Seven years of evaluation of ectoparasites and vector-borne pathogens among ring-tailed coatis in an urban park in southeastern Brazil.

Wild animals have been recognized as potential reservoirs of vector-borne pathogens. Proximity between these animals and urban areas increases the need to know which pathogens these are and whether they can infect domestic animals and humans. In Mangabeiras Municipal Park in Belo Horizonte, Brazil, coatis live near the urban area, which is mainly occupied by human residents and their domestic animals. Therefore, the objective of this study was to detect, through molecular and direct methods, the presence of ectoparasites and hemoparasites in coatis. A total of 216 samples were collected, of which 209 samples were from first-captures and seven were from recaptures. The following parasites were found: ticks of the genus Amblyomma, lice of the species Neotrichodectes pallidus and fleas of the species Rhopalopsyllus lutzi lutzi and Ctenocephalides felis felis. All the samples were negative for the family Anaplasmataceae and the species Leishmania sp. and Trypanosoma cruzi. The hemoparasites Trypanosoma evansi, Hepatozoon procyonis, Babesia sp. and Sarcocystis neurona were found. The area of the present study is not endemic for T. evansi, which therefore suggests that these coatis may be acting as reservoirs or sentinels of this parasite. This finding is of great epidemiological importance and should be investigated more closely. Thus, this study showed that there is a great variety of pathogens in the park that transit among coatis and, probably, among other animals that inhabit or live close to the park.

Detecção direta e evidência de exposição à Anaplasma Phagocytophilum em equinos de Minas Gerais, Brasil / Anaplasma Phagocytophilum direct detection and exposure evidence in equines from Minas Gerais, Brazil

A Anaplasmose Granulocítica Equina (AGE) é causada pelo Anaplasma phagocytophilum, bactéria intracelular obrigatória, Gram-negativa, membro da família Anaplasmataceae incluída na ordem das Rickettsiales. Pouco se sabe sobre a doença, dinâmica de transmissão, cepa e prevalência da doença no território brasileiro. O presente trabalho teve por objetivo verificar, por métodos diretos e sorológicos, a ocorrência de A. phagocytophilum em equinos no municípios de Belo Horizonte, Minas Gerais. Encontrou-se uma ocorrência ao exame da reação de imunofluorescência indireta (RIFI) de 53,57% (120/224). Ao exame de capa leucocitária foi encontrado um percentual de 3,14% (7/223) de animais positivos. A maioria das amostras (41,47%) apresentou uma diluição de 1:160, tais amostras foram então submetidas à titulação nas diluições de 1:320, 1:640 e 1:1280. Pode-se concluir que a infecção por A. phagocytophilum está presente em cavalos pertencentes ao Projeto Carroceiros no município de Belo Horizonte. Para que se entenda melhor qual a importância epidemiológica desta infecção para o município mais levantamentos sorológicos e moleculares, em equinos, devem ser realizados a fim de se entender melhor a dinâmica do agente na população equina.(AU)

Equine granulocytic anaplasmosis is caused by Anaplasma phagocytophilum, a gram negative, obligatory intracellular bacteria, member of Anaplasmataceae family, included in the Rickettsiales order. Little is known about the disease, transmission dynamics, strain and prevalence in Minas Gerais State. This work aimed to verify, using direct and serologic methods, the occurrence of A. phagocytophilum in equines of the municipality of Belo Horizonte, Minas Gerais. It was found a positive reaction in the IFA in a frequency of 53.57% (120/124). In the buffy coat evaluation, it was found a percentage of 3.14% (7/223) of positive animals. Most of the samples reacted at a 1:160 dilution and were submitted to titration in the dilutions of 1:320, 1:640 and 1:1280. It is possible to conclude that A. phagocytophilum infection is present in horses of “Projeto Carroceiros” of the municipality of Belo Horizonte. In order to better understand the epidemiological importance of this infection more studies are needed to comprehend the dynamic of the agent in the equine population.(AU)

Detecção direta e evidência de exposição à Anaplasma Phagocytophilum em equinos de Minas Gerais, Brasil / Anaplasma Phagocytophilum direct detection and exposure evidence in equines from Minas Gerais, Brazil

A Anaplasmose Granulocítica Equina (AGE) é causada pelo Anaplasma phagocytophilum, bactéria intracelular obrigatória, Gram-negativa, membro da família Anaplasmataceae incluída na ordem das Rickettsiales. Pouco se sabe sobre a doença, dinâmica de transmissão, cepa e prevalência da doença no território brasileiro. O presente trabalho teve por objetivo verificar, por métodos diretos e sorológicos, a ocorrência de A. phagocytophilum em equinos no municípios de Belo Horizonte, Minas Gerais. Encontrou-se uma ocorrência ao exame da reação de imunofluorescência indireta (RIFI) de 53,57% (120/224). Ao exame de capa leucocitária foi encontrado um percentual de 3,14% (7/223) de animais positivos. A maioria das amostras (41,47%) apresentou uma diluição de 1:160, tais amostras foram então submetidas à titulação nas diluições de 1:320, 1:640 e 1:1280. Pode-se concluir que a infecção por A. phagocytophilum está presente em cavalos pertencentes ao Projeto Carroceiros no município de Belo Horizonte. Para que se entenda melhor qual a importância epidemiológica desta infecção para o município mais levantamentos sorológicos e moleculares, em equinos, devem ser realizados a fim de se entender melhor a dinâmica do agente na população equina.

Equine granulocytic anaplasmosis is caused by Anaplasma phagocytophilum, a gram negative, obligatory intracellular bacteria, member of Anaplasmataceae family, included in the Rickettsiales order. Little is known about the disease, transmission dynamics, strain and prevalence in Minas Gerais State. This work aimed to verify, using direct and serologic methods, the occurrence of A. phagocytophilum in equines of the municipality of Belo Horizonte, Minas Gerais. It was found a positive reaction in the IFA in a frequency of 53.57% (120/124). In the buffy coat evaluation, it was found a percentage of 3.14% (7/223) of positive animals. Most of the samples reacted at a 1:160 dilution and were submitted to titration in the dilutions of 1:320, 1:640 and 1:1280. It is possible to conclude that A. phagocytophilum infection is present in horses of “Projeto Carroceiros” of the municipality of Belo Horizonte. In order to better understand the epidemiological importance of this infection more studies are needed to comprehend the dynamic of the agent in the equine population.

In vitro culture of a novel genotype of Ehrlichia sp. from Brazil.

Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the haemolymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were continuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. However, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis.

Cross-protection between geographically distinct Anaplasma marginale isolates appears to be constrained by limited antibody responses.

The rickettsia Anaplasma marginale causes the haemolytic disease bovine anaplasmosis, an economic problem in tropical and subtropical areas worldwide. The closely related but less pathogenic Anaplasma centrale is commonly used as a live vaccine to prevent anaplasmosis, but it can only be produced from infected blood. UFMG1 is a low pathogenic Brazilian strain of A. marginale, which has been shown to protect cattle against a high pathogenic Brazilian isolate. As UFMG1 can be grown in tick cells, the strain was proposed as a possible cell culture-derived vaccine. We have evaluated whether UFMG1 could protect cattle against a geographically distant heterologous strain, using A. centrale vaccination as a standard for comparison. Trial calves were infected with UFMG1, A. centrale or PBS. UFMG1-infected animals were more symptomatic than those infected with A. centrale, but none required treatment. All calves were then challenged with the Israeli A. marginale Gonen strain (one of the most prevalent strain in Israel). The A. centrale group had the mildest symptoms, while UFMG1 and control groups both had a more severe response. Nevertheless, the challenge did not cause life-threatening disease in any group. Animals infected with A. centrale had a significantly higher IgG response than UFMG1, when measured in an ELISA against initial bodies from their homologous strain or Gonen. The level of cross-reactivity of the response to initial infection correlated significantly with reduced symptoms after challenge. In conclusion, UFMG1 had limited effect in preventing disease by the geographically distant heterologous Gonen strain. While the low pathogenicity of the Gonen strain in this trial makes it impossible to conclusively state that UFMG1 would have given no protective effect against more serious disease, the comparatively low IgG response to UFMG1 suggests it would not have been as effective as A. centrale.

Factors associated with epidemiology of Anaplasma platys in dogs in rural and urban areas of Minas Gerais State, Brazil.

This epidemiological survey of Anaplasma platys was carried out in rural and urban areas of three distinct regions of the State of Minas Gerais, Brazil. EDTA blood samples were collected during the dry season from dogs living on farms with an attempt to resample the same dogs in the subsequent rainy season. Samples were also taken from dogs in urban areas. DNA was extracted from blood samples for real time PCR. Risk factors, such as age, breed, sex, presence of ticks, and packed cell volume were analyzed. During the rainy season, the prevalence of infection by A. platys in dogs in the rural areas was significantly higher (13.9%) than that observed in dogs in the urban areas (5.1%). Dogs in the Nanuque region were 3.74 times (p=0.001) more likely to be real-time PCR positive than dogs in the other two studied regions. Dogs infested with ticks showed higher rates of positivity. The results showed that in rural areas of Minas Gerais A. platys infection is influenced by climatic conditions. In areas of higher temperature and higher humidity, transmission occurs during both the dry and rainy seasons, while in areas with lower temperature and humidity transmission occurs mainly during the dry season.

Use of a Real Time PCR for detecting subspecies of Babesia canis.

This paper reports the development and use of a Real Time PCR for detection of Babesia canis canis, B. canis rossi, and B. canis vogeli in endemic areas of Brazil. The sequences of the internal transcribed spacer (ITS) of several organisms were aligned and five primers and four probes were designed for amplification of a fragment (around 125 bp) which differentiates subspecies of B. canis. Blood samples collected from dogs living in farms in three distinct rural regions within the State of Minas Gerais (Lavras, Belo Horizonte and Nanuque) were tested. Blood samples had been collected during a dry season (Lavras, n=100; Belo Horizonte, n=50; Nanuque, n=102); the dogs were re-sampled in the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=29; Nanuque, n=66). From each sample, DNA was extracted and Giemsa stained smears were microscopically examined for direct detection of Babesia parasites. B. canis vogeli was the only subspecies found, with an overall prevalence of 9.9% during the dry season and 10.8% during the rainy season. Dogs living in Nanuque and Belo Horizonte showed significantly higher prevalence rates than those living in Lavras (13.7%, 12.0% and 5.0%, respectively). The Real Time PCR developed proved to be appropriate to detect B. canis subspecies in endemic areas.

Molecular detection of tick-borne pathogens of the family Anaplasmataceae in Brazilian brown brocket deer (Mazama gouazoubira, Fischer, 1814) and marsh deer (Blastocerus dichotomus, Illiger, 1815).

Deer are important natural reservoir hosts of Anaplasmataceae. The present study used nested PCR and nucleotide sequencing to evaluate the occurrence of Anaplasmataceae species in 23 free-living and six captive specimens of the cervids Mazama gouazoubira and Blastocerus dichotomus in Minas Gerais State, Brazil. Blood samples were tested for the presence of Ehrlichia and Anaplasma spp. using nPCR assays and sequencing of the msp4, msp1 and 16S rRNA genes. The identity of each sequence was confirmed by comparison with sequences available from GenBank using BLAST software. Of the animals investigated, 93.1% (27/29) were infected with haemoparasites including Anaplasma marginale (79.3%), Ehrlichia chaffeensis (3.4%), Anaplasma bovis (3.4%) and Anaplasma spp. (assigned to A. platys and A. phagocytophilum) (17.2%). Co-infection occurred in 20% (6/29) of the deer examined. Four (13.8%) were infected with A. marginale and Anaplasma sp., one (3.4%) was infected with A. marginale and E. chaffeensis, and one (3.4%) was infected with A. marginale and A. bovis. The results of the present study suggest that cross-protection does not occur in these deer. Immunological cross-reaction occurs when sera are tested diagnostically because these bacteria are closely related taxonomically, reinforcing the importance of molecular diagnosis followed by nucleotide sequencing.

Epidemiologic aspects of an outbreak of Trypanosoma vivax in a dairy cattle herd in Minas Gerais state, Brazil.

The aim of this study was to assess the epidemiological situation of bovine trypanosomiasis caused by Trypanosoma vivax in a dairy cattle herd from Igarapé, Minas Gerais state, Brazil. The herd was monitored from September 2007 to February 2009 by sampling blood for determination of packed cell volume (PCV), microhaematocrit centrifugation test of parasitaemia (MHCT), serology (IFA), morphological identification of T. vivax and molecular diagnosis by polymerase chain reaction (PCR). During all the experimental period, 25 animals were MHCT and PCR positive, considering that in each sample collection a mean of 70 animals was evaluated. The morphometric characteristics of trypomastigote forms confirmed the infection by T. vivax. The seroprevalence ranged from 7.4% in September 2007 to 48% in February 2009, and the highest incidence observed could be correlated with an increased population of Stomoxys calcitrans flies in that region. Anaemia was the most important change found in infected animals, which showed lower averages of PCV than parasitologically negative animals (p<0.0001). Infected individuals showed lower averages of PCV than parasitologically negative animals (p<0.0001), indicating higher anaemia in the former compared with the latter group.

Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves.

This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75 days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2 weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus.

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface.

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence.

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n=92; Belo Horizonte, n=50; Nanuque, n=102) and the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=28; Nanuque, n=66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.

Ocorrência de Trypanosoma vivax no estado de Minas Gerais / Occurrence of Trypanosoma vivax in Minas Gerais state, Brazil

A cow from Igarapé city, Minas Gerais state, Brazil, showing vision damage was referred at the Veterinary Hospital of the Universidade Federal de Minas Gerais. Clinical examination revealed anemia and a flagellated protozoan was observed in the blood smears obtained from the animal. The morphometric analysis demonstrated that the protozoan was Trypanosoma vivax. This is the first report of T. vivax occurrence in Minas Gerais state. An epidemiological investigation is currently being conducted on the farm and neighbor areas in order to estimate the parasite prevalence and the distribution of this trypanosomiasis in the region.

Ocorrência de Amblyomma longirostre (Koch, 1844) em Chaetomys subspinosus (Olfers, 1818) oriundos da Mata Atlântica / Occurrence of Amblyomma longirostre (Koch, 1844) in Chaetomys subspinosus (Olfers, 1818) from Atlantic Forest

Chaetomys subspinosus (Olfers, 1818) or bristle-spined porcupine is a species of rodent restricted to the Atlantic Forest of the Eastern Coast of Brazil, vulnerable to extinction. Four specimens captured in the State of Espírito Santo, at the Biological Station of Santa Lúcia (EBSL) and at the State Park Paulo César Vinha (PEPCV), were examined for the presence of ectoparasites. One female and three males of Amblyomma longirostre were collected from a single C. subspinosus female in three independent captures carried out in 2004 at the EBSL. No ticks were found on bristle-spined specimens from the PEPCV. This constitutes the first report of A. longirostre infesting this rodent species in the State of Espírito Santo and reinforces that these mammals from the family Erethizontidae act as hosts for adults stages of this ixodic tick species.

Ocorrência de Amblyomma longirostre (Koch, 1844) em Chaetomys subspinosus (Olfers, 1818) oriundos da Mata Atlântica / Occurrence of Amblyomma longirostre (Koch, 1844) in Chaetomys subspinosus (Olfers, 1818) from Atlantic Forest

Chaetomys subspinosus (Olfers, 1818) or bristle-spined porcupine is a species of rodent restricted to the Atlantic Forest of the Eastern Coast of Brazil, vulnerable to extinction. Four specimens captured in the State of Espírito Santo, at the Biological Station of Santa Lúcia (EBSL) and at the State Park Paulo César Vinha (PEPCV), were examined for the presence of ectoparasites. One female and three males of Amblyomma longirostre were collected from a single C. subspinosus female in three independent captures carried out in 2004 at the EBSL. No ticks were found on bristle-spined specimens from the PEPCV. This constitutes the first report of A. longirostre infesting this rodent species in the State of Espírito Santo and reinforces that these mammals from the family Erethizontidae act as hosts for adults stages of this ixodic tick species.(AU)

Ocorrência de Trypanosoma vivax no estado de Minas Gerais / Occurrence of Trypanosoma vivax in Minas Gerais state, Brazil

A cow from Igarapé city, Minas Gerais state, Brazil, showing vision damage was referred at the Veterinary Hospital of the Universidade Federal de Minas Gerais. Clinical examination revealed anemia and a flagellated protozoan was observed in the blood smears obtained from the animal. The morphometric analysis demonstrated that the protozoan was Trypanosoma vivax. This is the first report of T. vivax occurrence in Minas Gerais state. An epidemiological investigation is currently being conducted on the farm and neighbor areas in order to estimate the parasite prevalence and the distribution of this trypanosomiasis in the region.(AU)

Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus.

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.

Manutenção in vitro de células IDE8 em dois tipos de soro bovino / In vitro maintenance of IDE8 cells using two types of bovine serum

The present study had the objective of defining the culture conditions, optimizing the maintenance and expansion of an IDE-8 cell line in Brazil, with the aim to propose its use as a model for in vitro infection and multiplication of Brazilian strains of rickettsia and other hemoparasites. The supplementation of IDE-8 cells with two distinct fetal bovine sera (a Brazilian and an imported) was evaluated. Culture media were changed weekly and subcultures were carried out every 15 days. The development of cultures and subcultures was evaluated by the percentage of viability and cellular morphology. The results indicate that the imported SFB can be replaced by the Brazilian SFB one, as no significant differences (P<0.05) were seen among culture viabilities.

Manutenção in vitro de células IDE8 em dois tipos de soro bovino / In vitro maintenance of IDE8 cells using two types of bovine serum

The present study had the objective of defining the culture conditions, optimizing the maintenance and expansion of an IDE-8 cell line in Brazil, with the aim to propose its use as a model for in vitro infection and multiplication of Brazilian strains of rickettsia and other hemoparasites. The supplementation of IDE-8 cells with two distinct fetal bovine sera (a Brazilian and an imported) was evaluated. Culture media were changed weekly and subcultures were carried out every 15 days. The development of cultures and subcultures was evaluated by the percentage of viability and cellular morphology. The results indicate that the imported SFB can be replaced by the Brazilian SFB one, as no significant differences (P<0.05) were seen among culture viabilities.(AU)