Temporary Immunity of Blackbelly Lambs Reinfected with Trichostrongylus colubriformis

Background: Trichostrongylus colubriformis is one of the most prevalent gastrointestinal nematodes of sheep in warm climates. In this nematode species, anthelmintic resistance (AR) has been reported, which has motivated the search for alternative methods to control it. One practice is to increase the immune response level through the selection of animals with natural resistance, such as Blackbelly sheep. The aim of this study was to investigate the response of IgA and IgG in serum and saliva and the level of cells involved in the acquired resistance in Blackbelly lambs after single artificial reinfection with a resistant strain of Trichostrongylus colubriformis.Materials, Methods & Results: Sixteen weaned lambs grazed for one month in nematode-contaminated grasslands. All the lambs were treated with Albendazole and Levamisole; later, they were stabled for two months. After that, eight lambs were re-infected with 6000 larvae of T. colubriformis and other eight lambs remained naturally infected. In addition, eight nematode-free lambs raised in cages, served as negative control. Blood samples were taken fortnightly to determine packed cell volume (PCV, %), plasmatic protein (g dL-1), as well as absolute and differential leukocytes. The eggs per gram of feces (EPG) were recorded. An indirect ELISA against T. colubriformis Ag was performed to determine the IgG and IgA levels. All data were analyzed by repeated measures. The EPG in the re-infected group increased after 34 days (625 ± 287), while in the naturally infected group it remained close to 200 ± 158 throughout the study. The control lambs did not show any infection. The PCV (29.4 ± 3.4 %), basophils (44 ± 74), lymphocytes (8443 ± 2845) and monocytes (77 ± 91) counts were not affected by infection. The IgA OD against T. colubriformis increased significantly after 15 days post-infection (dpi) in the re-infected lambs (1.69 ± 0.5, 80% with regards to standard). With IgG was not possible to distinguish the effect of infection.Discussion: Highly persistent infection of T. colubriformis was observed for at least four months in the naturally infected group despite having been treated with anthelmintics. It is a significant problem because this species has a high prevalence in the region of study and shows anthelmintic resistance. For such reason, is important to evaluate the ability of the animal to develop an immune response. In other study, a natural reduction in the fecal egg count (FEC) was indicated at 100 dpi in the same species, but in the present work, the FEC could not be reduced until around 120 days when the naturally infected lambs were evaluated. The larger number of neutrophils and leukocytes in both the naturally infected and re-infected groups implies a sustained response for a long time. While the higher counts of eosinophils only in the re-infected group were produced as an immediate response to infection with T. colubriformis. Similarly, with a single infection of T. colubriformis, in Santa Ines sheep the infected lambs showed significantly higher levels of serum IgA than the control group. In this case, differences in IgA activity were observed between the re-infected lambs and the other groups at fifteen dpi, but the basal levels of IgA in the naturally pre-infected lambs were maintained at least five weeks after infection. In conclusion, an increase in the peripheral immune response in lambs re-infected with T. colubriformis resulted from the increase in IgA levels in serum and saliva, and increase in the number of peripheral eosinophils, total leukocytes and neutrophils. Re-infected lambs had a higher level of IgA than naturally infected lambs and control lambs, making possible to promote the immune response with a single re-infection with Trichostrongylus colubriformis

In vitro larvicidal effect of a hydroalcoholic extract from Acacia cochliacantha leaf against ruminant parasitic nematodes

Larvicidal effect was evaluated using acacia cochliacantha leafThe aim of this study was to evaluate the in vitro lethal effect of a hydroalcoholic extract (HAE) from Acacia cochliacantha leaf against three gastrointestinal nematodes species (Haemonchus contortus, H. placei and Cooperiapunctata) of domestic ruminants. The HAE was assessed using five concentrations: 100, 125, 175, 150 and 200 mg/ml; 0.5% Ivermectin was used as a positive control and distilled water, as negative control. The data were normalized using the square root and analysed with a completely randomized design through ANOVA analysis using the general linealmodel (GLM) of the SAS program. The HAE tannin conten was determined through spectrophotometry (UV-visible) and the other major phenols, were identified by chromatographic processes. The results showed an in vitro larvicidal activity of the HAE against the three assessed nematode species with all assessed concentrations. A clear HAE increased concentration dependence effect was observed. The highest activity of the HAE was obtained at the highest concentration (close to 100%, P < 0.05). This result was similar to the one obtained with Ivermectin. On the other hand, the chemical analysis of HAE showed the presence of tannins, caffeoyls and coumaroyl derivates and quercetin as the main compounds. The results suggest that the HAE from this plant species pos- sess in vitro anthelmintic properties. The identified com- pounds in this study would good candidates for further in vivo researches.This study received financial support from CONACYT-Mexico (Project number 251244), INIFAP (Project number 9454133183, through the Researcher Retaining Program)

Efecto del hipoclorito de sodio y extracto de cítricos en la reducción de la infestación con nematodos gastrointestinales resistentes a antihelmínticos en ovinos de pelo

The study included two experiments. The objective of experiment one was to learn about the in vitro effect of different sodium hypochlorite concentrations and a commercially-available citric extract on the survival of infestant larvae of gastrointestinal nematodes by observation at different time points. With >0.05% sodium hypochlorite concentrations, larvae lost their sheaths in two minutes, while at 1.3% concentration larvae died within one hour. Larval mortality also occurred as late as 12 h after exposure to the concentrations tested of citric extract.El estudio se realizó en dos experimentos. En el primero el objetivo fue conocer el efecto in vitro de diferentes concentraciones de hipoclorito de sodio y de un extracto de cítricos comercial sobre la sobrevivencia de larvas infestantes de nematodos gastrointestinales, mediante observación a diferentes tiempos. A concentraciones mayores al 0.05 % de hipoclorito de sodio, las larvas se desenvainaron en dos minutos y con concentraciones de 1.3 % las larvas murieron en una hora. Con el extracto de cítricos se observó mortalidad larvaria hasta las 12 h en todas las concentraciones utilizadas

Evaluación antihelmíntica de cuatro extractos de árboles forrajeros contra el nematodo Haemonchus contortus bajo condiciones in vitro

The objective was to evaluate the nematocidal effect of four hydroalcoholic extracts (HAE) of Brosimum alicastrum (HAE-Ba), Guazuma ulmifolia (HAE-Gu), Erythrina americana (HAE-Ea) and Leucaena leucocephala (HAE-Ll) against Haemonchus contortus. The tests of egg hatching inhibition (EHI) and larval (infective larvae) mortality were used. The treatments were HAEs at concentrations of 6.25-50 mg/mL for EHI and 25-100 mg/mL for larval mortality, ivermectin (5 mg/mL, positive control) and distilled water (negative control). Data were analyzed using an ANOVA and treatments with a concentration-dependent effect were subjected to a regression analysis to determine lethal concentrations (LC50 and LC90). In addition, a phytochemical analysis was performed on the extracts to identify the presence of the main secondary metabolites. The best ovicidal and larvicidal activity was observed in HAE-Gu with 96.78 % EHI at 6.25 mg/mL and 57.2 % larval mortality at 75 mg/mL. Followed by HAE-Ba showing 90 % EHI at 6.25 mg/mL and 58.0 % larval mortality at 75 mg/mL. The LC50 and LC90 of HAE-Gu on EHI were 2.7 and 4.4 mg/mL, respectively. While the LCs of this same extract on larvae were LC50= 64 and LC90= 125 mg/mL. The phytochemical analysis indicates that all extracts contain tannins, coumarins, flavonoids and terpenes. The fodder species G. ulmifolia and E. americana could be candidate plants for the control of H. contortus.El objetivo del presente estudio fue evaluar el efecto nematicida de cuatro extractos hidroalcohólicos (EHA) de Brosimum alicastrum (EHA-Ba), Guazuma ulmifolia (EHA-Gu), Erythrina americana (EHA-Ea) y Leucaena leucocephala (EHA-Ll) contra Haemonchus contortus. Se usaron las pruebas de inhibición de la eclosión de huevos (IEH) y mortalidad larval (larvas infectantes). Los tratamientos fueron los EHA´s a concentraciones de 6.25-50 mg/ml para IEH y de 25-100 mg/mlpara mortalidad larval, ivermectina (5 mg/ml, control positivo) y agua destilada (control negativo). Los datos se analizaron mediante un ANOVA y los tratamientos con efecto dependiente a la concentración se sometieron a un análisis de regresión para determinar las concentraciones letales (CL50 y CL90).  Además, se realizó un análisis fitoquímico a los extractos para identificar la presencia de los principales metabolitos secundarios.  La mejor actividad ovicida  y larvicida fue observada en  EHA-Gu con  un 96.78 % de IEH a 6.25 mg/ml y 57.2 % de mortalidad larval a 75 mg/ml. Seguido de EHA-Ba mostrando 90 % IEH a 6.25 mg/mly un 58.0 % de mortalidad larval a 75 mg/ml. Las CL50 y CL90 del EHA-Gu sobre la IEH fueron 2.7 y 4.4 mg/ml, respectivamente. Mientras que las CL´s de este mismo extracto sobre larvas fue de CL50=64 y CL90=125 mg/ml. El análisis fitoquímico indica que todos los extractos contienen taninos, cumarinas, flavonoides y terpenos. Las especies forrajeras G. ulmifolia y E. americana podrían ser plantas candidatas para el control de H. contortus

Digestibility of Duddingtonia flagrans chlamydospores in ruminants: in vitro and in vivo studies

<p>Abstract</p> <p>Background</p> <p>The use of <it>Duddingtonia flagrans </it>as a tool for the biological control of gastrointestinal nematodes (GIN) is a promising alternative to anthelmintics. The chlamydospores of <it>D. flagrans </it>are orally dosed and their thick cell wall gives them the capacity to resist digestion and pass through the gastrointestinal tract (GIT). Chlamydospores reaching the faeces are able to germinate and trap nematode larvae. The efficacy of this control method is based on reducing the numbers of infective larvae leaving the faeces. Techniques have recently been developed for quantifying the numbers of chlamydospores in faeces. As the number of non-digested spores could be relevant in the design and optimization of dosing programmes for the control of GIN infective larvae, the aim of the present study was to estimate the loss of <it>D. flagrans </it>chlamydospores during their passage through the ruminant gastrointestinal tract using <it>in vitro </it>and <it>in vivo </it>techniques.</p> <p>Results</p> <p>After <it>in vitro </it>rumen digestion, chlamydospore recovery was not different from the quantity originally incubated (undigested spores) (P > 0.05). <it>In vitro </it>rumen+abomasum digestion caused nearly 36% loss of the chlamydospores originally incubated (P < 0.05). Germination of chlamydospores classified as viable was 24.3%. Chlamydospores classified as non-viable did not germinate. Rumen digestion resulted in more spore germination (R1 = 35.7% and R2 = 53.3%) compared to no digestion (time 0 h = 8.7%). Subsequent abomasal digestion reduced germination (R1+A = 25%) or stopped it (R2+A = 0%). <it>In vivo </it>apparent chlamydospore digestibility in sheep showed a loss of 89.7% of the chlamydospores (P < 0.05).</p> <p>Conclusions</p> <p>The loss of chlamydospores was evident under <it>in vitro </it>and <it>in vivo </it>conditions. Negligible amounts of spores were lost during the <it>in vitro </it>rumen digestion. However, <it>in vitro </it>rumen+abomasum digestion resulted in a chlamydospore loss of approximately 36%. <it>In vivo </it>passage through the sheep GIT resulted in a total loss of 89.7% of the orally administered spores.</p

First report of multiple anthelmintic resistance in goat farm in Cuba

This study determined the efficacies of four classes of anthelmintics (AH) in a goat flock where anthelmintic resistance (AR) to ivermectin was suspected. We selected and randomly distributed 105 animals with >500 eggs per gram of feces into seven groups of 15 animals: an untreated con- trol group and groups treated with ivermectin, eprinomectin, albendazole sulfoxide, albendazole, levamisole, and closantel. The goats were individually weighed and treated with the recommended dose. Fecal samples were collected 14 days post-treatment to determine the fecal egg counts and for a fecal egg count reduction test (FECRT). Coprocultures were also performed for identifying any resistant genera. A molecular assay (polymerase chain reaction, PCR) was used to confirm benzimidazole resistance. The FECRTs for the ivermectin, eprinomectin, albendazole sulfoxide, and albendazole treatments were <90 %, indicating multiple anthelmintic resistance, all in Haemonchus spp. Levamisole had a FECRT confidence interval <90 %, indicating a moderate level of AR. The PCR detected the ß-tubulin alleles responsible for benzimidazole resistance, confirming AR. This study is the first monospecific report of AR in goats in Cuba, with a total failure of macrocyclic lac- tones anthelmintic class.The project was funded by the Cuban Ministry of Agriculture (Pro- ject No. P131LH001-0072)

Actividad antihelmíntica in vivo de hojas de Acacia cochliacantha sobre Haemonchus contortus en cabritos Boer

This study aimed to evaluate the effect of supplementing the maintenance diet of Boer goat kids with Acacia cochliacantha leaves. The endpoints evaluated were Haemonchus contortus fecal egg count (FEC) and water and dry matter intake. Two experimental treatments were evaluated on ten recently weaned goat kids (16.850 ± 1.630 kg of initial live weight and three months of age) experimentally infested with H. contortus larvae (L3) (350 larvae per live weight kilogram). Treatment 1 (T1) served as the control and consisted of infested animals without diet supplementation with A. cochliacantha leaves. Treatment 2 (T2) consisted of infested animals fed diets supplemented with 5% of A. cochliacantha leaves. Animals were grouped from highest to lowest based on their FEC. The two animals groups with the highest values were randomly assigned to T1 or T2; this was repeated until completing five repetitions per treatment. The evaluated variables were: FEC (per gram of feces), water intake, and dry matter intake (DMI). The results show that goat kids fed diets with 5% of A. cochliacantha leaves have lower (P 0.05). Se concluye que la adición de hojas de A. cochliacantha en dietas para cabritos tienen actividad antihelmíntica, por lo que esta leguminosa arbórea podría representar una opción en el manejo integral de la nematodiasis de cabritos Boer en crecimiento

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1–7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40μg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1–7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6μg/mL) compared to older larvae (P0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥−0.83; P <0.05) and EC50 (r≥−0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age

Fungal Antagonism Assessment of Predatory Species and Producers Metabolites and Their Effectiveness on Haemonchus contortus Infective Larvae

The objective of this study was to assess antagonism of nematophagous fungi and species producers metabolites and their effectiveness on Haemonchus contortus infective larvae (L 3 ). Assay A assesses the synergistic, additive, or antagonistic effect on the production of spores of fungal isolates of the species Duddingtonia flagrans, Clonostachys rosea, Trichoderma esau, and Arthrobotrys musiformis; Assay B evaluates in vitro the effect of intercropping of these isolates grown in 2% water-agar (2% WA) on L 3 of H. contortus. D. flagrans (Assay A) produced 5.3 × 10 6 spores and associated with T. esau, A. musiformis, or C. rosea reduced its production by 60.37, 45.28, and 49.05%, respectively. T. esau produced 7.9 × 10 7 conidia and associated with D. flagrans, A. musiformis, or C. rosea reduced its production by 39.24, 82.27, and 96.96%, respectively. A. musiformis produced 7.3 × 10 9 spores and associated with D. flagrans, T. esau, or C. rosea reduced its production by 99.98, 99.99, and 99.98%, respectively. C. rosea produced 7.3 × 10 8 conidia and associated with D. flagrans, T. esau, or A. musiformis reduced its production by 95.20, 96.84, and 93.56%, respectively. These results show evidence of antagonism in the production of spores between predators fungi