Description of Amblyomma monteiroae n. sp. (Acari: Ixodidae), a parasite of the great horned owl (Strigiformes: Strigidae) in southern Brazil.

In 2020, adult hard ticks (males and females) were collected from great horned owls [Bubo virginianus (Gmelin, 1788)] in the coastal region in southern Brazil. The engorged females were allowed to oviposit in the laboratory and hatched larvae could be obtained. Analyses of the external morphology of the adult ticks revealed that they represent a new species, which was named Amblyomma monteiroae n. sp. Partial sequences of the mitochondrial 16S rRNA gene and the nuclear second internal transcribed spacer (ITS2) were generated from a male and a female. Their 16S rRNA haplotypes were identical to each other and closest (96% identity) to corresponding sequences of Amblyomma parvitarsum Neumann, 1901, and 90% identical to Amblyomma neumanni Ribaga, 1902. Their ITS2 haplotypes were 95.8 to 96.0 identical to the single ITS-2 partial sequence of A. parvitarsum available in GenBank. In the phylogenetic trees inferred by both 16S rRNA and ITS2 partial sequences, A. monteiroae n. sp. formed a clade with A. parvitarsum, with A. neumanni branching sister to this clade. Amblyomma monteiroae n. sp. is genetically and morphologically related to A. parvitarsum. Both tick species are unique in combining the following morphological characters: scutum extensively ornate; eyes rounded and bulging; coxa I with two moderate pointed spurs, the external longer than the internal; a single triangular short spur on coxae II-III; presence of two spines on the tibia of legs II-IV; hypostomal dentition 3/3, trochanters without spurs. However, the males of the two species can be separated by specific features in palps and festoons, whereas the females differ in specific features of the coxal spurs. The larva of A. monteiroae n. sp. can be morphologically distinguished from A. parvitarsum only by morphometry, with the former species being slightly smaller. Currently, A. monteiroae n. sp. is restricted to southern Brazil, and the only known host is B. virginianus (Strigiformes: Strigidae). The present study increases the Amblyomma Brazilian fauna to 34 species.

Sharing of living area and exposure of domestic and wild canids to ticks, Rickettsia parkeri and Rangelia vitalii in the Atlantic Forest of southern Brazil.

Spotted fever caused by the bacterium Rickettsia parkeri, and canine rangeliosis caused by the protozoan Rangelia vitalii, are emerging or re-emerging tick-borne diseases in Brazil, where the main tick vectors are Amblyomma ovale and Amblyomma aureolatum, respectively. In the present study, we evaluated exposure to R. parkeri and R. vitalii infection among domestic (Canis lupus familiaris) and wild (Cerdocyon thous) canids sharing living areas within an Atlantic Forest area of southern Brazil. Four different domestic dogs and three different wild canids were captured. Most of the ticks collected from either domestic or wild canids were identified as A. ovale and A. aureolatum. DNA of R. parkeri was amplified from 14% of the A. ovale specimens. Two domestic dogs and two wild canids were seroreactive to R. parkeri antigens with high endpoint titers (>1024). Rangelia vitalii DNA was detected in two wild canids; one of them was resampled 93 days later, again infected by R. vitalii. We report exposure/infection of domestic dogs and wild canids to R. vitalii and/or R. parkeri-infected ticks in an Atlantic Forest area shared by both canid species, indicating that they also shared the same populations of the tick vectors, A. aureolatum and A. ovale. While A. ovale, A. aureolatum, R. parkeri, R. vitalii and C. thous, are all native to the Atlantic Forest, further studies are needed to evaluate the impact of an exotic canine species - C. lupus familiaris - on the enzootic cycles of R. vitalii and R. parkeri, as well as the possible role of domestic dogs in emergence and re-emergence of R. parkeri-spotted fever in humans and canine rangeliosis, respectively.

Rehabilitation and release of confiscated songbirds into the wild: A pilot study.

Songbirds are currently the most prevalent animals in illegal trafficking in Brazil and other countries, so they are often confiscated, and this poses legal, ethical, and conservation challenges. Returning them to nature requires complex and expensive management, a topic that is sparingly addressed in the literature. Here, we described the processes and costs associated with an attempt to rehabilitate and release confiscated songbirds into the wild. A total of 1,721 songbirds of several species were quarantined, rehabilitated, and released, primarily on two farms located within their typical geographical distribution. Health assessments were performed on samples from 370 birds. Serology revealed no antibodies against Newcastle disease, and Salmonella spp. cultures were negative. Real-time polymerase chain reactions detected M. gallisepticum in samples from seven birds. Atoxoplasma spp. and Acuaria spp. infections, sepsis, and trauma were the top causes of bird death. About 6% of the released birds were recaptured, within an average period of 249 days after release, and at a mean distance of 2,397 meters from the release sites. The majority of these birds were found with free-living mates within or close to fragments of transitional ecoregions with native or cultivated grasslands, and native groves/forests, and shrublands. However, eucalyptus plantations with rich understory regeneration provided a suitable environment for the released forest species to settle, since they were recaptured during the defense of these sites. Over half of the recaptured birds presented behavioral profiles with both dominant and tame traits. Birds with dominant traits are more likely to settle in habitats and face the live decoys during fieldwork, whereas birds with tame characteristics tend to accept close contact with humans. Ultramarine grosbeak (Cyanoloxia brissonii), the least common species among those released, at the release sites showed an almost 2-fold recapture rate in the shortest mean distances from the release sites. This suggests less territory competition, perhaps a major factor of bird re-establishment here. The total per-bird cost was USD 57. Our findings suggested suitable survival and re-establishment of confiscated songbirds in the wild, when managed as we describe.

Verminotic pneumonia in South American fur seal (Arctocephalus australis) in Southern Brazil.

Verminotic pneumonia caused by Parafilaroides spp. nematodes is an underreported disease in beached South American fur seals, with scant literature available on the characteristics of parafilaroidiasis, the nematode itself, as well as its occurrence in pinnipeds in Brazil. The present work aims to identify, describe and detail the histological features of the infection and molecular characteristics of verminotic pneumonia in the South American fur seal. Twenty-six specimens of Arctocephalus australis, found dead on the northern coast of Rio Grande do Sul in 2021, were analysed. These animals were identified and submitted to necropsy and histology. For the molecular identification of metastrongylids, lung fragments were subjected to DNA extraction, polymerase chain reaction targeting the Internal transcribed spacer 2 (ITS-2) gene and subsequent sequencing. In total, 12 animals presented with parasites in the lung parenchyma on histological evaluation, and only 1 showed a granulomatous lung lesion at necropsy. Microscopically, the nematodes were found mainly in the alveoli, associated with little or no inflammatory response, and they had morphological characteristics compatible with metastrongylids. Six ITS-2 gene quality sequences were obtained; after comparative analysis via BLAST, they showed similarity with sequences obtained from Parafilaroides sp. Therefore, verminotic pneumonia caused by Parafilaroides represents an important differential diagnosis of lung disease in South American fur seals found on the northern coast of Rio Grande do Sul.

Life cycle of Ixodes schulzei (Acari: Ixodidae) in the laboratory, and demonstration of reproduction by parthenogenesis.

Ixodes schulzei is an ixodid tick that parasitizes Cricetidae rodents, chiefly the South American water rat, Nectomys squamipes, in Brazil and Argentina. In the present study, we evaluated the life cycle of I. schulzei by exposing larvae and nymphs to feed on two rodent species, N. squamipes and Calomys callosus (large vesper mouse),while adult ticks were exposed to feed on N. squamipes. Off-host developmental periods were observed in an incubator at 27 °C, 95% relative humidity, and 0:24 (light:dark) regimen. Larvae and nymphs successfully fed on either C. callosus or N. squamipes. Mean larval and nymphal feeding periods were 8.8 and 8.7 days on N. squamipes and 8.5 and 9.7 days on C. callosus. The majority of engorged larvae (79.0-80.8%) and nymphs (67.0-86.0%) successfully molted to nymphs and adults, respectively. Mean premolt periods were 11.5-11.7 days for engorged larvae and 22.5-23.7 days for engorged nymphs. Only adult females emerged from engorged nymphs, regardless of host species, i.e., none of 120 engorged nymphs molted to male. Around 18% of the unfed females presented teratologies compatible with the metagynander type of gynandromorphism. Ixodes schulzei adult females successfully fed (mean feeding period, 9.4 days), oviposited, and presented high reproductive performance (high engorged weight, egg mass weight, and % egg mass hatching), in the absence of male ticks. Our results showed that I. schulzei successfully reproduces by parthenogenesis, and corroborate field data that indicate N. squamipes as the most important host for this tick species. The male of I. schulzei remains unknown.

Ticks (Parasitiformes: Ixodida) on new world wild primates in Brazil

Brazil concentrates the largest number of primate species in the world. In the present study, an extensive literature review of ticks on New World wild monkeys has been carried out, demonstrating that between the years 1912 to 2018, 182 larvae, 137 nymphs and 31 adult ticks (10 males and 21 females) were collected on 78 primates (from 12 different species) in 28 distinct localities in the Brazilian territory. Additionally, examination of allotments of 11 tick collections of Brazil revealed that from 1919 to 2019, 93 larvae, 91 nymphs and 175 adult ticks (62 males and 113 females) were collected from 100 monkeys (among 20 different species) from 43 localities in distinct Brazilian biomes. Overall, 19 tick species were identified on wild primates in the country: Amblyomma aureolatum (Pallas, 1772), Amblyomma cajennense (Fabricius, 1787) sensu stricto, Amblyomma coelebs Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma geayi Neumann, 1899, Amblyomma incisum Neumann, 1906, Amblyomma longirostre (Koch, 1844), Amblyomma naponense (Packard, 1869), Amblyomma nodosum Neumann, 1899, Amblyomma ovale Koch, 1844, Amblyomma parkeri Fonseca & Aragão, 1952, Amblyomma romarioi Martins, Luz & Labruna, 2019, Amblyomma rotundatum Koch, 1844, Amblyomma sculptum Berlese, 1888, Haemaphysalis juxtakochi Cooley, 1946, Ixodes fuscipes Koch, 1844, Rhipicephalus microplus (Canestrini, 1888), Rhipicephalus sanguineus (Latreille, 1806) sensu lato,and Ornithodoros rostratus Aragão, 1911. The presence of A. incisum, A. naponense, A. nodosum, A. rotundatum and I. fuscipes on monkeys is recorded for the first time. Thisresearch is therefore a significant contribution to the knowledge of tick speciesassociated with non-human primates in the Neotropical region.

Ticks (Acari: Ixodidae) on wild raptors in Brazil

This research reports ticks on wild raptors in Brazil. Between the years 1936 and 2019, 127 larvae, 230 nymphs and 34 adult ticks were collected on 92 raptors (among 27 different species) from 35 localities in distinct Brazilian biomes. Additionally, an extensive literature review on ticks on wild raptors has been carried out, demonstrating that from 1993 to 2016, 29 larvae, 81 nymphs, 29 adults and 186 indeterminate immature ticks (larvae or nymphs) were collected on 41 raptors (16 different species) in 17 distinct localities in the Brazilian territory. The following tick species were identified on wild raptors in the country: Amblyomma aureolatum (Pallas, 1772), Amblyomma auricularium (Conil, 1878), Amblyomma brasiliense Aragão, 1908, Amblyomma cajennense (Fabricius, 1787) sensu stricto, Amblyomma calcaratum Neumann, 1899, Amblyomma coelebs Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma ovale Koch, 1844, Amblyomma parkeri Fonseca & Aragão, 1952, Amblyomma sculptum Berlese, 1888, Haemaphysalis juxtakochi Cooley, 1946, Rhipicephalus microplus (Canestrini, 1888), and Rhipicephalus sanguineus (Latreille, 1806) sensu lato. This study is therefore a significant contribution to our knowledge of the ticks associated with Brazilian raptors.

Caracterização das lesões parasitárias de ovinos observadas na linha de abate / Characterization of parasitic lesions of sheep observed at slaughter line

Considerando a possibilidade de erros na identificação das lesões e a necessidade de melhorar o diagnóstico, o objetivo desse trabalho foi caracterizar macroscopicamente e microscopicamente as principais lesões parasitárias observadas em ovinos na linha de abate. Os materiais foram colhidos durante duas visitas a um matadouro frigorífico de ovinos no estado do Rio Grande do Sul. Totalizaram-se 161 amostras com lesões parasitárias de ovinos em diferentes órgãos. As alterações observadas incluíam hidatidose, cisticercose por Cysticercus ovis, cisticercose por Cysticercus tenuicollis, sarcocistose (morfologia compatível com Sarcocystis gigantea), fasciolose (Fasciola hepatica) e esofagostomose. Das 161 amostras, 25,5% correspondiam a hidatidose, e os cistos hidáticos foram observados, predominantemente, nos pulmões (46,3%) e fígado (41,5%). Ao corte, os cistos demonstraram três padrões morfológicos: cistos uniloculares viáveis (34%); cistos multivesiculares viáveis (31,7%); e cistos hidáticos (uniloculares e multivesiculares) degenerados (34%). As lesões de cisticercose por C. ovis (22,4%) foram visualizadas no coração (63,9%), língua (13,9%), músculo masseter (11,1%) e diafragma (11,1%). Morfologicamente os cisticercos foram classificados em vivos (viáveis), degenerados e mineralizados. Lesões provocadas por S. gigantea (19,2%) estavam presentes na túnica muscular do esôfago, na língua e na laringe. Macroscopicamente, observaram-se múltiplas estruturas nodulares brancas que continham uma cápsula fibrosa e lúmen preenchido por material gelatinoso translúcido. Cisticercose por C. tenuicollis representaram 18,6% das lesões. Os cistos foram observados aderidos no omento, no mesentério, na cápsula do fígado e na vesícula biliar. Morfologicamente os cistos foram classificados como vivos (viáveis) e degenerados. Os cistos vivos apresentavam a parede translúcida ou levemente opaca, contendo um escólex no seu interior. Os cistos degenerados eram brancacentos, firmes, com espessa cápsula fibrosa e centro mineralizado. Lesões provocadas por F. hepatica corresponderam a 7,4% dos casos. As lesões hepáticas caracterizavam-se macroscopicamente por espessamento variável dos ductos biliares por fibrose e ocasionalmente havia exemplares de F. hepatica no lúmen dos ductos. Em oito casos, observaram-se áreas de acentuada necrose do parênquima hepático. Lesões provocadas por Oesophagostomum spp. perfizeram 6,8% dos casos. As alterações foram observadas no intestino delgado e intestino grosso de todos os ovinos e em dois casos, havia também envolvimento dos linfonodos mesentéricos. Nos intestinos, as lesões caracterizavam-se por nódulos bem delimitados, salientes na serosa, firmes e que invadiam também a camada muscular. Nos linfonodos havia obliteração do parênquima nodal por acentuada mineralização. É extremamente importante a identificação morfológica das diferentes lesões parasitárias encontradas nas linhas de abate em frigoríficos de ovinos, para posterior destino correto das mesmas. As alterações devem ser avaliadas com o intuito principal de reconhecer a sua capacidade infecciosa. Além disso, é fundamental o conhecimento dos locais anatômicos mais comuns em que cada alteração geralmente costuma ocorrer.(AU)

Considering the possibilities of mistaken diagnoses in identifying lesions at meat inspection this study was designed to provide data for a better-educated diagnosis by the meat inspectors through the gross and microscopic characterization of parasitic lesions observed in slaughtered sheep at the inspection line. One hundred and sixty-one samples of parasitic lesions were sampled from various organs of slaughtered sheep during two visits to a sheep abattoir located in the state of Rio Grande do Sul, Brazil. Lesions observed included hydatid cysts, cysticercosis due to Cysticercus ovis and to Cysticercus tenuicollis, sarcocystosis (morphology compatible with Sarcocystis gigantea), fasciolosis (Fasciola hepatica) and oesophagostomosis. Twenty-five point five percent of the 161 samples corresponded to hydatidosis and the hydatid cysts were observed predominantly in the lungs (46.3%) and liver (41.5%). On cut surface, the cysts had three different morphological patterns: viable unilocular cysts (34%); viable multivesicular cysts (31.7%); and degenerate (unilocular and multivesicular) hydatid cysts (34%). Cysticercosis by C. ovis (22.4%) was observed in the myocardium (63.9%), tongue (13.9%), masseter (11.1%), and diaphragm (11.1%). Morphologically the cysticerci were classified as viable, degenerated or mineralized. Lesions caused by S. Gigantea (19.2%) were observed in the muscle layer of the esophagus, tongue, and larynx. Grossly there were multiple white nodular structures that contained a fibrous capsule with the lumen filled by translucent and gelatinous material. Cysticercosis by C. tenuicollis accounted for 18.6% of observed parasitic lesions; the cysts adhered to the omentum, mesentery, liver capsule, and serosal surface of gall bladder; grossly the cysts were classified as viable and degenerated. Viable cysts had translucent or slightly opaque walls and contained a single scolex. Degenerated cysts were white, firm and with a thick fibrous capsule and mineralized center. Lesions caused by F. hepatica accounted for 7.4% of the cases and were grossly characterized by variable fibrous thickening of bile ducts which occasionally contained the adult flukes in their lumina. In eight cases there were marked areas of necrosis in the hepatic parenchyma. Lesions caused Oesophagostomumspp. accounted for 6.8% of the observed parasitic cases and the changes were observed in all cases in the walls of the small and large intestine; in two cases mesenteric lymph nodes were also involved. In the intestines, lesions were characterized by firm well-circumscribed nodules prominent in the serosal surface and also invading the muscle layer. In the lymph nodes marked mineralization obliterated the nodal parenchyma. The correct identification of the various parasitic lesions found in the viscera of sheep in the abattoir inspection line it is important to dictate the proper destination of affected organs and carcasses. The lesions should be evaluated aiming to determine their infective capacity and to acquire knowledge about their more frequent anatomical sites.(AU)

Evaluation of the vector competence of six ixodid tick species for Rangelia vitalii (Apicomplexa, Piroplasmorida), the agent of canine rangeliosis.

Rangelia vitalii is the etiologic agent of canine rangeliosis, a severe piroplasmosis that affects domestic dogs in Brazil, Uruguay and Argentina. While R. vitalii is one of the most pathogenic tick-borne pathogens for dogs in the world, its tick vector has remained unknown. The present study evaluated the vector competence of Rhipicephalus sanguineus sensu lato (both tropical and temperate species), Amblyomma aureolatum, Amblyomma ovale, Amblyomma tigrinum, and Amblyomma sculptum for R. vitalii. These six tick species were selected for the study because they comprise the main tick species infesting dogs within the distribution area of canine rangeliosis in South America. Acquisition feeding of the above six tick species was performed on domestic dogs showing clinical signs of canine rangeliosis, after being experimentally infected through intravenous inoculation or infestation with R. vitalii-infected ticks. Thereafter, engorged ticks were evaluated for transstadial and transovarial passages of R. vitalii through molecular analysis after molting or oviposition and egg hatching. The resultant ticks were evaluated for their competence to transmit R. vitalii to susceptible dogs. Among the six tick species, only A. aureolatum was able to acquire and perpetuate R. vitalii by transstadial and transovarial passages, as demonstrated by >5% infection rates of ticks after hatching or molting. When exposed to transmission feeding, only A. aureolatum ticks were competent to transmit R. vitalii to dogs, which became severely ill, and the results confirmed by molecular methods and blood smear examination to have acquired rangeliosis. Results of the present study, coupled with epidemiological data, indicate that A. aureolatum is a natural vector of R. vitalii. Our results also indicate that R. vitalii is the first Piroplasmorida agent to be transovarially transmitted in Amblyomma ticks.

Aspectos macroscópicos e histológicos da cisticercose bovina / Macroscopic and histological findings of bovine cysticercosis

Cisticercose bovina é uma importante doença parasitária de caráter zoonótico, com elevada ocorrência em algumas regiões do Brasil. Considerando a possibilidade de erro na identificação das lesões, bem como a dificuldade de classificação dos cistos e a necessidade de melhorar o diagnóstico, o objetivo desse trabalho foi caracterizar e correlacionar as lesões macroscópicas e microscópicas da cisticercose bovina, além de utilizar a técnica da PCR para auxiliar na identificação do agente. Amostras de lesões císticas e nodulares de bovinos, compatíveis macroscopicamente com cisticercose, foram coletadas em abatedouros frigoríficos do Estado do Rio Grande do Sul. Os cistos foram divididos em três grupos: Grupo 1, cisticercos vivos (viáveis); Grupo 2 (subdividido em 2a e 2b), cisticercos degenerados com potencial escólex viável; e Grupo 3 cisticercos mortos (mineralizados). Após a obtenção das lâminas histológicas dos cisticercos de cada grupo, foi realizada a correlação macroscópica e microscópica. Para a realização da técnica da PCR foram utilizadas lesões císticas de 26 bovinos. Foram analisados cisticercos de 127 bovinos, totalizando 232 cistos. Dos 127, 46 bovinos (36,2%) apresentaram mais de um cisticerco e 81 (63,8%) um cisticerco cada. Em relação a localização anatômica dos cistos, o coração demonstrou o maior envolvimento (55,9%), seguido do músculo masseter (22,8%). Quando houve o envolvimento de dois órgãos em um mesmo bovino, coração e músculo masseter juntos, totalizaram 11 casos (8,6%). De maneira geral a média da frequência de cisticercose foi de 10% a 15% de bovinos acometidos por lote. Entretanto, a média isolada de alguns lotes demonstrou condenações acima de 50%, 80% e 90%. Morfologicamente, os 232 cisticercos foram classificados dentro de três grupos. No Grupo 1, 23 cistos (9,9%) foram considerados como vivos (viáveis), e eram caracterizados por lesões císticas com parede translúcida ou levemente opaca, contendo líquido claro e um ponto esbranquiçado no interior (escólex). Na histologia, os cistos eram compostos por uma membrana de onde invaginava um escólex de Taenia saginata. No segundo grupo (Grupo 2), foram incluídos 156 (67,2%) cisticercos degenerados com potencial escólex viável e macroscopicamente os cistos demonstraram dois padrões morfológicos distintos. No primeiro deles (Grupo 2a), visualizado em 111 casos (71,1%), observaram-se lesões nodulares com aspecto caseoso. Microscopicamente, os cistos caracterizavam-se por formações nodulares compostas por área central contendo escólex e membrana, ambos degenerados, e necrose caseosa. No segundo padrão (Grupo 2b), observado em 45 cisticercos (28,9%), as lesões também eram caseosas, entretanto ao corte os cistos demonstravam na área central um orifício em meio ao material caseoso. Os aspectos microscópicos dos 45 cistos incluídos no segundo padrão macroscópico assemelhavam-se aos cisticercos do primeiro padrão. Entretanto, oito cistos (17,8%) demonstraram somente a membrana parasitária viável e em um cisto notou-se a membrana com o escólex viável. No restante dos 36 cistos (80%), observou-se área central contendo escólex e membrana, ambos degenerados, e necrose caseosa. No terceiro grupo de classificação morfológica dos cisticercos (Grupo 3), foram inseridos os cistos mineralizados (mortos), totalizando 53 cistos (22,9%). O aspecto macroscópico desses cisticercos caracterizava-se por lesões nodulares, amarelas, firmes ao corte, que se fragmentavam. Histologicamente observaram-se formações nodulares com área central de acentuada mineralização, rodeadas por infiltrado inflamatório granulomatoso. Dos 127 bovinos, foi realizado PCR a partir do DNA extraído dos cisticercos de 26 bovinos, no qual 24 foram positivos para cisticercose. Em relação aos dois cisticercos negativos, um deles fazia parte do Grupo 2a e o outro do Grupo 3. A correlação entre os aspectos macroscópicos e microscópicos do segundo padrão morfológico observado dentro do Grupo 2, demonstrou que esse subgrupo representa o maior problema na interpretação, pois alguns cistos apresentaram características de viabilidade. Macroscopicamente esses cisticercos podem ser identificados quando cortados, porque possuem um orifício na área central que pode auxiliar no diagnóstico.(AU)

Bovine cysticercosis is an important zoonotic parasitic disease with high prevalence in several regions of Brazil. Considering the need of improvement of the accuracy of diagnosis of these lesions, as well as the difficulty of classification of the cysts, this study aimed to correlate gross and histopathological changes of bovine cysticercosis and to use polymerase chain reaction (PCR) as an aid in their identification. Cystic and nodular lesions from cattle, grossly compatible with cysticercosis, were sampled in slaughterhouses from Rio Grande do Sul State. Lesions were allotted in one of the following groups. Group 1: viable cysticercus; Group 2 (subdivided 2a e 2b): degenerating cysticercus with a potentially viable scolex; and Group 3: dead cysticercus (mineralized). The gross and microscopic aspects of every cysticerci of each group were compared. Two hundred and thirty two cysts and nodules compatible with cysticercus were sampled from 127 bovine. Twenty six of those lesions were tested with PCR. Out of 127 cattle, 46 (36.2%) had more than one cyst and the remaining 81 (63.8%) had on cyst each. Myocardium was the most frequently involved anatomical site (55.9%), followed by masseter muscle (22.8%). When there was more than one organ involved in the same bovine, myocardium a master muscle sum up 11 cases (8.6%). In general, the average of cysticercosis frequency was 10-15%. However the average in some cattle lots was in excess of 50%, 80% and 90%. Morphologically, 232 cysticerci were classified in three groups. In Group 1, 23 cysticerci (9.9%) were considered viable and were characterized by cysts of translucent or slightly opaque wall, containing clear and a white point (scolex) within the cyst. Histologically, the cysts consisted of a membrane from which a scolex of Taenia saginata invaginated. One hundred and fifty six cysts (67.2%) were allotted in Group 2; grossly these cysts revealed two different morphological patterns. In 111 (71.1%) cases of Group 2 (Group 2a) nodular caseous lesions were observed. Histologically, the cysts were characterized by nodules consisting by a central area containing the scolex and membrane, both degenerated, and caseous necrosis. In the remaining 45 (28.9%) cases of Group 2 (Group 2b), lesions were also caseous; however, at cut surface the cysts had a central hole amidst the caseous material. The microscopic aspect of the 45 cysts included in the second was similar to that of the first pattern. However in eight (17.8%) of the 45 cysts only a viable parasitic membrane was observed and in one cyst the membrane and viable scolex were observed. In the remaining 36 cases (80%), the cysts consisted of a central area containing both degenerated membrane and scolex, and caseous necrosis. In Group 3, 53 dead cysts (mineralized) (22.9%) were found among the total of 232 cysts. The gross aspect of these cysticerci was characterized by yellow form nodules which crumbled when cut. Histologically nodules were observed with marked central area of mineralization surrounded by granulomatous inflammatory response. Twenty four of the twenty cysts examined by PCR were positive for Cysticercus bovis and two of them were negative. One of the negatives was part of Group 2 (degenerated cysts) and the other one of the Group 3 (dead mineralized cysts). The correlation between gross and microscopic aspects of the second morphologic aspect of the Group 2 demonstrated that this subset represents a major complicating factor in interpretation, since a large number of these cysts reveal characteristics of viability. Grossly, these cysticerci might be identified when cut, since a hole in the central area will be observed aiding in recognizing his lesions.(AU)

Experimental infection with Rickettsia rickettsii in an Amblyomma dubitatum tick colony, naturally infected by Rickettsia bellii.

Amblyomma dubitatum engorged females, naturally infected by Rickettsia bellii, were used to establish a laboratory colony. Larvae, nymphs, and adults were exposed to two strains of Rickettsia rickettsii by feeding on needle-inoculated guinea pigs, and thereafter reared on uninfected guinea pigs. After acquisition feeding, engorged larvae and nymphs molted to nymphs and adults, respectively, which were shown to be infected (confirming transstadial perpetuation), and were able to transmit both strains of R. rickettsii to uninfected animals, as demonstrated by clinical, serological, and molecular analyses. However, the larval, nymphal, and adult stages of A. dubitatum showed to be only partially susceptible to R. rickettsii infection, since in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. While transovarial transmission of R. rickettsii was inefficient in the A. dubitatum engorged females of the present study, 100% of these females passed R. bellii transovarially. Because it has been reported that a primary infection by a Rickettsia species would preclude transovarial transmission of a second Rickettsia species, it is likely that the ineffectiveness of A. dubitatum to perpetuate R. rickettsii by transovarial transmission was related to its primary infection by R. bellii; however, it could also be related to unknown factors inherent to A. dubitatum. The relevance of A. dubitatum as a natural vector of R. rickettsii to humans or animals is discussed.

Natural infection of the wild canid, Cerdocyon thous, with the piroplasmid Rangelia vitalii in Brazil.

Canine rangeliosis, caused by the piroplasmid protozoon Rangelia vitalii, is currently recognized as a reemerging disease that affects domestic dogs in Brazil. In the present study, piroplasmid infection was searched in wild canids (20 Cerdocyon thous and 4 Lycalopex gymnocercus) in Brazil. Molecular analysis, based on PCR and DNA sequencing of a portion of the 18S rRNA gene, revealed that 30% (6/20) C. thous were infected by R. vitalii. Blood and bone marrow samples from one of the R. vitalii-infected C. thous were inoculated into a domestic dog, which developed clinical rangeliosis that was confirmed by molecular tests. However, the C. thous donor showed no clinical, hematological or biochemical alterations, even though its R. vitalii infection status was confirmed for at least 80 days. These observations suggest that R. vitalii is not as highly pathogenic for C. thous as it is for domestic dogs. Phylogenetic analysis inferred by the 18S rRNA gene placed R. vitalii embedded in the clade 'Babesia sensu stricto', consisting of a number of species that represent truly the genus Babesia. It is proposed that the species R. vitalii should be transferred to the genus Babesia. The present study expands our knowledge on the natural history of R. vitalii, suggesting that it might have a natural cycle involving the wild canid C. thous. Further studies are needed to confirm that C. thous is a natural reservoir of R. vitalii in Brazil.

Rickettsial infection in Amblyomma cajennense ticks and capybaras (Hydrochoerus hydrochaeris) in a Brazilian spotted fever-endemic area.

BACKGROUND: Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is the deadliest spotted fever of the world. In most of the BSF-endemic areas, capybaras (Hydrochoerus hydrochaeris) are the principal host for the tick Amblyomma cajennense, which is the main vector of BSF. METHODS: In 2012, a BSF case was confirmed in a child that was bitten by ticks in a residential park area inhabited by A. cajennense-infested capybaras in Itú municipality, southeastern Brazil. Host questing A. cajennense adult ticks were collected in the residential park and brought alive to the laboratory, where they were macerated and intraperitoneally inoculated into guinea pigs. A tick-inoculated guinea pig that presented high fever was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). Tissue samples from guinea pig passages were also used to inoculate Vero cells through the shell vial technique. Infected cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB). Blood serum samples were collected from 172 capybaras that inhabited the residential park. Sera were tested through the immunofluorescence assay using R. rickettsii antigen. RESULTS: A tick-inoculated guinea pig presented high fever accompanied by scrotal reactions (edema and marked redness). These signs were reproduced by consecutive guinea pig passages. Rickettsia was successfully isolated in Vero cells that were inoculated with brain homogenate derived from a 3rd passage-febrile guinea pig. Molecular characterization of this rickettsial isolate (designated as strain ITU) yielded DNA sequences that were all 100% identical to corresponding sequences of R. rickettsii in Genbank. A total of 83 (48.3%) out of 172 capybaras were seroreactive to R. rickettsii, with endpoint titers ranging from 64 to 8192. CONCLUSIONS: A viable isolate of R. rickettsii was obtained from the tick A. cajennense, comprising the first viable R. rickettsi isolate from this tick species during the last 60 years. Nearly half of the capybara population of the residential park was seroreactive to R. rickettsii, corroborating the findings that the local A. cajennense population was infected by R. rickettsii.

Natural blood feeding and temperature shift modulate the global transcriptional profile of Rickettsia rickettsii infecting its tick vector.

Rickettsia rickettsii is an obligate intracellular tick-borne bacterium that causes Rocky Mountain Spotted Fever (RMSF), the most lethal spotted fever rickettsiosis. When an infected starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. Using customized oligonucleotide microarrays and high-throughput microfluidic qRT-PCR, we analyzed the effects of a 10°C temperature elevation and of a blood meal on the transcriptional profile of R. rickettsii infecting the tick Amblyomma aureolatum. This is the first study of the transcriptome of a bacterium in the genus Rickettsia infecting a natural tick vector. Although both stimuli significantly increased bacterial load, blood feeding had a greater effect, modulating five-fold more genes than the temperature upshift. Certain components of the Type IV Secretion System (T4SS) were up-regulated by blood feeding. This suggests that this important bacterial transport system may be utilized to secrete effectors during the tick vector's blood meal. Blood feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. The modulated genes identified in this study, including those encoding hypothetical proteins, require further functional analysis and may have potential as future targets for vaccine development.

Hematologic and bone marrow changes in dogs experimentally infected with Rangelia vitalii.

BACKGROUND: Rangelia vitalii is a tick-transmitted piroplasm that causes both hemolytic and hemorrhagic disease in dogs in Brazil. OBJECTIVE: The aim of this study was to evaluate the response of the bone marrow in dogs experimentally infected with R vitalii during the acute stage of the disease. METHODS: For this study, 2 groups of a total of 12 young dogs were used. Group A was composed of healthy dogs (n = 5), and group B consisted of animals infected with R vitalii (n = 7). Blood samples were collected on days 0, 10, 20, and 30 post-inoculation and stored in EDTA tubes for a full hematology profile, including a reticulocyte count. On days 10 and 20, bone marrow samples were collected, stained, and examined. RESULTS: In infected dogs anemia was identified on days 10 and 20 post-inoculation (P < .01), and on day 20 reticulocytosis was present. Infected dogs had leukopenia due to neutropenia and eosinopenia, along with lymphocytosis and monocytosis, when compared with control animals. In bone marrow, the myeloid:erythroid ratio was significantly decreased (P < .05) in infected dogs due to increased erythroid precursors. CONCLUSIONS: Dogs experimentally infected with R vitalii develop regenerative extravascular hemolytic anemia accompanied by erythroid hyperplasia in the bone marrow. During the acute phase of the disease, leukopenia due to neutropenia and eosinopenia suggests intense tissue recruitment of these cells in response to the endothelial damage caused by this parasite.

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii.

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia.

Experimental infection with Rangelia vitalii in dogs: acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment.

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n=5), and Group B consisted of animals infected with R. vitalii (n=7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n=6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs.

Experimental infection of Amblyomma aureolatum ticks with Rickettsia rickettsii.

We experimentally infected Amblyomma aureolatum ticks with the bacterium Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever (RMSF). These ticks are a vector for RMSF in Brazil. R. rickettsii was efficiently conserved by both transstadial maintenance and vertical (transovarial) transmission to 100% of the ticks through 4 laboratory generations. However, lower reproductive performance and survival of infected females was attributed to R. rickettsii infection. Therefore, because of the high susceptibility of A. aureolatum ticks to R. rickettsii infection, the deleterious effect that the bacterium causes in these ticks may contribute to the low infection rates (<1%) usually reported among field populations of A. aureolatum ticks in RMSF-endemic areas of Brazil. Because the number of infected ticks would gradually decrease after each generation, it seems unlikely that A. aureolatum ticks could sustain R. rickettsii infection over multiple successive generations solely by vertical transmission.

Detection and molecular characterization of a canine piroplasm from Brazil.

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name "nambiuvú", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvú. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp. sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of "nambiuvú" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia.

Cross-mating experiments with geographically different populations of Amblyomma cajennense (Acari: Ixodidae).

The present study evaluated the reproductive compatibility of the crosses between adult ticks of the following three geographically different populations of Amblyomma cajennense: State of São Paulo (SP), southeastern Brazil; State of Rondônia (RO), northern Brazil; and Colombia (CO). In addition, crosses between A. cajennense ticks from Argentina (AR) and SP ticks were also performed. The Argentinean population (AR) was compatible with SP because their crosses resulted in high % egg hatching (mean values ranging from 71.5 to 93.5%), similarly to all homologous (intrapopulational) crosses. In contrast, the tick populations SP, RO, and CO were shown to be incompatible with each other, since their heterologous (interpopulational) crosses always resulted in very low % egg hatching (range: 0-5%). The F(1) larval offspring derived from some of these females that yielded 5% egg hatching were reared until the F(1) adult stage. In all cases, only adult females molted from engorged nymphs. These F(1) females were likely to be a product of thelytokous parthenogenesis of the SP, RO, and CO females that were used in the heterologous crosses. Reproductive incompatibility is not expected to occur between different populations of a single species. Thus, our results suggest that the taxon A. cajennense might be represented by a complex of different species, whereas SP and AR ticks might represent a single species. Further populational genetic studies, coupled with extensive morphological analyses, are needed to clarify and determine a possible complex of valid species that might have been classified under the taxon A. cajennense.