Prevalence of Endoparasites in Urban Stray Dogs from Brazil Diagnosed with Leishmania, with Potential for Human Zoonoses.

BACKGROUND: In tropical environments, abandoned animals can be an important source for human zoonotic infections, such as human visceral leishmaniasis and other vector-borne diseases. Here, we report the frequency of protozoan and helminth intestinal parasites in stray dogs, which might have an implication for human health in urban Brazilian settings. MATERIAL AND METHODS: We performed necropsies on 93 animals, euthanized due to canine visceral leishmaniasis control program, and examined their intestines looking for the presence of helminths; we determined the parasite load, and the elimination of eggs and cysts of protozoan parasites in fecal samples. Further, we performed serology tests for the detection of specific antibodies against Toxoplasma gondii. RESULTS: Overall, a high prevalence of intestinal parasites with potential for human zoonoses resulted and only 8.6% of examined intestines remained negative. The most prevalent helminths were Ancylostoma caninum and Dipylidium caninum. For nematodes, high worm burdens were detected for A. caninum and Trichuris vulpis. Additionally, we analyzed worm burdens and quantitative stool examinations, but found no significant association between positive serology for Leishmania infection and intestinal parasite burden. Interestingly, serology for T. gondii infection revealed a prevalence of 33.3% and a positive result was significantly associated with a higher A. caninum adult worm recovery (p = 0.0087). CONCLUSION: Our results showed stray dogs living in urban areas are heavily parasitized, which presents a potential risk for humans. Beyond the control of canine visceral leishmaniasis, we propose an improvement of the control program to reduce the risk for other parasitic diseases in dogs and humans.

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii.

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4days of infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.

Risk factors for infection by Toxoplasma gondii in herds of goats in Ceará, Brazil / Fatores de risco para infecção pelo Toxoplasma gondii em rebanhos de caprinos no Ceará, Brasil

In order to identify possible risk factors for T. gondii infection in goat herds in Ceará, Brazil, 2362 serum samples were tested by ELISA. The serological prevalence was 25.1 percent. The risk factors identified for Toxoplasma gondii infection in goat herds were age, number of cats, use of wooden feeding troughs and absence of feeding troughs. Goats older than 37 months had 2.01 (CI 95 percent; 1.55 - 2.61) higher risk of infection than younger animals. Greater risk of infection was observed in farms with more than 10 cats (OR = 1.73; CI 95 percent; 1.01 - 3.33). The use of wooden feeding troughs represented a high probability of infection (OR = 7.81; CI 95 percent; 1.66 - 36.67). The lack of feeding troughs also represented a high probability of infection (OR = 5.50; CI 95 percent; 1.24 - 24.39).

Com o objetivo de se identificarem os fatores de risco associados à presença de infecção por toxoplasmose em rebanhos caprinos no estado do Ceará, Brasil, soros sanguíneos de 2362 caprinos foram testados por meio de ELISA. A prevalência estimada pelo ELISA foi 25,1 por cento. Os fatores de risco identificados nas propriedades foram: idade dos animais, número de gatos, comedouro manufaturado de madeira e ausência de comedouro. Em caprinos com mais de 37 meses de idade o risco de estarem infectados pelo T. gondii foi 2,01 vezes maior (IC 95 por cento; 1,55 - 2,61) que em animais mais jovens. Maior risco de infecção foi observado em fazendas com mais de 10 gatos (OR = 1,73; IC 95 por cento 1,01 - 3,33). Quando a propriedade utilizava comedouros de madeira, o risco de estarem infectados foi também maior (OR = 7.81; IC 95 por cento; 1.66 - 36.67). Animais oriundos de propriedades sem comedouro também apresentaram alto risco de infecção (OR = 5.50; IC 95 por cento; 1,24 - 24,39).

Biological and molecular characterization of Besnoitia akodoni n.sp. (Protozoa: Apicomplexa) from the rodent Akodon montensis in Brazil.

The diversity among coccidian parasites of the genus Besnoitia is incompletely known. Of the eight currently described members of the genus, only B. jellisoni is known to parasitize a rodent host. Here, we propose a new name, Besnoitia akodoni, for the species initially isolated form the rodent Akodon montensis in Brazil. The tissue cysts of B. akodoni were up to 442 microm in diameter and bradyzoites were 8.4 x 1.4 microm in size. The bradyzoites contained enigmatic bodies, micronemes and rhoptries. Tachyzoites were 5.8 x 1.5 microm in size and they could be grown in vitro in bovine monocytes and African Green monkey cells where they divided by endodyogeny. Besnoitia akodoni was infective to laboratory-raised mice (Mus musculus) and gerbils (Meriones unguiculatus) but not to cats (Felis catus). Comparison of the conserved sequences of the small subunit rDNA clearly established the close relationship of B. akodoni with other members of the genus. However, sequences of the more variable first internal transcribed spacer portion of the ribosomal DNA repeat support its differentiation from the other species of the genus.

Activity of natural and synthetic naphthoquinones against Toxoplasma gondii, in vitro and in murine models of infection.

The effect of 14 natural and synthetic naphthoquinones in the replication of Toxoplasma gondii was evaluated. In vitro studies were accomplished in cultures of 2C4 fibroblasts infected with RH-strain. Enzyme-linked immunosorbent assay was used to quantify parasite growth. For the studies in vivo, mice were infected with tachyzoites of the RH strain or cysts of the T. gondii EGS strain. In vitro, seven naphthoquinones demonstrated significant inhibition of intracellular T. gondii growth at concentrations of 1 and 5 micrograms/ml. Only three compounds were significantly protective when tested in animals: 2-hydroxy-3'-(3'-pentenyl)-1,4-naphthoquinone (PHNQ4), 2-hydroxy-3-(1'-vinylphenyl)-1,4-naphthoquinone (PHNQ5), and 2-hydroxy-3-(1'-propen-3'-phenyl)-1,4-naphthoquinone (PHNQ6). In animals infected with the EGS strain and treated with PHNQ4 (50 mg/kg/day orally), a 7-day prolongation of the time to death was observed. Treatment with 100 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ5 resulted in a 5-day and 16-day prolongation of the time to death, respectively. Treatment with 50 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ6 resulted in a 4-day prolongation of the time to death or up to 30 days after treatment, respectively. Our results suggest that the naphthoquinones may be important therapeutic agents for the treatment of toxoplasmosis.

Experimental toxoplasmosis in Balb/c mice: prevention of vertical disease transmission by treatment and reproductive failure in chronic infection

In a study of congenital transmission during acute infection of Toxoplasma gondii, 23 pregnant Balb/c mice were inoculated orally with two cysts each of the P strain. Eight mice were inoculated 6-11 days after becoming pregnant (Group 1). Eight mice inoculated on the 10th-15th day of pregnancy (Group 2) were treated with 100 mg/kg/day of minocycline 48 h after inoculation. Seven mice inoculated on the 10th-15th day of pregnancy were not treated and served as a control (Group 3). Congenital transmission was evaluated through direct examination of the brains of the pups or by bioassay and serologic tests. Congenital transmission was observed in 20 (60.6 per cent) of the 33 pups of Group 1, in one (3.6 per cent) of the 28 pups of Group 2, and in 13 (54.2 per cent) of the 24 pups of Group 3. Forty-nine Balb/c mice were examined in the study of congenital transmission of T. gondii during chronic infection. The females showed reproductive problems during this phase of infection. It was observed accentuated hypertrophy of the endometrium and myometrium. Only two of the females gave birth. Our results demonstrate that Balb/c mice with acute toxoplasmosis can be used as a model for studies of congenital T. gondii infection. Our observations indicate the potential of this model for testing new chemotherapeutic agents against congenital toxoplasmosis.

Infecçäo experimental de caprinos com Strongyloides papillosus / Experimental strongyloidiasis in goats

Acompanhou-se a evoluçäo da infecçäo por Strongyloides papillosus em 14 caprinos sem raça definida, de três a seis meses de idade, inoculados com 3.000 larvas infectantes por kg de peso vivo. Os resultados mostraram que a suscetibilidade ao parasita näo está relacionada com a idade do animal, mas que o período de eliminaçäo de ovos de S. papillosus foi mais longo nos animais mais jovens. A necrópsia foram observadas alteraçöes histopatológicas que caracterizam enterite catarral aguda com pronunciada exulceraçäo simples da mucosa. A pesquisa de anticorpos da classe IgC, pelo ELISA, mostrou que esses anticorpos foram detectados a partir do 20§ dia após a inoculaçäo (DAI) até o 200§ DAI, quando os animais foram necropsiados. Os níveis máximos de anticorpos (título variando entre 1:5.120 a 1:40.960) foram observados entre o 30§ e o 130§ DAI

Toxoplasma gondii em caprinos inoculados experimentalmente / Toxoplasma gondii in tissues of experimentally infected goats

Toxoplasma gondii foi demonstrado em órgäos de 10 entre 12 caprinos inoculados pela via subcutânea com 10 elevado à sétima potência taquizoítos de uma cepa de virulência moderada do T. gondii (C4). O parasito näo foi demonstrado em órgäos de duas cabras näo gestantes que já apresentavam anticorpos anti-T. gondii antes do inóculo, mas foi evidenciado em uma cabra gestante que apresentava estes anticorpos previamente. Os órgäos mais frequentemente parasitados foram cérebro, coraçäo, músculo esquelético e diafragma. Um animal sacrificado no 60§ dia após a inoculaçäo (DAI) apresentou 91 por cento dos órgäos positivos, enquanto outro sacrificado no 561§ DAI, apresentou 17 por cento dos órgäos positivos. A presença do parasita em órgäos de caprinos com 505 a 561 dias de infecçäo indica a importância da carcaça destes animais na transmissäo da toxoplasmose devido ao costume comum de consumo desta carne no Brasil e em outras partes do mundo

Diagnosis of caprine toxoplasmosis by a dot enzyme-linked immunosorbent assay

Foi avaliada a reaçäo imunoenzimática dot-ELISA, no imunodiagnóstico da toxoplasmose caprina, considerando a reaçäo de Imunofluorescência Indireta (RIFI) como reaçäo de referência. Para a avaliaçäo da dot-ELISA discos de nitrocelulose foram sensibilizados com 0,1 microgramas de proteína/ml de uma soluçäo antigênica obtida através da sonicaçäo de taquizoitas de Toxoplasma gondii. Esta concentraçäo antigênica foi considerada ótima na discriminaçäo de soros reagentes e näo reagentes. Foram testadas 169 amostras de soro, destas 115 (68 por cento) foram reagentes a antígenos de T. gondii, com títulos de anticorpos variando entre 1/16 e 1/16:384, quando testadas através da reaçäo de Imunofluorescência Indireta (RIFI); na dot-ELISA, 119 (70 por cento) foram positivas, com título variando entre 1/16 e 1/65.536. A comparaçäo entre as técnicas mostra os seguintes índices: co-positividade (sensibilidade) 0,89, co-negatividade (especificidade) 0,98, e Youden (concordância) 0,93, quando foi utilizado o título de 1/16 como discriminatório de reaçöes positivas e negativas. Utilizando como discriminatório o título de 1/64, os índices calculados foram co-positividade 0,97, co-negatividade 0,96 e Youden 0,93. Os resultados mostram a possibilidade da utilizaçäo da dot-ELISA no imunodiagnóstico da toxoplasmose caprina. A introduçäo desta reaçäo no imunodiagnóstico da toxoplasmose seria de grande importância, principalmente em estudos soro-epidemiológicos, já que a técnica pode ser realizada sob condiçöes de campo, pois nenhum equipamento especial é requerido para a sua execuçäo

Toxoplasmose experimental em cabras gestantes / Experimental toxoplasmosis in pregnant goats

Onze cabras gestantes sem raça definida foram inoculadas pela via subcutânea com 10 elevado à sétima potência taquizoítos de uma cepa de virulência moderada do Toxoplasma gondii (CA). Duas cabras gestantes foram usadas como controle näo inoculados. Foi observado transmissäo congênita do T. gondii em sete das oito cabras inoculadas entre o 52§ e o 67§ dia de gestaçäo (cinco abortos e dois partos normais com filhotes debilitados). Três cabras inoculadas entre o 100§ e o 133§ dia de gestaçäo pariram crias aparentemente normais, apesar da ocorrência de transmissäo congênita em um dos animais. Foi possível ainda evidenciar a transmissäo congênita do parasita em uma entre quatro cabras avaliadas em duas gestaçöes sucessivas. As cabras controle pariram crias aparentemente normais, não infectadas pelo T. gondii