Efficacy of a single-dose albendazole against lancet liver fluke Dicrocoelium dendriticum and liver enzymes activity in naturally infected sheep.

Infections with D. dendriticum are distributed worldwide and mostly associated with ruminant livestock. Depending on the length and strength of the infection it can be manifested with losses in milk production, reductions in milk and wool quality, decreased weight gains, reproductive performance and poor carcass quality. The objective of this study was to determine the efficacy of albendazole (ABZ) against the lancet liver fluke Dicrocoelium dendriticum in naturally infected sheep using parasitological methods. Twenty-four sheep were divided into four groups: two untreated control groups (C1, C2) and two treated groups (T1, T2), with six animals in each group. The sheep in the treated groups were administered a single oral dose (15 mg/kg bwt) of ABZ suspension. After ABZ treatment the animals were slaughtered on Day 14 (groups C1, T1) and Day 30 (groups C2, T2) and were necropsied. Coprological therapeutic ABZ efficacy reached 92.4% on Day 14 (P < 0.001) and 88.5% on Day 30 (P < 0.001). On Day 30, the serum activities of hepatic and cholestatic enzymes including serological analysis of total protein concentration (TP) and protein fractions were evaluated. Significant decrease of aspartate aminotransferase (AST) (P < 0.01) and gamma-glutamyltransferase (GGT) (P < 0.05) activity by 36.9% and 34.6%, respectively, were detected for sheep in T2 group. These enzymes showed a strong positive correlation to fluke burden: AST (r = 0.654) and GGT (r = 0.768), respectively (P < 0.05). Additionally, the electrophoretic analysis of serum total protein and protein fraction concentrations revealed minimal hypoproteinemia and hyperalbuminemia after ABZ treatment. The decrease of liver enzyme activities and their correlation with fluke burden may indicate recovery of hepatocellular and biliary damage following the reduction of fluke burdens after ABZ therapy. A decline in AST and GGT activity could serve as a valuable adjunct bioindicator of liver damage and fluke reduction after treatment of dicrocoeliosis in naturally infected sheep.

Equine tapeworm (Anoplocephala spp.) infection: evaluation of saliva- and serum-based antibody detection methods and risk factor analysis in Slovak horse populations.

A lack of accurate information on the prevalence and distribution of Anoplocephala spp. infections on horse farms has led to insufficient attention to tapeworm control and increasing horse anoplocephaloses in Europe. Our study aimed to examine the occurrence of Anoplocephala spp. infection using coprological, serum- and saliva-based antibody detection methods and to analyze the risk factors associated with tapeworm infection in domestic horses in Slovakia. Fecal, serum, and saliva samples were collected from 427 horses from 31 farms in Slovakia. Additionally, a questionnaire study was conducted to collect information on tapeworm distribution on horse farms and analyze risk factors associated with infection. Fecal samples were examined by the mini-FLOTAC and the double centrifugation/combined sedimentation-flotation techniques. Serum and saliva samples were analyzed by ELISA to determine antibody levels against Anoplocephala spp. The effects of variables associated with an individual horse were tested for the positive result of the saliva ELISA test on Anoplocephala spp. Cestode eggs were detected in 1.99% of fecal samples (farm prevalence 12.90%), with no differences between the two coprological methods. Serum-based tapeworm ELISA results revealed that 39.39% of horses tested positive (farm prevalence 83.87%); while saliva-based tapeworm ELISA results revealed 56.95% positive horses (farm prevalence 96.77%). Binary logistic regression analysis revealed four meaningful predictors that significantly impacted the likelihood of detecting tapeworm infection in horses: horse age, pasture size, anthelmintic treatment scheme, and access to pasture. The influences of other variables associated with an individual horse were not significantly associated with detecting tapeworm infection.

Cage trapping and field anaesthesia of brown bears as part of nuisance bear management.

The population and distribution of the European brown bear (Ursus arctos) in Slovakia are expanding as bears were observed beyond the southern border of the country in Hungary. This study presents the authors' experience with field anaesthesia of wild brown bears trapped in a custom-made container trap and of free-ranging individuals. A total of 25 bears were captured and translocated using a specially designed metal cage trap. The study compared the effectiveness of three anaesthetic protocols in managing both free-ranging and trapped bears. For juveniles, or small adults up to 70 kg body weight (BW), ketamine-xylazine mixture was used at doses of 3.0-4.0 mg kg-1 ketamine and 1.0-1.5 mg kg-1 xylazine BW. The immobilisation of free-ranging bears, which are usually attracted by municipal solid garbage, was performed remotely using PneuDart darts with 2-3 ml of anaesthetics. For this purpose, tiletamine-zolazepam-detomidine (T-Z-D) was preferred at a dose of 1.7-2.5 (T) mg kg-1, 1.7-2.5 (Z) mg kg-1, and 0.1-0.2 (D) mg kg-1 BW. Induction time was from 7 to 18 min post darting with the average of 12.04 min. The same combination was applied to bears trapped in a container trap, with anaesthesia lasting from 40 to 150 min. If T-Z-D was used, no further anaesthetic was needed. In all cases, anaesthesia was antagonised by atipamezole at a dose of 0.15-0.225 mg kg-1 BW. Atipamezole was injected at a half dose intramuscularly and a half dose subcutaneously at the time when the palpebral reflex reappeared and the bear was able to move his tongue. It was shown that the T-Z-D mixture is a safe, low-volume anaesthetic darting protocol that is reversible, has minimal adverse effects on physiological parameters, and has a sufficient duration. The results can be used to manage large carnivore populations in the Carpathian region.

Resistance of strongylid nematodes to anthelmintic drugs and driving factors at Czech goat farms.

BACKGROUND: Strongylid nematode infections may negatively affect both animal health and welfare, with deleterious consequences for livestock productivity. Many farmers in recent decades have relied on anthelmintics as the sole strategy of control, but the intensive use of these chemotherapeutics has led to the development of anthelmintic resistance (AR). Knowledge of both the efficacy of anthelmintics and factors promoting AR are essential to effectively control nematode infections, but no information on these topics for goats in the Czech Republic (CR) is available. This survey aimed to determine the occurrence of AR at Czech goat farms and to identify risk factors for the development of AR. A total of 24 herds of dairy goats across the CR were evaluated using in vitro tests for detecting AR, and a questionnaire survey was carried out to evaluate factors associated with AR. RESULTS: Resistance against benzimidazoles was confirmed at 18 (75%) farms, and the level of resistance was high in four (22%) of the affected herds based on the egg hatch test. Ivermectin-resistant nematodes were detected in 13 (54%) herds using the larval development test; Teladorsagia/Trichostrongylus and Haemonchus were the predominant types of resistant larvae. Eight (62%) of the affected herds were evaluated as highly resistant to ivermectin. Eleven (46%) of the herds were resistant to both benzimidazoles and ivermectin. This report is the first on dual AR in the CR. A univariate logistic regression analysis indicated that a high stocking rate and farmer inexperience were significantly associated with ivermectin and benzimidazole resistance, respectively. CONCLUSIONS: The results of our survey suggest that AR is widespread amongst herds of dairy goats in the CR, likely due to inappropriate practices of pasture and health management. AR may be an issue for expanding dairy-goat production in the CR in the near future unless both veterinary practitioners and farmers widely adopt strategies to prevent the development of AR.

Prevalence of anthelmintic resistance of gastrointestinal nematodes in Polish goat herds assessed by the larval development test.

BACKGROUND: Helminthic infections, in particular those caused by gastrointestinal nematodes (GIN), are found worldwide and are among the most economically important diseases of goats. Anthelmintic resistance (AR) in GIN of goats is currently present worldwide, and single- or multidrug resistant species are widespread. The aim of this study was to determine the prevalence of AR to benzimidazoles (BZ), macrocyclic lactones (ML) and imidazothiazoles represented by levamisole (LEV) in the Polish goat herds by using an in vitro larval development test, which is useful especially in large-scale epidemiological surveys. RESULTS: This cross-sectional study was conducted from September 2018 to June 2019 and enrolled 42 dairy goat herds scattered over the entire country. The most commonly used anthelmintic class in goat herds in Poland were BZ (92%), followed by ML (85%) and LEV (13%). BZ-resistant GIN populations were found in 37 herds (88%, CI 95%: 75 to 95%), ML-resistant GIN populations in 40 herds (95%, CI 95, 84 to 99%), and LEV-resistant GIN populations in 5 herds (12%, CI 95%: 5 to 25%). Multidrug resistance involving all three anthelmintic classes was found in 5 herds (12%, CI 95, 5 to 25%). Based on the morphological features of stage 3 larvae the main resistant GIN turned out to be Haemonchus contortus and Trichostrongylus spp. The use of BZ and frequency of anthelmintic treatments were significantly related to the presence of AR to BZ in Polish goat herds. CONCLUSIONS: This cross-sectional study demonstrates the existence of AR to BZ, ML and LEV on Polish goat farms. Resistance to BZ and ML is widespread, while AR to LEV is currently at a low level. A considerable proportion of herds harbours multidrug resistant GIN, which requires further consideration. An effective anthelmintic treatment strategy, reasonable preventive measures and better understanding of the resistance-related management practices by farmers and veterinarians may delay further development of AR.

Experimental evidence for the lack of sensitivity of in vivo faecal egg count reduction testing for the detection of early development of benzimidazole resistance.

The objective of this study was to compare the results of an in vitro egg hatch test (EHT), micro-agar larval development test (MALDT) and in vivo faecal egg count reduction test (FECRT) between worm strains obtained from goats and sheep identically infected with the gastrointestinal parasitic nematode Haemonchus contortus. Results from the in vivo and in vitro tests were compared with benzimidazole (BZ)-resistance-associated ß-tubulin allele frequencies determined using Pyrosequencing™. BZ resistance was not detected by the in vivo FECRT, where reductions of > 99% for both the resistant and the susceptible H. contortus strains were detected in both species. Discriminating doses in EHT and MALDT for the resistant strain indicated a low level (approx. 25%) of resistant individuals. Genotyping indicated that the susceptible strain had 10% BZ-resistant ß-tubulin codon 200 alleles and the resistant strain had 26% respective resistant alleles. The in vitro tests and allele-frequency distribution suggested low levels of resistance in both strains; however, the FECRT did not support the evidence of resistant individuals of either strain in either species, suggesting a potential underestimation of low-level resistance in sheep and goats when employing this test.

The first report of multidrug resistance in gastrointestinal nematodes in goat population in Poland.

BACKGROUND: Prophylactic anthelmintic treatment with one of three basic classes of anthelmintics (benzimidazoles, macrocyclic lactones and imidazothiazoles) is still the mainstay of control of gastrointestinal nematode infections in small ruminants worldwide. As a consequence, anthelmintic resistance is a serious threat to small ruminant health and production. While the resistance to one class of anthelmintics has already been reported in most of countries, the newly-emerging problem is the resistance to two or even all of classes referred to as multidrug resistance. This study aimed to evidence the presence of multidrug resistance of gastrointestinal nematodes in goats in Poland. RESULTS: The combination of one in vivo method (fecal egg count reduction test) and two in vitro methods (egg hatch test and larval development test) performed in two goat herds in the southern Poland showed the presence of gastrointestinal nematodes resistant to fenbendazole and ivermectin in both herds. Moreover, in one herd it revealed the development of resistance to the last effective anthelmintic, levamisole, in response to one-year intensive use. Haemonchus contortus was the most prevalent gastrointestinal nematode in samples in which resistance to benzimidazoles and ivermectin was found, whereas Trichostrongylus colubriformis predominated when resistance to levamisole was observed. CONCLUSION: This study shows for the first time that multidrug resistance of gastrointestinal nematodes to three basic classes of anthelmintics is already present in goat population in Poland. Moreover, it may indicate that different species or genera of gastrointestinal nematodes are responsible for the resistance to specific anthelmintics.

Assessment of the F200Y mutation frequency in the ß tubulin gene of Haemonchus contortus following the exposure to a discriminating concentration of thiabendazole in the egg hatch test.

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.

Natural chemotherapeutic alternatives for controlling of haemonchosis in sheep.

BACKGROUND: Parallel in vitro and in vivo experiments were designed to evaluate promising chemotherapeutic alternatives for controlling haemonchosis in ruminants. In vitro anthelmintic activities (egg hatch test - EHT; larval development test - LDT) of aqueous and methanolic herbal extracts Mix1 and Mix2 were investigated. The in vivo effects of dietary supplementation with Mix1 and Mix2 on the parasitological status, inflammatory response, antioxidant parameters and microbial community of the lambs infected experimentally with Haemonchus contortus were investigated. Lambs were divided into four groups for the in vivo study: uninfected control lambs (C), infected lambs (I), infected lambs supplemented with Mix1 (I + Mix1) and infected lambs supplemented with Mix2 (I + Mix2). The experimental period was 70 days. RESULTS: The number of eggs per gram (EPG) of feces was quantified 22, 30, 37, 44, 51, 58, 65 and 70 days post-infection, and mean abomasal worm counts were assessed 70 days post-infection. Quantitative analyses identified 57.3 and 22.2 mg/g phenolic acids, 41.5 and 29.5 mg/g flavonoids and 1.4 and 1.33 mg/g protoberberine-type alkaloids in Mix1 and Mix2, respectively. The methanolic extracts of the herbal mixtures in both in vitro tests had higher anthelmintic effects (P < 0.01) than the aqueous extracts, but the effects did not differ significantly between Mix1 and Mix2 (P > 0.05). I + Mix1 and I + Mix2 lowered mean EPGs between 44 and 70 d by 58.1 and 51.6%, respectively. The level of IgG antibodies against H. contortus increased significantly after infection in each infected group. CONCLUSION: These results represent the first monitoring of the in vitro anthelmintic effects of herbal mixtures on H. contortus. The in vivo experiment indicated that the anthelmintic effect was not sufficient for the elimination of parasites, but this herbal treatment may affect the host over a longer term, reducing the parasitic infection in the host.

Effects of herbal nutraceuticals and/or zinc against Haemonchus contortus in lambs experimentally infected.

BACKGROUND: The gastrointestinal parasitic nematode Haemonchus contortus is a pathogenic organism resistant to several anthelmintics. This study assessed the efficacy of a medicinal herbal mixture (Herbmix) and organic zinc, as an essential trace element for the proper functioning of both unspecific and specific immune defensive mechanisms, against experimental infections with H. contortus in lambs. All lambs were infected orally with approximately 5000 third-stage larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Twenty-four female lambs 3-4 months of age were divided into four groups: unsupplemented animals (control), animals supplemented with Herbmix (Hmix), animals supplemented with organic zinc (Zn) and animals supplemented with Herbmix and organic zinc (Hmix+Zn). Eggs per gram (EPG) of faeces were quantified 20, 28, 35, 42, 49, 56, 62 and 70 d post-infection and mean abomasal worm counts were assessed 70 d post-infection. Samples of blood were collected from each animal 7, 35, 49 and 70 d post-infection. RESULTS: Quantitative analyses of the bioactive compounds in Herbmix identified three main groups: flavonoids (9964.7 µg/g), diterpenes (4886.1 µg/g) and phenolic acids (3549.2 µg/g). Egg counts in the lambs treated with Hmix, Zn and Hmix+Zn decreased after 49 d. The EPGs in the Zn and Hmix+Zn groups were significantly lower on day 56 (P < 0.05 and P < 0.01, respectively), and the EPGs and mean worm counts were significantly lower on day 70 in all supplemented groups (P < 0.05 and P < 0.01). Hemograms of complete red blood cells of each animal identified clinical signs of haemonchosis after day 35. Serum calprotectin concentrations and IgA levels were significantly affected by treatment. The treatment influenced serum malondialdehyde concentrations (P < 0.05) and sulfhydryl groups (P < 0.01) of antioxidant status. The mineral status was unaltered in all lambs. CONCLUSION: A direct anthelmintic impact on the viability of nematodes was not fully demonstrated, but the treatments with herbal nutraceuticals and zinc likely indirectly contributed to the increase in the resistance of the lambs to nematode infection.

Ovicidal and larvicidal activity of extracts from medicinal-plants against Haemonchus contortus.

The use of medicinal plants (MP) containing bioactive compounds is an alternative strategy to control of parasitic nematode of small ruminants Haemonchus contortus at various stages of their life cycle. The aims of this study were to determine the in vitro anthelmintic activity of both aqueous and methanolic extracts from 13 medicinal plants typical for Central Europe, and to determine quantity of selected plant secondary metabolites (PSMs) in the methanolic extracts. In vitro egg hatch test and larval development tests were conducted to determine the possible anthelmintic effects of methanolic and aqueous extracts of the roots of Althaea officinalis L., Petasites hybridus L. and Inula helenium L.; flowers of Malva sylvestris L. and Chamomilla recutita L.; leaves of Plantago lanceolata L. and Rosmarinus officinalis L.; seeds of Foeniculum vulgare Mill. and stems of Solidago virgaurea L., Fumaria officinalis L., Hyssopus officinalis L., Melisa officinalis L. and Artemisia absinthium L. on eggs and larvae of H. contortus. Ultra-performance liquid chromatography and tandem mass spectroscopy was used for quantifying six PSMs: gallic acid (GA), rutin (RU), diosmin (DI), hesperidin (HE), quercetin (QU) and kaempferol (KA). RU content of the most effective methanolic extracts was in the order: M. sylvestris (9.33 mg/g DM) > A. absinthium (6.10 mg/g DM) > C. recutita (0.42 mg/g DM). The highest concentration of QU (44.8 mg/g DM) and KA (6.59 mg/g DM) were detected in stems of F. officinalis comparing to the other evaluated plants. The most significant (p < 0.05) anthelmintic effects exhibited methanolic extracts of A. absinthium in both in vitro tests (i.e., egg hatch test and larval development test). Additionally, only two methanolic extracts of C. recutita and M. sylvestris were comparable to activity of A. absinthium using the larval development test. Wider spectrum of aqueous extracts exhibited stronger ovicidal activity in comparison to methanolic extracts. The similar trend was observed in evaluating of larvicidal activity of aqueous and methanolic plant extracts.

Molecular evidence of infection with air sac nematodes in the great tit (Parus major) and the captive-bred gyrfalcon (Falco rusticolus).

Serratospiculiasis is a parasitic disease caused by filariid nematodes of the genus Serratospiculum that parasitise the air sacs of various species of falcons, bald eagles and Cooper's hawks around the world. An infection with Serratospiculum was recently confirmed in a nonspecific host, the great tit, in Slovakia. Parasitic material from this host was fixed for molecular analysis. Nematode found in the air sacs from a captive-bred gyrfalcon was also stored. Analysis of small subunit (18S) ribosomal DNA (18S rDNA) gene indicated that sequences from Serratospiculum sp. and Serratospiculoides amaculata were closely related to a reference sequence from Serratospiculum tendo, in agreement with morphology. This study is the first to generate molecular data and infer the phylogenetic position of S. amaculata as the first representative of the genus Serratospiculoides.

Anthelmintic resistance in sheep gastrointestinal nematodes in Slovakia detected by in-vitro methods.

BACKGROUND: The intensive use of anthelmintics for the control of helminthic infections has resulted in the development of anthelmintic resistance, which has become a major practical problem in many countries. A variety of tests are available to monitor anthelmintic resistance but most of them are expensive, laborious and time consuming and therefore unpractical for large field surveys. The main aim of this survey was thus to detect the occurrence of benzimidazole (BZ) and macrocyclic lactone resistance on sheep farms in Slovakia by using novel and modified in vitro methods that are inexpensive, easy to use and quick and therefore practical for large surveys. RESULTS: BZ-resistant gastrointestinal nematodes were found on all 27 farms. Two farms (7.4%) had high levels of resistance (>40% of hatching), and 22 farms had low levels (<20% of hatching) of resistant nematodes. IVM-resistant populations were found on 14 of 49 sheep farms. The prevalence of BZ and IVM resistance has slightly increased on Slovak sheep farms during the last two decades. CONCLUSIONS: Both the BZ and IVM surveys indicated that resistance against anthelmintics was present on Slovak sheep farms. Resistance against the BZ class of anthelmintics had been stable for two decades, but a slight increase on IVM resistance was confirmed. Farmers must thus observe the preventive measures to avoid a faster onset of IVM resistance, otherwise the presence of resistant parasites and ineffective treatment may harm the economy of their farms.

Gastrointestinal nematodes of dairy goats, anthelmintic resistance and practices of parasite control in Northern Italy.

BACKGROUND: Gastrointestinal nematodes (GINs) are one of the main constraints to ruminant production worldwide. Anthelmintic resistance (AR) has been reported in goats throughout Europe, yet little is known about the AR status in Italy. The aims of the study were: i) determine the frequency of AR in GINs in goat flocks in Northern Italy, Italy, ii) survey goat farmers on the current practices of parasite control, iii) update the species composition of the gastrointestinal helminthofauna. Thirty three flocks were enrolled and 1288 individual fecal samples were collected. Based on the egg per gram (EPG), 15 flocks were selected to evaluate the presence of AR in GINs with the Fecal Egg Count Reduction Test (FECRT). A questionnaire surveyed 110 dairy goat farmers to acquire information about farm management and drenching practices against GINs. Further, the gastrointestinal tracts of 42 goats were analyzed. RESULTS: The FECRs indicated that five of the 15 flocks had problems of AR, which was identified in all two of the anthelmintic classes tested. Resistance and suspected resistance was found in 40% of the flocks selected for AR testing that were treated with benzimidazoles while 20% of the flocks treated with eprinomectin had resistant GINs. Teladorsagia/Trichostrongylus L3 were isolated from the post-treatment coprocultures of all flocks with resistance but not from the flock with suspected oxfendazole resistance. Treatments against helminths were performed once annually in 73.63% of the flocks, but 20.00% of farmers declared not regularly treating their goats every year. Annual treatments usually occurred in autumn or winter at dose rate for sheep. Te. circumcincta, H. contortus, Tr. colubriformis, Skrjabinema caprae and Oesophagostomum venulosum were the most abundant and prevalent species of the gastrointestinal tract. CONCLUSIONS: Strategies to prevent the development of AR should be widely adopted in Northern Italy. Further, farmers and practitioners should be educated about the importance of using the correct dose rates in goats. In addition, the presence of highly pathogenic GINs coupled with high worm burden in all sector of gastrointestinal tract and the prevalence values further suggest that improved diagnosis and active surveillance of GINs infection is needed.

Variation in allele frequencies in benzimidazole resistant and susceptible isolates of Haemonchus contortus during patent infection in lambs.

We evaluated the variation in the frequency of benzimidazole (BZ) resistance-associated alleles at codons 200, 167 and 368 (F200Y, F167Y, V368L) of the ß-tubulin isotype 1 gene during the patent period in isolates of Haemonchus contortus susceptible and resistant to BZ using pyrosequencing. Four lambs 5-6 months old were infected with 5000-6000 infective third-stage larvae (L3) of the susceptible MHco1 and the multi-resistant MHco4 isolates, respectively. Faecal samples were collected 28-times during 20-90 days post-infection (dpi). Coprocultures were subsequently prepared to produce L3 for genotyping. The frequency of the resistant allele (TAC) at codon 200 in MHco1 was lowest at 43 and 76 dpi with at each time point 0% and highest at 36 dpi with 10.85%, with a mean of 6.47% ± 2.39 and a coefficient of variation of 37.01%. The frequency of the TAC at codon 200 in MHco4 was lowest at 76 dpi with 25.6% and highest at 90 dpi with 49.25%, with a mean of 35.7% ± 4.42 and a coefficient of variation of 12.39%. No resistance alleles were detected in MHco1 at either codon 167 or 368. For MHco4 isolate, resistance alleles were detected only on codon 167 with a mean of 8.00% ± 4.83 and a mean coefficient of variation of 60.40%. Our results demonstrate the considerable variation in the frequency of resistant alleles in the susceptible and resistant isolates during the patent period. This variation should be considered when testing for the presence of BZ resistance in populations of gastrointestinal parasites, especially those with a low frequency of TAC.

Multidrug resistance in Haemonchus contortus in sheep - can it be overcome?

Introduction: Gastrointestinal nematodes pose a threat to animal health and affect farmers by negatively impacting farm management. Material and Methods: The study was conducted on a sheep farm with suspected reductions in the efficacies of anthelmintics. Efficacy was determined using in vivo faecal egg count reduction, in vitro egg hatch (EHT) and larval development (LDT) tests. In the first phase, 60 sheep were equally split into six groups. Group 1 received the recommended dose of albendazole (ALB), group 2 received the same after fasting for 24 h, group 3 received the dose divided into two halves at 6 h intervals, group 4 received a double dose of ALB, and group 5 received the recommended dose of ivermectin (IVM). Group 6 served as a control. The second phase of the experiment had two groups: one treated with levamisole (LEV) and a control group. Faecal samples were collected from all sheep. Results: No reduction of egg output was observed in the groups treated with single, double, or divided doses of ALB, but one of 13.7-16.9% was noted in the fasting group. Efficacy in the IVM group ranged from 31.50 to 39.97%. The mean concentrations sufficient to prevent 50% of the eggs from hatching in the in vitro EHT and the mean concentrations in which the development of larvae to the L3 stage was inhibited by 50% in the LDT exceeded established thresholds for benzimidazoles and IVM. Haemonchus contortus was the only species identified after treatment. The LDT did not indicate the presence of resistance to LEV. All animals treated with LEV were negative for eggs 10 d after treatment. Conclusion: Resistance to ALB and IVM in Haemonchus contortus was confirmed. Alternative approaches to improve the efficacies of benzimidazole did not sufficiently increase the efficacy, but LEV was an efficient anthelmintic treatment.

Antibody response and abomasal histopathology of lambs with haemonchosis during supplementation with medicinal plants and organic selenium.

We evaluated the effects of dietary supplementation with medicinal plants (Herbmix) or organic selenium (Selplex) on the immune responses and histopathology of lambs infected with Haemonchus contortus. Twenty-seven lambs were infected and reinfected with approximately 11,000 third-stage larvae of H. contortus during the experiment (on days 0, 49 and 77). Lambs were divided into two supplemented experimental groups (Herbmix and Selplex) and unsupplemented group (Control). The abomasal worm counts at necropsy on day 119 were lower for Herbmix (4230) and Selplex group (3220) than to the Control (6613) which resulted in 51.3% and 36.0% of reduction, respectively. The mean length of adult female worms was in the order Control > Herbmix > Selplex (2.1, 2.08, and 2.01 cm, respectively). The specific IgG response against adults was significantly affected by time (P < 0.001). Serum-specific and total mucus levels of IgA in the Herbmix group were highest on day 15. Mean levels of serum IgM against adults were influenced by treatment (P = 0.048) and time (P < 0.001). The Herbmix group had strong local inflammation in the abomasal tissue, with the formation of lymphoid aggregates and the infiltration of immune cells, but the tissues of the Selplex group had higher numbers of eosinophils, globule leukocytes, and plasma cells. The lymph nodes of each animal had reactive follicular hyperplasia due to the infection. Dietary nutritional supplementation with a mixture of medicinal plants or organic selenium could improve local immune responses and thus enhance the resistance of animals to this parasitic infection.

The metabolism of flubendazole and the activities of selected biotransformation enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics.

Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g. sheep and goat). The treatment of haemonchosis is complicated because of recurrent resistance of H. contortus to common anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug flubendazole (FLU) and the activities of selected biotransformation enzymes towards model xenobiotics in 4 different strains of H. contortus: the ISE strain (susceptible to common anthelmintics), ISE-S (resistant to ivermectin), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (resistant to all common anthelmintics). H. contortus adults were collected from the abomasums from experimentally infected lambs. The in vitro as well as ex vivo experiments were performed and analysed using HPLC with spectrofluorimetric and mass-spectrometric detection. In all H. contortus strains, 4 different FLU metabolites were detected: FLU with a reduced carbonyl group (FLU-R), glucose conjugate of FLU-R and 2 glucose conjugates of FLU. In the resistant strains, the ex vivo formation of all FLU metabolites was significantly higher than in the susceptible ISE strain. The multi-resistant WR strain formed approximately 5 times more conjugates of FLU than the susceptible ISE strain. The in vitro data also showed significant differences in FLU metabolism, in the activities of UDP-glucosyltransferase and several carbonyl-reducing enzymes between the susceptible and resistant H. contortus strains. The altered activities of certain detoxifying enzymes might protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.

Is the micro-agar larval development test reliable enough to detect ivermectin resistance?

To aid the diagnosis of anthelmintic resistance, a range of in vivo and in vitro techniques have been developed. Amongst in vitro techniques, the larval development test is the most widely employed. Six lambs were infected with susceptible (three) and ivermectin-resistant (three) isolates of Haemonchus contortus. The micro-agar larval development test (MALDT) was able to easily distinguish between susceptible and resistant isolates. Different proportions of resistant and susceptible eggs were subsequently incubated, i.e. development to the third larval stage occurred only in the resistant isolate. The percentage of resistant eggs ranged from 2 to 20.0 % of all eggs in the wells. In all cases, the MALDT was able to detect the presence of a minimum of 10 % of resistant worms amongst a susceptible background population. The probability was approximately 87 % of positively diagnosing a proportion of resistant worms of only 2-4 % within the population.

Anthelmintic resistance of horse strongyle nematodes to fenbendazole in Lithuania.

BACKGROUND: Control of strongyle infections presents a global challenge for horse practitioners due to the development of anthelmintic resistance (AR), however comprehensive information on AR in Lithuania is still lacking. The aim of this study was to assess the current situation of fenbendazole (FBZ) AR in horses at stable level in Lithuania. RESULTS: Faecal samples from 121 horses from six stables were examined using the Mini-FLOTAC method. Of these, 89 horses met the inclusion criteria that included strongyle faecal egg counts (FEC) exceeding 200 eggs per gram (EPG). Faecal egg count reduction tests (FECRTs) were performed in these. AR was evaluated at horse stable level based on faecal egg count reduction (FECR) and the lower limit of the 95% credible interval (LLCI) using the Bayesian hierarchical model. This study confirmed that strongylids (Cyathostominae (CYA)) resistant to FBZ are pervasive in Lithuania. FBZ was ineffective in three of the six stables (FECR 77.1-79.0%; 49.8-99.8 LLCI), was suspected to be ineffective in one stable (FECR 93.6%; 85.4-100 LLCI) and was effective (FECR 99.8-100%; 99.8-100 LLCI) in two stables. FEC showed a significant (P < 0.01) difference between the treatment and control groups. Only CYA larvae were detected in larval cultures derived from strongyle-positive faecal samples collected 14 days after treatment of a test group with FBZ. CONCLUSION: This in vivo study showed that resistance to FBZ in the treatment of strongyle nematodes is prevalent in horse stables in Lithuania. These findings should guide the implementation of more sustainable management of strongyle infections in horses in Lithuania.