Brucella Infection Investigation in Cetaceans and Manatees in Northeast Brazil.

Among the bacterial infections that impair the health status of marine mammals, those caused by Brucella spp. are the most reported worldwide. Brucella infections in marine mammals can result in acute or chronic disease and are associated with variable clinical outcomes, depending on the organ involved during the infectious process, infection route, host immunity, and strain pathogenicity. Asymptomatic infections may also occur. The current study expands the investigation of Brucella infection in northeast Brazil by analyzing 19 dead, stranded cetaceans and 52 Antillean manatees Trichechus manatus manatus. The manatees included 8 dead, captive manatees and 44 live specimens, of which 10 were analyzed only after reintroduction into the wild as part of a rehabilitation program, 9 were analyzed both while in captivity or semi-captivity and after reintroduction, 20 were sampled only in captivity or semi-captivity, and 5 were free-living manatees. Serological tests were used to screen for antibodies against smooth Brucella spp. Whole blood, swabs, and tissue samples were screened for Brucella spp. DNA by PCR. Samples with positive PCR results were cultured for Brucella spp. isolation. All manatees yielded negative results in serological and molecular tests. Brucella spp. DNA was detected in the kidney of one adult Guiana dolphin Sotalia guianensis exhibiting necrosis in the liver. No growth of Brucella spp. was observed via microbiological culturing. This study is the first report of Brucella spp. DNA detection in cetaceans in the state of Pernambuco, and it highlights the importance of conducting systematic monitoring for the presence of Brucella infection in marine mammals along the Brazilian coast, especially in the northeast region, where several cases have been reported.

Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for canine brucellosis diagnosis.

Canine brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of canine brucellosis.

Experimental infection of inbred BALB/c and A/J mice with Massachusetts and Brazilian strains of infectious bronchitis virus (IBV).

The ability of avian coronaviruses to replicate in mice was investigated to investigate interspecies transmission. Two inbred mouse strains (BALB/c and A/J) with different genetic backgrounds were inoculated with the avian coronavirus strains Mass and BR-I and monitored for at least 10 days. Analysis of viral RNA, histopathological examinations, immunohistochemistry and serology were performed. After virus inoculation, neither clinical signs nor evident gross lesions were observed. Viral RNA, histopathological changes, and viral nucleoprotein were observed in the lung, trachea and sinus of all inoculated mice. Our study demonstrates the importance of elucidating the epidemiology of coronaviruses, including in rodents that are pests in poultry production.

Multilocus amplification of genomic DNA from single cysts of Giardia duodenalis separated using micromanipulation technique.

Giardia duodenalis is divided into at least eight groups, named assemblages A to H. Assemblages A and B are the only ones able to infect humans and other mammals. The species status for these assemblies is a moot point, but has not gained general acceptance because sexual activity in Giardia is not completely understood. Heterozygosity in G. duodenalis can be detected through simultaneous identification of multiple loci in single cysts or trophozoites. In this paper, we describe a technique that enables simultaneous detection of fragments from four genes from single cysts of G. duodenalis recovered from stool samples. Each cyst from a fecal sample of human origin was separated, the DNA was extracted and amplified by means of multiplex PCR directed to four genes and the multiplex PCR product was further re-amplified using four single PCR (one for each gene). The following loci were detected: beta giardin (bg), GLORF-C4 (orfC4), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh). This procedure should make it possible to investigate multiple genes from a single cyst of G. duodenalis assemblage A or B.

Detection of Leishmania infantum DNA in blood samples of horses (Equus caballus) and donkeys (Equus asinus) by PCR.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by the Leishmania infantum parasite. The protozoan is able to infect several domestic and wild mammals. Since the first report on Leishmania spp. infection in horses in South America, leishmaniasis in equids has been highlighted in Brazil. A molecular epidemiological survey was carried out to verify the occurrence of Leishmania spp. DNA in horses and donkeys, in leishmaniases endemic areas in Sao Paulo State, Brazil. To this end, blood samples were obtained from 107 horses and 36 donkeys and subjected to DNA extraction followed by PCR targeting the ITS-1 region. Among the horses and donkeys, 1.87% (2/107) and 8.33% (3/36) were positive by PCR, respectively. The DNA sequencing of the ITS-1 amplification products confirmed L. infantum DNA in these animals. Our results suggest that horses and donkeys from non-VL and VL endemic areas of São Paulo State may be infected by the parasite.

Detection of Brucella ovis in ovine from Paraíba State, in the Northeast region of Brazil.

To determine the presence of Brucella ovis in ovine from Paraíba State, in the Northeast region of Brazil, 80 animals slaughtered in the public slaughterhouse of Patos city were used. Before slaughter, blood samples were collected by jugular venopuncture from each animal, and after slaughter, testicles, epidydimus and uterus were aseptically collected. For the serological diagnosis of B. ovis and B. abortus infections, the agar gel immunodiffusion (AGID) and Rose Bengal (RBT) tests were carried out, respectively. In addition, microbiological culture and polymerase chain reaction (PCR) were performed on testicle, epidydimus and uterus samples. Six animals (7.5%) tested positive for the presence of B. ovis antibodies and all animals tested negative for the presence of B. abortus antibodies. One AGID-positive animal tested positive at uterine swab culture. PCR was able to amplify DNA of Brucella spp. from the pool of testicle, epidydimus and uterus samples from AGID-positive animals. This is the first report of isolation and detection of B. ovis DNA by PCR in ovine from the Northeast region of Brazil.

MALDI-TOF MS and genomic analysis can make the difference in the clarification of canine brucellosis outbreaks.

Brucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of canine brucellosis.

Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1.

Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Molecular detection of Leishmania infantum DNA according to clinical stages of leishmaniasis in dog.

The aim of this study was to compare molecular tests used to diagnose Leishmania spp. in dogs with different stages of infection. Blood and conjunctival swab (CS) samples from dogs classified in four clinical stages were subjected to different PCR protocols (13A/13B, MC1/MC2, LITSR/L5.8S and LEISH-1/LEISH-2 primers). To the study, 22.3% (48/215) of dogs were classified as without clinical signs, 67.5% (145/215) stage I (mild disease), 7.0% (15/215) stage II (moderate disease) and 3.2% (7/215) stage III (severe disease). The results showed that in blood samples, 13A/13B detected a significant higher number of positive dogs in stage I (25/145) and in total (42/215) (p≤0.05). However, when CS samples were tested, no difference was observed (p>0.05). On the other hand, in blood samples, MC1/MC2 detected significantly fewer positive dogs classified as without clinical signs (0/48), in stage I (0/145) and in total (1/215) (p≤0.05). Likewise, in CS samples, this primers showed also lower detection (1/215) (p≤0.05). So than, we can conclude that PCR on blood samples with 13A/13B primers has greater capacity to detect positive dogs, mainly at the initial of clinical disease than do other primers and MC1/MC2 are not a good choice to detect Leishmania infantum infection in dogs.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences.

Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genus Hammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well.

Detection of canine visceral leishmaniasis by conjunctival swab PCR.

INTRODUCTION: Conjunctival swab PCR was evaluated as a tool to diagnose visceral leishmaniasis in dogs. METHODS: Conjunctival swab PCR was compared to indirect immunofluorescence antibody test and blood PCR. RESULTS: Indirect immunofluorescence was significantly correlated with conjunctival swab PCR (p < 0.05), but not with blood PCR (p > 0.05). In addition, conjunctival swab PCR was significantly associated with presence of clinical symptoms (p < 0.05), whereas blood PCR was associated with absence of clinical symptoms (p < 0.05). CONCLUSIONS: Results indicate that conjunctival swab PCR is useful in epidemiological surveys of canine visceral leishmaniasis.

Evidence of heterozygosity and recombinant alleles in single cysts of Giardia duodenalis.

Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.

Infection of mice with oocysts of Toxoplasma gondii by oral route showed differences of virulence from Brazilian RFLP genotypes BrI and BrIII.

South American strains of Toxoplasma gondii present higher genetic diversity than classical European strains. We compared the virulence of two non-archetypal Brazilian genotypes of T. gondii to mice. Oocysts of four isolates, two genotype BrI (TgCatBr71 and TgShBr11) and two BrIII (TgCatBr74 and TgCatBr60) were obtained from cats fed experimentally infected mice. After sporulation, 5.0×10(1) and 1.0×10(2) oocysts were orally administrated to Swiss albine mice in Experiments #1 and #2, respectively (4-10 mice/group). Humoral response from dead and surviving mice was analyzed on days 9 to 35 post-infection. Microscopic observations of lungs and brains were performed for tachyzoites and cysts visualization in fresh preparations. Negative results were tested by PCR. Virulence after infection with oocysts is dose dependent for genotype BrIII isolates, but not for BrI. Differences in mortality were observed among isolates from genotype BrIII on Experiment #1. Intra-genotype phenotypic variation related to the parasite stage of infection was demonstrated and this characteristic should be further studied and may influence future work regarding the role of virulence amid hosts.

Leishmania infantum in the reproductive organs of dogs / Leishmania infantum em órgãos reprodutivos de cães

ABSTRACT: Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.

RESUMO: Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.

Molecular detection of Leishmania infantum DNA according to clinical stages of leishmaniasis in dog / Deteção molecular de DNA de Leishmania infantum em diferentes estágios clínicos da leishmaniose em cães

Abstract The aim of this study was to compare molecular tests used to diagnose Leishmania spp. in dogs with different stages of infection. Blood and conjunctival swab (CS) samples from dogs classified in four clinical stages were subjected to different PCR protocols (13A/13B, MC1/MC2, LITSR/L5.8S and LEISH-1/LEISH-2 primers). To the study, 22.3% (48/215) of dogs were classified as without clinical signs, 67.5% (145/215) stage I (mild disease), 7.0% (15/215) stage II (moderate disease) and 3.2% (7/215) stage III (severe disease). The results showed that in blood samples, 13A/13B detected a significant higher number of positive dogs in stage I (25/145) and in total (42/215) (p≤0.05). However, when CS samples were tested, no difference was observed (p>0.05). On the other hand, in blood samples, MC1/MC2 detected significantly fewer positive dogs classified as without clinical signs (0/48), in stage I (0/145) and in total (1/215) (p≤0.05). Likewise, in CS samples, this primers showed also lower detection (1/215) (p≤0.05). So than, we can conclude that PCR on blood samples with 13A/13B primers has greater capacity to detect positive dogs, mainly at the initial of clinical disease than do other primers and MC1/MC2 are not a good choice to detect Leishmania infantum infection in dogs.

Resumo O objetivo deste estudo foi comparar testes moleculares usados para diagnosticar Leishmania spp., em cães apresentando diferentes estágios de infecção. Amostras de sangue e suabe conjuntival (SC) de cães classificados em quatro estágios clínicos foram submetidas a diferentes PCRs (primers 13A/13B, MC1/MC2, LITSR/L5.8S e LEISH-1/LEISH-2). Para o estudo, 22,3% (48/215) dos cães foram classificados como sem sinais clínicos, 67,5% (145/215) estágio I (doença leve), 7,0% (15/215) estágio II (doença moderada) e 3,2% (7/215) estágio III (doença grave). Os resultados mostraram que, em amostras de sangue, 13A/13B detectou número significativamente maior de cães positivos no estágio I (25/145) e no total (42/215) (p≤0,05). No entanto, quando as amostras de SC foram testadas, nenhuma diferença foi observada (p>0,05). Por outro lado, no sangue, MC1/MC2 detectou significativamente menos cães positivos sem sinais clínicos (0/48), em estágio I (0/145) e no total (1/215) (p≤0,05). Da mesma forma, em amostras de SC, MC1/MC2 também apresentou menor detecção (1/215) (p≤0,05). Assim, a PCR em amostras de sangue com 13A/13B tem maior capacidade de detectar cães positivos, principalmente no início da doença do que outros primers, e o par de primers MC1/MC2 não é uma boa escolha para detectar infecção por Leishmania infantum em cães.

Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1 / Detecção molecular de Leishmania spp. em gado no Brasil por PCR com espaçador interno transcrito 1

Abstract Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Resumo Leishmania spp. são agentes causadores das leishmanioses em humanos e em animais, gerando grande impacto à saúde pública. Este estudo objetivou detectar a circulação de Leishmania spp. em área não endêmica para leishmaniose visceral de São Paulo, Brasil. Foram extraídas amostras de DNA de 100 novilhas da cidade de Pirassununga. Estas amostras foram amplificadas com os iniciadores específicos para tripanosomatídeos Internal Transcriber Spacer 1 (ITS1). Os ensaios revelaram uma amostra com bandas entre 300 e 350 pares de base (pb). A amostra demonstrou 100% de identidade com Leishmania infantum (314 pb). Os resultados sugerem que o gado pode ser infectado por L. infantum no Brasil.

Comparison between tests for tuberculosis diagnosis in slaughtered bovines / Comparação entre testes de diagnóstico para tuberculose em bovinos abatidos

Our goal for this article is to compare several different diagnosis tests for bovine tuberculosis identification. We have performed bacterial isolation, histopathological characterization, acid-fast bacilli (AFB) identification and M. bovis DNA detection. Lesions suggestive of Tuberculosis were sampled from bovine lymph nodes during slaughtering of bovines at an abattoir that operates under federal inspection. The bacterial isolation was performed in solid culture mediums, the histopathological characterization was made by Hematoxylin-eosinstaining, and AFB identification by Ziehl-Neelsen staining. Bacterial DNA detection was performed by Polymerase Chain Reaction (PCR) using DNA from two different sources, directly collected from the tuberculosis-like lesions (PCR followed by nested PCR) and from isolated bacteria. We have concluded that the multi-step approach, including histopathological characterization, bacterial isolation and AFB identification, is strongly recommended to diagnose tuberculosis in bovines. Furthermore, PCR assays using specimens of lesions suggestive of tuberculosis are a faster and more promising way to diagnose the disease. However, it should not be used alone due to the low sensitivity shown in this study.(AU)

O objetivo deste estudo foi a comparação entre diferentes testes de diagnóstico para tuberculose bovina. Foram realizados o isolamento bacteriano, a caracterização histopatológica, a identificação de bacilos álcool-ácido resistentes e a detecção do DNA de M. bovis pela reação em cadeia da polimerase, em bovinos adultos abatidos em matadouros frigoríficos sob o Serviço de Inspeção Federal, valendo-se de amostras de linfonodos com lesões macroscópicas sugestivas de tuberculose, identificadas e coletadas durante o abate. O isolamento bacteriano foi realizado pelo cultivo em meios de cultura sólidos; a caracterização histopatológica, pela coloração com hematoxilina-eosina; e a identificação de bacilos álcool-ácido resistentes foi feita pela coloração de Ziehl-Neelsen. A detecção de DNA foi realizada em amostra extraída das lesões sugestivas de tuberculose pela reação em cadeia da polimerase, seguida da nested reação em cadeia da polimerase e por meio das colônias isoladas para identificação do M. bovis, utilizando-se também da reação em cadeia da polimerase . Os resultados obtidos permitiram concluir que os testes histopatológicos, o isolamento bacteriano e a identificação de bacilos álcool-ácido resistentes são aconselháveis para o diagnóstico da tuberculose bovina. Além disso, ensaios de reação em cadeia da polimerase utilizando amostras de lesões sugestivas de tuberculose são um modo mais rápido e promissor para diagnosticar a enfermidade, no entanto não deve ser utilizado sozinho, em virtude da baixa sensibilidade apresentada neste estudo.(AU)

Molecular discrimination of Mycobacterium bovis in São Paulo, Brazil.

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, is the most common agent of cattle tuberculosis, a zoonosis that causes losses in meat and milk production in several countries. In order to support epidemiological studies aimed at controlling the disease, several methods for molecular discrimination of M. bovis isolates have recently been developed. The most frequently used are spacer oligonucleotide typing (spoligotyping), mycobacterial interspersed repetitive units (MIRU), and exact tandem repeat (ETR), but they all have different discriminatory power. In the present study, allelic diversity was calculated for each MIRU and ETR locus, and the Hunter-Gaston discriminatory index (HGI) was calculated for spoligotyping, 10 MIRUs, and 3 ETRs, in 116 isolates of M. bovis obtained from cattle. The analysis of allelic diversity indicated that MIRUs 16, 26, and 27, and ETRs A, B, and C, showed the greatest diversity between the assayed loci. The HGIs for each of the techniques were: spoligotyping=0.738381; MIRU=0.829835; and ETR=0.825337. The associations of the methods' improved discriminatory power were: spoligotyping+MIRU=0.930585; spoligotyping+ETR=0.931034; and MIRU+ETR=0.953373. The greatest discriminatory power was obtained when the three techniques were associated (HGI=0.98051). Considering the analyses of the present study, spoligotyping should be the first method to be used because it differentiates M. bovis from the other members of the Mycobacterium tuberculosis complex. As the associations of MIRU and ETR with spoligotyping resulted in nearly identical HGIs, ETR seems to be the best choice after spoligotyping, because it is faster and more economical than MIRU. Finally, MIRU should be the last method used. In spite of this finding, the choice of the method used should be based on the discriminatory power necessary for the objective at hand.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H. heydorni RAPD fragment AP10.

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs.