%). Although the occurrence of H. canis infection was low, significative hematological alterations were observed in most infected dogs. Coinfection with Babesia spp. and E. canis was detected in two dogs and the hematological alterations cannot be attributed exclusively to H. canis in these animals. Longitudinal studies would be of fundamental importance to determine the causality of these alterations. These results highlight the importance of differential diagnosis in dogs when there is clinical suspicion of infection by hemoparasites, since the hematological changes in dogs infected by H. canis are quite variable.(AU)

Realizou-se estudo retrospectivo de laudos hematológicos de nove cães detectados com infecção por Hepatozoon canis, por meio de exame microscópico de esfregaços sanguíneos, em um laboratório no município de Dourados, Mato Grosso do Sul, Brasil. Este estudo objetivou avaliar o perfil hematológico dos cães infectados, além da ocorrência de coinfecções com outros agentes que infectam células sanguíneas, tendo em vista que estudos a respeito da hepatozoonose canina no Brasil são escassos e que existem algumas divergências a respeito da infecção por H. canis que ainda requerem esclarecimento. Os nove casos de infecção por H. canis foram identificados dentre todos os cães examinados no laboratório estudado, em 2009 e 2010, com uma ocorrência de 7/1.192 (0,59%; IC 95% 0,15 - 1,02%) cães positivos no primeiro ano e de 2/1.313 (0,15%; IC 95% 0,02 - 0,55%) em 2010. A análise dos laudos hematológicos dos nove cães evidenciou a ocorrência de coinfecção entre H. canis e outros agentes em dois (2/9; 22,22%; IC 95% 2,81 - 60,00%) desses cães, um deles com E. canis e outro com Babesia spp. (1/9; 11,11%; IC 95% 0,28 - 48,24%). Apenas o exame sanguíneo de um cão não evidenciou alterações, com base nos valores de referência. Anemia foi a alteração hematológica mais frequentemente observada (6/9; 66,67%; IC 95% 29,93 - 92,51%). Embora a ocorrência de infecções por H. canis neste estudo tenha sido baixa, alterações hematológicas significativas foram observadas na maioria dos cães infectados. Coinfecções com Babesia spp. e E. canis foram observadas em dois cães, não sendo possível atribuir exclusivamente a H. canis as alterações hematológicas detectadas nesses animais. Estudos longitudinais seriam fundamentais para determinar a causalidade dessas alterações. Os resultados ressaltam a importância de realizar diagnóstico diferencial em cães quando há suspeita clínica de infecção por hemoparasitas, uma vez que as alterações hematológicas em cães infectados por H. canis são bastante variáveis.(AU)

Hepatozoon spp. infections in wild rodents in an area of endemic canine hepatozoonosis in southeastern Brazil.

Hepatozoon canis is a tick-borne parasite that occurs worldwide. In rural areas of Brazil, H. canis vectors remain unknown, which has led to speculation about alternative routes of transmission. Small rodents can play a role in the transmission (via predation) of Hepatozoon americanum, which led us to question whether predation might be an alternative mode of transmission for H. canis. Thus, this study investigated whether Hepatozoon spp. are present in wild small rodents in forest fragments that surround rural areas in Botucatu County, São Paulo, Brazil, where canine hepatozoonosis is endemic. The study included blood samples from 158 dogs, which were screened by microscopy and molecular analysis. Blood samples and tissues from 67 rodents were obtained for histopathology and molecular detection. The prevalence of H. canis was high (66.45%) in dogs from rural areas of Botucatu, São Paulo, Brazil. The molecular analysis showed that wild rodent species in Brazil were infected with Hepatozoon spp. other than H. canis. Therefore, although the hypothesis that sylvatic rodents act as reservoirs for H. canis was not supported, the presence of monozoic cysts in the rodents suggests that, in addition to intermediate hosts, wild small rodents in Brazil might act as paratenic hosts of Hepatozoon spp. because they harbor infective stages for intermediate host predators.