Detecção de riquétsias em carrapatos do gênero Amblyomma (Acari: Ixodidae) coletados em parque urbano do município de Campinas, SP

O Município de Campinas situa-se em região endêmica para febre maculosa brasileira do Estado de São Paulo, onde vários casos desta doença vem ocorrendo. Capivaras têm sido associadas ao ciclo dessa riquetsiose por apresentarem sorologia positiva e serem hospedeiras de carrapatos Amblyomma spp principais vetores da doença. Carrapatos foram coletados no parque urbano do Lago do Café, Campinas, SP, local associado a casos humanos suspeitos de febre maculosa brasileira, sobre a vegetação e das capivaras ali presentes, e pesquisados quanto à presença de riquétsias pela reação em cadeia da polimerase e pelo teste de hemolinfa. Adultos de Amblyomma cajennense e Amblyomma cooperi albergavam Rickettsia bellii, não patogênica, identificada pela análise das seqüências de nucleotídeos do gene gltA, porém, não foram constatadas riquétsias do Grupo da Febre Maculosa. Estes resultados associados à ausência de um isolado de riquétsias do Grupo da Febre Maculosa de capivaras indicam que seu papel, enquanto reservatório, necessita de maior investigação.

Risk factors associated with the transmissionof Brazilian spotted fever in the Piracicaba river basin, State of São Paulo, Brazil

INTRODUCTION : Brazilian spotted fever (BSF) is a disease transmitted by ticks for which the etiological agent is Rickettsia rickettsii. The present essay evaluates the risk factors associated with the transmission of cases of BSF in the time period between 2003 and 2013 in the Piracicaba river basin, state of São Paulo. METHODS : This essay presents a retrospective study to identify the factors associated with the transmission of cases of BSF among all suspected cases identified by the System for Epidemiological Surveillance of São Paulo (CVE). After the description of temporal distribution (onset of symptoms) and the environmental and demographic variations of the confirmed and discarded cases, a multiple logistic regression model was applied. RESULTS : We searched 569 probable locations of infection (PLI) with 210 (37%) confirmed cases of BSF and 359 (63%) discarded cases. The associated variables for the confirmation of BSF in the multiple logistic model using a confidence interval (CI) of 95% were age (OR = 1.025 CI: 1.015-1.035), the presence of Amblyomma sculptum in the environment (OR = 1.629 CI: 1.097-2.439), the collection of ticks from horses (OR = 1.939 CI: 0.999-3.764), the presence of capybaras (OR = 1.467 CI: 1.009-2.138), an urban environment (OR = 1.515 CI: 1.036-2.231), and the existence of a dirty pasture (OR = 1.759 CI: 1.028-3.003). CONCLUSIONS : The factors associated with the confirmation of BSF cases included an urban environment, age, presence of the A. sculptum vector, the collection of ticks from horses, the presence of a capybara population, and a dirty pasture environment.

Notas de Ixodologia: II - uma nova espécie do gênero Amblyomma e uma nova espécie do gênero Ixodes (Acari Ixodidae)

A new tick, Amblyomma parkeri, n. sp., is described as a parasite of Coendu sp. from S. Paulo, Brazil. Female holotype, nymph and larva are described (Figs. 2 e 3). The n. sp. differs completly from Koch's species Amblyomma longirostre, the common parasite of the Erethizotidae. Standard data for measures of the female dorsal scutum ixodidae are proposed as follows (fig. 1): PA = Antero-posterior; PB = Postero-basal; PM = Postero-median; TT = Transversal; OO = Inter-ocular; OT= Occulo-transversal; SS = Inter-scapular; CC = Cervical; PT = Postero-transversal; ST = Scapulo-transversal; NPT = Normal to the postero-transversal; NST = Normal to the scapulo-transversal. In the female holotype the standard data are as follows: PA = 2.00 mm; PB = 2.26 mm; PM = 1.10 mm; TT = 2.20 mm; OO = 2.26 mm; SS = 0,84 mm; CC = 0.63 mm; SC = 0.12 mm; NPT = 0.20mm; STN = 0.1 mm. Peritrema 0.80 x 0.42 mm with a narrow postero-internal angle and a large, elongated macula. Coxa I with two short spines and all other coxae with only one shorter spine, shortest in coxa IV. Hypostoma spatulated with formula 3/3. Gnathosoma 1.42 mm long and basis 0.63 mm long by 0.84 greatest wide. Palpi with smoth external surface, 1.00 mm long. Type lot No. 4458 from Cotia, S. Paulo, Brazil; in the acarological collection of the Escola Paulista de Medicina, S. Paulo. Ixodes didelphidis, n. sp., differing from Ixodes loricatus Neumann by the shape of the peritremata (figs. 4 a 5) of the male and female and by the number of the punctations in this organ is described form Didelphidae, Muridae and Cavidae. Twenty eight lots were obtained from Anápolis, Goiás, Brasil, where I. loricatus is subtituded by the n. sp. under description. Comparison with NEUMANN'S types of I. loricatus was possible through the courtesy of Prof. A. BRIZARD from Toulouse, who kindly loaned NEUMANN'S material. Female cotypes N° 40 and male allotype N° 531 in the Collection of Ixodidae of the Oswaldo Cruz Institute.

Contribuição para o conhecimento das doenças do grupo "Tifo Exantemático" do Brasil: (Tifo Exantemático Neotrópico)

O A. recapitula os dados já conhecidos sôbre a filtrabilidade de muitas Rickettsias e sôbre o que êle chama o ciclo evolutivo das mesmas, citando os trabalhos de S. B. Dulky e E. Gordon sôbre a Coxiella papilliae e a observação de A. Donatien e F. Lestoquard bem como os de P. Giraud. Refere-se à filtrabilidade do agente da febre "Q". Reporta-se depois aos trabalhos de Mme. Ruth Rein Gutfreund sôbre os achados de Rickettsia prowazedi em animais domésticos na Etiópia e a transmissão dos mesmos pelos carrapatos, dizendo que isto confirma suas próprias idéias, de há muito emitidas, de que não há no grupo tifo exantemático, de regra, especificidade estrita para os transmissores, não podendo êste carácter servir de base para classificações racionais. Descreve um caso clínico, mostrando a dificuldade de diagnóstico cofundido com a febre tifóide, mesmo com longa prática do exame na doença e que só a inoculação em animais sensíveis - aqui os cobaios - pode decidir a questão. Estuda depois dois surtos epidêmicos de tifo exantemático neotrópico - em 1950 em Carmópolis (Minas Gerais); outro em 1956, Mucuri (Bahia). Em ambos, a percentagem de mortes dos casos graves, não tratados, foi elevada e a terapêutica pelos antibióticos, principalmente a Cloromicetina e Terramicina foi brilhante. Nestes dois surtos epidêmicos com já se vira em 1941 (4 casos na mesma residência) e 1948 (6 casos na mesma moradia), apuraram-se 2 e 3 casos da doença na mesma casa. As reações de Fixação de Complemento (R.F.C.), Weil-Feliz (W.F.) e Widal - confirmam o diagnóstico de tifo exantemático neotrópico.

A new species of Boettcheria (Diptera: Sarcophagidae) from the Dominican Republic

Boettcheria solo sp. n. from the Dominican Republic is described and it is argued that its most probable sister group is B. parkeri Aldrich, 1916 from Jamaica. Boettcheria Parker, 1914 is briefly diagnosed and a list of included species is provided.

Primary isolation of spotted fever group rickettsiae from Amblyomma cooperi collected from Hydrochaeris hydrochaeris in Brazil

This paper reports the first isolation of a spotted fever group rickettsia from an Amblyomma cooperi ixodid collected from a capybara (Hydrochaeris hydrochaeris) in an endemic area of spotted fever in the County of Pedreira, State of São Paulo, Brazil. Isolation was performed in Vero cell culture and submitted to immunofluorescence, using antibody from Rickettsia rickettsii-positive human serum.

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

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of São Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis - 63.7 and 56.4% and B. bigemina - 53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Outbreak of Rocky Mountain spotted fever in Córdoba, Colombia

Rocky Mountain spotted fever (RMSF) is a tick-borne disease caused by the obligate intracellular bacterium Rickettsia rickettsii. Although RMSF was first reported in Colombia in 1937, it remains a neglected disease. Herein, we describe the investigation of a large cluster of cases of spotted fever rickettsiosis in a new area of Colombia.

Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis

Individual, naturally occurring Phlebotomus mongolensis and Phlebotomus caucasicus from Iran were screened for infections with the maternally inherited intracellular Rickettsia-like bacterium Wolbachia pipientis via targeting a major surface protein gene (wsp). The main objective of this study was to determine if W. pipientis could be detected in these species. The sandflies were screened using polymerase chain reaction to amplify a fragment of the Wolbachia surface protein gene. The obtained sequences were edited and aligned with database sequences to identify W. pipientis haplotypes. Two strains of Wolbachia were found. Strain Turk 54 (accession EU780683) is widespread and has previously been reported in Phlebotomus papatasi and other insects. Strain Turk 07 (accession KC576916) is a novel strain, found for first time in the two sister species. A-group strains of W. pipientis occur throughout much of the habitat of these sandflies. It is possible that Wolbachia is transferred via horizontal transmission. Horizontal transfer could shed light on sandfly control because Wolbachia is believed to drive a deleterious gene into sandflies that reduces their natural population density. With regard to our findings in this study, we can conclude that one species of sandfly can be infected with different Wolbachia strains and that different species of sandflies can be infected with a common strain.