Flábio R. Araújo

Molecular survey and genetic characterization of tick-borne pathogens in dogs in metropolitan Recife (north-eastern Brazil)

To identify DNA of the main tick-borne pathogens in dogs from Recife (Brazil), polymerase chain reactions were carried out on blood samples of dogs treated at the Veterinary Hospital of the Universidade Federal Rural de Pernambuco from March 2007 to June 2008. The detection of DNA was performed using specific primers. Amplicons were analyzed through electrophoresis and sequencing. A phylogenetic tree was constructed using the UPGMA method, revealing that the sequences were closely related to those of strains from other geographic regions. Among the 205 blood samples analyzed, 48.78% was positive for Anaplasma platys; 38.04% was positive for Ehrlichia canis; 7.31% was positive for Babesia canis vogeli; and 0.49% was positive for Hepatozoon canis and Mycoplasma haemocanis. Coinfection of two or three pathogens was found in 23.9% (49/205) of the dogs. The subspecies B. canis vogeli was identified. Infection by H. canis and M. haemocanis is reported for the first time in dogs in the state of Pernambuco (Brazil). The data indicate that the main tick-borne pathogens in dogs in this region are E. canis and/or A. platys, followed by B. canis vogeli.

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.

Larva migrans cutânea em crianças de uma escola em área do Centro-Oeste do Brasil

Relata-se a ocorrencia de larva migrans cutânea em criancas de uma escola de educacao infantil de Campo Grande, MS (Brasil). Dos 16 alunos que frequentam a escola, seis (37,5%) adquiriram essa dermatite parasitaria em duas areas de recreacao com areia contaminada por fezes de gatos, cujo exame parasitologico revelou a presenca de larvas de ancilostomideos. As lesoes serpiginosas e/ou papulares essavam localizadas nas maos, pes, nadegas, coxas, vulva e saco escrotal. Sao discutidas medidas de controle dessa parasitose.

Molecular detection of Anaplasma platys in a naturally-infected cat in Brazil

Following the accidental finding of inclusion bodies similar to Anaplasma platys in a stained blood smear from a cat, DNA analysis of the 16S rRNA gene was performed and 100% identity was found with different strains of A. platys. These data confirm that cats are susceptible to parasitism by A. platys.

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.

Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará, Brazil

Babesiosis, anaplasmosis, and trypanosomosis are relevant diseases, potentially causing morbidity in cattle, leading to economic losses. Borreliosis is import as a potential zoonosis. The objective of this study was to determine, by indirect enzyme-linked immunosorbent assay (ELISA), the frequency of seropositive cattle to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of Pará, Brazil. Sera samples from 246 female adult cattle from municipalities of Castanhal and São Miguel do Guamá were used. Crude antigens ELISAs were used to detect antibodies to all agents, except to A. marginale, to which an indirect ELISA with recombinant major surface 1a protein (MSP1a) antigen was used. Overall frequencies of seropositive animals were: B. bigemina--99.2%; B. bovis--98.8%; A. marginale--68.3%; T. vivax--93.1% and B. burgdorferi--54.9%. The frequencies of seropositive cattle to B. bovis and B. bigemina suggest a high rate of transmission of these organisms by tick in the studied region, which can be classified as enzootically stable to these hemoprotozoans. The low frequency of seropositive cattle to A. marginale may be attributed to a lower sensitivity of the recombinant antigen ELISA utilized or a distinct rate of inoculation of this rickettsia by ticks, as compared with Babesia sp. transmission. The high frequency of seropositive cattle to T. vivax indicates that this hemoprotozoan is prevalent in herds from the Northeastern region of Pará. The rate of animal that showed homologues antibodies to B. burgdorferi indicates the presence of the tickborne spirochaetal agent in the cattle population in the studied region.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

Comparação de nested-PCR com o diagnóstico direto na detecção de Ehrlichia canis e Anaplasma platys em cães

The clinical signs of Ehrlichia canis and Anaplasma platys infection are similar, and the diagnosis of these pathogens made by stained blood smears is poor due sensibility and specificity. On the other hand, the molecular diagnosis is highly sensitive and specific and nested-PCR have been optimized for accurate diagnosis these pathogens in dogs. At the veterinary teaching hospital, whole-blood samples with EDTA were obtained from 100 dogs and smears were made from blood samples for evaluation for intracellular parasites. For each sample, DNA was extracted and submitted to nPCR analysis for detection of E. canis and A. platys. The results of stained blood smears showed 9% of the animals were positive for E. canis and 21% for A. platys. Regarding of nPCR analysis, 57 and 55% of dogs were positive for E. canis and A. platys respectively. As compared to a nested PCR, the stained blood smears revealed false-negative results for both E. canis and A. platys. The results indicate that the nPCR is highly sensitive and specific for detection of both pathogens and the molecular diagnosis could be more useful at veterinary hospital.

Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis

This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection.

Avaliação clínico-laboratorial de bovinos Nelore infectados experimentalmente com Trypanosoma vivax

In order to evaluate the clinical-laboratorial alterations, six Nellore calves were inoculated with 107 Trypanosoma vivax isolated from Pocone region, Mato Grosso, Brazil. The animals were evaluated daily for rectal temperature, packed cell volume (PCV), parasitemia, antibody production, color of mucous membranes, behavior and appetite. Blood and serum samples for biochemical evaluation for aspartate aminotransferase (AST), alkaline phosphatase (AF), gamma glutamyltransferase (GGT), cholesterol, urea, creatinine, creatine kinase (CK), calcium, phosphorus and proteinogram were collected on days 4, 8, 12, 16, 23 and 30 post inoculation (DPI). During the following 6 months rectal temperature, PCV and parasitemia were evaluated weekly. T. vivax was evidenced from 1 DPI in all calves and persisted until day 30 in five of six animals. A remarkable decrease (p<0.05) of PCV mean value (25%) was observed on 10 DPI. The animals presented no alterations in their clinical or serum biochemical state during the trial. Seroconversion took place 6 and 8 DPI, and all the animals remained seropositive during the 30 days of experiment. In all the experimental animals the occurrence of T. vivax infection was verified, characterized by the increase of corporal temperature, presence of the blood protozoa and reduction of the globular volume, without alterations in the other variables analyzed. Nellore calves, when experimentally inoculated with T. vivax, are able to establish a balance between host-parasite relationship.