Guacyara T. Cavalcante

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.

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.

Shedding of Neospora caninum oocysts by dogs fed different tissues from naturally infected cattle

Abstract Neospora caninum is one of the most important causes of abortion in dairy cattle worldwide. The distribution of N. caninum in tissues of adult cattle is unknown and the parasite has not been demonstrated histologically in tissues of cows. In the present study the distribution of N. caninum in different tissues of adult cattle was evaluated by bioassays in dogs. Seventeen dogs (2–3 month-old) were fed different tissues of 4 naturally exposed adult cattle (indirect fluorescent antibody test N. caninum titer ≥400): 5 were fed with masseter; 5 with heart, 3 with liver, 4 with brain, and 3 pups were used as non-infected control. Two dogs fed masseter, 2 fed heart, 1 fed liver, and 3 fed brain shed oocysts, and all dogs presented no seroconvertion to N. caninum during the observation period of 4 weeks. The oocysts were confirmed as N. caninum based on the detection of N. caninum-specific DNA by PCR and sequencing. The results indicate that dogs can be infected by N. caninum with different tissues of infected cattle.

Seroprevalence of Leptospira spp in cattle from Monte Negro municipality, western Amazon

The prevalence of anti-Leptospira spp antibodies was investigated in 2,109 female cattle from 86 herds of Monte Negro municipality, Rondonia, Brazil. Sera samples were evaluated by Microscopic Agglutination Test against 24 leptospira serovars. Titers =100 for at least one of 24 leptospira serovars were detected in 1,114 cows (52.8%) from 82 (95.3%) herds. The adjusted overall prevalence for Monte Negro municipality was 53.9% (49-58.7%; CI: 95%). The most prevalent serovars were Hardjo (14.5%), Wolffi (12.3%), Shermani (10.8%), Patoc (7.9%), and Hebdomadis (6.1%). Other serovars worldwidely reported like Bratislava, Pomona and Grippotyphosa were detected in low levels.

Experimental infection of dogs (Canis familiaris) with sporulated oocysts of Neospora caninum

Abstract Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.

Anti-Leptospira spp and anti-Brucella spp antibodies in humans from rural area of Monte Negro municipality, state of Rondônia, Brazilian Western Amazon

Sera from 276 humans living in 71 farms located in Monte Negro Municipality, RO., Western Amazon, Brazil were examined for anti-Leptospira spp antibodies by Microscopic Agglutination Test and for anti-Brucella spp antibodies by Tube Agglutination Test. Leptospira spp antibodies were detected in 28 (10.2%) of them with at least one positive case in 23 farms (32.4%). The most frequent leptospira serovars were Patoc (46.7%), Autumnalis (10.0%) and Shermani (10.0%). The proportion of positive males (14.5%) were higher than females (5.0%; P <0.05) and the contact with river water presented association with Leptospira spp infection (OR: 27; P=0.01). A total of 04 (1.4%) humans reacted against Brucella antigens; three farms (4.2%) presented at least one positive case of brucellosis.

Experimental infection with Neospora caninum in pregnant bitches

In this study, transplacental transmission of Neospora caninum in bitches at different stages of pregnancy was evaluated. Three bitches were inoculated in the 3rd week and three in the 6th week of gestation with 10(8) tachyzoites of N. caninum (Nc-1 strain). All the infected bitches and at least one of their offspring presented anti-N. caninum antibodies according to the indirect fluorescent antibody test (IFAT > 400). The pups and their mothers were sacrificed and tissues from the central nervous system (CNS), popliteal lymph nodes, skeletal muscle, brain, lungs, heart and liver were analyzed for the presence of N. caninum using the nested polymerase chain reaction (nested PCR), restriction fragment length polymorphism (RFLP) and immunohistochemistry (IHC). The parasite was found in the pups in lymph node, CNS, heart and liver tissues using nested PCR. There was no difference in perinatal mortality between the offspring from bitches infected in the 3rd week of gestation (60%) and in the 6th week (53.8%).

Ocorrência de anticorpos anti-Brucella abortus e anti-Brucella canis em cães rurais e urbanos do Município de Monte Negro, Rondônia, Brasil

Serum samples from 304 dogs living in urban or rural areas of Monte Negro County, Rondonia State, Brazil, were evaluated for Brucella abortus reactivity using the Rose-Bengal Test (RBT), Standard Tube agglutination Test (SAT), and Mercaptoethanol Test (MET), and for Brucella canis reactivity using the Immunodiffusion test (AGID) and Immunodiffusion test with 2-Mercaptoethanol (ME-AGID). Serum was considered positive if reacted in the MET or ME-AGID. Fifty-six (18.4%) dogs reacted by the RBT, and 12 (4.0%) by the SAT. One (0.3%) dog was positive by the MET. Eleven (3.6%) dogs reacted by the AGID, but with no confirmation by the ME-AGID. It the small occurrence of dogs reacting by the MET and the absence of reactivity by ME-AGID is emphasized.