Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

Evidence of spotted fever group rickettsiae in state of Rio de Janeiro, Brazil

Ticks were obtained from dogs from February to September of 1999 at weekly intervals, in the County of Piraí, State of Rio de Janeiro. Four hundred seventy four ixodids were taxonomically identified, 103 Amblyomma cajennense, seven Amblyomma ovale, 209 Rhipicephalus sanguineus, and 155 Amblyomma sp. An hemolymph test associated with Giemsa's stain revealed two specimens in 163 ticks tested (R. sanguineus and Amblyomma sp), containing rickettsia-like organisms. Direct immunofluorescence verified the presence of spotted fever group rickettsia in one specimen of R. sanguineus. Considering the limited information on rickettsiosis in Brazil, principally in relation to the vectors involved in perpetuating it in foci, these preliminary results give us an idea on the importance of infection in ticks, allowing to expand our knowledge on this zoonosis.

Rickettsia parkeri: a Rickettsial pathogen transmitted by ticks in endemic areas for spotted fever rickettsiosis in southern Uruguay

At first Rickettsia conorii was implicated as the causative agent of spotted fever in Uruguay diagnosed by serological assays. Later Rickettsia parkeri was detected in human-biting Amblyomma triste ticks using molecular tests. The natural vector of R. conorii, Rhipicephalus sanguineus, has not been studied for the presence of rickettsial organisms in Uruguay. To address this question, 180 R. sanguineus from dogs and 245 A. triste from vegetation (flagging) collected in three endemic localities were screened for spotted fever group (SFG) rickettsiosis in southern Uruguay. Tick extracted DNA pools were subjected to PCR using primers which amplify a fragment of the rickettsial gltA gene. Positive tick DNA pools with these primers were subjected to a second PCR round with primers targeting a fragment of the ompA gene, which is only present in SFG rickettsiae. No rickettsial DNA was detected in R. sanguineus. However, DNA pools of A. triste were found to be positive for a rickettsial organism in two of the three localities, with prevalences of 11.8% to 37.5% positive pools. DNA sequences generated from these PCR-positive ticks corresponded to R. parkeri. These findings, joint with the aggressiveness shown by A. triste towards humans, support previous data on the involvement of A. triste as vector of human infections caused by R. parkeri in Uruguay.

SEROLOGICAL SURVEY OF Ehrlichia SPECIES IN DOGS, HORSES AND HUMANS: ZOONOTIC SCENERY IN A RURAL SETTLEMENT FROM SOUTHERN BRAZIL

SUMMARY The aims of this study were to determine the seroprevalence of Ehrlichia spp. and risk factors for exposure in a restricted population of dogs, horses, and humans highly exposed to tick bites in a Brazilian rural settlement using a commercial ELISA rapid test and two indirect immunofluorescent assays (IFA) with E. canis and E. chaffeensis crude antigens. Serum samples from 132 dogs, 16 horses and 100 humans were used. Fifty-six out of 132 (42.4%) dogs were seropositive for E. canis. Dogs > one year were more likely to be seropositive for E. canis than dogs ≤ one year (p = 0.0051). Ten/16 (62.5%) and 8/16 (50%) horses were seropositive by the commercial ELISA and IFA, respectively. Five out of 100 (5%) humans were seropositive for E. canis and E. chaffeensis. Rhipicephalus sanguineus (n = 291, 97.98%) on dogs and Amblyomma cajennense (n = 25, 96.15%) on horses were the most common ticks found. In conclusion, anti-Ehrlichia spp. antibodies were found in horses; however, the lack of a molecular characterization precludes any conclusion regarding the agent involved. Additionally, the higher seroprevalence of E. canis in dogs and the evidence of anti-Ehrlichia spp. antibodies in humans suggest that human cases of ehrlichiosis in Brazil might be caused by E. canis, or other closely related species.

PARTICIPATION OF TICKS IN THE INFECTIOUS CYCLE OF CANINE VISCERAL LEISHMANIASIS, IN TERESINA, PIAUÍ, BRAZIL

In this study, we detected Leishmania spp. infection in R. sanguineus collected from dogs that were naturally infected with L. (L.) infantum. We examined 35 dogs of both sexes and unknown ages. The infected dogs were serologically positive by the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Quick Test-DPP (Dual Path Platform), as well as parasitological examination of a positive skin biopsy or sternal bone marrow aspiration. Ten negative dogs were included as controls. The ticks that infested these dogs were collected in pools of 10 adult females per animal. The PCR was performed with specific primers for Leishmania spp., which amplified a 720-bp fragment. Of the 35 analyzed samples, a product was observed in eight samples (8/35; 22.9%). We conclude that the presence of parasite DNA suggests that ticks participate in the zoonotic cycle of canine visceral leishmaniasis, in the city of Teresina, Piauí.

Rickettsial Infection Caused by Accidental Conjunctival Inoculation

The most common transmission route of tick-borne Rickettsia is through tick bite; nevertheless, other transmission routes should also be considered. We report a case of rickettsial infection in a 15-year-old boy caused by accidental contamination of the conjunctiva through the infected fluid of a crushed engorged tick removed from a dog. Right eye pain, conjunctival hyperaemia with mucopurulent exudate, chemosis and eyelid oedema were the first signs and symptoms. Two days later, the boy developed fever, myalgia, headache, abdominal pain and was vomiting; physical examination showed multiple cervical adenopathies but no rash. He was treated with doxycycline (200 mg/day) for 7 days with progressive resolution of clinical signs. Rickettsial infection was confirmed by immunofluorescence assay with serological seroconversion in two consecutive samples. Rickettsia conorii or Rickettsia massiliae were the possible causal agents since they are the Rickettsia spp found in the Rhipicephalus sanguineus dog tick in Portugal.

Epidemiological aspects of the Brazilian spotted fever: seasonal activity of ticks collected in an endemic area in São Paulo, Brazil

Ticks were collected from vegetation and animals at monthly intervals during one year (1993-1994) in an endemic area of Brazilian spotted fever in the County of Pedreira, State of São Paulo. Six species of ticks were identified Amblyomma cajennense, Amblyomma cooperi, Amblyomma triste, Anocentor nitens, Rhipicephalus sanguineus and Boophilus microplus. Only the first species was sufficiently numerous to permit a quantitative study with seasonal activity, although the distribution and source of capture of other species were observed and are reported. This information is correlated with the epidemiology of tick-borne rickettsiosis.

Report on ticks collected in the Southeast and Mid-West regions of Brazil: analyzing the potential transmission of tick-borne pathogens to man

Specimens of ticks were collected in 1993, 1996, 1997, and 1998, mostly from wild and domestic animals in the Southeast and Mid-West regions of Brazil. Nine species of Amblyommidae were identified: Anocentor nitens, Amblyomma cajennense, Amblyomma ovale, Amblyomma fulvum, Amblyomma striatum, Amblyomma rotundatum, Boophilus microplus, Boophilus annulatus, and Rhipicephalus sanguineus. The potential of these tick species as transmitters of pathogens to man was analyzed. A Flaviviridade Flavivirus was isolated from Amblyomma cajennense specimens collected from a sick capybara (Hydrochaeris hydrochaeris). Amblyomma cajennense is the main transmitter of Rickettsia rickettsii (=R. rickettsi), the causative agent of spotted fever in Brazil. Wild mammals, mainly capybaras and deer, infested by ticks and living in close contact with cattle, horses and dogs, offer the risk of transmission of wild zoonosis to these domestic animals and to man.

Identificação de genoespécies do complexo Borrelia burgdorferi sensu lato (s.l.) circulantes na fauna ixodideológica em portugal

Dissertação para obtenção do grau de Mestre em Microbiologia Médica

Ectoparasitos de cães e gatos da cidade de Manaus, Amazonas, Brasil

São apresentados resultados da coleta de ectoparasitos em cães e gatos entre agosto de 2001 e maio de 2002 em diferentes bairros da cidade Manaus. No cão foram encontrados: Ctenocephalides f. felis (Bouché, 1835) (Siphonaptera, Pulicidae), Heterodoxus spiniger (Enderlein, 1909)(Phthiraptera, Boopidae), Trichodectes canis (De Geer, 1778) (Phthiraptera, Trichodectidae) e Rhipicephalus sanguineus (Latreille,1806) (Acari, Ixodidae). No gato foi coletado C. f. felis. A prevalência de ectoparasitos foi de 80,8% para cães e 72,7% para gatos. Para a pulga C. f. felis foi de 28,7% para cães e 72,7% para gatos. Para o piolho H. spiniger foi de 12,3% para cães. Para o piolho T. canis foi de 0,1% para cães e para o carrapato R. sanguineus foi de 63% para cães. A média de infestaçãode pulga foi de 1,26 para cães e 1,27 para gatos. A proporção sexual fêmea/macho foi de 1,96:1 no cão e de 3,66:1 no gato. A pulga C. canis (Curtis, 1826), registrada em 1922, não foi coletada.

Atividade biológica de extratos acetato de etila, etanólico e aquoso de timbó (Lonchocarpus floribundus) sobre carrapato bovino

Os extratos acetato de etila, etanólico e aquoso de raízes de Lonchocarpus floribundus foram utilizados, a fim de avaliar a atividade biológica sobre carrapato bovino. Carrapatos adultos foram coletados em bovinos infestados artificialmente, separados em grupos de dez indivíduos, pesados e imersos, separadamente, nos extratos de raízes de L. Floribundus, nas concentrações de 5, 25, 50, 75 e 100 mg mL-1. Para a avaliação em larvas, foram utilizados indivíduos de 14 a 21 dias, os quais foram imersos nos extratos nas concentrações de 1, 5, 10, 15 e 20 mg mL-1. Após o tratamento, cada grupo foi colocado em placa de Petri e incubado a 27 ± 1 ºC e umidade relativa de 80 ± 5%. Os extratos avaliados não foram eficazes para induzir, acima de 50%, a mortalidade de fêmeas ingurgitadas. Os extratos acetato de etila e etanólico induziram 100% de mortalidade de larvas. Entretanto, quanto aos valores de concentração letal mediana (CL50), o extrato etanólico (CL50 = 2,1 mg mL-1) foi mais tóxico que o extrato acetato de etila (CL50 = 4,1 mg mL-1). O extrato etanólico estimou concentração inibitória mediana (CI50) de 3,0 mg mL-1 e foi mais tóxico que os demais extratos quanto a este parâmetro de avaliação. Entre os três extratos avaliados, os extratos acetato de etila e etanólico apresentaram os melhores resultados quanto ao controle de reprodução de R. (B.) microplus, atingindo 100% na concentração de 5 mg mL-1. Os extratos de raízes de L. Floribundus apresentaram atividade biológica sobre carrapato bovino.

Ixodidas brasileiros e de alguns paizes limitrophes

The knowledge of the Ixodidae becomes every day, more and more important owing to the fact of the increasing number of diseases of man and animals they can transmit. In Brasil besides transmitting treponemosis, piroplasmosis and anaplasmosis to several domestic animals, the ticks are also responsible fo the transmission of the brazilian rocky mountain spotted fever (A. cajennense and Amblyomma striatum) and they can also harbour the virus of the yellow fever and even to transmit it in laboratory experiments (A. cajennense, O. rostratus). The Brazilian fauna of ticks is a small one and has no more than 45 well-established species belonging to the genus Argas, Ornithodoros, Ixodes, Haemaphysalis, Rhipicephalus, Boophilus, Amblyomma and Spaelaeorhynchus. The genus Amblyomma is the best represented one, with 67% of all species of ticks known in Brazil. One of the most important species in the Amblyomma cajennense owing to its abundance and its wide parasitism in many vertebrates: reptiles, birds and mammals, incluing man, who is much attacked by the larva, the nymph and the adult of this species. The other ticks who attack the man are the Amblyomma brasiliense (the pecari tick), in the forests, and the Ornithodoros, especially the species. O. rostratus and brasiliensis. Other species can bite the man, but only occasionally, like Amblyomma fossum, striatum, oblongogutatum etc. Argas persicus, Rhipicephalus sanguineus and Boophilus are very important species not only as parasites but specially because they transmit several diseases to animals. Some of the ticks of the brazilian wild animals are now also parasites of the domestic ones and vice-versa. Arga persicus var. dissimilis is very common among the poultry and transmits the Treponema anserinum (gallinarum). Boophilus microplus is very abundant on our domestic and wild ruminants (Bos, Cervus, Mazama etc.) and can also ben found on horse, dogs, Felis onca, Felis concolor etc., and it transmits to cattle piroplasmosis and anaplasmosis. Rhipicephalus sanguineus (an introduced species) is now very common on the dog, over all the country. The author recommend to give popular names to some brazilian ticks in order to make them more acquainted with the non scientific people. The author gives a classification of the superfamilia Ixoidoidea and keys to the determination of the different species of brazilian ticks. He creates a new family of Nuttallielidae to the so interesting tick, described by Bedford with the name of Nuttaliella namaqua in South Africa, a new variety of Argas persicus, the Argas persicus var. dissimilis nov. var. owing to the differences on the segment and on the size and morphology of the peritrema. He describes also the female of Amblyomma fuscum Nn. A great part of the author's work deals with the biology, life conditions and parasitism of many of the brazilian ticks in accordance with his personal and from other author's researches, especially in reference to Argas persicus, Ornithodoros rostratus, O. brasiliensis, Boophilus microplus, Rhipicephalus sanguineus, Amblyomma cajennense, A. pseudoconcolor, A. auriculare, A. rotundatum (= A. agamum) etc. The author gives a detailed report upon the parthenogenesis of A. rotundatum (A. agamum) that he first described in 1912 and gives also many references to other species of brazilian ticks, to teratological forms etc. He also gives a detailed report of the geographical distribution of brazilian ticks and of the peculiar conditions of its parasitism. The last part of this article deals with references to the species of ticks of some of the South American Republics namely Argentina, Bolivia, Colombia, Paraguay and Venezuela. Amblyomma testudinis Conil, A. neumanni Ribaga 1902 (= A. furcula Dõnitz 1909) and A. parvitarsum Nn. 1899 (= A. altiplanum Dios 1917), are found only in Argentina. It is given a special bibliography dealing with the brazilian ticks and four text figures and one plate.

Verificação da existencia de uma fase negativa do sangue no periodo prepatente da babesiose canina transmitida por carrapatos

Foram infestados dois cães com carrapatos Rhipicephalus sanguineus infectados pela Babesia canis, tendo sido colocadas 1500 imaqos no primeiro animal e 100 no segundo. Ambos os cães apresentaram infecção das hemácias após o período prepatente de 6 dias. Durante o período prepatente foram diariamente retirados de cada cão 5 ml de sangue, que foram inoculados em 10 cães jovens, por via intravenosa. Os cães inoculados no 1°. 2° e 3° dias do período prepatente permanecerarn negativos. Aqueles inoculados no 4° e 5° dias apresentaram infecção pela babésia. Estes resultados são interpretados, de acordo com os conhecimentos existentes sobre a evolução preeritrocitária dos outros hemosporídeos, como indicadores da existência, no hospedador vertebrado, de uma fase inicial de evolução da babésia em células de tecido, que corresponde a uma fase negativa do sangue e que tem a duração mínima de 3 dias.

Transmission and diagnosis of equine babesiosis in South Africa

The transmission and prevalence of Babesia equi and B. caballi are being studied. Rhipicephalus evertsi mimeticus an ixodid tick from Namibia was identified as a new vector of B. equi, however, R. turanicus, previously reported to be a vector, failed to transmit both B. equi and B. caballi in the laboratory. The accurate diagnosis of B. caballi is being investigated because the nature of its low level parasitaemia does not allow easy detection in thin blood smears, routinely used for diagnosis, by clinicians. Consequently its role as a pathogen remains obscure. The importance of identifying infected horses, destined for export to Babesia-free coutries, is also stressed. Thock and thin blood smears, serology (IFAT) and DNA probes are currently employed to study disease prevalence. To date 293 healthy, adult, throughbred horses have been screened by all three methods. The percentage positives are as follows: B. equi 4.4%, 70.6%, 13% and B. caballi 0.7%, 37%, 18.4% respectively. The DNA probes were more sensitive than blood smear examination for diagnosing carrier infections but are probably not sensitive enough to identify all carrier infections. A poor correlation was found between detection of the parasites' DNA and seropositivity. However, polymerase chain reaction could be used to amplify parasite DNA in a particular sample and its could result in more accurate diagnosis.

Rickettsiae-infected Ticks in an Endemic Area of Spotted Fever in the State of Minas Gerais, Brazil

A study on tick-borne rickettsiosis was developed in the county of Santa Cruz do Escalvado, State of Minas Gerais, Brazil, where a clinical case of the disease, confirmed by necropsy, had been reported. Of the 1,254 ticks collected, 1,061 belonged to the Amblyomma genus, 57 to the Rhipicephalus sanguineus species, 81 to Boophilus microplus, and 46 to Anocentor nitens. The hemolymph test associated with Giménez staining showed that 18 of the 221 A. cajennense specimens, 1 of the 16 R. sanguineus, 1 of the 22 B. microplus, 3 of the A. nitens, and 1 of the A. ovale contained rickettsia-like microorganisms. Only 3 A. cajennense ticks were positive under direct immunofluorescence. A. cajennense was the only species found on humans