Eficácia do dipropionato de imidocarb, da enrofloxacina e do cloridrato de oxitetraciclina no tratamento de bovinos naturalmente infectados por Anaplasma marginale / Efficacy of imidocarb dipropionate, enrofloxacin and oxytetracycline chlorydrate on the treatment of cattle naturally infected by Anaplasma marginale

O agente de maior importância, em relação à anaplasmose bovina, é o Anaplasma marginale. Os principais sinais clínicos dessa enfermidade são anemia hemolítica, icterícia, dispneia, taquicardia, febre, fadiga, lacrimejamento, sialorreia, micção frequente, anorexia, perda de peso, aborto e morte. A terapia antimicrobiana é o principal protocolo terapêutico. O objetivo do presente trabalho foi avaliar a eficácia do dipropionato de imidocarb, da enrofloxacina e do cloridrato de oxitetraciclina no tratamento de bovinos leiteiros naturalmente infectados por Anaplasma marginale. Para isso, foram avaliados 48 zebuínos mestiços que apresentavam os sinais clínicos sugestivos da doença. Os animais foram submetidos à coleta de sangue para a realização de hemograma e à extração de DNA para a confirmação da presença de A. marginale, por meio da reação em cadeia pela polimerase (PCR). Os animais foram divididos em três grupos experimentais, para realização dos protocolos terapêuticos, utilizando-se dipropionato de imidocarb, oxitetraciclina e enrofloxacina. Trinta e seis animais (75%) apresentaram reação positiva ao PCR. Os animais positivos não apresentaram diferenças significativas quanto ao hemograma e ao leucograma quando comparados com os negativos, no entanto os níveis de proteínas séricas foram inferiores nos animais positivos (P<0,05). Os três protocolos terapêuticos foram capazes de reduzir a infecção ao longo do tratamento (P<0,01), porém, após cinco dias de tratamento, a enrofloxacina apresentou maior efetividade em relação aos demais (P<0,01). Após o final do tratamento, nenhum protocolo foi capaz de eliminar totalmente a infecção pelo A. marginale em bovinos naturalmente infectados e manejados a campo.

Anaplasma marginale is the most important agent regarding cattle anaplasmosis. The main clinical signs of this disease are hemolitic anemia, jaundice, dyspnea, tachycardia, fever, fatigue, lacrimation, salivation, frequent urination, anorexia, weight loss, abortion and death. Antimicrobial therapy is the main therapeutic protocol. The aim of this paper was to assess the efficacy of this therapy frequently used in field conditions. In order to do so, 48 crossbred zebu cattle presenting suggestive clinical signs of the disease were assessed. The animals were submitted to blood sample collection to perform a CBC and DNA extraction to confirm the presence of A. marginale by the polymerase chain reaction (PCR) test. The animals were divided into three experimental groups to perform the therapeutic protocols, using imidocarb dipropionate, enrofloxacin and oxytetracycline. Thirty-six animals (75%) presented positive reaction to PCR. The positive animals do not present significant differences in the CBC and WBC when compared to the negative ones. However, the serum protein levels were lower in positive animals (P<0.05). All the treatments were able to reduce the infection throughout the treatment (P<0.01). However, in time 1, enrofloxacin presented greater effectiveness in relation to the other ones (P<0.01). After the end of the treatment no protocol was able to totally eliminate the infection by A. marginale in cattle naturaly infected and handled on the field.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinantclone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post- immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Dairy cow abortion associated with Neospora caninum and other infectious agents

Trinta e cinco vacas de rebanhos leiteiros da região Norte do estado do Paraná, com histórico de abortamento, foram pesquisadas sorologicamente para verificar a presença de anticorpos contra Neospora caninum, Toxoplasma gondii, Leptospira spp., Brucella abortus, BHV-1 e BVDV. Vinte e uma vacas apresentaram títulos sorológicos compatíveis com infecção. Todas elas, soropositivas para N. caninum, foram também soropositivas para outros agentes infecciosos, sugerindo a possibilidade de associação desses agentes nos problemas reprodutivos de bovinos, no estado do Paraná.

Occurrence of antibodies to Neospora caninum and Toxoplasma gondii in dairy cattle from the northern region of the Paraná State, Brazil

Foram analisadas, por meio da imunofluorescência indireta, 385 amostras de soros de vacas, pertencentes a 90 propriedades leiteiras de 12 municípios da região Norte do estado do Paraná. Foram observados 45 (12%) sororeagentes ao Neospora caninum e 102 (26%) ao Toxoplasma gondii. Apenas quatro animais apresentaram títulos de anticorpos para ambos os coccídios. Não foi observada diferença significativa na associação entre a sorologia do N. caninum e as variáveis relacionadas ao manejo, produção de leite, problemas reprodutivos, alimentação, presença de cães, gatos e roedores. Os resultados sugerem que neosporose e toxoplasmose estão disseminadas nos rebanhos leiteiros da região Norte do estado do Paraná, e a freqüência simultânea de anticorpos anti-N. caninum e anti-T. gondii, demonstra sua ocorrência independente em vacas leiteiras.

Transmissäo congênita de Babesia bovis / Prenatal Babesia bovis infection

A case of prenatal Babesia bovis infection in Brazil in a 17 year-old Holstein X Brown Swiss cow which aborted at approximately eight months of gestation is described and discussed. The newborn calf outlived for few minutes and then died. At necropsy, the thoracic and abdominal cavities were filled by a great volume of a transparent liquid and petechial hemorrhages in oral mucosa and epicardium were observed. Histopathologic examination stained by Haematoxylin-Eosin of lungs, spleen, liver, kidneys, brain and cerebellum revealed variable degrees of congestion and edema, particularly in the liver and brain. In the liver, inflammatory multi-nucleated cells were seen surrounding the portal area and a reasonable degeneration was noted. The brain also revealed endothelium reaction, multi-located hemorrhagic areas in blood vessels and neuronal degeneration. The diagnosis was based on necropsy and microscopic examination of brain that showed B. bovis in the capillary vessels in imprints by Giemsa

Enteroparasites prevalence among daycare and elementary school children of municipal schools, Rolândia, PR, Brazil

Coproparasitological analyses were performed on 191 daycare children and 434 elementary school children from urban and rural areas in Rolândia, Parana State, Brazil. The overall prevalence of enteroparasites was 15.2 % for daycare children and 52.5% for elementary school children. Risk factors are discussed.

Exames coproparasitológicos realizados em 191 crianças de creches e em 434 alunos da primeira à quarta série das áreas urbana e rural da rede municipal de Rolândia, PR, evidenciaram enteroparasitas em prevalência de 15,2% nas creches e de 52,5% entre os escolares. Fatores de risco são discutidos.

Toxoplasma gondii: I.Avaliaçäo da virulência de oito amostras / Toxoplasma gondii: I.Evaluation of the virulence of eight strains

Oito amostras T. gondii - LIV IV, LIV V e S 11 isoladas de suínos, RH e VPS de seres humanos, AS 28 de camundongos, HV III de cäo e CN de gato - foram inoculadas em camundongos suíços albinos e em coelhos com o objetivo de avaliar a virulência e a patogenicidade. As oito amostras apresentaram-se altamente virulentas para camundongos, matando todos os animais que receberam inóculo, via intraperitoneal, de 10 elevado a quarta potência taquizoitas, entre 6,0 e 7,8 dias, em média, após a inoculaçäo. As amostras isoladas mais recentemente, LIV V e HV III (DL50 subscrito de 7 e 15 taquizoítas, respectivamente) foram as mais virulentas. A amostra RH foi a que apresentou a menor virulência, com DL50 subscrito de 3.160 taquizoítas. A amostra LIV V também se mostrou mais virulenta para coelhos, porém, como foram inoculados apenas 2 animais, estudos posteriores devem ser realizados para confimar este achado

Toxoplasma gondii: II.Caracterizaçäo antigênica de taquizoíto de oito amostras / Toxoplasma gondii: II.Tachyzoite antigenic characterization of eigth strains

O amostras de T. gondii - LIV IV, LIV V e S 11 isoladas de suínos, RH e VPS de seres humanos, AS 28 de camundongos, HV III de cäo e CN de gato - foram analisadas com o objetivo de verificar a existência de possíveis diferenças na resposta imune quando inoculadas em coelhos. Através da técnica de ELISA, näo foram constatadas diferenças entre as oito amostras estudadas. Todas as amostras reagiram de forma semelhante com soros homólogos e heterólogos. A suspensäo antigênica, constituída de extrato celular total, mostrou-se eficiente no ELISA teste indireto, já que os soros positivos reagiram fortemente e os soros negativos näo apresentaram reaçäo contra os antígenos testados. A análise das amostras, pela técnica de Western blot, revelou que os isolados de T. gondii compartilham vários antígenos com algumas variaçöes. Dentre as bandas reconhecidas no Western blot, três foram comuns a todas as amostras: a p33 (33-37 kDa), p54 (52-55 kDa) e a p66 (66 kDa). A amostra HV III, isolada recentemente de um cäo, foi a que mais diferiu no perfil antigênico. Essa amostra näo apresentou três antígenos (50, 70 e 75 kDa) presentes nas demais amostras. Apenas dosi antígenos, um de 62 kDa da CN e outro de 67 kDa da LIV IV, foram amostra-específicos

Freqüência de anticorpos contra Babesia bigemina, B. bovis e Anaplasma marginale em rebanhos leiteiros da regiäo de Londrina, Paraná / Frequency of antibodies against Babesia bigemina, B. bovis and Anaplasma marginale in dairy farms of Parana State, Brazil

Sera collected from 417 dairy cows from Londrina, PR, Brazil, were analysed by the indirect fluorescent antibody test (IFAT) for the presence of anitbody against Babesia bigemina, Babesia bovis e Anaplasma marginale. All samples were tested diluted 1:80 using rabbit anti-bovine IgG FITC conjugate diluted 1:100. Among the samples, 289 (67.30 per cent), 251 (60.19 per cent) and 281 (67.38 per cent) showed reactivity to B. bigemina, B. bovis and A. marginale, respectively. Although the number of reactive animals as relatively high, considerable percentages of negative animals (30.70 per cent, B. bigemina, 39.18 per cent, B. bovis and 32.62 per cent, A. marginale) were susceptible, characterizing the situation as enzootically instable

Levantamento soroepidemiológico da toxoplasmose em ovinos na regiäo de Londrina, Paraná / Epidemiological study of ovine toxoplasmosis in Londrina region, Parana State, Brazil

Three hundred and seventy serum samples collected from ovine herds in Londrina, Parana State, were tested by indirect immunofluorescence for Toxoplasma gondii. The overal percentage of reagents (>=1:64) was 47.83 per cent. There were no significant differences amongst age groups. The percentage of reagents was: 48.55 per cent amongst ewes, 36.84 per cent amongst rams, 47.37 per cent amongst ram lambs and 48.31 per cent amongst lambs. Is was concluded that T. gondii infection is highly pervalent in ovine herds of Londrina region