Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati.

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15­120 min), and different concentrations of malachite green dye (0.004­0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10­100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples.

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

Development of a copro-LAMP assay for detection of several species of Echinococcus granulosus sensu lato complex.

Cystic echinococcosis represents a significant problem in human and animal health and constitutes one of the most severe Neglected Tropical Diseases prioritized by the World Health Organization. The etiological agent is the complex Echinococcus granulosus sensu lato (s. l.), composed of several species/genotypes. Diagnosis in the definitive host and molecular epidemiology studies are important points for cystic echinococcosis control. Here we developed a new copro-LAMP assay, LAMP EGSL, for diagnosis in the definitive host for simultaneous detection of Echinococcus granulosus sensu stricto (s. s.), Echinococcus ortleppi, and Echinococcus canadensis species. Also, the analytical sensitivity, specificity and plausibility of performance in a rural context of a previously reported species-specific LAMP reaction, was evaluated. Both reactions showed high analytical sensitivity values (10 fg-100 fg DNA) and did not show cross reaction with DNA from host or other helminthic parasites. LAMP EGSL was performed with samples from an endemic area. In addition, the alkaline hydrolysis of one E. granulosus s. s. adult parasite followed by specific LAMP to E. granulosus s. s. was performed in a laboratory with low resources from another cystic echinococcosis endemic area. The results obtained suggest that LAMP EGSL represents a potential tool for canine diagnosis that could be useful for cystic echinococcosis control programs. In addition, we showed that LAMP reaction for E. granulous s. s., E. ortleppi and E. canadensis specific detection, could be useful for molecular epidemiology studies applicable to the definitive host. Both reactions were performed in endemic, rural areas without sophisticated equipment.

Identification and characterization of the major pseudocoelomic proteins of the giant kidney worm, Dioctophyme renale.

BACKGROUND: The giant kidney worm, Dioctophyme renale, is a debilitating and potentially lethal parasite that inhabits and destroys, typically host's right kidney, and may also be found in ectopic sites. It is circumglobally distributed, mainly in dogs, and is increasingly regarded as a threat to other domestic animals and humans. There is little information on the parasite's true incidence, or immune responses to it, and none on its biochemistry and molecular biology. RESULTS: We characterised the soluble proteins of body wall, intestine, gonads and pseudocelomic fluid (PCF) of adult parasites. Two proteins, P17 and P44, dominate the PCF of both male and females. P17 is of 16,622 Da by mass spectrometry, and accounts for the intense red colour of the adult parasites. It may function to carry or scavenge oxygen and be related to the 'nemoglobins' found in other nematode clades. P44 is of 44,460 Da and was found to associate with fatty acids by thin layer chromatography. Using environment-sensitive fluorescent lipid probes, P44 proved to be a hydrophobic ligand-binding protein with a binding site that is highly apolar, and competitive displacement experiments showed that P44 binds fatty acids. It may therefore have a role in distributing lipids within the parasites and, if also secreted, might influence local inflammatory and tissue responses. N-terminal and internal peptide amino-acid sequences of P44 indicate a relationship with a cysteine- and histidine-rich protein of unknown function from Trichinella spiralis. CONCLUSIONS: The dominant proteins of D. renale PCF are, like those of large ascaridids, likely to be involved in lipid and oxygen handling, although there is evidence of strong divergence between the two groups.

Occurrence of dioctophymosis in canines within a riparian zone of the Río de La Plata watercourse, in Ensenada, Buenos Aires Province, Argentina.

Dioctophymosis is a parasitic disease occasioned by the so-called "giant kidney worm", Dioctophyme renale, a nematode with an indirect life cycle. This parasite's definitive host is the mink, Mustela vison, though numerous wild and domestic mammals as well as man can serve as final hosts. The worms also can be in ectopic locations in the body. We surveyed 692 canines by ecography, urine sampling, surgery, necropsy, and clinical examination and diagnosed 244 cases of dioctophymosis (35.3%). Of the cases of dioctophymosis identified, 30.7% were obtained by ecography, 45.9% by urinalysis, and 17.6% by both those techniques -in addition to positive findings through surgery (2.5%), necropsy (2.5%), and the spontaneous elimination of the parasites (0.8%). Cases of dioctophymosis were observed in animals as young as 4months of age up to 15years. The frequency of D. renale diagnosis throughout the sampling period varied significantly. There was a statistically significant association between risk factors (swimming in the river, eating frogs, fish or eels, drinking ditch water) and the prevalence of infection. It was discussed the period missing after infection in canines.

Toxocara canis en humanos, perros y suelos en ribera del Río de la Plata, provincia de Buenos Aires / Toxocara canis in humans, dogs and soil on the river side of Rio de la Plata, province of Buenos Aires / Toxocara canis em humanos, cães e solos nas margens do Rio da Prata, província de Buenos Aires

El objetivo de este trabajo fue describir la situación epidemiológica de toxocariosis en un hábitat ribereño. Se determinaron anticuerpos anti-toxocara por ELISA en sueros de 34 niños y 64 adultos, y se hallaron seroprevalencias de 32,3% y 45,3%, respectivamente. Esta fue alta en adultos y en niños de 2 a 3 años. Se realizaron 217 análisis coproparasitológicos de caninos y 23,04% fueron positivos para huevos de Toxocara canis. La distribución de caninos positivos por rango etario fue de 66% entre 1 y 6 meses; de 20,7% entre 6 y 12 meses; y de 10,3% en mayores de 12 meses. El porcentaje de animales parasitados por T. canis fue significativamente menor en relación a otros parásitos y disminuyó marcadamente con el aumento de la edad. Se analizaron 104 muestras de suelo y 1,92% de las mismas fueron positivas para huevos de T. canis. La escasa cantidad de huevos en suelos podría deberse a que los cachorros no se encontraban libres en los espacios públicos. En este barrio podría inferirse que el suelo no actuó como diseminador de esta parasitosis, sino que fueron de mayor relevancia factores como la tenencia de caninos menores de 1 año, el contacto estrecho con los mismos en ámbitos domiciliarios y las condiciones higiénico-sanitarias poco saludables.

The aim of this study was to describe the epidemiological situation of toxocariasis in a coastal habitat. Blood samples of 34 children and 64 adults were analyzed in order to determinate antibodies anti-toxocara by ELISA method. Prevalences of 32.3% and 45.3% were obtained respectively. A total of 217 coproparasitological canine analyses were performed, and 104 soil samples were analyzed. In humans, the seroprevalence of 32.4% in children and 45.3% in adults was found. In dogs, 50 samples were positive for T. canis eggs (23.04%). The distribution of canine positive for each age range yielded the following results: from 1 to 6 months, 66%; from 6 to 12 months, 20.7% and over 12 months, 10.3%. In soils, only 2 samples (1.92%) were positive for T. canis eggs. Seroloprevalence in humans was high, especially in adults and children aged 2 to 3 years. The percentage of parasitized animals by Toxocara was significantly lower in relation to other canine parasites and the percentage of dogs parasitized diminished significantly by increasing the age range. The low number of eggs found in soils could be due to the absence of puppies in public spaces. In this neighborhood, it can be inferred that the ground of public spaces did not act as a disseminator of this parasitosis, but that there were more relevant factors such as the possession of canines under 1 year of age, the close contact with them in residential areas, and unhealthy sanitary conditions.

O objetivo deste trabalho foi descrever a situação epidemiológica da toxocaríase em um habitat ripícola. Os anticorpos anti-toxocara foram determinados por ELISA em soros de 34 crianças e 64 adultos, com soroprevalência de 32,3% e 45,3%, respectivamente. Ela foi alta em adultos e em crianças de 2 a 3 anos de idade. Foram realizadas 217 análises coproparasitológicas caninas e 23,04% foram positivas para os ovos de Toxocara canis. A distribuição de caninos positivos por faixa etária foi de 66%, entre 1 e 6 meses, de 20,7%, entre 6 e 12 meses, e de 10,3% em maiores de 12 meses. A porcentagem de animais parasitados por T. canis foi significativamente menor em relação a outras parasitas e diminuiu marcadamente com o aumento da idade. Foram analisadas 104 amostras de solo e 1,92% delas foram positivas para ovos de T. canis. A escassa quantidade de ovos nos solos pode ser o resultado do fato de que os filhotes não estivessem livres nos espaços públicos. Poderia ser inferido que, nesse bairro, o solo não atuou como disseminador dessa parasitose, mas fatores como a posse de caninos menores de 1 ano de idade, um contato próximo com eles em ambientes domésticos e condições higiênico-sanitárias insalubres foram fatores de maior relevância.