Identification of third stage larvae of strongyles and molecular diagnosis of Strongylus vulgaris in the feces of Thoroughbred horses kept in training centers in Rio de Janeiro, Brazil.

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals.

Eliminación de huevos de los pequeños estróngilos en la materia fecal de los equinos y su importancia práctica en la posibilidad de tratamientos selectivos / Fecal shedding of equine small strongyle eggs and its practical importance for selective treatment

Resumen Los pequeños estróngilos son los parásitos de mayor prevalencia e importancia en los equinos de todo el mundo, y el desarrollo generalizado de la resistencia de estos nematodos a los antihelmínticos está impulsando la aplicación de tratamientos selectivos (TS) para disminuir o demorar el desarrollo de estos fenómenos. Esta estrategia se basa en conteos de huevos de nematodos en la materia fecal de todos los integrantes del grupo animal para tratar solamente aquellos que sobrepasan un determinado umbral o cut off. Este principio de TS se ve favorecido por la consistencia de los conteos de huevos por gramo de heces (hpg) en los equinos y está siendo empleada en algunos países del hemisferio norte, especialmente en los animales adultos. En la Argentina, existe una ausencia de información básica en relación a las condiciones climáticas, de pastoreo y manejo que podrían resultar en cargas parasitarias y transmisión de nematodos diferentes a las observadas en el hemisferio norte. En este contexto, el objetivo de este estudio fue caracterizar la eliminación de huevos de pequeños estróngilos en 436 equinos de diferentes edades pertenecientes a 19 establecimientos del área central de la Argentina. El 90% de los equinos mostró huevos de estróngilos en las heces y los valores de hpg fueron menores a 200 y mayores a 1.000 en el 32% y 22% de los equinos respectivamente. La distribución de los huevos de estróngilos entre individuos de todas las edades fue sobre-dispersa o agregada, con una media aritmética general de 671,59 ± 789,76 y un valor de k o parámetro de agregación inversa de 0,72 (distribución de la binomial negativa). La edad de los animales (potrillos vs adultos) y los biotipos (deportivo vs trabajo) en la categoría adultos influenciaron la magnitud de los valores del hpg (p<0,001). En esta última categoría el 35% de los animales fue responsable de la excreción de aproximadamente el 80% de los huevos eliminados en las pasturas. Si bien existen algunas diferencias con lo observado en países del hemisferio norte con poblaciones parasitarias más agregadas o sobre-dispersas, los TS para el control de los pequeños estróngilos en equinos adultos podrían también ser una estrategia válida bajo nuestras condiciones para disminuir la presión de selección sobre el genoma parasitario y permitir el desarrollo de poblaciones en refugio.

Abstract Small strongyles are the most prevalent and important parasites in grazing horses throughout the world and the widespread development of antihelminthic resistance has prompted in some countries of the northern hemisphere the application of selective treatments (ST) to reduce or delay the development of these phenomena. This strategy is based on determining strongyle fecal egg counts of all herd members and treating only those exceeding a certain threshold or cut off. However, there is an absence of basic information on horses maintained under climatic, grazing or management conditions different from those observed in the northern hemisphere. In this context, the aim of this study was to characterize the elimination of eggs of small strongyles in horses of different ages from 19 farms in the central area of Argentina. Out of a total of 436 horses, 90% showed positive strongyles egg counts and in these animals the count of eggs per gram of feces (epg) was under 200 and over 1,000 in 32% and 22% of the horses respectively. The distribution of strongyle eggs among individuals of all categories was over-dispersed or aggregated with a general epg arithmetic mean of 671.59 ± 789.76 and a value of the inverse aggregation parameter of 0.72 (negative binomial distribution). The age of the animals (foals vs. adults) and the biotypes in the adult category (sports vs. work) influenced the magnitude of the epg values (P <0.001). In the latter category, approximately 35% of the animals were responsible for the excretion of approximately 80% of the eggs in the pastures. Although some differences on magnitude and distribution of strongyle fecal egg counts were observed in countries of the northern hemisphere, ST of small strongyles in adult equines could also be a valid strategy under Argentinian conditions to reduce the selection pressure on the parasitic genome and allow the development of refugia populations.

Resistencia antihelmíntica en equinos: Observaciones sobre el período de reaparición de huevos luego del tratamiento con ivermectina en potrancas y yeguas madres

En los equinos parasitados por los pequeños estróngilos, un menor período requerido luego del tratamiento con antihelmínticos para la reaparición de huevos (PRH) de los nematodes en la materia fecal, puede ser utilizado como un indicador de la presencia de resistencia o pérdida de eficacia a estas drogas. En el presente trabajo se evaluó la eficacia clínica y el PRH luego de tratamientos con ivermectina (0,2 mgr/kg) en equinos adultos (yeguas madres) y jóvenes (potrancas) naturalmente parasitados por pequeños estróngilos en un establecimiento de la provincia de Tucumán. La eficacia clínica determinada en la segunda semana post-tratamiento y utilizando un test de reducción en el conteo de huevos, osciló entre el 99,8 y el 100% en yeguas y potrancas respectivamente. Por su parte el PRH (considerado como el período post- tratamiento en que se alcanza el 10% del hpg pre-tratamiento o una reducción del mismo < 90%) fue de seis y siete semanas para las potrancas y yeguas madres respectivamente (p=0,001). En la sexta semana post- tratamiento, las potrancas mostraron poseer 11,74 veces más riesgo (IC 95% = 2,58-53,38) de reaparición de huevos que las yeguas madres. Estas diferencias ocasionadas por la edad de los animales deberían considerarse cuando se realizan estudios para establecer el status de susceptibilidad o resistencia a los antihelmínticos basados en el PRH para evitar asunciones incorrectas sobre el mismo.

In horses parasitized by small strongyles, a shortened strongyle egg reappearance period (ERP) after treatment with anthelmintics is considered an early indicator of the presence of resistance or loss of efficacy to these drugs. In the present work, clinical efficacy and ERP were evaluated after treatments with ivermectin in adult horses (broodmares) and juveniles (its fillies) naturally parasitized by small strongyles in an farm of Tucumán province. The clinical efficacy determined in the second week after treatment, and using a test of reduction in the egg count, ranged between 99.8 and 100% in mares and fillies respectively. The ERP (considered as the post- treatment period in which 10% of the pre-treatment epg was reached or a reduction of it < 90%) was six and seven weeks for the fillies and mother mares respectively (p= 0.001). In the sixth week after treatment, the fillies showed 11.74 times more risk (95% CI = 2.58-53.38) of reappearance of eggs than the brood mares. These differences caused by the age of the animals should be considered when conducting studies to establish the status of susceptibility or resistance to anthelmintics based on the ERP to avoid incorrect assumptions.

Factors associated with the prevalence of helminths in Mangalarga Marchador horses in southern of Minas Gerais, Brazil / Fatores associados com a prevalência de helmintos em cavalos da raça Mangalarga Marchador em propriedades do Sul de Minas Gerais

Horses are highly susceptible to parasitism. Helminth infections cause great harm to the animals and to their breeders. This study aimed at evaluating socioeconomic, cultural and management factors associated with the prevalence of gastrointestinal helminths of horses. A total of 40 farmas the Mangalarga Marchador horse breed were visited in southern Minas Gerais, Brazil, where interviews were conducted. Horse feces were collected on the farms and coproparasitological laboratory tests were conducted to quantify the infection and to identify parasites. Data were tabulated in Epidata and analyzed using the SPSS 20.0 software. A great similarity between breeds was observed, specifically in their profiles, as well as in their animal management techniques and in their parasite control habits. The cyathostome was the most prevalent helminth, followed by Oxyuris and large strongyles. The farms which prioritize only equine production are less likely to have animals with massive helminth infection.(AU)

Os equinos são animais muito susceptíveis ao parasitismo. As helmintoses causam grandes prejuízos tanto para os animais, quanto para os criadores. Este estudo teve como objetivo avaliar os fatores sócio-econômicos-culturais e de manejo associados à prevalência de helmintos gastrointestinais de equinos. Foram visitados 40 criatórios de equinos Mangalarga Marchador no Sul de Minas Gerais, nos quais foram realizadas entrevistas. Foram coletadas fezes dos animais nas propriedades e realizados exames laboratoriais coproparasitológicos a fim de quantificar a infecção dos animais e identificar os parasitos. Os dados foram tabulados no Epidata e analisados no software SPSS 20.0. Observou-se uma grande semelhança no perfil dos criadores, assim como na caracterização da propriedade, manejo e controle de parasitos. Os ciatostomíneos foram os helmintos mais prevalentes, seguidos pelo Oxyuris e os grandes estrôngilos. As propriedades que priorizam a equinocultura têm menor chance de apresentar animais com infecção maciça.(AU)

Factors associated with the prevalence of helminths in Mangalarga Marchador horses in southern of Minas Gerais, Brazil / Fatores associados com a prevalência de helmintos em cavalos da raça Mangalarga Marchador em propriedades do Sul de Minas Gerais

Horses are highly susceptible to parasitism. Helminth infections cause great harm to the animals and to their breeders. This study aimed at evaluating socioeconomic, cultural and management factors associated with the prevalence of gastrointestinal helminths of horses. A total of 40 farmas the Mangalarga Marchador horse breed were visited in southern Minas Gerais, Brazil, where interviews were conducted. Horse feces were collected on the farms and coproparasitological laboratory tests were conducted to quantify the infection and to identify parasites. Data were tabulated in Epidata and analyzed using the SPSS 20.0 software. A great similarity between breeds was observed, specifically in their profiles, as well as in their animal management techniques and in their parasite control habits. The cyathostome was the most prevalent helminth, followed by Oxyuris and large strongyles. The farms which prioritize only equine production are less likely to have animals with massive helminth infection.(AU)

Os equinos são animais muito susceptíveis ao parasitismo. As helmintoses causam grandes prejuízos tanto para os animais, quanto para os criadores. Este estudo teve como objetivo avaliar os fatores sócio-econômicos-culturais e de manejo associados à prevalência de helmintos gastrointestinais de equinos. Foram visitados 40 criatórios de equinos Mangalarga Marchador no Sul de Minas Gerais, nos quais foram realizadas entrevistas. Foram coletadas fezes dos animais nas propriedades e realizados exames laboratoriais coproparasitológicos a fim de quantificar a infecção dos animais e identificar os parasitos. Os dados foram tabulados no Epidata e analisados no software SPSS 20.0. Observou-se uma grande semelhança no perfil dos criadores, assim como na caracterização da propriedade, manejo e controle de parasitos. Os ciatostomíneos foram os helmintos mais prevalentes, seguidos pelo Oxyuris e os grandes estrôngilos. As propriedades que priorizam a equinocultura têm menor chance de apresentar animais com infecção maciça.(AU)

Applicability of FLOTAC® technique in recovering equine strongyle larvae in the pasture: A comparison study.

The FLOTAC® technique represents a highly sensitive method for the isolation of oocysts, eggs, and larvae of parasites in faeces. This assay could be used for detecting free-living stages of nematodes in the pasture but no attempt has been assessed so far. Therefore, the performance of FLOTAC® technique for isolating infective larvae of nematodes in the environment was investigated and compared with the spontaneous sedimentation (SST) and centrifugal sedimentation (CST) techniques. The study was conducted in a horse farm located in northeastern Brazil, where the occurrence of strongyle larvae had been previously reported. Pasture samplings were collected monthly from January to May 2016 in a 376 m2 crop area harvested with the Guinea grass Panicum cultivar Massai. The recovery of third-stage larvae (L3) was performed using the FLOTAC®, SST and CST techniques. Values of Cohen's kappa coefficient, sensitivity, specificity, positive and negative predictive values, and accuracy of each technique were assessed. Although strongyle larvae were evenly detected, with the FLOTAC® technique yielded the highest number of positive samples (i.e., 41%, 41/100, p < .0001). The main parasites isolated belonged to the Cyathostominae and Strongylinae subfamilies. Based on these results, the FLOTAC® technique should be considered as practical and safe method for the isolation of nematode larvae in the pasture, thus opening a new potential use for this tool in the field.

Factors associated with the prevalence of helminths in Mangalarga Marchador horses in southern of Minas Gerais, Brazil

ABSTRACT: Horses are highly susceptible to parasitism. Helminth infections cause great harm to the animals and to their breeders. This study aimed at evaluating socioeconomic, cultural and management factors associated with the prevalence of gastrointestinal helminths of horses. A total of 40 farmas the Mangalarga Marchador horse breed were visited in southern Minas Gerais, Brazil, where interviews were conducted. Horse feces were collected on the farms and coproparasitological laboratory tests were conducted to quantify the infection and to identify parasites. Data were tabulated in Epidata and analyzed using the SPSS 20.0 software. A great similarity between breeds was observed, specifically in their profiles, as well as in their animal management techniques and in their parasite control habits. The cyathostome was the most prevalent helminth, followed by Oxyuris and large strongyles. The farms which prioritize only equine production are less likely to have animals with massive helminth infection.

RESUMO: Os equinos são animais muito susceptíveis ao parasitismo. As helmintoses causam grandes prejuízos tanto para os animais, quanto para os criadores. Este estudo teve como objetivo avaliar os fatores sócio-econômicos-culturais e de manejo associados à prevalência de helmintos gastrointestinais de equinos. Foram visitados 40 criatórios de equinos Mangalarga Marchador no Sul de Minas Gerais, nos quais foram realizadas entrevistas. Foram coletadas fezes dos animais nas propriedades e realizados exames laboratoriais coproparasitológicos a fim de quantificar a infecção dos animais e identificar os parasitos. Os dados foram tabulados no Epidata e analisados no software SPSS 20.0. Observou-se uma grande semelhança no perfil dos criadores, assim como na caracterização da propriedade, manejo e controle de parasitos. Os ciatostomíneos foram os helmintos mais prevalentes, seguidos pelo Oxyuris e os grandes estrôngilos. As propriedades que priorizam a equinocultura têm menor chance de apresentar animais com infecção maciça.

Pequeños estróngilos de los equinos: Eficacia clínica y periodo de reaparición de huevos luego del tratamiento con moxidectina y pirantel

El control de los pequeños estróngilos de los equinos (grupo Ciatostoma) se basa casi exclusivamente en la aplicación de antihelmínticos. En nuestro país, el desarrollo de resistencia generalizada a los bencimidazoles, está limitando las alternativas químicas disponibles a las lactonas macrocíclicas (ivermectina y moxidectina) y al pirantel, consideradas como drogas de larga y corta acción respectivamente. La información actualizada sobre la actividad de estas drogas en el campo es crítica para determinar su eficacia y detectar el desarrollo de fenómenos de resistencia a los antihelmínticos. En los equinos el período de reaparición de huevos (PRH) luego del tratamiento es considerado como un indicador temprano de la presencia de resistencia. En el presente trabajo se evaluó la eficacia clínica y el PRH luego de tratamientos con moxidectina y pirantel en equinos adultos de cinco establecimientos de las provincias de Santa Fe y Córdoba naturalmente parasitados por pequeños estróngilos. La eficacia clínica determinada al día 15 pos tratamiento utilizando un test de reducción en el conteo de huevos, osciló entre el 99,8 y el 100% para la moxidectina y entre el 98,9 y el 98,8% para el pirantel. Por su parte en el presente estudio el PRH fue de al menos 100 días para la moxidectina y de 35 días para el pirantel. Estos resultados indican que ambas drogas se muestran activas para el control de estos nematodes y que las poblaciones estudiadas (alguna de ellas resistentes a bencimidazoles) permanecen actualmente susceptibles a la moxidectina así como al pirantel. Esta última droga es de uso limitado en nuestro país, pero su inclusión en los programas de control contra los pequeños estróngilos podría reducir la dependencia y la presión de selección sobre las lactonas macrocíclicas y contribuir a mantener la vida útil de las mismas.

The control of the small strongyles (Ciathostoma group) in horses is based on the application of anthelmintics. In our country, the development of generalized resistance to benzimidazoles is limiting the chemical alternatives available to macrocyclic lactones (ivermectin and moxidectin) and to pyrantel, considered as long-acting and short-acting drugs respectively. Updated information on the activity of these drugs in the field, is critical for determining its efficacy and detecting the development of anthelmintic resistance. In these horse nematodes the period of egg reappearance (ERP) after treatment is considered as an early indicator of the presence of resistance. The present study evaluated the clinical efficacy and ERP after moxidectin and pirantel treatments in adult horses naturally parasitized by small strongyles from five farms from Santa Fe and Córdoba provinces. Clinical efficacy determined at day 14 or 15 post treatment using a test of reduction in the egg count ranged from 98.9 to 98.8% for the pirantel and 99.8 to 100% for moxidectin. The ERP was at least 100 days for moxidectin and 35 days for the pirantel. These results indicate that both drugs are active for the control of these nematodes and that the populations studied (some of them resistant to benzimidazoles) remain currently susceptible to moxidectin as well as to pyrantel. This last drug is of limited use in Argentina, but its inclusion in the control programs against the small strongyls could reduce the dependence and the selection pressure on the macrocyclic lactones and contribute to maintain the useful life of the same ones.

Helminth egg excretion in horses kept under tropical conditions-Prevalence, distribution and risk factors.

Increasing levels of anthelmintic resistance observed in equine cyathostomin parasites have led to recommendations of selective anthelmintic treatment strategies to lower the selection pressure favoring resistant populations. This principle is based on determining strongyle fecal egg counts from all herd members, and treating those exceeding a predetermined treatment cutoff. However, epidemiological information is lacking from horses kept under tropical conditions, where parasite burdens may be of a different composition and magnitude compared to those of horses kept under temperate climate conditions. The aim of the present work was to characterize the strongylid fauna in horses kept in tropical Camagüey, Cuba and identify risk factors associated with strongylid and ascarid egg counts. A total of 396 horses from eight different establishments were included in the study. Coprocultures revealed that Strongylus vulgaris and cyathostomins, sensu lato, were detected in all of those establishments. Prevalence and mean value of strongylid eggs per gram of feces were 97% and 1436, respectively. Eggs of Parascaris spp. were observed in 10% of horses. A multivariate mixed linear model identified sex (p=0.022), month (p=0.044), operation type (p=0.037) and time since last deworming (p<0.001) to be significantly associated in with the magnitude of strongylid fecal egg counts. A multivariate logistic regression identified horses less than two years of age (p=0.010) and horses not receiving anthelmintic treatment (p<0.001) to be significantly more likely to harbor Parascaris spp. parasites. The high magnitude and prevalence of strongylid fecal egg counts observed and the common occurrence of S. vulgaris suggest that strongylid parasite burdens are substantially different from those typically observed in managed equines kept under more temperate conditions.

The route of administration drastically affects ivermectin activity against small strongyles in horses.

The goal of the current study was to evaluate the comparative efficacy of ivermectin (IVM) against small strongyles (cyathostomins) following its oral and intramuscular (IM) administration, in naturally parasitized horses. The parasitological data were complemented with the assessment of the plasma disposition kinetics of IVM. The trial included two different experiments. In experiment I, 40 horses naturally infected with small strongyles were randomly allocated into four experimental groups (n=10) and treated with IVM (0.2mg/kg) as follows: IVM oral paste, animals were orally treated with Eqvalan® (IVM 1.87% paste, as the reference formulation) by the oral route; IVM oral solution, animals were orally treated with Remonta® (IVM 2% solution, as a test formulation); IVM IM solution, animals were IM treated with the test product (Remonta® IVM 2% solution); and control, animals were kept without treatment as untreated controls. In experiment II, 24 horses naturally parasitized with small strongyles were randomly allocated into the same four experimental groups (n=6) described for experiment I. Faecal samples were individually collected directly from the rectum of each horse prior (day -1) and at 7 and 15 (Experiment I) or 7, 15 and 21 (Experiment II) days after-treatment, to assess the eggs per gram (epg) counts and estimate the efficacy of the treatments. Additionally, the comparative plasma disposition kinetics of IVM in treated animals was assessed in experiment II. In both experiments, an excellent (100%) IVM efficacy was observed after its oral administration (test and reference formulations). However, the IM administration of IVM resulted in a low efficacy (36-64%). Similar IVM plasma concentration was observed after its oral administration as a paste or as a solution. The higher IVM plasma profiles observed after the IM administration accounted for an enhanced systemic availability. The improved IVM efficacy observed against adult cyathostomins after its oral administration can be explained by an enhanced drug exposure of the worms located at the lumen of the large intestine. These findings may have a direct impact on the practical use of macrocyclic lactones in horses.

Diagnosis of resistance alleles in codon 167 of the beta-tubulin (Cya-tbb-1) gene from third-stage larvae of horse cyathostomins.

Anthelmintic resistance is a serious problem for the control of equine gastrointestinal nematodes. In the present survey, 173 third stage larvae of cyathostomins were investigated from three different locations for the presence of the resistant genotype at codon 167 of the beta-tubulin gene, as this is the most prevalent mutation. The larvae from the state of Parana (n=67), Sao Paulo (n=54) and Santa Catarina (n=52), showed 61.2; 31.5 and 38.5% of the heterozygous resistant genotype - TTC/TAC, respectively. An unpublished mutation at codon 172 that results in a serine (S) to threonine (T) substitution was found in 17.9% (12/67) of samples from Parana; and 13.0% (7/54) of samples from Sao Paulo. We have compared the molecular diagnostic with the fecal egg count data (R2=-0.79) from the same farms, and consider that the use of routine molecular diagnostic in individual larva may help to determine the population genetic distribution that is associated with drug failure.

Efficacy of two extra-label anthelmintic formulations against equine strongyles in Cuba.

Equine cyathostomin parasites are ubiquitous in grazing horses and have been shown to cause severe inflammatory disease in the large intestine of horses. Decades of intensive anthelmintic therapy have led to widespread anthelmintic resistance in cyathostomins across the world. In Cuba, no anthelmintic products are formulated and sold for equine usage and little is known about anthelmintic efficacy of ruminant and swine formulations used. A strongyle fecal egg count reduction test was used to assess the efficacy of a liquid formulation of ivermectin labelled for use in swine, ruminants and carnivores and a pelleted formulation of albendazole labelled for usage in ruminants. Nine farms in the province Camagüey were enrolled in the study comprising 149 horses in total. Albendazole efficacy was reduced on five farms and with the other four farms having no signs of reduced efficacy. Mean farm efficacies were ranging from 41.7% to 100% on the tested farms. Coprocultures found large strongyle larvae present on all farms, but all larvae identified post-treatment were cyathostomins. Ivermectin was found 100% efficacious on all studied farms. This study provided evidence of reduced albendazole efficacy in the study population. Further work is needed to evaluate whether these findings reflect true resistance or if they are due to pharmacokinetic or pharmacodynamic characteristics of the pelleted formulation tested here.

Nematocidal activity of extracellular enzymes produced by the nematophagous fungus Duddingtonia flagrans on cyathostomin infective larvae.

Duddingtonia flagrans produces chitinases, however, optimization of the production of these enzymes still needs to be explored, and its nematocidal activity should still be the subject of studies. The objective of the present study was to optimize chitinase production, and evaluate the nematocidal activity of extracellular enzymes produced by the nematophagous fungus D. flagrans on cyathostomin infective larvae. An isolate from D. flagrans (AC001) was used in this study. For the production of enzymes (protease and chitinase), two different culture media were inoculated with AC001 conidia. Both enzymes were purified. The statistical Plackett-Burman factorial design was used to investigate some variables and their effect on the production of chitinases by D. flagrans. After that, the design central composite (CCD) was used in order to determine the optimum levels and investigate the interactions of these variables previously observed. Only two variables (moisture and incubation time), in the evaluated levels, had a significant effect (p<0.05) on chitinase production. The conditions of maximum chitinase activity were calculated, with the following values: incubation time 2 days, and moisture 511%. The protease and chitinase derived from D. flagrans, individually or together (after 24h), led to a significant reduction (p<0.01) in the number of intact cyathostomin L3, when compared to the control, with following reduction percentage values: 19.4% (protease), 15.5% (chitinase), and 20.5% (protease+chitinase). Significant differences were observed (p<0.05) between the group treated with proteases in relation to the group treated with proteases+chitinases. In this study, the assay with the cyathostomins showed that chitinase had a nematocidal effect, suggesting that this enzyme acts on the "fungus versus nematodes" infection process. It is known that nematode eggs are rich in chitin, and in this case, we could think of a greater employability for this chitinase.

Evaluating the effectiveness of a formulation containing neem's pie in control of equine gastrointestinal nematode / Avaliação da eficácia de uma formulação comercial contendo torta de nim no controle de nematóides gastrintestinais de equinos

Challenges to control of gastrointestinal nematodes of equine is due, largely, to antihelminthic resistance observed in populations of cyathostominae and large strongyles. Few effort are accomplished to seek a alternative to the control this agents therefore of the employment of drugs. The present work had as aim test the efficacy of a phytoterapic formulation of neems pie on gastrointestinal nematodes of equine. For this, a formulation comprise 20 grams of neem for animal per six months added at cornmeal (100g per animal, daily) plus water ad libidum at animals in the treated group (A). The same was supplied at control animals (group B) except neems pie. The mineral mix was supplies at animals at dose 1g per animal daily. They were harvested sample of faeces directly from the rectum of the animals that were submitted at parasitological examination of flotation. After coprocultures, recovery larvae were identified by light microscopy. Mean number eggs of gastrointestinal nematodes per grams of faeces of equine of treated group was 89.50 and, in the control, was 255.71 (P< 0.001). It was counted and identified 100 recovered larvae of coprocultures per samples of faeces andit do not verified differences (P >0.05) among two groups (A and B) in the frequency of cyathostominae andlarge strongyles. After six months of application of neems pie, it was possible verify a reduction in number eggsper grams of faeces of the treated equine (group A) compared to animals of control group (B). (AU)

Os desafios para o controle dos nematóides gastrintestinais de equinos é devido, em grande parte, à resistência a antihelmínticos observada em populações dos ciatostomíneos e dos grandes estrôngilos. Poucos esforços são realizados na investigação de tratamento alternativo para o controle desses agentes em equinos, além do emprego de fármacos. Esse trabalho objetivou testar a eficácia de uma formulação de torta de nim no desses parasitos. Assim, foi fornecida, diariamente, uma formulação contendo 20 gramas de nim por animal por seis meses misturados a farinha de milho (100g/animal) ao grupo tratado (A), ao grupo controle foi fornecida mistura mineral na mesma quantidade que ao grupo tratado. A mistura mineral foi fornecida na dose de 1g por animal/dia por seis meses. Foram coletadas semanalmente amostras de fezes diretamente do reto desses animais que foram submetidas a exame parasitológico quantitativo de flutuação. Após a coprocultura e a recuperação das larvas, essas foram submetidas à identificação em microscopia de luz. A média do número de ovos de nematóides gastrintestinais por gramas de fezes dos equinos do grupo tratado foi de 89,50 ovos por gramas de fezes, e no controle, de 255,71 (P<0.001). Foram contadas e identificadas 100 larvas recuperadas das coproculturas por amostra de fezes e não foi verificada diferença (P>0.05) entre os dois grupos (A e B) na frequência de ciatostomíneos e grandes estrôngilos. Após seis meses de aplicação desse produto foi possível verificar redução no número de ovos por gramas de fezes dos equinos tratados (A) em relação ao controle (B). (AU)

Evaluating the effectiveness of a formulation containing neem's pie in control of equine gastrointestinal nematode / Avaliação da eficácia de uma formulação comercial contendo torta de nim no controle de nematóides gastrintestinais de equinos

Challenges to control of gastrointestinal nematodes of equine is due, largely, to antihelminthic resistance observed in populations of cyathostominae and large strongyles. Few effort are accomplished to seek a alternative to the control this agents therefore of the employment of drugs. The present work had as aim test the efficacy of a phytoterapic formulation of neem’s pie on gastrointestinal nematodes of equine. For this, a formulation comprise 20 grams of neem for animal per six months added at cornmeal (100g per animal, daily) plus water ad libidum at animals in the treated group (A). The same was supplied at control animals (group B) except neem’s pie. The mineral mix was supplies at animals at dose 1g per animal daily. They were harvested sample of faeces directly from the rectum of the animals that were submitted at parasitological examination of flotation. After coprocultures, recovery larvae were identified by light microscopy. Mean number eggs of gastrointestinal nematodes per grams of faeces of equine of treated group was 89.50 and, in the control, was 255.71 (P0.05) among two groups (A and B) in the frequency of cyathostominae andlarge strongyles. After six months of application of neem’s pie, it was possible verify a reduction in number eggsper grams of faeces of the treated equine (group A) compared to animals of control group (B).

Os desafios para o controle dos nematóides gastrintestinais de equinos é devido, em grande parte, à resistência a antihelmínticos observada em populações dos ciatostomíneos e dos grandes estrôngilos. Poucos esforços são realizados na investigação de tratamento alternativo para o controle desses agentes em equinos, além do emprego de fármacos. Esse trabalho objetivou testar a eficácia de uma formulação de torta de nim no desses parasitos. Assim, foi fornecida, diariamente, uma formulação contendo 20 gramas de nim por animal por seis meses misturados a farinha de milho (100g/animal) ao grupo tratado (A), ao grupo controle foi fornecida mistura mineral na mesma quantidade que ao grupo tratado. A mistura mineral foi fornecida na dose de 1g por animal/dia por seis meses. Foram coletadas semanalmente amostras de fezes diretamente do reto desses animais que foram submetidas a exame parasitológico quantitativo de flutuação. Após a coprocultura e a recuperação das larvas, essas foram submetidas à identificação em microscopia de luz. A média do número de ovos de nematóides gastrintestinais por gramas de fezes dos equinos do grupo tratado foi de 89,50 ovos por gramas de fezes, e no controle, de 255,71 (P0.05) entre os dois grupos (A e B) na frequência de ciatostomíneos e grandes estrôngilos. Após seis meses de aplicação desse produto foi possível verificar redução no número de ovos por gramas de fezes dos equinos tratados (A) em relação ao controle (B).

Parasitismo por estrongilos en caballos salvajes Venezolanos del hato el frío (Edo. Apure, Venezuela): Estudio preliminar / Strongyles parasitism in wild Venezuelan horses from hato el frío, State of Apure, Venezuela: A preliminary study

Los estróngilos son los parásitos más comunes en los caballos, causando serios problemas aún en aquellos animales que reciben tratamiento antihelmíntico. Existe en el país escaso conocimiento del comportamiento de este parasitismo en caballos salvajes cuyas poblaciones de helmintos no están influenciadas por el uso de fármacos antiparasitarios, por lo que el objetivo del presente estudio fue determinar las características de la infección por nematodos Strongylidae en caballos salvajes venezolanos no sometidos a tratamiento antihelmíntico en base a examen coprológico. El estudio se realizó en Abril 2006 en el Hato El Frío, ubicado cerca de Mantecal, estado Apure, Venezuela. Se tomó una muestra de heces directamente del recto a un total de 35 caballos salvajes (21 machos, 14 hembras, representando el 15% de la población), las cuales se procesaron por la técnica de Mc Master modificada y se realizaron cultivos a partir de 4 grupos (pool) de muestras positivas para la identificación de las larvas Strongylidae de tercer estado. Se obtuvo una prevalencia general de 82,86% (29/35) con un promedio general de 818,57 huevos por gramo de heces (hpg) [0-2.300]. De los animales examinados, el 25,71% tenía entre 500 y 1.000 hpg, mientras que 37,14% tenía más de 1.000 hpg. Strongylus vulgaris fue encontrado en 3 de 4 coprocultivos (75%) en porcentajes de 2, 12 y 7%, respectivamente. S. edentatus fue encontrado en 1 de 4 coprocultivos (25%) en porcentaje del 2%. La presencia de grandes estróngilos (S. vulgaris, S. edentatus) encontrados en los coprocultivos fue bastante marcada, así como el significativo número de animales con contajes superiores a 1.000 hpg.

Strongyles are the most common parasites in horses, which can cause serious health problems, even in regularly dewormed horses. There is practically no information in this country on this parasitic infection in wild horses which helminth populations are not influenced by using of antiparasitic drugs. So, the aim of this research was to determinate the caracteristics of Strongylidae nematode infection in wild Venezuelan horses which have never received anthelmintic therapy, on the basis of coprological examination. Study was performed in April 2006 in “Hato El Frío” near to Mantecal, State of Apure, Venezuela. Faecal samples were taken from the rectum, once from each of thirty five randomly selected wild horses (21 males, 14 females), representing approximately 15% of the total population of the herd, and processed using the McMaster modified technique. Four cultures of bulked positive samples for identification of Strongylidae larvae were also performed. General prevalence was 82.86% (29/35), with a general epg mean of 818,57 epg (0-2,300). From the animals sampled 25,71% showed epg counts between 500-1,000 epg but 37,14% had more than 1,000 epg. Strongylus vulgaris was found in 3 out of 4 cultures (75%) in a proportion of 2, 12 and 7%, respectively. Strongylus edentatus was found in 1 out of 4 cultures (25%) in a proportion of 2%. There must be remarkable the presence of large strongyles in cultures as well as the important number of animals harboring more than 1,000 epg.

Seasonal abundance of equine strongyles (Nematoda: Strongylidae) in the State of Rio de Janeiro, Brazil / Abundância sazonal de estrongilídeos de eqüinos (Nematoda: Strongylidae) no Estado do Rio de Janeiro

The seasonal abundance of lumenal and larval forms of equine strongyles in the State of Rio de Janeiro, Brazil was evaluated. Thirty horses referred to the Federal Rural University of Rio de Janeiro were necropsied and their gastrointestinal tracts were separated into stomach, small intestine, cecum, ventral colon, dorsal colon and rectum. Two aliquots of 5% of the content were collected and examined for the presence of small strongyles and the rest of the content was examined for the presence of large strongyles. The mesenteric artery, liver, pancreas and peritoneum were examined for the presence of strongyle larvae. The mucosa of intestine segments was cut into parts of equal length, and each one was examined by the mural transillumination technique. Higher abundances of larvae of Strongylus equinus (P<0.10) and of adults Strongylus vulgaris (P<0.05) were observed during the dry season. Encysted cyathostome larvae had the highest abundance during the rainy season (P<0.02).(AU)

Estudou-se a ocorrência sazonal de formas larvares e adultas de estrongilídeos nas mucosas do estômago, intestino delgado, ceco, colon ventral, colon dorsal e reto de 30 eqüídeos no Estado do Rio de Janeiro. Os grandes estrongilídeos foram encontrados em todas as mucosas e duas alíquotas de 5% do conteúdo foram retiradas para a procura de pequenos estrongilídeos. Artéria mesentérica, fígado, pâncreas e peritônio foram também examinados para larvas de estrongilídeos. As mucosas foram examinadas pela técnica de transiluminação. Larvas de Strongylus equinus (P<0,10) e adultos de Strongylus vulgaris (P<0,05) foram mais abundantes na estação seca. Larvas escistadas de ciatostomíneos o foram na estação chuvosa (P<0,02).(AU)

Hematologic parameters in horses naturally infected with strongyles / Valores hematológicos em eqüinos naturalmente infectados por estrongilídeos

In order to verify the possible hematologic alterations related to intestinal parasitism in horses, 84 mixed breed animals (42 males, 42 females) were selected, with age varying from 2 to 15 years, used in the daily routine work of urban traction, with history of weight loss and less resistance to physical effort, and with a diagnoses of intense natural infection with strongyles. The fecal egg count per grams of feces (EPG) showed a variation from 350 to 6.500 EPG (Md 1.425 EPG) characterizing a considerable degree of infection with adult strongyles. The hematologic parameters were erithrocytes 6.43 ± 0,90 x 106/µL; hemoglobin 10,62 ± 1,63 g/dL; PCV 30,98 ± 4,08 %; MCV 48,38 ± 3,14 fL; MCHC 34,51 ± 4,00 %; leucocytes 12.495 ± 2.776,26 /µL; band neutrophils 28,98 ± 114,43/µL; mature neutrophils 7.970,77 ± 2.534,33/µL; lymphocytes 3.921 ± 1.585,29/µL; monocytes 128,60 ± 187,10/µL and eosinophils 434,33 ± 398,07/µL. These values showed a leucocytosis with neutrophilia, as well as a tendency of erythrocytic values in staying close to the normal inferior limits of the reference range for the specie. No alterations regarding eosinophylic values were observed, thus partly contradicting the concept that eosinophilia should occur with intestinal parasitism in horses.

Com objetivo de verificar as possíveis alterações hematológicas relacionadas ao parasitismo intestinal em eqüinos selecionaram-se 84 animais (42 machos, 42 fêmeas), mestiços, com idade entre 2 e 15 anos, utilizados em trabalho diário de tração urbana, com histórico de emagrecimento e menor resistência ao esforço físico, diagnosticando-se apenas elevada infecção natural por estrongilídeos. A contagem de ovos por gramas de fezes (OPG) mostrou uma variação de 350 a 6.500 OPG (Md = 1.425 OPG) caracterizando um grau considerável de infecção por formas adultas de estrongilídeos, sendo o quadro hematológico representado pelos valores médios a seguir; Eritrócitos: 6,43 ± 0,90 x 106/µL; Hb: 10,62 ± 1,63 g/dL; Ht: 30,98 ± 4,08 %; VCM: 48,38 ± 3,14 fL; CHCM: 34,51 ± 4,00 %; Leucócitos: 12.495,83 ± 2.776,26/µL; Bastonetes: 28,98 ± 114,43/µL; Neutrófilos: 7.970,77 ± 2.534,33/µL; Linfócitos: 3.921,62 ± 1.585,29/µL; Monócitos: 128,60 ± 187,10/µL e Eosinófilos: 434,33 ± 398,07/µL. Tais achados apontam a existência de leve leucocitose por neutrofilia, assim como uma tendência do quadro eritrocitário em se manter próximo aos limites inferiores de normalidade considerados para a espécie. Não se observou, por fim, qualquer alteração quanto aos valores de eosinófilos, contradizendo em parte o conceito de que a eosinofilia deva acompanhar os quadros de parasitismo intestinal na espécie eqüina.

Hematologic parameters in horses naturally infected with strongyles / Valores hematológicos em eqüinos naturalmente infectados por estrongilídeos

In order to verify the possible hematologic alterations related to intestinal parasitism in horses, 84 mixed breed animals (42 males, 42 females) were selected, with age varying from 2 to 15 years, used in the daily routine work of urban traction, with history of weight loss and less resistance to physical effort, and with a diagnoses of intense natural infection with strongyles. The fecal egg count per grams of feces (EPG) showed a variation from 350 to 6.500 EPG (Md 1.425 EPG) characterizing a considerable degree of infection with adult strongyles. The hematologic parameters were erithrocytes 6.43 ± 0,90 x 106/µL; hemoglobin 10,62 ± 1,63 g/dL; PCV 30,98 ± 4,08 %; MCV 48,38 ± 3,14 fL; MCHC 34,51 ± 4,00 %; leucocytes 12.495 ± 2.776,26 /µL; band neutrophils 28,98 ± 114,43/µL; mature neutrophils 7.970,77 ± 2.534,33/µL; lymphocytes 3.921 ± 1.585,29/µL; monocytes 128,60 ± 187,10/µL and eosinophils 434,33 ± 398,07/µL. These values showed a leucocytosis with neutrophilia, as well as a tendency of erythrocytic values in staying close to the normal inferior limits of the reference range for the specie. No alterations regarding eosinophylic values were observed, thus partly contradicting the concept that eosinophilia should occur with intestinal parasitism in horses.

Com objetivo de verificar as possíveis alterações hematológicas relacionadas ao parasitismo intestinal em eqüinos selecionaram-se 84 animais (42 machos, 42 fêmeas), mestiços, com idade entre 2 e 15 anos, utilizados em trabalho diário de tração urbana, com histórico de emagrecimento e menor resistência ao esforço físico, diagnosticando-se apenas elevada infecção natural por estrongilídeos. A contagem de ovos por gramas de fezes (OPG) mostrou uma variação de 350 a 6.500 OPG (Md = 1.425 OPG) caracterizando um grau considerável de infecção por formas adultas de estrongilídeos, sendo o quadro hematológico representado pelos valores médios a seguir; Eritrócitos: 6,43 ± 0,90 x 106/µL; Hb: 10,62 ± 1,63 g/dL; Ht: 30,98 ± 4,08 %; VCM: 48,38 ± 3,14 fL; CHCM: 34,51 ± 4,00 %; Leucócitos: 12.495,83 ± 2.776,26/µL; Bastonetes: 28,98 ± 114,43/µL; Neutrófilos: 7.970,77 ± 2.534,33/µL; Linfócitos: 3.921,62 ± 1.585,29/µL; Monócitos: 128,60 ± 187,10/µL e Eosinófilos: 434,33 ± 398,07/µL. Tais achados apontam a existência de leve leucocitose por neutrofilia, assim como uma tendência do quadro eritrocitário em se manter próximo aos limites inferiores de normalidade considerados para a espécie. Não se observou, por fim, qualquer alteração quanto aos valores de eosinófilos, contradizendo em parte o conceito de que a eosinofilia deva acompanhar os quadros de parasitismo intestinal na espécie eqüina.