Immunological studies on bovine babesiosis with particular reference to Brazil, using In vitro culture-derived antigens

The aim of the present study was to develop enzyme linked immunosorbent assays (ELISAs) for detection of specific antibodies against Babesia bovis and B.bigemina for use in epidemiological studies of bovine babesiosis in Brazil. These tests were developed using purified proteins of each parasite, which were identified as species-specific in a comprehensive immunochemical characterisation of different stocks of Babesia parasites.The review of the literature on bovine babesiosis covers the geographical distribution, transmission and life cycle, and in vitro cultivation of both species, as well as immunology, diagnosis, epidemiology and control ofthe disease and its current situation in Brazil.The thesis first describes a series of studies on the in vitro culture of B. bovis, which include the evaluation of the effect of different sera on in vitro growth, the incorporation of feeder cells (bovine aortic endothelial and mouse peritoneal cells) into cultures, cloning of one isolate by in vitro limiting dilutions, initiation of cultures from low parasitaemia blood, and several attempts to establish African isolates ofB.bigemina in vitro.Methods for concentration of Babesia infected red blood cells and free parasites were then examined. These included the use of differential hypotonic lysis, density gradient centrifugation, induction of free merozoites into culture supernatant and techniques for fractionation of exoantigens present in culture supernatant, namely high performance liquid chromatography.Somatic components and exoantigens of different stocks of parasites were characterised immunochemically by Western immuno-blotting, and immunoprecipitation of -^S-methionine labelled proteins by acrylamide gel electrophoresis, using a wide variety of sera which included calf sera experimentally raised against different stocks of each parasite, sera collected in the field in Brazil and a panel of monoclonal antibodies previously produced against B. bovis and characterised by IFAT, Western immuno-blotting and ELISA in the present study. The aim of the immunochemical characterisation was to identify antigens common to all stocks yet species-specific with potential use for the development of ELISAs. Antigenic diversity amongst stocks of B.bovis is reported.For the development of the ELISAs, calf serum samples of known status and serum samples collected in endemic areas in Brazil, Malawi and Mozambique were utilised to compare assays using crude antigen preparations of each Babesia species and purified specific proteins. Four B.bovis stockconserved proteins from somatic components were electro-eluted from acrylamide gels and used to develop an ELISA. Amongst these, a 56 kDa protein was found to be a potential candidate to replace the crude parasite extract in immunodiagnostic assays.Several B.bigemina specific proteins were also identified in the immunochemical analysis and four candidates were electro-eluted from acrylamide gels. However all purified B.bigemina proteins crossreacted with B.bovis antisera in ELISA. The complementary use of a crude and cross-reactive B.bigemina antigen together with the 56 kDa B.bovis-specific protein in ELISA to elucidate the epidemiology of these diseases is recommended.Future prospects and potential application in Brazil of these ELISA antigens for use in the detection of anti-B.bovis and anfi-B.bigemina antibodies are discussed with particular reference to their role in defining the epidemiology of bovine babesiosis in the dairy cattle of Minas Gerais State, Brazil

Survey of pyrethroid and organophosphate resistance in Brazilian field populations of Rhipicephalus (Boophilus) microplus: Detection of C190A mutation in domain II of the para-type sodium channel gene

AbstractThe cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2–15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82–100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected

New species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus shows an ortholog of the E. canis major immunogenic glycoprotein gp36 with a new sequence of tandem repeats

Background: Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses that inflict serious and fatal infections in companion animals and livestock. The aim of this paper was to phylogeneticaly characterise a new species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus from Minas Gerais, Brazil. Methods: The agent was isolated from the hemolymph of Rhipicephalus (B.) microplus engorged females that had been collected from naturally infested cattle in a farm in the state of Minas Gerais, Brazil. This agent was then established and cultured in IDE8 tick cells. The molecular and phylogenetic analysis was based on 16S rRNA, groEL, dsb, gltA and gp36 genes. We used the maximum likelihood method to construct the phylogenetic trees. Results: The phylogenetic trees based on 16S rRNA, groEL, dsb and gltA showed that the Ehrlichia spp isolated in this study falls in a clade separated from any previously reported Ehrlichia spp. The molecular analysis of the ortholog of gp36, the major immunoreactive glycoproteins in E. canis and ortholog of the E. chaffeensis gp47, showed a unique tandem repeat of 9 amino acids (VPAASGDAQ) when compared with those reported for E. canis, E. chaffeensis and the related mucin-like protein in E. ruminantium. Conclusions: Based on the molecular and phylogenetic analysis of the 16S rRNA, groEL, dsb and gltA genes we concluded that this tick-derived microorganism isolated in Brazil is a new species, named E. mineirensis (UFMG-EV), with predicted novel antigenic properties in the gp36 ortholog glycoprotein. Further studies on this new Ehrlichia spp should address questions about its transmissibility by ticks and its pathogenicity for mammalian hosts

Rickettsia felis in Fleas, Germany

Among 310 fleas collected from dogs and cats in Germany, Rickettsia felis was detected in all specimens (34) of Archaeopsylla erinacei (hedgehog flea) and in 9% (24/226) of Ctenocephalides felis felis (cat flea). R. helvetica was detected in 1 Ceratophyllus gallinae (hen flea)

New species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus shows an ortholog of the E. canis major immunogenic glycoprotein gp36 with a new sequence of tandem repeats

Background: Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses that inflict serious and fatal infections in companion animals and livestock. The aim of this paper was to phylogeneticaly characterise a new species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus from Minas Gerais, Brazil. Methods: The agent was isolated from the hemolymph of Rhipicephalus (B.) microplus engorged females that had been collected from naturally infested cattle in a farm in the state of Minas Gerais, Brazil. This agent was then established and cultured in IDE8 tick cells. The molecular and phylogenetic analysis was based on 16S rRNA, groEL, dsb, gltA and gp36 genes. We used the maximum likelihood method to construct the phylogenetic trees. Results: The phylogenetic trees based on 16S rRNA, groEL, dsb and gltA showed that the Ehrlichia spp isolated in this study falls in a clade separated from any previously reported Ehrlichia spp. The molecular analysis of the ortholog of gp36, the major immunoreactive glycoproteins in E. canis and ortholog of the E. chaffeensis gp47, showed a unique tandem repeat of 9 amino acids (VPAASGDAQ) when compared with those reported for E. canis, E. chaffeensis and the related mucin-like protein in E. ruminantium. Conclusions: Based on the molecular and phylogenetic analysis of the 16S rRNA, groEL, dsb and gltA genes we concluded that this tick-derived microorganism isolated in Brazil is a new species, named E. mineirensis (UFMG-EV), with predicted novel antigenic properties in the gp36 ortholog glycoprotein. Further studies on this new Ehrlichia spp should address questions about its transmissibility by ticks and its pathogenicity for mammalian hosts

In vitro cultivation of Anaplasma marginale and A. phagocytophilum in tick cell lines: a review

Continuous cell lines have been established from several ixodid and argasid tick species, representing an excellent tool suitable for the isolation of pathogens and their subsequent propagation, which in turn allows the production of antigenic material for diagnostic tests, antibody and vaccine production, and also for studies on host-vector-pathogen relationships. This paper reviews the use of tick cells for culture initiation and maintenance of two obligate intracellular bacterial pathogens, Anaplasma marginale and Anaplasma phagocytophilum. These in vitro cultivation systems have been used in a wide range of studies, covering morphological ultrastructural analysis, genetics, proteomics and biological differences between strains, including genome transcriptional and protein expression approaches, enabling comparisons between host and vector cells. Thus, such systems open a new window for a better understanding of interactions between pathogens and tick cells. Last but not least, such systems contribute to the reduction in usage of animals for experimental research, as antigenic material can be produced in reasonably large quantities without the use of in vivo species-specific systems

Detection of Ehrlichia canis in bone marrow aspirates of experimentally infected dogs Detecção de Ehrlichia canis em aspirados de medula óssea de cães experimentalmente infectados

The present work describes the detection of infected cells in the bone marrow aspirates of dogs experimentally infected with a Brazilian isolate of Ehrlichia canis. Dogs were monitored twice a day by clinical evaluation and peripheral blood smear examination. Every three days, blood samples were collected for cell counts. Weekly, aspirates from the bone marrow were examined and serum samples were tested by IFAT. The clinical signs observed were fever, pallid membranes, lymphadenopathy, serous nasal secretions, and pronounced weight loss. Hematological alterations included normocytic normochromic anemia, decrease of neutrophils and lymphocytes, and thrombocytopenia. Few E. canis infected cells were seen in blood smears. However, stages of E. canis were visualized in bone marrow aspirates 15 days post infection.<br>O presente trabalho descreve a detecção de células infectadas em aspirados de medula óssea de cães experimentalmente infectados com uma amostra brasileira de Ehrlichia canis. Os cães foram monitorados duas vezes por dia através de avaliação clínica e exames de esfregaços de sangue periférico. A cada três dias, amostras de sangue foram coletadas para contagem celular. Semanalmente foram feitas punções de medula óssea para exame microscópico direto do material aspirado e coleta de sangue para exames sorológicos através da reação de imunofluorescência indireta. Os sinais clínicos observados foram febre, membranas pálidas, linfadenopatias, secreções nasais serosas e acentuada perda de peso. As alterações hematológicas incluíram anemia normocítica normocrômica, redução de neutrófilos e linfócitos e trombocitopenia. Poucas células infectadas com E. canis foram observadas em esfregaços sanguíneos, entretanto várias formas de desenvolvimento de E. canis foram visualizadas em aspirados de medula óssea 15 dias após a infecção

Occurrence of ectoparasites on dogs in rural regions of the state of Minas Gerais, Brazil Ocorrência de ectoparasitos de cães de área rural do estado de Minas Gerais, Brasil

The present study examined occurrences of ectoparasites and identified them on dogs in rural regions in Brazil, and assessed the influence of climate on these parasites. Ectoparasites were randomly collected from 194 dogs living on farms located in Lavras (n = 92) and Nanuque (n = 102) during the dry season. During the subsequent rainy season, the same dogs in Lavras (n = 71) and Nanuque (n = 66) were resampled. During the experiment, fleas, ticks, lice and fly larvae were collected. The flea species Ctenocephalides felis was the most common ectoparasite collected from these dogs. The main tick species that infested the dogs in rural areas of Nanuque and Lavras was Amblyomma cajennense. In Lavras, the dogs had high levels of flea infestation (80.4 and 88.7% in the dry and rainy seasons, respectively) and low levels of tick infestation (19.6 and 28.2% in the dry and rainy seasons, respectively), without any significant differences in infestation rates between the seasons. In Nanuque, moderate levels of flea infestation (68.6 and 43.9% in the dry and rainy seasons, respectively) and A. cajennense (65.7 and 47.0% in the dry and rainy seasons, respectively) were observed, with significantly lower prevalence in the rainy season (p O presente estudo examinou a ocorrência e identificação de ectoparasitas em cães de áreas rurais no Brasil, e a influência do clima sobre esses parasitas. Ectoparasitas foram aleatoriamente coletados de 194 cães provenientes de fazendas localizadas em Lavras (n = 92) e Nanuque (n = 102) durante o período seco. No período chuvoso subsequente, os mesmos cães de Lavras (n = 71) e Nanuque (n = 66) foram re-amostrados. Durante o experimento, pulgas, carrapatos, piolhos e larvas de diptera foram coletados. A espécie de pulga Ctenocephalides felis foi o ectoparasita mais comumente encontrado desses cães. A principal espécie de carrapato que infestava os cães nas áreas rurais de Nanuque e Lavras foi Amblyomma cajennense. Em Lavras, os cães tinham altos níveis de infestação de pulgas (80,4 e 88,7% nas estações seca e chuvosa, respectivamente) e baixos níveis de infestação por carrapatos (19,6 e 28,2% nas estações seca e chuvosa, respectivamente), não sendo observada diferença significativa nas taxas de infestação entre as estações. Em Nanuque, níveis moderados de infestação por pulgas (68,6 e 43,9% nas estações seca e chuvosa, respectivamente) e A. cajennense (65,7 e 47,0% na estação seca e chuvosa, respectivamente) foram observadas, com prevalência significativamente menor na estação chuvosa (p < 0,05). A presença de ectoparasitas foi evidente em ambas as épocas do ano, mas as diferentes temperaturas poderiam ter influenciado a ocorrência de parasitas em Lavras e Nanuque

Ehrlichia minasensis sp. nov., isolated from the tick Rhipicephalus microplus

Recently, we obtained a rickettsial isolate (Ehrlichia sp. UFMG-EV) from the haemolymph of engorged Rhipicephalus microplus tick females. On the basis of maximum-likelihood phylogenetic analysis using 16S rRNA gene, groEL, dsb, gltA and trp36 sequences we showed that Ehrlichia sp. UFMG-EVbelongs to the α-Proteobacteria, family Anaplasmataceae, genus Ehrlichia. Ehrlichia sp. UFMG-EVis a sister taxon of Ehrlichia canis with 16S rRNA gene, groEL, dsb, gltA and trp36 sequence similarities of 98.3%, 97.2%, 94.7%, 94.3% and 49.1%, respectively. Ehrlichia sp. UFMG-EVT has been maintained in the laboratory by continuous passage in the IDE8 tick cell line where the ultrastructure was characterized using electron microscopy and was found to resemble that of E. canis, Ehrlichia muris and Ehrlichia chaffeensis, but not Ehrlichia ruminantium and Ehrlichia ewingii. We propose the name Ehrlichia minasensis sp. nov. for this bacterium to acknowledge the place from where it was initially isolated, Minas Gerais, Brazil. The type strain is strain Ehrlichia sp. UFMG-EV(=DSM 100393=TCB-TBB-0018).This research was supported by the EU FP7 ANTIGONE project number 278976. ACC was supported by a grant from the Ministere de l’Education Superieure et de la Recherche of France.Peer Reviewe

Ehrlichia minasensis sp. nov., isolated from the tick Rhipicephalus microplus

Contribution: Conceived the idea and drafted the manuscriptInternational audienceRecently, we obtained a rickettsial isolate (Ehrlichia sp. UFMG-EVT ) from the haemolymph of engorged Rhipicephalus microplus tick females. On the basis of maximum-likelihood phylogenetic analysis using 16S rRNA gene, groEL, dsb, gltA and trp36 sequences we showed that Ehrlichia sp. UFMG-EVT belongs to the a-Proteobacteria, family Anaplasmataceae, genus Ehrlichia. Ehrlichia sp. UFMG-EVT is a sister taxon of Ehrlichia canis with 16S rRNA gene, groEL, dsb, gltA and trp36 sequence similarities of 98.3 %, 97.2 %, 94.7 %, 94.3 % and 49.1 %, respectively. Ehrlichia sp. UFMG-EVT has been maintained in the laboratory by continuous passage in the IDE8 tick cell line where the ultrastructure was characterized using electron microscopy and was found to resemble that of E. canis, Ehrlichia muris and Ehrlichia chaffeensis, but not Ehrlichia ruminantium and Ehrlichia ewingii. We propose the name Ehrlichia minasensis sp. nov. for this bacterium to acknowledge the place from where it was initially isolated, Minas Gerais, Brazil. The type strain is strain Ehrlichia sp. UFMG-EVT (5DSM 100393T 5TCB-TBB-0018T )