Cross-disciplinary approaches for measuring parasitic helminth viability and phenotype

Parasitic worms (helminths) within the Phyla Nematoda and Platyhelminthes are responsible for some of the most debilitating and chronic infectious diseases of human and animal populations across the globe. As no subunit vaccine for any parasitic helminth is close to being developed, the frontline strategy for intervention is administration of therapeutic, anthelmintic drugs. Worryingly, and unsurprising due to co-evolutionary mechanisms, many of these worms are developing resistance to the limited compound classes currently being used. This unfortunate reality has led to a renaissance in next generation anthelmintic discovery within both academic and industrial sectors. However, a major bottleneck in this process is the lack of quantitative methods for screening large numbers of small molecules for their effects on the whole organism. Development of methodologies that can objectively and rapidly distinguish helminth viability or phenotype would be an invaluable tool in the anthelmintic discovery pipeline. Towards this end, we describe how several basic techniques currently used to assess single cell eukaryote viability have been successfully applied to parasitic helminths. We additionally demonstrate how some of these methodologies have been adopted for high-throughput use and further modified for assessing worm phenotype. Continued development in this area is aimed at increasing the rate by which novel anthelmintics are identified and subsequently translated into everyday, practical applications.

Vermes parasíticos (helmintos) dos filos Nematoda e Platelmintos são responsáveis por algumas das doenças infecciosas crônicas e mais debilitantes das populações humana e animal em todo o globo. Já que nenhuma vacina está prestes a ser desenvolvida para nenhum parasita helmíntico, a frente estratégica de intervenção é a administração de drogas terapêuticas anti-helmínticas. De maneira preocupante, e não surpreendente devido a mecanismos coevolutivos, muitos destes vermes estão desenvolvendo resistência às limitadas classes de compostos que têm sido usados no momento. Esta infeliz realidade levou a um renascimento na descoberta de uma nova geração de anti-helmínticos tanto no setor acadêmico quanto no industrial. Contudo, um importante gargalo neste processo é a falta de métodos quantitativos para testar um grande número de pequenas moléculas em relação aos efeitos sobre o organismo inteiro. O desenvolvimento de metodologias que possam distinguir objetiva e rapidamente a viabilidade dos helmintos ou o fenótipo seria uma ferramenta valiosa para canalizar a descoberta de anti-helmínticos. Para este fim, descrevemos aqui como muitas técnicas básicas, correntemente usadas para avaliar a viabilidade de células únicas de eucariotos, têm sido aplicadas com sucesso para helmintos parasíticos. Adicionalmente demonstramos como algumas destas metodologias foram adotadas para uso em larga escala e além disso modificadas para avaliar o fenótipo de vermes. O desenvolvimento contínuo nesta área está voltado para aumentar a taxa com que novos anti-helmínticos são identificados e subsequentemente traduzidos em aplicações práticas cotidianas.

Atividade anti-helmíntica do extrato aquoso das folhas de Zanthoxylum rhoifolium Lam. (Rutaceae) / Anthelmintic activity of aqueous extract of Zanthoxylum rhoifolium Lam. leaves (Rutaceae)

O estudo avaliou a atividade anti-helmíntica do extrato aquoso das folhas de Zanthoxylum rhoifolium em dois experimentos. O teste in vitro utilizou cultivos de fezes de caprinos tratados com diferentes concentrações do extrato: (134,5 a 335,0 mg.mL-1). No teste in vivo, utilizaram-se 20 ovelhas: G1 tratado durante quatro dias (0,63 g.kg-1 PV); G2, mesma dose, por oito dias; G3, ivermectina (200 μg.kg-1); e G4, sem tratamento. In vitro, observou-se redução de larvas de Haemonchus, Trichostrongylus e Oesophagostomum superior a 95% nas concentrações de 193,7 a 335,0 mg.mL-1. In vivo, a redução de ovos por grama de fezes foi de 51, 56 e 90%, respectivamente, para G1, G2 e G3, no oitavo dia de tratamento, enquanto para os estágios imaturos e adultos variou de 0 a 91% no G1 e 26 a 94% no G2. A eficácia da ivermectina alcançou 99% para L4 e L5 de H. contortus e 100% para as demais espécies de nematoides. Parâmetros clínicos e bioquímicos permaneceram na faixa de normalidade, e as análises histopatológicas não revelaram alterações sugestivas de toxicidade. Embora altamente efetivo in vitro, o extrato aquoso de folhas de Z. rhoifolium foi pouco eficaz in vivo na redução de nematoides gastrintestinais.

The study evaluated the anthelmintic activity of aqueous extract of Zanthoxylum rhoifolium leaves in two experiments. In vitro test, cultures of goat fecal samples were treated with different concentrations of extract (134.5 to 335.0 mg.mL-1). In vivo test was composed of 20 sheep: G1: treated with 0.63 g.kg-1, during four days; G2: same dose, for eight days; G3: ivermectin (200 μg.kg-1) and G4 untreated group. In vitro results showed a reduction of Haemonchus spp, Trichostrongylus spp. and Oesophagostomum spp. larvae greater than 95% in the concentrations between 335.0 and 193.7 mg.mL-1. Faecal egg counting reduction was 51, 56 and 90% in G1, G2 and G3, respectively, while immature stages and adults ranged from 0 to 91% in G1 and from 26 to 94% in G2. Ivermectin effectiveness was 99% for L4 and L5 of H. contortus and 100% for other nematodes species. Clinical and biochemical parameters have remained in the normality and histophatologic analyses did not show alteration suggesting absence of toxicity. Although the great effectiveness of Z. rhoifolium leaves extract in vitro test, it displayed poor efficiency in vivo regarding gastrointestinal nematodes reduction.

Praziquantel, levamisol e diflubenzuron no controle de Dolops carvalhoi (Crustacea: Branchiura) e Anacanthorus penilabiatus (Monogenea: Dactylogyridae) em Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae) / Praziquantel, levamisole and diflubenzuron in the control of Dolops carvalhoi (Crustacea: Branchiura) and Anacanthorus penilabiatus (Monogenea: Dactylogyridae) in Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae)

Neste trabalho, avaliou-se a eficácia antiparasitária do praziquantel, levamisol e diflubenzuron administrados via oral, adicionados à ração, para pacus (Piaractus mesopotamicus) infectados por Anacanthorus penilabiatus e Dolops carvalhoi. Foram utilizadas 19 caixas d'água de 300 L de capacidade, comportando 28 peixes cada. Os tratamentos foram feitos misturando os princípios ativos nas dietas. A intensidade parasitária e eficácia foram avaliadas 1 dia antes e 3, 7 e 15 dias após o início da alimentação com ração contendo diflubenzuron, levamisol e praziquantel isolados ou associados em diferentes concentrações por 7 dias. Os resultados da eficácia terapêutica sugerem que, isoladamente ou associado com levamisol e praziquantel, o diflubenzuron é eficiente contra o crustáceo D. carvalhoi, demonstrando que a eficácia dos tratamentos nos dias 3, 7 e 15 variou de 96,2 a 100 por cento. Contra os monogenóides, as drogas não apresentaram eficácia satisfatória. Os resultados sugerem o uso do diflubenzuron para o controle de D. cavalhoi em peixes de cativeiro e em condições de quarentenário.

This assay evaluated the control efficacy of diflubenzuron, praziquantel and levamisole added to the diet of pacu (Piaractus mesoptamicus) infected with Anacanthorus penilabiatus and Dolops carvalhoi. 19 water tanks of 300 L capacity were utilized with 28 fish in each one. The treatments were made by mixing the active principles in the diet. The experiment was evaluated in four harvests done 1 day before and 3, 7 and 15 days after the treatment. The medicated feeding was applied for 7 days. The results of efficacy suggest that the diflubenzuron alone or associated with levamisole and praziquantel was efficient against the crustacean D. carvalhoi and the efficacy in the 3, 7 and 15 days evaluations ranged from 96,2 to 100 percent. Against the monogenean the drugs did not present efficacy. The results suggest the use of diflubenzuron for the control of D. carvalhoi in captive fishes in special conditions.

Detoxifying and anti-oxidant enzymes of Fasciola gigantica worms under triclabendazole sulphoxide [TCBZ-SX]: An in vitro study

Triclabendazole [TCBZ] is an efficient fasciolicide that affects both juvenile and adult worms. After oral administration it is rapidly metabolized to TCBZ sulphoxide and sulphone that were found responsible for its fasciolicidal activity. Parasite defense mechanisms include detoxifying and anti-oxidant enzymes that would suppress its oxidative killing. The present work aimed at evaluating these enzymes under TCBZ-SX. Thirty juvenile and 30 adult F. gigantica worms collected from the liver parenchyma and bile ducts formed the subject of the study. Levels of superoxide dismutase [SOD], glutathione peroxidase [GPx], glutathione reductase [GR], glutathione S-transferase [GST], and glutathione [GSH] were measured in juvenile and adult worms incubated, without and with 15 and 50 micro g/ml TCBZ-SX for 18 hours at 37°C. Results demonstrated a significant decrease in SOD activity and an increase in GST level in both juvenile and adult worms after incubation in the two concentrations. The remaining enzymes and GSH revealed variable levels

Evaluation of coverage of deworming interventions in Vietnam.

In 2004-2005, the school deworming program in Vietnam targeted 2,400,000 school-children in 25 provinces. A questionnaire survey of a random sample of schools throughout the country was organized to validate the teacher feedback, assess the real coverage of the program and estimate the occurrence of side-effects. A total of 91 schools in four different provinces were visited few days after drug distribution. A total of 2,323 children and 80 teachers were questioned. The total cost of the monitoring has been estimated at US dollar 7,000 (less than 10% of the deworming program). The survey demonstrated a coverage of 95% for school-children and of 86.3% for teachers (on average a coverage 2% lower than the one reported by the teachers through forms on the day of drug administration). Approximately 0.4% of those interviewed reported side-effects after deworming, while the occurrence of side effects through routine reports was 0.15%. In both cases, the side effects were described as mild forms of nausea, abdominal pain, or headaches that did no required pharmacological treatment. The survey confirmed the positive results of routine reporting; we consider the survey a cost-effective activity to maintain the control activities under monitoring.

Appreciation of school deworming program by parents in Ha Giang Province (Vietnam).

School-based deworming programs are one of the most cost-effective public health strategies to reach children with anthelminthics and health education. However, despite their low cost and high health impact, they are not implemented on a large scale. The present survey investigated a school-based deworming program in Ha Giang Province, Vietnam, to assess the community perception and support towards these programs. Information from 81 households, 88 primary school students, 25 teachers and 9 people in charge of commune health stations was collected by structured questionnaires. The survey displayed complete satisfaction and a high level of support within all 4 questioned groups. In addition 96.1% of all parents noticed an improvement in their children's health. The great appreciation of the deworming program by the community is an additional argument to convince decision makers to implement and scale up these programs. Even teachers that did not receive any specific training were able to distribute the drug successfully following the page of written instructions accompanying the drug.

Effect of closantel on some blood parameters, histopathological changes and fetal development in pregnant female rabbits

The effect of closantel on some blood parameters, histopathological changes and fetal development was studied on 3 groups of 10 pregnant rabbits each. The first group was used as a control whereas, the other groups were injected subcutaneously with closantel in a dose of 10 mg/kg body weight twice with one week apart at days 0 [day of mating] and 7of pregnancy [group A] and days 6 and 13 of pregnancy [group B]. The results revealed increased gamma-globulin concentration in the 20[th] and 28[th] day of pregnancy in both groups. Esinophil and monocyte numbers were significantly increased in the 20[th] and 28[th] day of pregnancy in group A and in the 28[th] of pregnancy in group B. There was no significant effect of the treatment on total leukocytic count, numbers of lymphocytes, neutrophils, basophiles and red blood cells. Hemoglobin concentration, packed cell volume [PCV], total protein, albumin, and globulin in the blood [alpha and beta] were within normal levels. Histopathological examination demonstrated activation in the mesenteric lymph nods, spleenic corpuscles and thymus gland in treated animals at both periods. Atretic oocytes in primary follicles in the ovary of the treated rabbits at day 0 and 7 of pregnancy and necrotic changes in hepatic parenchyma and urinefrous tubules were clearer in rabbits treated at days 6 and 13 of pregnancy. The anthelmentic closantel did not cause embryonic or teratogenic effects in rabbits. It is concluded that closantel has immune stimulatory effects which may contribute to the death of the parasite. It decreased the ovulation rate so it is highly advisable to avoid exposure of females to the drug during ovulation time. The drug also caused some necrotic changes in liver and urineferous tubules. It is contraindicated in animals having liver and kidney problems

Doramectin e Levamizole no controle dos helmintos de bovinos no inicio da estaçäo seca / Doramectin and levamizole in the control bovines helminths in the begining dry season

Sessenta bovinos da raça Canchim, com idade aproximada de 15 meses e mantidos em pastagem de Brachiaria decumbens Stapf, foram distribuídos aleatoriamente em três tratamentos (T) com 20 animais cada, para controle de nematódeos gastrintestinais (T1: controle; T2: Doramectin, 1ml/50kg/peso vivo, correspondendo a 200µg/kg; e T3: Levamizole e 1ml/40kg/peso vivo, equivalente a 3,75mg do princípio ativo por quilograma de peso). A aplicaçäo foi feita via parenteral, subcutânea, sendo as infecçöes verminóticas obtidas naturalmente a pasto. O experimento foi realizado na fazenda Canchim, base física do Centro de Pesquisa de Pecuária do Sudeste (CPPSE), em Säo Carlos, SP, de abril a junho de de 1995. Foram avaliados o peso corporal (PC), número de ovos por grama de fezes (OPG) e volume corpuscular médio (VCM), por seis períodos (P): 0, 14, 28, 42, 56 e 70 dias pós-dosificaçäo. O modelo de análise incluiu, além da média, os efeitos de T, de P e da interaçäo T x P. A partir dos 14 dias de avaliaçäo, o Doramectin foi o que apresentou o menor OPG (P<0,01), comparado aos tratamentos controle e Levamizole, näo havendo diferença (P>0,05) entre esses dois grupos a partir dos 42 dias. Os animais tratados apresentaram ganho de peso semelhantes (P>0,05) até o 42§ dia. Quanto ao controle, observou-se reduçäo no ganho de peso (P<0,05), obtendo-se, inclusive, valores negativos aos 42 e 70 dias. Quanto aos valores do VCM, os animais tratados foram semelhantes (P>0,05) entre si em toda a fase experimental, observando-se, no entanto, valores inferiores (P<0,05) desta variável a partir dos 28 dias no grupo controle.

Desenvolvimento de metodologia para o estudo da estabilidade de suspensöes orais de albendazol / Metodology development for the study of albendazole oral suspensions stability

O albendazol é um potente anti-helmíntico benzimidazólico largamente utilizado em helmintíase intestinal humana, sendo também o medicamento de escolha no tratamento de cisto hidático e neurocisticercose. Apesar de amplamente estudado do ponto de vista clínico, existem poucas referências a respeito da metodologia analítica, sendo que nenhuma delas é oficial, como também näo há informaçöes sobre sua estabilidade na forma de suspensäo oral. O presente trabalho padronizou dois métodos para determinaçäo do teor albendazol em suspensöes farmacêuticas e estudou a estabilidade química, física e sensorial de formulaçöes contendo diferentes agentes suspensores, armazenadas a temperaturas de 4ñ 1ºC, 26ñ 1ºC, 37ºC, 50ºC e 65ºC e avaliado nos tempos 0, 1, 3 e 6 meses...

Efficacy of pyrantel pamoate either alone or combined with trichlorfon for control of cyathostomes of horses

Através de estudo comparativo, objetivou-se avaliar a eficiência anti-helmíntica de pamoato de pirantel, isolado ou associado ao triclorfon, no controle de estrongilídeos de equinos da raça Puro Sangue Inglês. As coproculturas realizadas antes e após os tratamentos levaram consistentemente ao encontro de populaçöes puras de ciatostomíneos com oito células intestinais. As reduçöes de ovos por grama de fezes foram avaliadas do 7§ ao 45§ dias pós-tratamento. Comparando os valores médios de OPG dos equinos tratados com pamoato de pirantel (13,2 mg/kg) associado ao triclorfon (30 mg/kg) e com pamoato de pirantel isolado (13,2 mg/kg), observou-se diferença näo significante (P<0,05) ao longo do experimento, verificando-se, 7 dias após o tratamento, reduçöes médias de OPG de quase 100 por cento e, do 7§ ao 20§ dias, reduçöes ainda superiores a 90 por cento. Os resultados indicam que os dois produtos säo eficazes na reduçäo de ovos por grama de fezes de ciatostomíneos de equinos mantidos sob condiçöes a campo