Immunodiagnosis of cystic echinococcosis in livestock: Development and validation dataset of an ELISA test using a recombinant B8/2 subunit of Echinococcus granulosus sensu lato.

The accuracy of screening tests for detecting cystic echinococcosis (CE) in livestock depends on characteristics of the host-parasite interaction and the extent of serological cross-reactivity with other taeniid species. The AgB8 kDa protein is considered to be the most specific native or recombinant antigen for immunodiagnosis of ovine CE. A particular DNA fragment coding for rAgB8/2 was identified, that provides evidence of specific reaction in the serodiagnosis of metacestode infection. We developed and validated an IgG Enzyme Linked Immunosorbent Assay (ELISA) test using a recombinant antigen B sub-unit EgAgB8/2 (rAgB8/2) of Echinoccocus granulosus sensu lato (s.l.) to estimate CE prevalence in sheep. A 273 bp DNA fragment coding for rAgB8/2 was expressed as a fusion protein (∼30 kDa) and purified by affinity chromatography. Evaluation of the analytical and diagnostic performance of the ELISA followed the World Organisation for Animal Health (OIE) manual, including implementation of serum panels from: uninfected lambs (n = 79); experimentally infected (with 2,000 E. granulosus s.l. eggs each) sheep with subsequent evidence of E. granulosus cysts by necropsy (n = 36), and animals carrying other metacestode/trematode infections (n = 20). The latter were used to assess the cross-reactivity of rAgB8/2, with these animals being naturally infected with Taenia hydatigena, Thysanosoma actinioides and/or Fasciola hepatica. EgAgB8/2 showed cross-reaction with only one serum sample from a sheep infected with Ta. hydatigena out of the 20 animals tested. Furthermore, the kinetics of the humoral response over time in five 6-month old sheep, each experimentally infected with 2,000 E. granulosus s.l. eggs, was evaluated up to 49 weeks (approximately one year) post infection (n = 5). The earliest detectable IgG response against rAgB8/2 was observed in sera from two and four sheep, 7 and 14 days after experimental infection, respectively. The highest immune response across all five animals was found 16 to 24 weeks post infection.

First report of cystic echinococcosis caused by Echinococcus granulosus sensu stricto/G1 in Felis catus from the Patagonian region of Argentina.

Two domestic cats from the Patagonia rural area in Argentina were found to be naturally infected with Echinococcus granulosus sensu stricto/G1 genotype; so far, the only species/genotype of E. granulosus sensu lato complex described to infect domestic cats. The felines developed abdominal disseminated larval disease; the diagnosis was performed by ultrasound, exploratory laparotomy, and molecular techniques. These results indicate that cystic echinococcosis must be considered for differential diagnosis of felines with abdominal distension and/or observation of vesicles through ultrasound, from endemic areas. Even though cats and dogs are carnivores, differences in digestive physiology and immunological characteristics between them could allow the development of larval or adult worm parasites. Domestic cats with cystic echinococcosis show to be environmentally infected with E. granulosus s. s./G1 eggs.

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.

Detection of Echinococcus granulosus sensu lato infection by using extracts derived from a protoscoleces G1 cell line.

Cystic echinococcosis (CE) can be diagnosed by means of several serological approaches, but their results vary among laboratories due to the molecular characteristics of the reference antigens used. Thus, this study aimed to address both the relevance of an EGPE cell line previously obtained from Echinococcus granulosus protoscoleces G1 and the complexity of the immune response by using two different in vitro growth stages as separate sources of parasite antigens. The serum reactivity was investigated by western blotting (WB) in 21 CE patients from an endemic area in a matched case-control design and also in seven experimentally infected sheep and five healthy control sheep. EGPE-antigen-human serum sensitivity by WB was higher than that of hydatid fluid (HF) WB, ELISA and DD5 (P < .05, Chi-square test). EGPE protein extract was immunogenic in mice and hyperimmune plasma reacted with HF proteins, and AgB2 expression was detected by molecular analysis. Proteins of 37 to 60 kDa were recognized by 95.24% of the CE patients' sera but, with poor specificity. Statistically significant differences were found between serum protein extract recognition at 7 and 20 days of cell growth. The EGPE cell line is a laboratory source of antigens for improvement of CE serological diagnosis.

Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America.

The aim of this work was to determine Echinococcus granulosus sensu lato species and genotypes in intermediate and definitive hosts and in human isolates from endemic regions of Argentina and Brazil including those where no molecular data is available by a combination of classical and alternative molecular tools. A total of 227 samples were isolated from humans, natural intermediate and definitive hosts. Amplification of cytochrome c oxidase subunit I gene fragment was performed and a combination of AluI digestion assay, High Resolution Melting analysis (HRM) assay and DNA sequencing was implemented for Echinococcus species/genotype determination. E. granulosus sensu stricto (G1) was found in sheep (n=35), cattle (n=67) and dogs (n=5); E. ortleppi (G5) in humans (n=3) and cattle (n=108); E. canadensis (G6) in humans (n=2) and E. canadensis (G7) in pigs (n=7). We reported for the first time the presence of E. ortleppi (G5) and E. canadensis (G6) in humans from San Juan and Catamarca Argentinean provinces and E. canadensis (G7) in pigs from Cordoba Argentinean province. In this work, we widened molecular epidemiology studies of E. granulosus s. l. in South America by analyzing several isolates from definitive and intermediate hosts, including humans from endemic regions were such information was scarce or unavailable. The presence of different species/genotypes in the same region and host species reinforce the need of rapid and specific techniques for accurate determination of Echinococcus species such as the ones proposed in this work.

Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts.

OBJECTIVE: To systematically review publications on Echinococcus granulosus sensu lato species/genotypes reported in domestic intermediate and definitive hosts in South America and in human cases worldwide, taking into account those articles where DNA sequencing was performed; and to analyse the density of each type of livestock that can act as intermediate host, and features of medical importance such as cyst organ location. METHODS: Literature search in numerous databases. We included only articles where samples were genotyped by sequencing since to date it is the most accurate method to unambiguously identify all E. granulosus s. l. genotypes. Also, we report new E. granulosus s. l. samples from Argentina and Uruguay analysed by sequencing of cox1 gene. RESULTS: In South America, five countries have cystic echinococcosis cases for which sequencing data are available: Argentina, Brazil, Chile, Peru and Uruguay, adding up 1534 cases. E. granulosus s. s. (G1) accounts for most of the global burden of human and livestock cases. Also, E. canadensis (G6) plays a significant role in human cystic echinococcosis. Likewise, worldwide analysis of human cases showed that 72.9% are caused by E. granulosus s. s. (G1) and 12.2% and 9.6% by E. canadensis G6 and G7, respectively. CONCLUSIONS: E. granulosus s. s. (G1) accounts for most of the global burden followed by E. canadensis (G6 and G7) in South America and worldwide. This information should be taken into account to suit local cystic echinococcosis control and prevention programmes according to each molecular epidemiological situation.

A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.

Diagnóstico de situación de la investigación en salud en el ámbito del Ministerio de Salud de la Nación y 10 ministerios provinciales / Situational diagnosis of the health research at the national and 10 Provincial Ministries fo Health in Argentina

INTRODUCCIÓN: El estudio y análisis de la investigaciónpara la salud reviste interés para los países desde el punto de vista sanitario y socioeconómico, ya que procura establecer información confiable y de calidad acerca de qué, cómo y a través de quiénes se investiga en este campo. OBJETIVO: Describir las actividades de gestión de la investigación, difusión o producción de conocimiento que se realizan en los ministerios y sus organismos dependientes, con la finalidad de efectuar un diagnóstico inicial de esa situación en 2010. MÉTODOS: Se realizó un estudio descriptivo transversal. Se analizaron 11 ministerios de salud y 215 organismos dependientes. El relevamiento se realizó a través de una encuesta estructurada a funcionarios responsables. Se relevaron los ministerios de Ciudad Autónoma de Buenos Aires (CABA), Provincia de Buenos Aires, Chaco, Chubut, Córdoba, Corrientes, Jujuy, Mendoza, Nación, Neuquén y Tucumán. RESULTADOS: CABA, Buenos Aires,Córdoba, Jujuy, Mendoza, Nación y Neuquén contaban con áreas específicas de investigación y realizaban actividades de regulación y auditorías de investigación. Se relevaron 1.019 investigaciones, de las cuales el 61% contaban con financiamiento. De los 126 hospitales relevados el 56% (71) efectuaban investigaciones. Entre los ministerios de CABA (424), Nación (177), Córdoba (125) y Buenos Aires(103) y sus hospitales concentraron el 81%. CONCLUSIONES: A pesar del alto porcentaje de investigaciones hospitalarias, solo el 55% de ellas recibieron financiamiento, lo que denotaría un gran esfuerzo institucional. Por otra parte, la mayoría de los ministerios relevados contaban con áreas específicas de investigación, lo que indicaría el interés del Estado en la investigación en salud.

INTRODUCTION: The study and analysis of health research is relevant for all countries from both health and socio-economic point of view - it intends to obtain trust worthy and quality information about what, how and by whom research is done in this field. OBJECTIVE: To describe the activities related to research management, diffusion and knowledge production with in the frame work of ministries and its agencies, in order to make an initial diagnosis of the situation during 2010. METHODS: A descriptive cross-sectional study was performed. It analyzed 11 health ministries and 215 dependent agencies. It was conducted through a structured survey to responsible officials in the Autonomous City of Buenos Aires (CABA), Buenos Aires Province, Chaco, Chubut, Córdoba,Corrientes, Jujuy, Mendoza, Nation, Neuquén and Tucumán. RESULTS: CABA, Buenos Aires, Córdoba, Jujuy, Mendoza, Nation and Neuquén had specific areas of research, carried out regulatory activities and research audits. Of the 1.019 investigations, 61% were funded. 126 hospitals were surveyed, and 56% (71) conducted researches. The ministries of CABA(424), Nation (177), Córdoba (125) and Buenos Aires (103) and the hospitals under their jurisdiction totaled 81% of the investigations. CONCLUSIONS: Despite the high percentage of hospital investigations, only 55% of them were funded, which would indicate a large institutional effort. More over,most of the ministries surveyed had specific research areas, that would indicate the State´s interest in health research.

New record and larval habitats of Culex eduardoi (Diptera: Culicidae) in an irrigated area of Patagonia, Chubut Province, Argentina

The object of the present work was to identify the larval habitats of Culex eduardoi and to determine the microenvironmental conditions related to their presence in different artificial freshwater environments (temporary, semi-permanent, irrigation ditches, and drainage ditches) in tillable areas of Chubut Province, Argentina. This report represents the first record of Cx. eduardoi from this Province and extends its range to latitude 45°S. Immature stages of Cx. eduardoi were found in 8 out of 109 (7.3 percent) freshwater habitats and were significantly more prevalent in semi-permanent water bodies. Positive sites had significantly larger surface areas and more vegetation cover than negative sites.

Viability and infectiousness of eggs of Echinococcus granulosus aged under natural conditions of inferior arid climate.

We studied the viability and infectiousness of aged Echinococcus granulosus eggs by in vivo evaluation in ovines. Our results demonstrated that after 41 months of ageing of the eggs under environmental conditions of an inferior arid climate (Patagonia, Argentina), they were still able to produce infection in 4/4 ovines challenged with 1200 eggs per ovine. In the ovines experimentally infected with these aged eggs, the occurrence of hepatic and pulmonary cysts was determined by necropsy, histologic and genetic studies. The eggs were found in a semi-senescent stage, thus keeping their capacity to generate an infection in the intermediary ovine host.

Diagnosis of cystic echinococcosis on sheep farms in the south of Argentina: areas with a control program.

In 2000 Guarnera et al. proposed using ELISA in canine faeces collected from the ground to detect dogs infected with Echinococcus granulosus, thus determining sheep farms with active transmission. The objective was to evaluate the prevalence of E. granulosus infection in sheep farms of the Patagonia. Sheep farms were randomly selected in the Provinces of Río Negro, Chubut, Neuquén, Santa Cruz and Tierra del Fuego (areas with control programs) and La Pampa (comparison area). From one to three samples of fecal matter were obtained for each sheep farm, which were processed by means of copro-ELISA test with confirmation of positive samples by copro-Western blot. A total of 1042 samples were obtained from 352 sheep farms, 26 (7.3%) proving positive. Of these 5 (6.3%) were from La Pampa, 9 (13.8%) from Neuquén, 4 (4.7%) from Río Negro, 2 (2.9%) from Chubut, 1 (5.9%) from Santa Cruz and 5 (13.9%) from Tierra del Fuego. The identification of parasitized dogs is an essential activity upon which rests the strategy of control and surveillance. Arecoline tests or coproantigen test with fecal matter obtained directly from the dog contribute information on individual prevalence, while the use of coproantigens detected in ground-collected samples transfers the dog unit of observation to units of greater epidemiological value. In the present experience, the technique employed seems promising for its application in systems of epidemiological surveillance of cystic echinococcosis and in drawing a baseline on which to measure the progress of control programs in the Argentine Patagonia in subsequent years.

Aportes morfológicos y morfométricos sobre Echinococcus granulosus para vacuna ovina EG95 / Morphological and morphometric contributions on Echinococcus granulosus for EG95 sheep vaccine

Se describe el estadío adulto de la cepa de Echinococus granulosus, utilizada en la validación de la Vacuna Ovina EG95 en el marco del Programa de Control de Hidatidosis (Provincia de Chubut, Argentina), con el propósito de medir la efectividad de la Vacuna Ovina EG95, elaborada por el Laboratorio de Parasitología Molecular de la Universidad de Melbourne de Australia. Se estudiaron treinta ejemplares adultos de E. granulosus (conservados en formol al 10%) cuyo tamaño osciló entre 1,6 y 5,1 mm de largo. Los resultados mostraron que, además de su escólex, el parásito adulto presenta entre 3 y 5 proglótidos. En los ejemplares observados la proglótida grávida siempre era la última, independientemente de la cantidad de proglótidas presentes. El promedio de huevos de cada proglótido grávida fue 525 por parásito (rango de 405-808).

The adult stage of the Echinococus granulosus strain used in Sheep Vaccine EG95 validation within the framework of the Hydatidosis Control Program (Chubut Province, Argentina) is described in this work in order to measure the effectiveness of this vaccine developed by the Molecular Parasitology Laboratory of Melbourne University, Australia. Thirty adult specimens of E. granulosus (in 10% formalin) were studied. The size of these specimens ranged from 1.6 to 5.1 mm long. The results showed that, in addition to its scolex, the adult parasite presents 3 and 5 proglottids. In the specimens observed, the gravid proglottid was always the last one, regardless of the number of proglottids present. The average number of eggs of each gravid proglottid was 525 per parasite (range 405- 808).

Contaminación por helmintos en espacios públicos de la provincia de Chubut, Argentina / Contamination by helminths in public places at Chubut province, Argentina

Canine-borne helminthiases constitute a worldwide problem. Some of these parasites can originate serious pathologies in humans. Given the potential existence of these zoonoses in Argentina, horizontal surveys on contamination degree evaluated as faeces per hectare and on parasite prevelence in faeces were carried out in six localities of the Province of Chubut. The behaviour of people visiting public places was also analyzed qualitatively. All of the survey sites were contaminated with faeces and with some parasitic forms. Contamination degrees ranged from 32 to 147 faeces/ha. Toxocara sp. (17.4 percent), Strongyloides sp. (5.1 percent), and Ancylostoma. sp (5.6 percent) were the main genera among other parasites detected. As a whole, the analysis revealed that contamintion degree, prevalence and human behaviour in relation to the exposure to the parasite would be the main factors to be taken into a account in formulating control measures. Transmission risk patterns in Chubut were not homogenous; therefore, studies and control strategies should be devised and implemented at the local scale