α-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention.

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/ß-exosites. The LC translocation mechanism has also been studied, but an effective, nontoxic inhibitor remains underexplored. In this work, we screened a library of salicylanilides as potential translocation inhibitors. Potential leads following a primary screen were further scrutinized to identify sal30, which has a cellular minimal concentration of a drug that is required for 50% inhibition (IC50) value of 141 nM. The inquiry of salicylanilide sal30's mechanism of action was explored through a self-quenched fluorogenic substrate conjugated to bovine serum albumin (DQ-BSA) fluorescence, confocal microscopy, and vacuolar H+-ATPase (V-ATPase) inhibition assays. The summation of these findings imply that endolysosomal proton translocation through the protonophore mechanism of sal30 causes endosome pH to increase, which in turn prevents LC translocation into cytosol, a process that requires an acidic pH. Thus, the inhibition of BoNT/A activity by salicylanilides likely occurs through disruption of pH-dependent endosomal LC translocation. We further probed BoNT inhibition by sal30 using additivity analysis studies with bafilomycin A1, a known BoNT/A LC translocation inhibitor, which indicated the absence of synergy between the two ionophores.

Identification of Potent and Safe Antiviral Therapeutic Candidates Against SARS-CoV-2.

COVID-19 pandemic has infected millions of people with mortality exceeding >1 million. There is an urgent need to find therapeutic agents that can help clear the virus to prevent severe disease and death. Identifying effective and safer drugs can provide more options to treat COVID-19 infections either alone or in combination. Here, we performed a high throughput screening of approximately 1,700 US FDA-approved compounds to identify novel therapeutic agents that can effectively inhibit replication of coronaviruses including SARS-CoV-2. Our two-step screen first used a human coronavirus strain OC43 to identify compounds with anti-coronaviral activities. The effective compounds were then screened for their effectiveness in inhibiting SARS-CoV-2. These screens have identified 20 anti-SARS-CoV-2 drugs including previously reported compounds such as hydroxychloroquine, amlodipine besylate, arbidol hydrochloride, tilorone 2HCl, dronedarone hydrochloride, mefloquine, and thioridazine hydrochloride. Five of the newly identified drugs had a safety index (cytotoxic/effective concentration) of >600, indicating a wide therapeutic window compared to hydroxychloroquine which had a safety index of 22 in similar experiments. Mechanistically, five of the effective compounds (fendiline HCl, monensin sodium salt, vortioxetine, sertraline HCl, and salifungin) were found to block SARS-CoV-2 S protein-mediated cell fusion. These FDA-approved compounds can provide much needed therapeutic options that we urgently need during the midst of the pandemic.

Repositioning of Anthelmintic Drugs for the Treatment of Cancers of the Digestive System.

Tumors of the digestive system, when combined together, account for more new cases and deaths per year than tumors arising in any other system of the body and their incidence continues to increase. Despite major efforts aimed at discovering and validating novel and effective drugs against these malignancies, the process of developing such drugs remains lengthy and costly, with high attrition rates. Drug repositioning (also known as drug repurposing), that is, the process of finding new uses for approved drugs, has been gaining popularity in oncological drug development as it provides the opportunity to expedite promising anti-cancer agents into clinical trials. Among the drugs considered for repurposing in oncology, compounds belonging to some classes of anthelmintics-a group of agents acting against infections caused by parasitic worms (helminths) that colonize the mammalian intestine-have shown pronounced anti-tumor activities and attracted particular attention due to their ability to target key oncogenic signal transduction pathways. In this review, we summarize and discuss the available experimental and clinical evidence about the use of anthelmintic drugs for the treatment of cancers of the digestive system.

Salicylanilide Analog Minimizes Relapse of Clostridioides difficile Infection in Mice.

Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembranous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota, which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple Clostridioides difficile strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized, and the most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and diminished disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the recovering patient.

A Novel Salicylanilide Derivative Induces Autophagy Cell Death in Castration-Resistant Prostate Cancer via ER Stress-Activated PERK Signaling Pathway.

Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. Cancer growth and progression is intimately affected by its interaction with host microenvironment. Cotargeting of the stroma and prostate cancer is therefore an emerging therapeutic strategy for metastatic CRPC. Cancer-induced osteoclastogenesis is known to contribute to CRPC bone metastasis. This study is to extend pharmacologic value of our synthesized LCC03, a derivative of 5-(2',4'-difluorophenyl)-salicylanilide that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. LCC03 was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. We demonstrated that LCC03 dose-dependently suppressed proliferation and retarded cell-cycle progression in CRPC cells. The classical autophagy features, including autophagosome formation and LC3-II conversion, were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy. Moreover, an expanded morphology of the endoplasmic reticulum (ER), increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the LCC03-induced autophagy. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. The dual action of 5-(2',4'-difluorophenyl)-salicylanilide on targeting both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC.

Copper oxide and closantel prevent alterations in hepatic energetic metabolism and reduce inflammation in Haemonchus contortus infection.

The aims of this study were to evaluate if the use of copper oxide wire particles, isolated or in association with closantel, in lambs infected with Haemonchus contortus enhances the anthelmintic efficacy of closantel, as well as to evaluate the effects of treatment in hepatic energy metabolism, inflammatory markers and hematological and biochemical tests. The lambs were randomly divided into five groups (6 animals each), as follows: uninfected animals (Control); animals infected with H. contortus (HC); infected and treated with closantel (HC + CL); infected and treated with copper oxide wire particles (HC + Cu); and infected and treated with closantel plus copper oxide wire particles (HC + CL + Cu). The animals of infected groups were infected orally with H. contortus (5,000 L3 -larvae) and on day 14 post infection (p.i) the treatments were initiated. The egg per gram of feces (EPG), butyrylcholinesterase (BuChE), myeloperoxidase (MPO), adenylate kinase (AK) and pyruvate kinase (PK) activities and hematological and biochemical tests were evaluated. Treatments with copper oxide (isolated and associated) were able to reduce the EPG count on days 28, 35, 42 and 49 p.i when compared to HC group, while closantel was able to reduce EPG only from day 35 p.i. Moreover, treatment with closantel (isolated or associated) was able to prevent the inhibition of hepatic AK and PK activities caused by H. contortus infection, which may contribute to efficient intracellular energetic communication in order to maintain the balance between cellular ATP consumption and production. Butyrylcholinesterase and MPO activities were higher in infected lambs compared to uninfected, while treated groups showed lower enzymatic activity compared to the group HC. The use of all therapeutic protocols was able to reduce the EPG count. Based on these evidences, the use of copper oxide plus closantel may be considered an alternative to treat lambs infected by H. contortus.

Zygocotyle lunata as a model for in vivo screening of anthelmintic activity against paramphistomes: Evaluation of efficacy of praziquantel, albendazole and closantel in experimentally infected mice.

Paramphistomes are important parasites in veterinary medicine. There are few anthelmintic drugs available against them. The development of new drugs is urgently needed and this process can be accelerated through the development of rodent models for in vivo testing. Among the few paramphistomes that develop in rodents is the caecal fluke Zygocotyle lunata, a species with which several biological studies have been performed over several decades. Nevertheless, its use as a model for evaluation of anthelmintic drugs had not yet been evaluated. In the present study, we evaluated the efficacy of praziquantel (PZQ 300 mg/kg 5x), albendazole (ABZ 200 mg/kg 5x) and closantel (CLO 50 mg/kg single dose, 50 mg/kg 3x and 25 mg/kg 3x) for treatment of mice experimentally infected with Z. lunata. The animals were infected with 20 metacercariae of the parasite and were treated 30 days post-infection. Untreated groups were maintained as controls. Seven days after the treatments, the animals were euthanized for recovery and counting of parasites. We found that PZQ and ABZ, at the dosages and therapeutic schedule employed here, did not cause significant alterations in worm burden [worm counts 16.0 ±â€¯2.8 (13-19), 17.6 ±â€¯2.1 (14-19) and 16.2 ±â€¯1.9 (13-18) (p = 0.51) in PZQ, ALB and control, respectively]. CLO 50 mg/kg in a single dose caused significant reduction in the number of parasites [treated: 1.8 ±â€¯0.9 (1-3); control: 15.6 ±â€¯2.5 (12-19)], although it did not result in complete elimination of the parasites in any animal. Despite the fact that three doses of CLO 50 mg/kg or CLO 25 mg/kg caused complete elimination of the parasites in most surviving animals, there was significant host mortality. In general, results here obtained are concordant with those of studies performed on ruminant paramphistomes. Given that Z. lunata can be maintained in laboratory rodents, it is a suitable model for screening anthelmintic drugs against paramphistomes.

Efficacy of herbal extracts and closantel against fenbendazole-resistant Haemonchus contortus.

This study assessed the efficacy of closantel vis-à-vis herbal extracts with known anti-parasitic properties, against fenbendazole-resistant nematodes in goats maintained under a semi-intensive system of management at the University goat farm, Jabalpur. Fifty goats were randomly assigned to five groups, each comprising 10 animals, irrespective of their breed, age and sex. Each animal in Group I, II and III was orally administered with aqueous leaf extracts of neem (Azadirachta indica) at 1 g/kg body weight, sitaphal (Annona squamosa) at 1.5 g/kg body weight and tobacco (Nicotiana tabacum) at 1 g/kg body weight, respectively, whereas Group IV was an untreated control group. Each animal in Group V was orally treated with closantel at 10 mg/kg body weight. During the course of the study, all animals were maintained under an identical semi-intensive system of management. Compared to the untreated control group (Group IV), there was no conspicuous reduction in post-treatment (day 10) faecal egg counts (FEC) in animals administered with the herbal extracts (Groups I, II and III), which is suggestive of poor anti-parasitic activity. However, using the faecal egg count reduction test (FECRT), the overall efficacy of closantel was recorded as 95.64%. This supports the rotational use of closantel as a preferred choice over the benzimidazole group of anthelmintics and/or herbal extracts to meet the acute challenge of in situ development of drug-resistant gastrointestinal nematodes, especially Haemonchus contortus.

Anthelmintic resistance in gastrointestinal nematodes of beef cattle in the state of Rio Grande do Sul, Brazil.

Gastrointestinal nematodes resistant to anthelmintics have been reported in several regions of Brazil, and they may be associated with economic losses for the cattle industry. This study aimed to evaluate the resistance status of gastrointestinal nematodes from naturally infected beef cattle to several commercially available anthelmintics, as well as to test the efficacy of combinations of anthelmintics against multi-resistant gastrointestinal nematodes. Ten farms located in Rio Grande do Sul state were selected by: farmers' consent; extensive raising system; availability of calves aged from 7 to 9 months naturally infected by gastrointestinal nematodes; absence of anthelmintic treatment for 60 days before the study; and presence of 70-100 calves or more of both genders with ≥ 200 eggs per gram of feces (EPG) (sensitivity of 50 EPG). These calves were distributed into 10 groups (of 7-10 animals) per farm and treated with ivermectin, doramectin, eprinomectin, fenbendazole, closantel, nitroxynil, disophenol, levamisole, albendazole, or moxidectin. Feces were collected 2 days before treatment and 14 days after treatment. Additional groups of 7-10 calves were used to test six different two-drug combinations at four of the studied farms. In general terms, fenbendazole was the most effective drug, followed by levamisole, disophenol, and moxidectin. However, parasite resistance to multiple drugs was found in all herds, especially in the genera Cooperia spp., Trichostrongylus spp., and Haemonchus spp.. Some of the two-drug combinations were effective against nematode populations identified as resistant to the same compounds when used as single drugs. The most effective combinations were moxidectin + levamisole, doramectin + fenbendazole, and levamisole + closantel. In this study, parasites resistant to the main commercially available anthelmintics were found in all herds, and some combinations of two active components belonging to different chemical groups were effective against multi-drug resistant gastrointestinal nematodes.

Fasciola hepatica: Histological changes in the somatic and reproductive tissues of liver fluke following closantel treatment of experimentally-infected sheep.

Lambs infected with the Cullompton isolate of Fasciola hepatica were treated orally or subcutaneously with 10mg/kg of closantel at 16 weeks post-infection. Adult flukes were recovered from the liver of individual animals at 12h, 24h, or 36h post-treatment. The flukes were processed for histological analysis. In general, degenerative changes in the reproductive and somatic tissues were progressive, and were most marked in flukes exposed to closantel in vivo for 36h. However, flukes from a 12h subcutaneously-treated lamb showed marked deterioration of the testis, possibly because a portion of the dose has been delivered intravenously. Fewer intact eggs were seen in the uterus of flukes exposed to closantel for longer times (whether administered subcutaneously or orally to the host). The most conspicuous closantel-induced effect in flukes from treated hosts was progressive damage to the tegumental syncytium. While the flukes from 24h-treated hosts showed relatively minor damage to limited areas of the syncytium, towards the posterior end, the flukes from 36h-treated hosts (and flukes from the lamb that putatively received intravenous dosage) had lost large areas of the surface syncytium from the posterior end and dorsal surface, although the syncytium over the anterior end and the anterior ventral surface was largely spared. In areas where the syncytium had sloughed, the underlying structures such as the vitelline follicles, gut profiles and testis profiles, showed marked degeneration and breakdown. Other changes included cell depletion and early stage apoptosis in the testis, ovary and vitelline follicles. This study establishes a model for histological changes in closantel-sensitive F. hepatica exposed to closantel in vivo. Histopathological studies could be complementary to the efficacy controlled test for for closantel resistance in fluke populations.

First report of closantel treatment failure against Fasciola hepatica in cattle.

Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests. Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12-16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT). Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65-77%) and 97% (95% CI: 95-98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively. Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12-16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle.

Novel insights into the pathogenic importance, diagnosis and treatment of the rumen fluke (Calicophoron daubneyi) in cattle.

Recently, sharp increases in the prevalence of rumen fluke infections have been recorded throughout Western Europe. However, scarce information is available on the diagnosis, pathogenic importance and control of this parasite. We undertook 3 pilot studies to gain more insights into these aspects of rumen fluke biology in cattle. First, we evaluated the diagnostic performance of mini-FLOTAC to detect adult rumen fluke infections based on faecal egg count in an abattoir survey and found high sensitivity (0.94) and specificity (0.98). Moreover, there was an association between ruminal fluke burden (assessed by visual scoring) and faecal egg count and a cut-off of 200 eggs per gram is proposed to detect highly infected animals (>200 flukes present in the rumen and/or reticulum). There was also a significant association between ruminal fluke burden and faecal consistency. However, in a second study, we performed a case-control field survey to investigate the association between rumen fluke infection and herd-level problems with diarrhoea and no association was found. Finally, we evaluated the use of closantel (Flukiver(®), Elanco Animal Health, subcutaneous administration at 10mg/kg) to treat rumen fluke infection on 3 herds, but no significant reduction in egg output post-treatment was found. Because this result is in contrast with a previous study using an oral dose of closantel, more research is required into the effect of administration route on the efficacy of closantel on rumen fluke.

Dual protonophore-chitinase inhibitors dramatically affect O. volvulus molting.

The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.

Integrating fasciolosis control in the dry cow management: the effect of closantel treatment on milk production.

The liver fluke Fasciola hepatica is a parasite of ruminants with a worldwide distribution and an apparent increasing incidence in EU member states. Effective control in dairy cattle is hampered by the lack of flukicides with a zero-withdrawal time for milk, leaving the dry period as the only time that preventive treatment can be applied. Here, we present the results of a blinded, randomized and placebo-controlled trial on 11 dairy herds (402 animals) exposed to F. hepatica to 1) assess the effect of closantel treatment at dry-off (or 80-42 days before calving in first-calving heifers) on milk production parameters and 2) evaluate if a number of easy-to-use animal parameters is related to the milk production response after treatment. Closantel treatment resulted in a noticeable decrease of anti-F. hepatica antibody levels from 3-6 months after treatment onwards, a higher peak production (1.06 kg) and a slightly higher persistence (9%) of the lactation, resulting in a 305-day milk production increase of 303 kg. No effects of anthelmintic treatment were found on the average protein and fat content of the milk. Milk production responses after treatment were poor in meagre animals and clinically relevant higher milk production responses were observed in first-lactation animals and in cows with a high (0.3-0.5 optical density ratio (ODR)), but not a very high (≥ 0.5 ODR) F. hepatica ELISA result on a milk sample from the previous lactation. We conclude that in dairy herds exposed to F. hepatica, flukicide treatment at dry-off is a useful strategy to reduce levels of exposure and increase milk production in the subsequent lactation. Moreover, the results suggest that treatment approaches that only target selected animals within a herd can be developed based on easy-to-use parameters.

The efficacy of trichlorphon and naphthalophos against multiple anthelmintic-resistant nematodes of naturally infected sheep in Argentina.

An anthelmintic efficacy trial was conducted in sheep harbouring anthelmintic-resistant worms in Argentina. Seventy lambs were selected from a flock that had been grazed on pastures infected with trichostrongyles previously shown to be resistant to the main anthelmintic groups. Lambs were allocated to comparable groups of ten animals each and treated with trichlorphon (50 mg/kg body weight (b.w.) orally); naphthalophos (50 mg/kg b.w. orally); ivermectin (0.2 mg/kg b.w. subcutaneously); fenbendazole (5 mg/kg b.w. orally); levamisole (8 mg/kg b.w. subcutaneously) and closantel (10 mg/kg b.w. orally). There was also an untreated group. The dose selection was based on manufacturer's recommendations.Faecal samples were collected 0 and 10 days post treatment to estimate efficacy (faecal egg count reduction). Six animals from each group were necropsied at day 10 for enumeration/identification of worms from the abomasum, small and large intestines to determine the absolute efficacy of each agent (controlled efficacy test). Trichlorphon and naphthalophos were effective (> 99 %) against Haemonchus contortus (p < 0.05).Naphthalophos also showed efficacy against Trichostrongylus axei (99.3 %), Teladorsagia circumcincta (97.8 %), Trichostrongylus colubriformis (99.2 %), Cooperia punctata/curticei/pectinata (90.4 %), Nematodirus spathiger (89.2 %) and Oesophagostomum venulosum/columbianum (93.7 %). Fenbendazole and levamisole showed efficacy (> 95 %) against all nematodes except T. colubriformis. The efficacy of ivermectin was low against H. contortus (23 %) and Cooperia spp. (46.3 %). Closantel showed low efficacy against T. axei (64.4 %), H. contortus (80.6 %) and T. colubriformis (59.5 %).When anthelmintic resistance is widespread, trichlorphon treatment is appropriate if H. contortus is present; however, naphthalophos represents an effective therapeutic alternative for incorporation into worm control programmes.

Resistance of Fasciola hepatica against triclabendazole in cattle in Patagonia (Argentina).

In the winter of 2008, cattle on a farm in the province of Neuquen, Argentina died from subacute and chronic liver fluke disease despite four previous treatments with Triclabendazole (TCBZ). In the spring of 2009, a preliminary efficacy test revealed good performance using nitroxynil, whereas TCBZ efficacy was only 18% by egg counts of Fasciola eggs in the faeces. Resistance to Fasciola hepatica to TCBZ has never been reported in South America, so in January of 2010 a controlled trial was conducted to confirm and to define the degree of resistance in this herd. In a clinical trial, the fluke egg output was monitored on Days 14 and 21 and serum enzymes gamma-glutamyl transpeptidase (GGT) and glutamic-oxaloacetic transaminase (GOT) on Days 0 and 21 in 36 calves treated with TCBZ or with closantel. The results showed a reduction of 100% in fluke egg output at Days 14 and 21 for closantel. The mean epg in the TCBZ-treated groups did not decrease. Because of the fact that in this study TCBZ treatment in cattle had no effect, even at double the recommended dose, it is highly indicative that resistance of F. hepatica against TCBZ is present on this farm. The GGT and GOT levels decreased in the closantel-treated group as a result of the treatment at 21 days after dosing. To evaluate the importance of TCBZ resistance in F. hepatica in Argentina, a study on more farms from endemic areas is needed.

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil.

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.