Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples

<p>Abstract</p> <p>Background</p> <p>The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology.</p> <p>Findings</p> <p>The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition.</p> <p>The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques.</p> <p>Conclusion</p> <p>These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies.</p

PHYTOHAEMAGGLUTININ’S EFFECT ON NEOSPORA CANINUM ANTIGEN PRODUCTION

The parasite Neospora caninum affects mainly cattle and dogs. This study aimed to evaluate the effect of phytohaemagglutinin (PHE) in antigen production of N. caninum NC-1 strain in gerbils (Meriones unguiculatus) and in vitro. 20 gerbils were used, 10 inoculated intraperitoneally with 1 x 106 tachyzoites and 10 with 1 x 106 tachyzoites plus 300 µL/mL of PHE. 16 bottles of Vero cell culture were inoculated, 8 with 1.5 x 105 tachyzoites and 8 with 1.5 x 105 tachyzoites plus 30 µL/mL of PHE. Serology of gerbils was performed on day 0 and before euthanasia. Tachyzoites present in peritoneal fluid and cell culture bottles were quantified by Neubauer chamber and by real-time PCR (qPCR). PHE has not interfered in the production of tachyzoites of N. caninum in intraperitoneal inoculated gerbils and the effect of PHE in cell culture had a negative impact, considering the qPCR technique as the gold standard

Prevalência e distribuição geográfica do Toxoplasma gondii em cães na área urbana de Botucatu, SP, Brasil

Toxoplasmose é uma zoonose de distribuição mundial causada pelo Toxoplasma gondii, que pode infectar uma grande variedade de animais, inclusive seres humanos. Animais domésticos podem ser sentinelas importantes para infecções na comunidade. Ocorrência de infecção pelo T. gondii foi avaliada em cães na área urbana de Botucatu (SP, Brasil). Para amostragem, considerou-se uma taxa de erro na estimativa de 10%, um intervalo de confiança de 95% e um nível de significância de 5%. As amostras de sangue dos cães foram coletadas durante uma campanha de vacinação antirrábica e processadas usando-se o teste de aglutinação modificado (MAT). Foram coletadas 670 amostras de sangue, com distribuição homogênea em cinco regiões da área urbana, representando 3.74% dos 17.910 cães vacinados. Dessa amostra, 17,3% (116/670) dos cães, sendo 58,6% (68/116) fêmeas e 41,4% (48/116) machos, foram positivos para infecção pelo T. gondii (p&lt;0,03). Entre os cães infectados 4,6%; (4/88) deles tinham idade menor e 95,4% (84/88) maior que um ano (p&lt;0,01); em 28 animais positivos a idade não era conhecida Os títulos séricos de anticorpos anti-T. gondii estavam distribuídos entre 16 (69,8%; 81/116), 64 (13,8%; 16/116), 256 (15,5%; 18/116) e 1024 (0,9%; 1/116). Os animais positivos estavam distribuídos nas regiões Norte 14,2% (19/134), Sul 18,0% (31/172), Leste 15,7% (19/121), Oeste 21,6% (27/125) e Centro 16,9% (20/118) do município (p=0,5). Em todas essas regiões, fêmeas e animais com mais de um ano de idade tiveram maior taxa de positividade para a infecção (p&lt;0.05).Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, and can infect a wide variety of animals including humans. Domestic animals can be an important sentinel population for infection in the community. Occurrence of T. gondii infection was assessed in dogs in the urban area of Botucatu city, SP, Brazil. In the sample, 10% rate for error estimate, 95% confidence interval, and 5% significance level were established. Serum samples were collected from dogs during a rabies vaccination campaign, and were processed using modified agglutination test (MAT). Blood samples were collected from 670 dogs, with homogeneous distribution in five regions in the urban area, representing 3.74% of 17,910 animals vaccinated. In this sample, 17.3% (116/670) dogs (68 – 58.6% female and 48 – 41.4% male) were positive for T. gondii infection (p&lt;0.03). Regarding age of the infected dogs 4.6% (4/88) were younger and 95.4% (84/88) were older than one year (p&lt;0.01); the age of 28 positive animals were undetermined. The serum titers of anti-T. gondii antibodies were: 16 (69.8%; 81/116), 64 (13.8%; 16/116), 256 (15.5%; 18/116), and 1024 (0.9%; 1/116). Prevalence was distributed among the North 14.2% (19/134), South 18.0% (31/172), East 15.7% (19/121), West 21.6% (27/125), and Central 16.9% (20/118) regions of the municipality (p=0.5). In all these regions, females and dogs aged more than one year showed a higher occurrence of T. gondii infection (p&lt;0.05)

Aspectos epidemiológicos nas leptospiroses: Pesquisa de anticorpos anti-Leptospira spp, isolamento e pesquisa biomolecular em bovinos, roedores e trabalhadores de propriedades rurais do Município de Botucatu, SP, Brasil

A leptospirose é uma infecção amplamente difundida pelo mundo, transmitida entre o homem e os animais que causa uma queda na produção de rebanhos bovinos, e oferece riscos relacionados à saúde pública sendo uma importante zoonose. Foram estudadas 27 propriedades de pecuária bovina leiteira em Botucatu-SP e limítrofes, com colheitas de amostras de sangue e rim de roedores, sangue e urina de bovinos e sangue dos trabalhadores. A sorologia foi realizada pela soroaglutinação microscópica (SAM). Foi tentado o isolamento de leptospiras por cultura da urina dos bovinos e dos rins dos roedores. Foi realizada a reação em cadeia pela polimerase (PCR) dos rins dos roedores. Para a SAM, 46/140 (32,85%) amostras de soro bovino e 8/34 (23,53%) humana foram positivas, respectivamente. Para as amostras humanas, o sorovar Bratislava (37,51%) apresentou maior ocorrência, enquanto que para os bovinos, os sorovares mais freqüentes foram Hardjo e Castellonis, com 26,08% cada. Todos os roedores foram negativos à sorologia. Nenhuma leptospira foi isolada, e as amostras de rim foram negativas à PCR. Nos bovinos, a água de açudes e as ordenhadas com má qualidade de higiene foram consideradas importantes riscos de infecção nas propriedades acometidas (p&lt;0,05), onde outros problemas reprodutivos, exceto aborto, podem estar relacionados. Por outro lado, para o homem o sistema de drenagem foi o mais importante fator de risco nas propriedades estudadas. Assim, enfatiza-se a necessidade de melhora no manejo zoosanitário das propriedades, principalmente com relação ao suprimento de água.Leptospirosis is a worldwide infection, transmitted between man and animals that causes a decrease in the production of bovine flocks, and offer risks for public health, as an important zoonosis. The rodents are the main reservoirs of leptospires. It was studied 27 dairy farm properties located in or near from Botucatu-SP, Brazil. In these farms were collected blood and kidney samples from rodents, blood and urine samples from bovines and blood samples from the workers. The serology was performed with microscopic agglutination test (MAT). Samples of bovine urine and rodent kidneys were cultivated searching for leptospires isolation. The polymerase chain reaction (PCR) of the kidneys of the rodents was performed. In MAT, 46/140 (32.85%) bovine and 8/34 (23.53%) human sera samples were positive, respectively. In human samples, the serovar Brastilava (37.51%) presented the highest occurrence, while in bovines, the serovars Hardjo and Castellonis were most frequent, with 26.08% each one. All of the rodents were negatives in serology. No leptospire was isolated, and kidney samples were negative in PCR. In bovines, the dam water and the bad hygiene quality of milking process were considered important risks of infection in the affected properties (p&lt;0.05), where other reproductive problems, except abortion, can be related. In other side, to human beings the drainage system was the most important risk factor in the studied properties. Thus, it was verified the necessity of an improvement in zoosanitary handling of the properties, mainly of water supply

Anti-leptospirosis agglutinins in Brazilian capybaras (Hydrochoerus hydrochaeris)

Abstract Background The interest in commercial use of wild animals is increasing, especially regarding raising of capybaras. Although this wild species is potentially lucrative for the production of meat, oil and leather, it is suggested as a probable reservoir of leptospires. Methods Due to the economic importance of this species and the lack of studies concerning leptospirosis, the presence of anti-leptospirosis agglutinins was assayed in 55 serum samples of capybaras (Hydrochoerus hydrochaeris) from commercial and experimental breeding flocks located in São Paulo state, Paraná state, and Rio Grande do Sul state, Brazil. Samples were obtained through cephalic or femoral venipunction (5 to 10 mL). Microscopic agglutination test was used according to the Brazilian Health Ministry considering as cut-off titer of 100. Results Out of the 55 samples analyzed, 23 (41.82 %) tested positive. The most prevalent serovar was Icterohaemorrhagiae (56.52 %) in 13 samples, followed by Copenhageni in nine samples (39.13 %), Pomona in four samples (17.39 %), Djasiman and Castellonis in three samples each (13.04 %), Grippotyphosa, Hardjo, Canicola, and Cynopteri in two samples each (8.7 %), and Andamana and Bratislava in one sample each (4.34 %). Conclusions These results suggest the evidence of exposure toLeptospira spp. and the need of new studies to evaluate a higher number of capybaras from different regions to better understand the importance of leptospirosis infection in these animals and verify the zoonotic role of this species as a possible source of infection to humans and other animals

Detection of antibodies to Toxoplasma gondii in wild animals in Brazil

Background Toxoplasmosis is a worldwide zoonosis caused by an obligate intracellular protozoan parasite, Toxoplasma gondii, that affects all warm-blooded animals, including wild animals. The increased number of cases of parasitic infections is mainly due to the destruction of environmental conservation areas, which is driving wild animals out of their habitats and towards urban areas. In this study, the occurrence of T. gondii infection was investigated by the modified agglutination test (MAT) in 26 different species of run over and injured wild animals that were treated at a Brazilian university veterinary hospital, from June 2007 to August 2008. Findings Of the studied animals, six (23.1%; CI95% 11.1-42.2%) had T. gondii antibodies, with titers equal to 10 (4; 66.7%) and 40 (2; 33.3%). The species Pseudalopex vetulus, Cerdocyon thous, Hydrochoerus hydrochaeris and Tapyrus terrestris had titers of 10, while Alouatta caraya and Puma concolor had titers of 40. There was no significant association regarding age, gender or purpose of care (p > 0.05). Conclusions Carnivorous, herbivorous and omnivorous wild animals are potential sentinels of human toxoplasmosis, especially when wild felids are present, maintaining the environmental contamination

Serological Monitoring for <em>Leptospira</em> Spp. and Monitoring of Productive and Reproductive Indices on Dairy Farm

Leptospirosis is a zoonosis caused by spirochetes of the genus Leptospira. It has a worldwide distribution with greater occurrence in tropical and subtropical countries. It is endemic in Brazil. It affects domestic, wild and production animals. The goal of this study was to assess dairy herd productive and reproductive indexes on a monthly basis by serologically monitoring the infection dynamics on two experimental groups: one with animals with negative results at study onset (G-1) and another with animals tested positive for at least one leptospira serovar (G-2). The serum microscopic agglutination test (MAT) was employed. Animals with titer equal to or greater than 100 IU were considered reactive. Animals were evaluated for productive and reproductive indexes based on data provided by the dairy’s IT system. Blood was collected from all animals in both groups once a month for nine months. Analysis showed interference between animals seroreactive to leptospirosis and both milk production and number of pregnancies for G-2 at collection moments 3, 4, 5, 6, 7 and 9 whereas for G-1 the same indexes showed decrease only in the 5th and 9th study months. The most prevalent serovars were Hardjoprajitino 59.5%, Pyrogenes 21.04%, Pomona 11.07%, Wollfi 11.07%, Hardjo 8.78%, Guaricura 6.55%, Copenhageni 5.09%, Icterohaemorrhagiae 1.11%, and Ctg 0.83%. Serovar Hardjoprajitino showed a relationship with herd milk production decrease

Production and purification of an immunogenic G protein of rabies virus from S2 insect cells

Rabies virus is still a major public health concern in under developed countries of Africa, Asia and Latin America. WHO estimates that almost 55.000 people die from rabies annually. Although Rabies virus strains from dogs still are the main responsible of causing human disease, the increasing case numbers of bat virus strains being transmitted to humans in America raises concerns. The called secondary transmission cycle of rabies involves cats or dogs which are exposed to contaminated bats and then transmit it to humans. Combating this type of transmission requires new strategies for disease control, including new vaccines. Particularly for cats, the current rabies vaccines have demonstrated to cause vaccine-associated sarcoma as a serious side effect. New rabies vaccines using recombinant technology have been proposed using the Rabies Glycoprotein (G) as antigen. The expression of this molecule in S2 insect cells has already proved the ability of this cell line to produce an immunogenic protein. Although this antigen is recognized as the main protein necessary for protection upon immunization, its utilization as a recombinant vaccine has been impaired by difficulties in its production and mainly purification steps. The G protein was expressed as a membrane bound protein in Drosophila melanogaster S2 cells. This membrane G protein was further solubilized using a standardized solution containing detergent and purified in a novel methodology using affinity chromatography resulting in protein recoveries up to 96 %. The membrane protein and the purified G were used for mice immunization, using or not ISCOM adjuvant resulting in good levels of specific antibodies and neutralizing antibodies. When immunized mice were submitted to lethal rabies virus challenge, the preparations showed to confer protection levels similar to that of commercially available veterinary rabies vaccine. A flow path from the production, purification, formulation and immunization against Rabies using a recombinant G protein is presented as a promisor veterinary vaccine candidate