Aline Diniz Cabral

Clostridium perfringens types A and D associated with enterotoxemia in an 18-month-old goat

Postmortem examination of a Boer buck that died peracutely revealed bowel and liver diffusely congested and edematous. Kidney was apparently edematous. Clostridium perfringens type A was isolated from bowel and type D from kidney. Microscopic examination revealed large areas of necrosis in the renal cortex and medulla (pulpy kidney disease), hyperemia and centrilobular necrosis of the liver, necrosis of the small-intestine wall, pulmonary edema and congestion, intense hyperemia of the cerebellum, hyperemia and edema of the brain.

Occurrence of antibodies against Toxoplasma gondii and its isolation and genotyping in donkeys, mules, and horses in Brazil.

The occurrence of antibodies against Toxoplasma gondii was determined in donkeys, mules, and horses from different regions of Brazil. Serum samples from 304 donkeys (67.11%), 118 horses (26.05%), and 31 mules (6.84%) were analyzed by means of the indirect fluorescent antibody test (cutoff=64). Antibodies against T. gondii were detected in 129 equids (28.47%) (82 donkeys, 32 horses, and 15 mules). Tissue samples from 19 seropositive and 50 seronegative animals were obtained in order to isolate the parasite by means of mouse bioassay, and T. gondii was isolated from a donkey. Through genotypic characterization of the isolate, by means of polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) using 11 genotypic markers, the genotype #163 (TgCkBr220), which has already been described in chickens in Brazil, was identified.

Fatal systemic toxoplasmosis in Valley quail (Callipepla californica)

Highlights • This is the first report of systemic fatal toxoplasmosis in Valley quail.• Toxoplasma gondii was molecularly identified as belonging to ToxoDB-PCR-RFLP #87.• It caused a generalized disease with necrotic lesion in liver, heart, spleen, lungs, trachea and bone marrow, with large numbers of tachyzoites stained positively with polyclonal antiserum to T. gondii.

Toxoplasma gondii in domestic and wild animals from forest fragments of the municipality of Natal, northeastern Brazil.

Toxoplasmosis stands out as a global disease that has felines as definitive hosts. In the municipality of Natal, Rio Grande do Norte State, Brazil, two parks are notable for their ecological and social importance. This study aimed to investigate the presence of Toxoplasma gondii in short hair cats, bats and small non-volant mammals in these two ecological reserves. Altogether, biological samples were obtained from 154 mammals, 92 wild animals from both areas and 62 domestic cats of the Parque da Cidade. In total, 22 (53.7%) non-volant wild mammals, 11 (21.5%) bats and 28 (52.8%) cats were positive for IgG anti-T. gondii antibodies using the Modified Agglutination Test (≥ 25). It was possible to detect the presence of T. gondii DNA, by means of a molecular amplification of a B1 gene fragment (155bp), in 92 tissue samples from wild animals, including Didelphis albiventris, Monodelphis domestica, Artibeus lituratus, Carollia perspicillata and Glossophaga soricina. Of the 62 cats examined by the same molecular method, T. gondii DNA could be detected in 4 cats. In this study, it was observed the circulation of T. gondii in wild species and domestic cats, demonstrating the involvement of wild and domestic animals in the cycle of T. gondii.

Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil.

Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report of Neospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

Neospora caninum as causative agent of bovine encephalitis in Brazil

For supporting the Brazilian bovine encephalitis surveillance program this study examined the differential diagnosis of Neospora caninum in central nervous system (CNS) by histological analysis (HE staining), immunohistochemistry (IHC), and nested-PCR using a set of primers from the Nc5 region of the genomic DNA and ITS1 region of the ribosomal DNA. A sample of 302 cattle presenting neurological syndrome and negative for rabies, aged 0 to 18 years, from herds in 10 Brazilian states was evaluated for N. caninum from January 2007 to April 2010. All specimens tested negative with IHC and nested-PCR using primers from the ITS1 region of ribosomal DNA, while two positive cases (0.66%) were found using primers from the Nc5 region of genomic DNA: a 20 month-old male and a 72 month-old female, both from São Paulo State. Only the male presented severe multifocal necrotizing encephalitis associated with mononuclear cell infiltration, a pathognomonic lesion caused by parasites of the family Sarcocystidae, and only this case was associated with N. caninum thus representing 0.33% positivity. Future studies should explore the association of IHC and nested-PCR with real-time PCR, a quantitative method that could be standardized for improving the detection of N. caninum in bovine CNS specimens.

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.

Nucleic Acid-based Diagnosis and Epidemiology of Infectious Diseases

In this chapter, the immense contribution of nucleic acid discovery to the diagnosis and molecular epidemiology of pathogenic microorganisms and its relevance for veterinary and human health will be discussed. The development of nucleic acid detection, amplification, and sequencing techniques, principally after the introduc‐ tion of polymerase chain reaction (PCR), allowed the improvement of different strategies to diagnose and to quantify infectious microbiological agents in a variety of organisms and biological samples. Pos-PCR associated techniques such as fragment enzyme restriction and sequence analysis permit the determination of nucleic acid sequence diversity to detect drug resistance, to associate pathogen genetic markers with disease outcome, and to predict temporal and spatial distribution of microorganisms which can be used to prevent and treat infectious diseases efficiently. The principal methods used in the detection of nucleic acids, the advantages and drawbacks of singleand multiple-copy genes for use in diagnosis by amplification, and the application of pos-PCR techniques in drug resistance identification are dis‐ cussed in Section 1.1. Section 1.2 discusses the sequencing methods used to recog‐ nize genetic variability, the implication of this variability to pathology and virulence, and the importance of genetic variability determination in disease control and vaccines. The contribution of molecular diagnosis and epidemiology for the treatment and prevention of infectious diseases is also considered.

A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies

There is a growing necessity to integrate morphological and genetic studies. This paper proposes a new technique that allows DNA extraction of arthropods while still keeping intact the entire morphology of the specimens. The technique uses Proteinase K to dissolve protein tissues and preserve the chitinous exoskeleton of specimens. The method is fast, cheap, non-toxic, and allows for good morphological preparations of specimens retaining much of their tridimensional structure. The methodology works fine with specimens preserved in different kinds of media, such as for dry (pinned) specimens, and specimens preserved in Ethanol. In addition, it allows the extraction of DNA from fresh specimens, as well as from specimens preserved for a long time. The technique works well for morphological studies alone, but allows the generation of an associated genomic library at an individual-scale. Among the advantages of the new technique is the possibility of extracting DNA from the entire specimen (necessary for the study of diseases transmitted by arthropod vectors), while still keeping the morphology intact for correct taxonomic identification. In addition, in comparison with methods that extract DNA from small tissue samples (e.g., from legs or wings), the method allows for the extraction of a larger amount of DNA and is better suited for small specimens.

A Highly Sensitive and Specific Conventional Molecular Diagnosis for Leishmania infantum Chagasi Based on a Single Copy Gene

Leishmaniasis are zoonotic diseases caused by a protozoa from Trypanosoma family and of the genus Leishmania, being transmitted by sandfly vectors. Leishmania genus comprise 30 species, including 20 species able to cause disease with different clinical manifestations in humans, ranging from asymptomatic, cutaneous and mucocutaneous lesions, to the severe visceral form. According to the World Health Organization, visceral leishmaniasis, caused by Leishmania infantum chagasi in the Americas, is the most severe form of the disease, and is lethal if not treated. Brazil is part of the group of countries with the highest prevalence of this disease, concentrating 90% of the cases registered in Latin America. A rapid and accurate diagnostic method is of great importance to detect and treat specifically L. i. chagasi in the American continent where others trypanosomiasis circulate, making a specific diagnostic difficult, due to cross reaction. In this work, a new conventional molecular diagnostic method was developed, based on the single copy L. infantum chitinase encoding gene, which presented high specificity and showed increased sensitivity when compared to the method based on the gene encoding the internal transcribed spacer 1 of the Leishmania rRNA (rDNA ITS-1) to diagnose L. i. chagasi on human clinical samples.