Serology, isolation, and molecular detection of Leptospira spp. from the tissues and blood of rats captured in a wild animal preservation centre in Brazil.

To elucidate the occurrence and epidemiology of leptospirosis in rats cohabitating with forest animals, 13 rats were captured at seven locations of the Centre for the Conservation of Wild Fauna (CCWF) in Sao Paulo state, Brazil, and samples of their blood, liver, and kidneys were collected. The diagnostic techniques utilized were the microscopic agglutination test (MAT), polymerase chain reaction (PCR) for Leptospira spp., and cultures of rat kidneys and liver in Fletcher's medium. The MAT results showed that 13 (100%) of the samples were reactive to 12 serovars among the 29 Leptospira spp. tested, and the Australis and Tarassovi serovars were the most frequently identified serovars. To research the agent in fragments of the liver and kidney, 13 samples from each tissue were cultured in Fletcher's medium, and the results revealed seven positive samples (53.8%; three from the kidneys and four from the livers). The analysis of the blood samples by PCR for Leptospira spp. showed that six animals (46.1%) were positive, whereas the analysis of the organs (kidneys and liver) by PCR revealed that nine animals (69.2%) were positive, and the culture of the organs revealed four positive animals (30.8%). These results suggest that the presence of Leptospira spp. infection in rats at the study site and the knowledge of the serovars that exist in this environment are important for the epidemiological comprehension of the disease and for the identification of control measures that should be considered to reduce the risk of transmission of the disease through this animal reservoir.

[Canine visceral leishmaniasis diagnosis by immunohistochemistry and PCR in skin tissues in association with IFAT and ELISA-test]. / Diagnóstico da Leishmaniose Visceral Canina pelas técnicas de imunoistoquímica e PCR em tecidos cutâneos em associação com a RIFI e ELISA-teste.

The purpose of the present study was to evaluate the immunohistochemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for Canine Visceral Leishmaniasis (CVL) diagnosis and compare the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitological test (microscopic direct examination of the parasite stained with haematoxylin and eosin--HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5%) but also healthy skins (31.8%) were positives by IMHC and confirmed by PCR in 97.8% of skin samples. In asymptomatic group, 87.5% dogs were negatives by serological tests, but positives by IMHC in 50% and by PCR in 100%. In oligosymptomatic group, 100%, 85.7% and 28.6% of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7% of polisymptomatic dogs were serum positive and had intact parasites in the skin. In general, PCR showed higher positivity (100%). The efficiency of each test varied with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR in inconclusive cases after HE and IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this canine disease.

Diagnóstico da Leishmaniose Visceral Canina pelas técnicas de imunoistoquímica e PCR em tecidos cutâneos em associação com a RIFI e ELISA-teste / Canine Visceral Leishmaniasis diagnosis by immunohistochemistry and PCR in skin tissues in association with RIFI and ELISA-test

O objetivo deste trabalho foi avaliar as técnicas de imunoistoquímica (IMIQ) e de PCR (Reação em Cadeia da Polimerase) em tecidos cutâneos para o diagnóstico da Leishmaniose Visceral Canina (LVC) e compará-los com os exames parasitológicos em tecidos corados histoquimicamente (hematoxilina-eosina, HE) e com testes sorológicos, como a Reação de Imunofluorescência Indireta (RIFI) e ensaio imunoenzimático (ELISA). Dos 34 cães naturalmente infectados, classificados em assintomáticos, oligossintomáticos e polissintomáticos, foram coletadas amostras de pele sadia ou com lesão para a realização da IMIQ, HE e PCR. Não somente peles lesionadas (56,5 por cento), mas também sadias (31,8 por cento) encontravam-se positivas pela IMIQ, confirmadas posteriormente pela PCR em 97,8 por cento das amostras. No grupo assintomático, 87,5 por cento estavam negativos pelos testes sorológicos, mas positivos em 50 por cento dos casos pela IMIQ e 100 por cento pela PCR. Entre os oligossintomáticos, 100 por cento, 85,7 por cento e 28,6 por cento encontravam-se positivos, respectivamente, pela PCR, sorologia e IMIQ. Os cães polissintomáticos eram 91,7 por cento soropositivos e tinham parasitas na pele. Em geral, a técnica PCR teve maior positividade (100 por cento). A eficiência dos testes variou de acordo com a evolução da doença, demonstrando a necessidade da associação de técnicas, usando-se IMIQ para confirmação da sorologia e a PCR apenas nos casos suspeitos após a IMIQ. Dessa forma, pode-se aumentar os níveis de positividade e contribuir para o controle desta zoonose.

The purpose of the present study was to evaluate the immunohistochemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for Canine Visceral Leishmaniasis (CVL) diagnosis and compare the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitological test (microscopic direct examination of the parasite stained with haematoxylin and eosin - HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5 percent) but also healthy skins (31.8 percent) were positives by IMHC and confirmed by PCR in 97.8 percent of skin samples. In asymptomatic group, 87.5 percent dogs were negatives by serological tests, but positives by IMHC in 50 percent and by PCR in 100 percent. In oligosymptomatic group, 100 percent, 85.7 percent and 28.6 percent of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7 percent of polisymptomatic dogs were serum positive and had intact parasites in the skin. In general, PCR showed higher positivity (100 percent). The efficiency of each test varied with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR in inconclusive cases after HE and IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this canine disease.

Diagnóstico da Leishmaniose Visceral Canina pelas técnicas de imunoistoquímica e PCR em tecidos cutâneos em associação com a RIFI e ELISA-teste / Canine Visceral Leishmaniasis diagnosis by immunohistochemistry and PCR

O objetivo deste trabalho foi avaliar as técnicas de imunoistoquímica (IMIQ) e de PCR (Reação em Cadeia da Polimerase) em tecidos cutâneos para o diagnóstico da Leishmaniose Visceral Canina (LVC) e compará-los com os exames parasitológicos em tecidos corados histoquimicamente (hematoxilina-eosina, HE) e com testes sorológicos, como a Reação de Imunofluorescência Indireta (RIFI) e ensaio imunoenzimático (ELISA). Dos 34 cães naturalmente infectados, classificados em assintomáticos, oligossintomáticos e polissintomáticos, foram coletadas amostras de pele sadia ou com lesão para a realização da IMIQ, HE e PCR. Não somente peles lesionadas (56,5%), mas também sadias (31,8%) encontravam-se positivas pela IMIQ, confirmadas posteriormente pela PCR em 97,8% das amostras. Nogrupo assintomático, 87,5% estavam negativos pelos testes sorológicos, mas positivos em 50% dos casos pela IMIQ e 100% pela PCR. Entre os oligossintomáticos, 100%, 85,7% e 28,6% encontravam-se positivos, respectivamente, pela PCR, sorologia e IMIQ. Os cães polissintomáticos eram 91,7% soropositivos e tinham parasitas na pele. Em geral, a técnica PCR teve maior positividade (100%). A eficiência dos testes variou de acordo com a evolução da doença, demonstrando a necessidade da associação de técnicas, usando-se IMIQ para confirmação da sorologia e a PCR apenas nos casos suspeitos apos a IMIQ. Dessa forma, pode-se aumentar os níveis de positividade e contribuir para o controle desta zoonose.

The purpose of the present study was to evaluate the immunohistochemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for Canine Visceral Leishmaniasis (CVL) diagnosis and compare the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitological test (microscopic direct examination of the parasite stained with haematoxylin and eosin - HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5%) but also healthy skins (31.8%) were positives by IMHC and confirmed by PCR in 97.8% of skin samples. In asymptomatic group, 87.5% dogs were negatives by serological tests, but positivesby IMHC in 50% and by PCR in 100%. In oligosymptomatic group, 100%, 85.7% and 28.6% of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7% of polisymptomatic dogs were serum positive and had intact parasites in the skin. In general, PCR showed higher positivity (100%). The efficiency of each test varied with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR in inconclusive cases after HE and IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this canine disease.

Eimeria spp. in Brazilian water buffalo.

Eimeria species are frequently found in water buffalo (Bubalus bubalis) in Brazil. Here, we report those Eimeria spp. that infect buffalos during their first year of life. Fresh fecal samples were examined from 2 groups (1 group/yr for 2 yr, 2000-2002), each with 18 water buffalo calves (both sexes), from birth through 12 mo of age, in Selvíria, MS, Brazil. Five oocyst morphotypes were observed, i.e., Eimeria ellipsoidalis and Eimeria zuernii, both previously described from water buffalo, and 3 other morphotypes consistent with descriptions of known Eimeria spp. from Artiodactyla hosts, but originally described from other genera than those in which we found them (referred to here as Eimeria species 1-3). Our results showed that buffalo calves started shedding oocysts in their feces between 6-29 days of age, with the highest concentration ranging from 188-292 oocysts/g of feces. The 3 unnamed oocyst morphotypes in the calf feces resembled E. auburnensis (Eimeria sp. 3), E. cylindrica (Eimeria sp. 1), and E. subspherica (Eimeria sp. 2). The most prevalent species were Eimeria sp. 1 and E. ellipsoidalis, which dominated in the youngest animals (6 to 133 days old). Eimeria zuernii oocysts, in contrast, were found only in low numbers in the feces of older calves (208 to 283 days old). Calves were infected more frequently during the rainy season (September to January) in both years, but cows were negative for Eimeria spp., whenever feces were collected (spring, winter, autumn, or summer seasons).

[Immune-humoral response of water buffalo (Bubalus bubalis) against Anaplasma marginale (Theiler, 1910)]. / Resposta imune-humoral de búfalos (Bubalus bubalus) contra Anaplasma marginale (Theiler, 1910).

The aim of the present study was to analyze the humoral-immune response of water buffalo (Bubalus bubalis) naturally infected against Anaplasma marginale. For this work, colostrums/milk and blood samples were sequentially collected from buffalo cows prior and after partum for a period of 335 days and from buffalo calves from birth to 365 days after. The antibodies in the colostrums/milk and serum samples of these animals were determined using an ELISA indirect method and the data were analyzed as a mean of a group of animals with the matched ages during the period of 1999/2000 or individually during the year of 2005. The data from animals analyzed in group showed that the antibodies against A. marginale were in low concentration (below the cut off point: D.O. = 0.265 and ELISA levels, EL < or =3), in the sera of buffalo, during the first 90 and 105 days, respectively for cows and calves. Then, the levels of antibodies in the serum samples of buffalo calves, slightly raised to above the cut-off point and kept in higher levels up to approximately 365 days after birth, indicating active acquired immunity. Furthermore, when the animals were individually examined, the buffalo cows showed high antibody levels in the colostrums, but low levels in the blood stream during the first seven days post-partum, suggesting antibody transference from blood to mammary gland. In addition to that, buffalo calves showed high antibody levels during the first 24 hours after suckling colostrum, indicating a colostral passive immunity. By conclusion, the buffaloes were able to arm a humoral immune response against A. marginale and were considered reservoir of this parasite.

Resposta imune-humoral de búfalos (Bubalus bubalis) contra Anaplasma marginale (Theiler, 1910) / Immune-humoral response of water buffalo (Bubalus bubalis) against Anaplasma marginale (Theiler, 1910)

O objetivo do presente estudo foi analisar a resposta imune humoral anti-Anaplasma marginale em búfalos (Bubalus bubalis) naturalmente infectados. Amostras de soro e de colostro/leite coletadas de búfalas adultas por um período de 335 dias após o parto e de soros dos seus bezerros do nascimento até 365 dias de vida, foram usadas para a realização do método ELISA indireto. Os dados foram analisados como a média de um grupo de animais, em diferentes faixas etárias, durantes os anos de 1999/2000 e individualmente (duas búfalas e dois bezerros), no ano de 2005. Os soros dos animais analisados em grupos apresentaram títulos de anticorpos abaixo do ponto de corte (D.O. = 0,265 e NE > 3) durante os primeiros 90 e 105 dias, para as búfalas e para os seus bezerros, respectivamente, mas em seguida, elevaram-se para níveis acima do ponto de corte até o final do trabalho, ou seja, um ano após, indicando uma imunidade ativa adquirida. Das duas búfalas examinadas individualmente, os anticorpos colostrais foram detectados em altos níveis, mas os sorológicos em baixos níveis durante os primeiros sete dias após o parto, sugerindo uma transferência de anticorpos do soro para a glândula mamária. Da mesma forma, os dois bezerros tiveram títulos de anticorpos detectáveis já nas primeiras 24 horas após mamarem o colostro, indicando uma imunidade colostral passiva. Em conclusão, os búfalos desenvolveram uma resposta imune humoral específica contra A. marginale e foram considerados portadores deste parasita.

The aim of the present study was to analyze the humoral-immune response of water buffalo (Bubalus bubalis) naturally infected against Anaplasma marginale. For this work, colostrums/milk and blood samples were sequentially collected from buffalo cows prior and after partum for a period of 335 days and from buffalo calves from birth to 365 days after. The antibodies in the colostrums/milk and serum samples of these animals were determined using an ELISA indirect method and the data were analyzed as a mean of a group of animals with the matched ages during the period of 1999/2000 or individually during the year of 2005. The data from animals analyzed in group showed that the antibodies against A. marginale were in low concentration (below the cut off point: D.O. = 0.265 and ELISA levels, EL > 3), in the sera of buffalo, during the first 90 and 105 days, respectively for cows and calves. Then, the levels of antibodies in the serum samples of buffalo calves, slightly raised to above the cut off point and kept in higher levels up to approximately 365 days after birth, indicating active acquired immunity. Furthermore, when the animals were individually examined, the buffalo cows showed high antibody levels in the colostrums, but low levels in the blood stream during the first seven days post-partum, suggesting antibody transference from blood to mammary gland n addition to that, buffalo calves showed high antibody levels during the first 24 hours after suckling colostrum, indicating a colostral passive immunity. By conclusion, the buffalos were able to arm a humoral immune response against A. marginale and were considered reservoir of this parasite.

Detection of antibody to Toxocara vitulorum perieneteric fluid antigens (Pe) in the colostrum and serum of buffalo calves and cows by Western blotting.

Toxocara vitulorum, a nematode parasite in the small intestine of cattle and water buffaloes, causes high morbidity and mortality of 1-3 months old buffalo calves. This research evaluated the specific perieneteric antigens (Pe) reactivity of anti-T. vitulorum-Pe antibody (Tv-Pe-Ab) in both immune sera and colostrum from buffalo cows immediately post-partum from buffalo cows. The presence of Tv-Pe-Ab in sera of buffalo newborn calves was also examined at 1 day before and after suckling the colostrum as well as in sera from naturally infected calves at the beginning and peak of the maximum infection and then again during the period of rejection and post-rejection of the parasite. Pe antigens were characterized for Tv-Pe-Ab by SDS-PAGE and Western blot (WB). The SDS-PAGE showed that Pe contained nine protein bands (11, 14, 31, 38, 58, 76, 88, 112 and 165 kDa). All Pe bands were recognized by Tv-Pe-Ab in sera and colostrum of buffalo cows. Only the serum antibodies of buffalo calves at 1 day of age after suckling the colostrum and during the beginning of T. vitulorum infection recognized Pe antigen's nine bands. In contrast, serum antibodies from 1-day-old buffalo calves, taken before suckling colostrum, did not react with any protein band. In suckling calves, which reached peak egg output, rejection and post-rejection stages of the infection, serum Tv-Pe-Ab reactivity with lower molecular weight protein bands (11-76 kDa) was lost and only reactivity with the Pe protein bands of higher molecular weight (88, 112 and 165 kDa) remained.

[Populational alteration of cells in the intestinal intraepithelial layer and morphological changes of the intestinal wall elicited by Toxocara vitulorum infection in buffalo calves (Bubalus bubalis)]. / Alteração populacional de células na camada intraepitelial e alterações morfológicas da parede intestinal causada pela infecção por Toxocara vitulorum en bezerros Búfalos (Bubalus Bubalis).

To understand the development of the inflammatory responses in the wall of the gut, during the process of expulsion of the parasites from the host, samples of tissues were removed from the small intestines from four groups of naturally infected buffalo calves with Toxocara vitulorum during the beginning of the infection, at the peak of egg output, during the period of expulsion and post-expulsion of the worms, as well as from uninfected calves. Cells (mast cells, eosinophils, intraepithelial lymphocytes - IEL and goblet cells) present in the epithelial layer (intraepithelial) of the small intestine were counted. In the duodenum, jejunum and ileum, the population of mast cells, eosinophils and lymphocytes increased significantly during the peak of the infection. Goblet cell numbers increased also during the beginning and at the peak of the infection. The decline of the number of these cells occurred during the periods of expulsion of the worms reaching to uninfected control counts at the post-expulsion period indicating a role of these cells in the process of expulsion of T. vitulorum by the buffalo calves. The layers of the intestinal wall (villus, crypt, submucosa and muscular) were also measured. Morphological examinations showed a significant vilar atrophy, particularly in the duodenum during the beginning, peak and during the period of expulsion of the worms, but smooth muscle hypertrophy or other alteration was not observed in any period of the infection.

Characterization of excretory/secretory antigen from Toxocara vitulorum larvae.

Toxocara vitulorum is a nematode parasite of the small intestine of cattle and water buffalo, particularly buffalo calves between one and three months of age, causing high morbidity and mortality. The purpose of this research was to characterize the excretory/secretory (ES) antigens of T. vitulorum larvae by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot (WB), using immune sera and colostrum of buffalo naturally infected by T. vitulorum. The parasitological status of the buffalo calves was also evaluated using sequential fecal examinations. The results showed that the ES antigen revealed eight (190, 150, 110, 90, 64, 56, 48, and 19 kDa) protein bands by SDS-PAGE. The majority of these bands were recognized in the sera and colostrum of infected buffalo with T. vitulorum when analyzed by WB. However, particularly fractions of high molecular weight (190, 150, 110, and 90 kDa) were represented in more prominent bands and persisted in the groups of buffalo calves at the peak of egg output, as well as during the period of rejection of T. vitulorum by the feces of the calves. During the period of post-rejection of the worms (between the day 118 and 210 of age) the serum antibodies did not react with any protein bands. On the other hand, sera from buffalo calves at one day of age (after suckling the colostrum and at the beginning of infection) reacted with the same bands detected in the serum and colostrum of the buffalo cows.

Mast cell and eosinophils in the wall of the gut and eosinophils in the blood stream during Toxocara vitulorum infection of the water buffalo calves (Bubalus bubalis).

Toxocara vitulorum is a pathogenic nematode from the small intestine of very young buffalo calves. To understand the development of the inflammatory responses in the wall of the gut, samples of tissues were removed from the duodenum, jejunum and ileum of buffalo calves naturally infected with T. vitulorum during the beginning of the infection, at the peak of egg output, as well as during the periods of rejection of the worms and post-rejection. Two additional control groups of uninfected calves (by anti-helminthic therapy of their mothers and after the birth) were also necropsied on days 30 and 50 after birth. Blood samples were fortnightly collected from birth to 174 days post-birth. Blood smears were prepared and stained with Giemsa for eosinophils. The parasitological status of buffalo calves was evaluated through weekly fecal egg counts (EPG) from 1 to 106 days after birth, which revealed that T. vitulorum egg shedding started on day 11, reached the peak of the infection on day 49 and finally expelled the parasites between days 50 and 85 after birth. In the infected buffalo calves, the mast cell population increased significantly, by two-fold in the mucosa (villus-crypt unit (VCU)) of the duodenum and four-fold in the proximal jejunum; but these increases were statistically significant only at the peak of the infection. Although mast cell numbers increased in the mucosa of the ileum as well as in both the submucosal and muscle tissues of the duodenum, proximal jejunum and ileum, the data was not significantly different from the controls. Eosinophil numbers increased in the mucosa of the duodenum (two-five times higher than the control) and proximal jejunum (three-five-fold) during the period of the infection (beginning, peak and rejection). The relative numbers of eosinophils increased in the blood stream from the second to the seventh week. In conclusion, T. vitulorum infection elicited mastocytosis and tissue eosinophilia in the duodenum and proximal jejunum, as well as eosinophilia in the blood stream, during the beginning, at the peak and during the rejection of the worm. After the rejection of the worms, the numbers of these cells returned to normal levels suggesting that these cells may have a role in the process of rejection of T. vitulorum by the host.