Daniel Moura de Aguiar

Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in Dogs and Rhipicephalus sanguineus (Acari: Ixodidae) Ticks from Brazil

Abstract The current study evaluated the prevalence of Ehrlichia canis Donatien and Lestoquard in domestic dogs, Canis familiaris L., and Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) ticks from different areas of Brazil. In Monte Negro County (state of Rondônia, Brazilian western Amazon), the indirect immunofluorescence assay detected E. canis-reactive antibodies (titer ≥40) in 58/153 (37.9%) and 40/161 (24.8%) dogs from the urban and rural areas, respectively. These values were significantly different between the two areas. Ticks from a household in the urban area of Monte Negro, and from households in three other localities (162–165 adult ticks per household) in the state of São Paulo (SP) were tested by polymerase chain reaction (PCR) targeting an erlichial dsb gene fragment. The prevalence of infected ticks (given as minimal infection rate) was 2.3, 6.2, and 3.7% for populations 1 (Monte Negro), 2 (Jundiaí, SP), and 3 (São Paulo I, SP), respectively, which were statistically similar. In contrast, no infected tick was detected in population 4 (São Paulo II, SP). DNA sequences were determined for some of the PCR products generated from ticks and dogs from populations 1–3, being all identical to each other and to available sequences of E. canis in GenBank. These results reinforce previous records of E. canis-infecting dogs in Brazil. Natural infection of R. sanguineus ticks by E. canis is reported for the first time in Brazil, where this tick is the commonest species infesting dogs. RESUMO O presente estudo avaliou a prevalência da infecção por Ehrlichia canis Donatien e Lestoquard em cães domésticos, Canis familiaris L., e carrapatos Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) em diferentes áreas do Brasil. Pela Reação de Imunofluorescência Indireta (RIFI) (título ≥40), foram detectados anticorpos reativos para E. canis em 37.9% de 153 cães da área urbana e 24.8% de 161 cães da área rural do município de Monte Negro, estado de Rondônia, Amazônia brasileira. Carrapatos R. sanguineus (162 a 165 carrapatos adultos por população) de uma residência da área urbana de Monte Negro e de outras três localidades do estado de São Paulo foram testados por PCR para um fragmento do gene dsb de Ehrlichia. A prevalência de carrapatos infectados [avaliada como Prevalência Mínima de Infecção (PMI)] foi de 2.3, 6.2, e 3.7% para as populações 1 (Monte Negro), 2 (Jundiaí, SP), e 3 (São Paulo I, SP), respectivamente, sendo estes resultados similares. Nenhum carrapato infectado foi detectado na população 4 (São Paulo II, SP). Os produtos da PCR de alguns carrapatos e cães das populações 1, 2 e 3 foram seqüenciados, sendo as seqüências obtidas idênticas entre si e à seqüência de E. canis disponível no GenBank. Estes resultados reforçam estudos anteriores que relataram a infecção por E. canis em cães do Brasil, contudo relata pela primeira vez no Brasil a infecção natural por E. canis em carrapatos R. sanguineus, tido como o principal carrapato de cães no país.

Diagnóstico sorológico de erliquiose canina com antígeno brasileiro de Ehrlichia canis

The present study describes a successful isolation of Ehrlichia canis and its establishment in DH82 cells, followed by the development of an Indirect Fluorescent Antibodies Test (IFAT). Leukocytes collected from an experimentally infected dog with the Jaboticabal strain of E. canis were used to inoculate a DH82 cell monolayer. Two weeks later, the inoculated culture was checked for infectivity, every 5-6 days by both cytological staining and PCR, targeting a fragment of the dsb gene. The cell culture showed to be infected by Ehrlichia on day 27 by PCR and on day 28 by cytological staining. By the day 33, the infection rate reached 20% and on day 53, 60%. Currently, the isolate is established in DH82 cells, with several passages reaching 90-100% of infected cells, within 7 to 10 days post inoculation. After sequencing, the amplicon was identical to other E. canis corresponding sequences available in the GenBank. DH82 infected cells were used to standardize an IFAT for the diagnosis of canine ehrlichiosis.

Seroprevalence and risk factors to Ehrlichia spp. and Rickettsia spp. in dogs from the Pantanal Region of Mato Grosso State, Brazil.

Sera of 320 dogs from urban and rural areas of a Pantanal region of Brazil were evaluated for rickettsial (Rickettsia rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, R. felis, and R. bellii) and ehrlichial (Ehrlichia canis) infection by the immunofluorescence assay (IFA). Risk factors for ehrlichiosis or rickettsiosis were also evaluated. Positive reaction against Ehrlichia spp. was detected in 227 (70.9%) dogs, 119 (74.3%) from an urban area and 108 (67.5%) from rural areas (P>0.05). For Rickettsia spp., 152 (47.5%) dogs were positive, 31 (19.3%) from urban and 121 (75.6%) from rural areas (P<0.05). Highest anti-Rickettsia spp. endpoint titers were observed for R. amblyommii, suggesting homologous reactions to this agent or a very closely related organism. While most of the urban dogs were found parasitized by the tick Rhipicephalus sanguineus, infestations on rural dogs were predominated by Amblyomma cajenennse. Rickettsial infection was significantly higher (P<0.05) in rural dogs, in dogs with hunting practice, or in A. cajennense-infested dogs.

Fatores de risco associados à ocorrência de anticorpos anti-Leptospira spp. em cães do município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira

The microscopic agglutination test was applied to 329 samples taken from 156 dogs from urban area and 173 from rural areas of Monte Negro County, Rondonia State, Brazil, to determine anti-Leptospira spp. antibodies. A survey was concomitantly done to detect eventual risk factors thru the use of logistic regression. Studied variables were age, sex, diet, management, environment, contact with other species and hunting habit. Titers >100 were detected in 27.3% (90/329) of the dogs, being 23.7% (37/156) urban and 30.6% (53/173) rural dogs. The most frequent serovars were Autumnalis (22.0%), Pyrogenes (12.0%), Canicola (10.0%) and Shermani (7.5%). Dogs older than 12 months showed higher positive frequencies than younger animals (P<0.05). Risk factor were associated with commercial food as compared to homemade food (odds ratio: 3.3; 95% confidence interval : 1.3 - 3.9; P=0.02) and associated with sex, with higher occurrence in males than in females (odds ratio: 2.3; 95% confidence interval: 1.3 - 3.9; P=0.003). It was emphasized the serological occurrence of serovars kept and spread in nature by wild animals.

Soroprevalência de anticorpos anti-Ehrlichia canis em cães de Cuiabá, Mato Grosso

Canine ehrlichiosis is a disease transmitted by ticks Rhipicephalus sanguineus and caused by Ehrlichia canis, obligatory intracellular bacteria. The present study examined the prevalence of anti-E. canis in 254 dogs from four administrative regions of Cuiabá, Mato Grosso, by indirect immunofluorescence assay. There was a prevalence of 42.5% (108/254) without significant difference between the studied regions. The variables age, breed, sex, habitat, access to rural and ticks were analyzed. The antibody titers ranged from 1:40 to 1:2,560. Only 32 (29.63%) seropositive dogs were infested with ticks, all R. sanguineus. The results confirm that do not have breed, sex or age predisposition to ehrlichiosis due E. canis, while the lowest occurrence of reactive dogs indoors probably related to low tick infestation, although no significant difference between dogs with or without infestation with the tick vector.

West Nile virus surveillance, Brazil, 2008-2010

BACKGROUND West Nile virus (WNV) is an emergent pathogen that is widely distributed in North and Central America. The recent introduction in South America has focused attention on the spread of WNV across Southern American countries. The transmission network involves mosquitoes, birds, horses and humans. METHODS The serological evaluation of sera from 678 equids and 478 birds was performed using a WNV-specific blocking ELISA, and only the positive results were confirmed by plaque reduction neutralisation tests (PRNTs). Molecular analysis was performed on sera from 992 healthy equids and on 63 macerates of brains from equids that died of encephalitis and had previously tested negative for other pathogens. We also tested swabs from 928 birds. The samples analysed were collected in different biomes of Brazil. RESULTS We identified WNV antibodies by ELISA in thirteen equids and five birds, and PRNT90 confirmed WNV positivity in four equid samples collected in 2009 in an area between the Amazon and the Pantanal. None of the ELISA positive bird samples were confirmed by PRNT90, and all samples tested by RT-PCR were negative. CONCLUSION WNV circulation is confirmed by this large scale survey even in the absence of detection of clinical cases.

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica.

A survey of tick-borne pathogens in dogs and their ticks in the Pantanal biome, Brazil.

Tick and blood samples collected from domestic dogs in the Brazilian Pantanal were tested by molecular methods for the presence of tick‐borne protozoa and bacteria. Among 320 sampled dogs, 3.13% were infected by Babesia vogeli (Piroplasmida: Babesiidae), 8.75% by Hepatozoon canis (Eucoccidiorida: Hepatozoidae), 7.19% by Anaplasma platys (Rickettsiales: Anaplasmataceae), and 0.94% by an unclassified Anaplasma sp. In three tick species collected from dogs, the following tick‐borne agents were detected: (a) B. vogeli, An. platys and Ehrlichia canis (Rickettsiales: Anaplasmataceae), infecting Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks; (b) H. canis, an unclassified Anaplasma sp. and Rickettsia amblyommii (Rickettsiales: Rickettsiaceae), infecting Amblyomma cajennense sensu lato (Ixodida: Ixodidae) ticks, and (c) Rickettsia sp. strain Atlantic rainforest, an emerging human pathogen, infecting Amblyomma ovale ticks. Molecular analysis, based on a mitochondrial gene, revealed that the Am. cajennense s.l. ticks of the present study corresponded to Amblyomma sculptum, a member of the Am. cajennense species complex, and that Rh. sanguineus s.l. belonged to the tropical lineage. Whereas dogs are exposed to a number of tick‐borne bacterial and protozoan agents in the Pantanal biome, humans are potentially exposed to infection by spotted fever group rickettsiae (e.g. R. amblyommii and Rickettsia sp. strain Atlantic rainforest) because both Am. sculptum and Am. ovale are among the most important human‐biting ticks in Brazil.

Isolation and Phylogenetic Relationships of Bat Trypanosomes from Different Biomes in Mato Grosso, Brazil

Abstract:  In the order Chiroptera, more than 30 trypanosome species belonging to the subgenera Herpetosoma, Schizotrypanum, Megatrypanum, and Trypanozoon have been described. The species Trypanosoma cruzi, Trypanosoma cruzi marinkellei, and Trypanosoma dionisii are the most common in bats and belong to the Schizotrypanum subgenus. Bats from 2 different biomes, Pantanal and Amazonia/Cerrado in the state of Mato Grosso, Brazil, were evaluated according to the presence of trypanosome parasites by means of hemoculture and PCR in primary samples (blood samples). A total of 211 bats from 20 different species were caught and the trypanosome prevalence, evaluated through hemoculture, was 9.0% (19), 15.5% (13), and 4.8% (6) in the municipalities of Confresa (Amazonia/Cerrado biome) and Poconé (Pantanal biome). Among the 123 primary samples obtained from the bats, only 3 (2.4%) were positive. Phylogenetic analysis using trypanosomatid barcoding (V7V8 region of SSU rDNA) identified all the isolates and primary samples as T. c. marinkellei. The sequences of the isolates were segregated according to the bat host genus or species and suggest that co-evolutionary patterns exist between hosts and parasites. Further studies in different Brazilian regions and biomes need to be conducted in order to gain real understanding of the diversity of trypanosomes in bats.

A novel Ehrlichia genotype strain distinguished by the TRP36 gene naturally infects cattle in Brazil and causes clinical manifestations associated with ehrlichiosis

A novel Ehrlichia genotype most closely related to E. canis was reported in North American cattle in 2010, and a similar agent was subsequently identified in the hemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks and isolated in 2012. The purpose of this study was to determine whether this or other novel ehrlichial agents naturally infect Brazilian cattle. Using PCR targeting the genus-conserved dsb gene, DNA from this novel ehrlichial agent in Brazilian cattle was detected. Attempts to isolate the organism in vitro were performed using DH82 cells, but morulae and ehrlichial DNA could only be detected for approximately one month. In order to further molecularly characterize the organism, PCR was performed using primers specific for multiple E. canis genes (dsb, rrs, and trp36). Sequence obtained from the conserved rrs and dsb genes demonstrated that the organism was 99-100% identical to the novel Ehrlichia genotypes previously reported in North American cattle (rrs gene) and Brazilian ticks (rrs and dsb genes). However, analysis of the trp36 gene revealed substantial strain diversity between these Ehrlichia genotypes strains, including divergent tandem repeat sequences. In order to obtain preliminary information on the potential pathogenicity of this ehrlichial agent and clinical course of infection, a calf was experimentally infected. The calf showed clinical signs of ehrlichiosis, including fever, depression, lethargy, thrombocytopenia, and morulae were observed in peripheral blood monocytes. This study reports a previously unrecognized disease-causing Ehrlichia sp. in Brazilian cattle that is consistent with the genotype previously described in North America cattle and ticks from Brazil. Hence, it is likely that this is the organism previously identified as Ehrlichia bovis in Brazil in 1982. Furthermore, we have concluded that strains of these Ehrlichia genotypes can be molecularly distinguished by the trp36 gene, which has been widely utilized to define E. canis strain diversity.