Rodrigo Martins Soares

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses.

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.

Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1

Porcine parvovirus (PPV) infection is associated with reproductive losses in swine and its causative agent, the PPV, has been isolated worldwide. Serological surveys and virus isolation studies throughout Brazil confirm the occurrence of PPV infection in this country. The most common methods to detect PPV infection are fluorescent antibody staining of fetal tissues, hemagglutination assay of tissue extracts and virus isolation from fetal tissues. Non-specificity and low sensitivity are the major drawbacks of these techniques. The development of a polymerase chain reaction (PCR) and nested-PCR assays for PPV DNA detection from infected cell lines and clinical samples is described. The primers were designed to a highly conserved region of the PPV genome which codes for the non-structural protein, NS-1. Results showed that PCR could detect PPV in titres at least 10(6) higher than the hemagglutination assay. The PCR and nested-PCR assays were used to detect successfully PPV DNA in clinical samples.

Prevalence of Toxoplasma gondii and Neospora caninum infections in sheep from Federal District, central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%<CI 0.95<49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%<CI 0.95<10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.

Biological studies and molecular characterization of a Cryptosporidium isolate from ostriches (Struthio camelus)

There are many reports of cryptosporidial infection in ostriches, but none with molecular characterization of the isolates. A study was undertaken for the characterization of a Brazilian Cryptosporidium sp. ostrich isolate by using molecular phylogenetic analysis of fragments of the 18S ribosomal DNA, heat-shock protein (hsp) 70 coding gene, and actin coding gene. Biological studies were accomplished by the experimental inoculation of chickens via oral or intratracheal routes with fresh ostrich Cryptosporidium sp. oocysts. Molecular analysis of nucleotide sequences of the 3 genes by using neighbor-joining and parsimony methods grouped the ostrich isolate as a sister taxon of Cryptosporidium baileyi and showed that the ostrich isolate is genetically distinct from all other known Cryptosporidium species or genotypes. None of the inoculated chickens developed infection as determined by mucosal smears, histology, and fecal screening for oocysts. Although biological and molecular studies indicate that the ostrich Cryptosporidium is a new species, further studies regarding morphological, biological, and molecular characteristics of other ostrich isolates are required to confirm the species status of the ostrich Cryptosporidium.

Genetic diversity among capybara (Hydrochaeris hydrochaeris) isolates of Toxoplasma gondii from Brazil

Recent studies indicate that Toxoplasma gondii isolates of many domestic hosts from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known about genetics of T. gondii isolates from wild mammals in Brazil. In this study, genotypes of 36 T. gondii isolates from capybaras (Hydrochaeris hydrochaeris) from six counties in São Paulo state, Brazil, were determined. Sixteen genotypes were identified using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. No classical clonal Type I and Type II isolates were found, confirming other findings that these lineages are rare in Brazil. Eight of these 36 isolates were grouped into the common clonal lineages in Brazil, previously designed as Types BrI, BrII and BrIII. Seven of the 16 genotypes were reported for the first time in this study. Three of the 36 isolates showed mixed infections. Analysis of mortality rates in infected mice indicated that Type BrI is highly virulent, Type BrII is intermediately virulent and Type BrIII is non-virulent, which is in agreement with previous report. The allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. These genotyping results support previous findings that the T. gondii population is highly diverse in Brazil.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

AMBLYOMMA LATEPUNCTATUM, A VALID TICK SPECIES (ACARI: IXODIDAE) LONG MISIDENTIFIED WITH BOTH AMBLYOMMA INCISUM AND AMBLYOMMA SCALPTURATUM

In 2000, we initiated an investigation on the tick fauna of Rondônia State, where we collected many specimens of Amblyomma scalpturatum Neumann, 1906 and Amblyomma incisum Neumann, 1906. In addition, we also collected a third group of ticks that were morphologically closely related to those 2 species, but sufficiently different to be considered a distinct species; members of this group were subsequently identified as Amblyomma latepunctatum Tonelli-Rondelli, 1939, through comparison with the type specimens of this taxon. Herein, we redescribe both sexes of A. scalpturatum and A. incisum, the female of A. latepunctatum, and provide the first description of the male of this latter species. Molecular analysis of the second internal transcribed spacer (ITS2) from rDNA of specimens of the 3 species supports morphological results. Examination of both A. scalpturatum and A. incisum deposited in different tick collections revealed that A. latepunctatum appeared relatively frequently in the vials believed to contain specimens of A. incisum or A. scalpturatum. Before this study, A. latepunctatum was considered a synonym of A. scalpturatum. Herein, we provide morphological and molecular evidence to validate the species A. latepunctatum. The South American tapir (Tapirus terrestris) seems to be the primary host for the adult stage of A. latepunctatum, A. scalpturatum, and A. incisum.

Molecular phylogenetic analysis in Hammondia-like organisms based on partial Hsp70 coding sequences

The 70 kDa heat-shock protein (Hsp70) sequences are considered one of the most conserved proteins in all domains of life from Archaea to eukaryotes. Hammondia heydorni, H. hammondi, Toxoplasma gondii, Neospora hughesi and N. caninum (Hammondia-like organisms) are closely related tissue cyst-forming coccidians that belong to the subfamily Toxoplasmatinae. The phylogenetic reconstruction using cytoplasmic Hsp70 coding genes of Hammondia-like organisms revealed the genetic sequences of T. gondii, Neospora spp. and H. heydorni to possess similar levels of evolutionary distance. In addition, at least 2 distinct genetic groups could be recognized among the H. heydorni isolates. Such results are in agreement with those obtained with internal transcribed spacer-1 rDNA (ITS-1) sequences. In order to compare the nucleotide diversity among different taxonomic levels within Apicomplexa, Hsp70 coding sequences of the following apicomplexan organisms were included in this study: Cryptosporidium, Theileria, Babesia, Plasmodium and Cyclospora. Such analysis revealed the Hammondia-like organism to be the lowest divergent group when compared to other groups within the phylum Apicomplexa. In conclusion, the Hsp70 coding sequences proved to be a valuable genetic marker for phylogenetic reconstruction and may constitute a good candidate to be used with other genes for species phylogeny within this group of organisms.

Evaluation of experimental Toxoplasma gondii (Nicolle and Manceaux, 1909) infection in pigs by bioassay in mice and polymerase chain reaction

The aim of the present experiment was to standardize a nested polymerase chain reaction (nPCR) for the detection of Toxoplasma gondii in tissues of experimentally infected pigs and to compare the performance of nPCR with the standard isolation technique, the bioassay in mice. Comparison between the two methods was done testing eight 4 month-old pigs orally inoculated with 5 x 104 oocysts of Toxoplasma gondii (AS-28 strain) and three non-infected pigs at the same age, kept as control. All animals were euthanatized 47 days after infection and samples of brain, heart, tongue and retina were collected from each animal for analysis by nPCR and bioassay in mice. By using the bioassay, Toxoplasma gondii was detected in 4 infected pigs, being two in the retina, one in the heart and one in the tongue. Toxoplasma gondii DNA was detected in five of the inoculated pigs, being: three in the tongue, two in the brain and heart and one in retina. The detection threshold of the nPCR on mouse brain suspension artificially infected with the RH strain of Toxoplasma gondii was 10 tachyzoites/ml. Although both techniques were unable to detect the parasite in all infected pigs, nPCR showed better performance as it was accomplished in a shorter period of time. When used concurrently, both techniques detected the agent in seven infected animals. The only way to increase sensitivity of either method is to increase the amount of tissue to be examined.