ABSTRACT The present study proposes to characterize the parasites isolated during the initial phase of production in fish farms located in Mato Grosso do Sul in the central-western region of Brazil, using histopathology analysis and molecular techniques. A total of 340 hybrid surubim fish (Pseudoplatystoma reticulatum × P. corruscans) from four farms were examined during the co-feeding phase. Histopathology analysis showed that 10.9% (n = 37) of the fish were infected with parasites. Branchitis, lifting epithelium, hypertrophy of epithelial cells, lamellar fusion, aneurisms and infection in the bone tissue of the gill filament was observed. The parasite species was determined by amplification of the 18S rRNA gene followed by sequencing. The phylogenetic analysis of nucleotide sequences indicates a close relationship (99.6%) with Henneguya pseudoplatystoma reported to be infecting the hybrid Pseudoplatystoma. This study demonstrates the occurrence of H. pseudoplatystoma in hybrid surubim (P. reticulatum × P. corruscans) during the co-feeding phase in fish farms in Mato Grosso do Sul. Also, molecular techniques provide a faster and sensitive method to identify fish parasites, and may assist in the development of new management techniques aimed at improving the sanitary conditions contributing to the reduction of mortality rates in these animals.

ABSTRACT Due to the growing population of pets, especially homeless dogs and cats, zoonoses still represent a significant public health problem. Toxoplasma gondii and Toxocara spp. are epidemiologically important zoonotic agents as they are etiological factors of human toxoplasmosis and toxocariasis, respectively. These parasites remain neglected even though they are substantially prevalent in rural areas. The aim of this study was to investigate T. gondii and T. canis seroprevalence and risk factors of seropositivity in a rural population in Pelotas municipality, Brazil. The study participants (n=344) were patients of a Basic Healthcare Unit (BHU) located in Cerrito Alegre. Blood samples were collected and tested for T. gondii antibodies by indirect immunofluorescence and T. canis antibodies by an indirect ELISA that targets an excreted-secreted antigen (TES). T. gondii seropositivity was 53.2%, with higher titers (1:256 - 1:1,024) in individuals who habitually eat pork, beef, or chicken, while T. canis seropositivity was 71.8% and concomitant T. gondii and T. canis seropositivity was 38.3%. Among the seropositivity risk factors assessed, only habitual undercooked meat consumption was significant (p = 0.046; OR = 3.7) for T. gondii and none of them were associated with T. canis seropositivity. Both parasites have a high prevalence in rural areas, which reinforces the need to invest in rural community education and health.

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

SUMMARY Trichomonas vaginalis infections have been associated with other diseases so that epidemiological studies of the parasite are important and help to prevent the spread of the disease. This study aimed to determine the prevalence of T. vaginalis in female patients of 19 counties in southwestern Rio Grande do Sul, Brazil. For diagnosis, was used direct examination, followed by applying a socio-epidemiological questionnaire. We analyzed 300 women and 9% were infected by Trichomonas vaginalis. The highest frequency occurred in women between 18 and 39 years old, single/divorced/widowed, whose family income was at one minimum wage or less, and they had not completed the primary school. Statistically significant risk factors were: women reporting two or more sexual partners in the last year were 3.3 times more likely to acquire the parasite, and those in use of oral contraceptives were 2.7 times more likely to have T. vaginalis. Importantly, 33% of the asymptomatic women were infected, and most of the negative results were from women presenting symptoms consistent with the infection. The findings emphasize that it is necessary to expand the knowledge of individuals about the disease, especially among women with the above mentioned risk factors and also to include the regular screening of Trichomonas vaginalis infections in health centers.

A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos; e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

A toxocaríase é uma zoonose na qual os títulos de IgM podem permanecer elevados por longos períodos, tornando difícil a determinação do estágio em que a doença se encontra. O objetivo deste estudo foi investigar a aplicabilidade de um teste indireto de ELISA, associado com ureia, para fazer a discriminação entre as fases aguda e crônica da toxocaríase. A avidez de IgG foi avaliada em 25 camundongos BALB/c experimentalmente infectados com 1000 ovos embrionados de Toxocara canis. A cada duas semanas, amostras de sangue foram coletadas, o soro tratado com ureia 6M e realizado o ensaio pela técnica de ELISA. O percentual de avidez de IgG foi determinado, usando-se a média das absorbâncias dos soros tratados com ureia dividida pela média das absorbâncias dos soros não tratados. Nos primeiros 15 dias pós-inoculação, foi observado um baixo percentual de avidez de IgG, entre 7,25 e 27,5%, característico da fase aguda da infecção. Após 60 dias de infecção, todos apresentaram avidez de IgG entre 31,4 e 58%, indicando a fase crônica da infecção.

Toxocariasis is a zoonotic disease in that IgM titers can remain high for long periods making difficult to determine the stage of the disease. The aim of this study is to investigate the applicability of indirect ELISA, associated with urea, to discriminate between the acute and chronic toxocariasis. IgG avidity was evaluated in 25 BALB/c mice experimentally infected with 1000 Toxocara canis eggs. Blood samples were collected, and sera treated with 6 M urea and assayed by ELISA every two weeks. The percent IgG avidity was determined using the mean absorbance of sera treated with urea, divided by the mean absorbance of untreated sera. In the first 15 days post-inoculation, was observed a low percentage, between 7.25 and 27.5%, IgG avidity, characteristic of an acute infection. After 60 days of infection, all the mice showed between 31.4 and 58% IgG avidity, indicating a chronic infection.

A toxocaríase visceral é uma zoonose negligenciada causada por larvas de Toxocara canis em hospedeiros não usuais. Em cães, os hospedeiros definitivos, a infecção ocorre normalmente por transmissão transplacentária e através do colostro. Estudos com modelos experimentais têm demonstrado a ocorrência de transmissão vertical durante a infecção aguda. Considerando que a toxocaríase é caracterizada como uma infecção crônica, com uma possível reativação das larvas presentes no cérebro, este estudo avaliou a presença de larvas no cérebro de camundongos Balb/C fêmeas e suas proles com infecção crônica durante três gestações sucessivas. Para avaliar os níveis de anticorpos foi utilizado ELISA-TES. Larvas de T. canis foram detectadas no encéfalo dos animais durante as três gerações sucessivas avaliadas. O nível de IgG das proles foi diminuindo gradualmente e as médias de absorbâncias (ABS) acima do ponto de corte (0,070) foram evidenciadas somente aos 30 (0,229) e 50 dias (0,096) de vida, enquanto que não foi detectada IgM. Infecções das proles confirmam a transmissão vertical de larvas de T. canis durante a toxocaríase crônica em três gerações sucessivas de camundongos.

Visceral toxocariasis is a neglected zoonosis caused by Toxocara canis larvae in unusual hosts. In dogs, the definitive host, the infection occurs mainly through transplacental and transcolostral transmission. Studies on experimental models have shown that vertical transmission may result from acute infections. Considering that toxocariasis is characterized as a chronic infection, with possible reactivation of larvae present in the brain, this study evaluated the presence of larvae in the brain of female BALB/c mice and their offspring with chronic infection during three successive pregnancies. ELISA-TES was used to evaluate the antibody levels. T. canis larvae were detected in the brain tissue of the mice during the three successive generations evaluated. The offspring's IgG level gradually decreased, and mean absorbance (ABS) above the cutoff point (0.070) was observed only at 30 (0.229) and 50 (0.096) days of age, while IgM was not detected. The infections in the offspring confirmed that vertical transmission of T. canis larvae occurred during chronic toxocariasis in three successive generations of mice.

The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.