Quantitative Analysis of Abamectin, Albendazole, Levamisole HCl and Closantel in Q-DRENCH Oral Suspension Using a Stability-Indicating HPLC-DAD Method.

Combination therapy of many anthelmintic drugs has been used to achieve fast animal curing. Q-DRENCH is an oral suspension, containing four different active drugs against GIT worms in sheep, commonly used in Australia and New Zeeland. The anti-parasitic drugs are Albendazole (ALB), Levamisole HCl (LEV), Abamectin (ABA), and Closantel (CLO). The main purpose of this study is to present a new simultaneous stability-indicting HPLC-DAD method for the analysis of the four drugs. The recommended liquid system was 1 mL of Triethylamine/L water, adjusting the pH to 3.5 by glacial acetic acid: acetonitrile solvent (20:80, v/v). Isocratic elusion achieved the desired results of separation at a 2 mL/min flow rate using Zorbax C-18 as a stationary phase. Detection was performed at 210 nm. The linearity ranges were 15.15 to 93.75 µg/mL for ALB, 25 to 150 µg/mL for LEV, 30 to 150 µg/mL for ABA, and 11.7 to 140.63 µg/mL for CLO. Moreover, the final greenness score was 0.62 using the AGREE tool, which reflects the eco-friendly nature. Moreover, the four drugs were determined successfully in the presence of their stressful degradation products. This work presents the first chromatographic method for simultaneous analysis for Q-DRENCH oral suspension drugs in the presence of their stressful degradation products.

Screening for potent and selective anticlostridial leads among FDA-approved drugs.

Clostridium difficile is a leading cause of morbidity and mortality particularly in hospital settings. In addition, treatment is very challenging due to the scarcity of effective therapeutic options. Thus, there remains an unmet need to identify new therapeutic agents capable of treating C. difficile infections. In the current study, we screened two FDA-approved drug libraries against C. difficile. Out of almost 3200 drugs screened, 50 drugs were capable of inhibiting the growth of C. difficile. Remarkably, some of the potent inhibitors have never been reported before and showed activity in a clinically achievable range. Structure-activity relationship analysis of the active hits clustered the potent inhibitors into four chemical groups; nitroimidazoles (MIC50 = 0.06-2.7 µM), salicylanilides (MIC50 = 0.2-0.6 µM), imidazole antifungals (MIC50 = 4.8-11.6 µM), and miscellaneous group (MIC50 = 0.4-22.2 µM). The most potent drugs from the initial screening were further evaluated against additional clinically relevant strains of C. difficile. Moreover, we tested the activity of potent inhibitors against representative strains of human normal gut microbiota to investigate the selectivity of the inhibitors towards C. difficile. Overall, this study provides a platform that could be used for further development of potent and selective anticlostridial antibiotics.

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.

Urine retention in cattle putatively associated with injection of an ivermectin and closantel anthelmintic formulation into the ischiorectal fossa.

CASE HISTORY: A group of 39, 19-22-month-old Friesian bulls were administered an ivermectin/closantel anthelmintic via intended S/C injection in the ischiorectal fossa on 15 June 2017 (Day 0). Over the next 50 days, 22 affected bulls presented various degrees of anorexia, abdominal pain and urine dribbling. Seventeen bulls were examined by transrectal ultrasonography which revealed urinary bladder distension in all 17, and peritoneal fluid accumulation in some. Overall, eight bulls died or were subjected to euthanasia. On-farm postmortem examination of three bulls revealed urinary bladder rupture. CLINICAL FINDINGS: On Day 50 one affected live bull was admitted to Massey University for further investigation. This bull continuously dribbled urine and had an overtly distended urinary bladder as determined by rectal palpation and ultrasonography. PATHOLOGICAL FINDINGS: Postmortem examination of this bull revealed a markedly distended urinary bladder, massive subcapsular and pericapsular renal oedema with retroperitoneal fluid accumulation, minimal hydronephrosis and no evidence of mechanical urinary outflow obstruction. The right ischiorectal fossa contained multifocal areas of tissue fibrosis that extended into areas innervated by the distal cutaneous branch of the pudendal nerve and the pelvic nerve. Histopathological changes consisted of extensive fibrosis, myonecrosis and neurodegeneration, and evidence of granulation tissue and inflammation at the putative injection site and in surrounding tissues. DIAGNOSIS: A local inflammatory reaction at the presumed injection site together with localised peripheral neurodegeneration and myelopathy may have led to detrusor-sphincter dyssynergia causing urine retention. CLINICAL RELEVANCE: These cases of urine retention and bladder rupture in cattle were of putative iatrogenic origin. Veterinarians should be aware of this rare complication after S/C injections in the ischiorectal fossa.

Target animal safety testing of an oral salicylanilide suspension, oxyclozanide, for the treatment of fascioliasis in bovine in China.

The aim of this study was to determine the potential toxicity risk of an oxyclozanide suspension to the target animal, bovine. In this experiment, 32 Simmental beef cattle were fattened and fed a full-price diet without antimicrobial agents. The test cattle were divided into 4 groups, which were treated with 0, 1, 3, and 5 times the recommended dosage through continuous intermittent oral administration at intervals of 2 days. The body weight of the cattle was recorded before and after the experiment, and the weight changes were calculated. The safety of the drugs was evaluated by weight gain, observation of clinical toxicity, haematology, clinical chemistry and histopathology. The results showed that the cattle had different degrees of diarrhoea, loss of appetite and depression after administration. The results of clinicopathology had no significant effect. The results of pathological examination showed that there was a certain degree of damage in the 5 times recommended dose group. The recommended dose was safe to use. Thus, the recommended dose should be given by a single oral administration to ensure the safe use of this drug in the clinic.

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.

Understanding the main route of drug entry in adult Fasciola hepatica: Further insights into closantel pharmacological activity.

Closantel (CLS) is highly effective against adult liver flukes after its oral or subcutaneous (sc) administration in ruminants. Trans-tegumental diffusion and oral ingestion are the two potential routes available for the entry of drugs into Fasciola hepatica. The work reported here contributes to improve the understanding of CLS pharmacology. The main goals of were: I) to determine the pattern of in vivo CLS accumulation into adult F. hepatica and relevant tissues in CLS-treated sheep; II) to investigate the influence of the physicochemical composition of the incubation medium on the CLS diffusion process into adult F. hepatica; III) to assess the ovicidal activity of CLS against F. hepatica eggs; and IV) to investigate the in vivo effect of CLS treatment on glutathione S-transferases activity in adult liver flukes exposed to CLS. Fourteen healthy sheep were each orally infected with 75 F. hepatica metacercariae. Sixteen (16) weeks after infection, animals were treated with CLS by oral (n = 6, 10 mg/kg) or sub-cutaneous (sc) (n = 6, 5 mg/kg) route. At 12, 24 and 36 h post-treatment, animals were sacrificed (n = 2) and samples of blood, bile and adult F. hepatica were collected. In addition, flukes recovered from non-treated sheep (n = 2) were ex vivo incubated (60 min) in the presence of CLS in either RPMI or bile as incubation medium. CLS concentration was measured by HPLC. The ovicidal activity of CLS was investigated using eggs obtained from the bile of untreated sheep. Finally, glutathione S-transferase activity in F. hepatica recovered from untreated and CLS-treated sheep was assessed. In the in vivo studies, the highest CLS concentrations were measured in plasma and adult liver flukes. A positive correlation was observed between CLS concentration in plasma and in F. hepatica. Results obtained in the current work indicate that the in vivo accumulation of CLS into adult liver flukes occurs mainly by the oral route. After ex vivo incubation, the uptake of CLS by the parasite was markedly diminished in the presence of bile compared with that observed in the presence of RPMI as incubation medium. CLS lacks ovicidal activity at therapeutically relevant concentrations. Lastly, CLS significantly increased glutathione S-transferase activity in flukes recovered at 12 h (oral treatment) and 24 h (sc treatment), compared to the control liver flukes.

Suppression of the Growth and Invasion of Human Head and Neck Squamous Cell Carcinomas via Regulating STAT3 Signaling and the miR-21/ß-catenin Axis with HJC0152.

Signal transducer and activator of transcription 3 (STAT3) is involved in the tumor growth and metastasis of human head and neck squamous cell carcinoma (HNSCC) and is therefore a target with therapeutic potential. In this study, we show that HJC0152, a recently developed anticancer agent and a STAT3 signaling inhibitor, exhibits promising antitumor effects against HNSCC both in vitro and in vivo via inactivating STAT3 and downstream miR-21/ß-catenin axis. HJC0152 treatment efficiently suppressed HNSCC cell proliferation, arrested the cell cycle at the G0-G1 phase, induced apoptosis, and reduced cell invasion in both SCC25 and CAL27 cell lines. Moreover, HJC0152 inhibited nuclear translocation of phosphorylated STAT3 at Tyr705 and decreased VHL/ß-catenin signaling activity via regulation of miR-21. Loss of function of VHL remarkably compromised the antitumor effect of HJC0152 in both cell lines. In our SCC25-derived orthotopic mouse models, HJC0152 treatment significantly abrogated STAT3/ß-catenin expression in vivo, leading to a global decrease of tumor growth and invasion. With its favorable aqueous solubility and oral bioavailability, HJC0152 holds the potential to be translated into the clinic as a promising therapeutic strategy for patients with HNSCC. Mol Cancer Ther; 16(4); 578-90. ©2017 AACR.

Estudos da resposta estringente de Bacillus subtilis e busca por pequenas moléculas moduladoras de RelA / Studies of the stringent response of Bacillus subtilis and search for small molecules capable of modulating RelA activity

Seja no meio ambiente, dentro de um hospedeiro ou em outro habitat, bactérias estarão frequentemente enfrentando condições adversas, como exposição a compostos antibacterianos ou carência nutricional. Em situações como essas, as bactérias são capazes de ativar a chamada resposta estringente, modulada pelo alarmônio (p)ppGpp. O acúmulo de (p)ppGpp promove a inibição da transcrição de rRNAs e tRNAs e a supressão do processo de tradução, e a ativação de operons de biossíntese de aminoácidos. Sabe-se também hoje que a resposta estringente está relacionada a outras importantes carências nutricionais em Escherichia coli, como a falta de ácidos graxos, porém não se sabe se o mesmo ocorre em Bacillus subtilis ou em outras Grampositivas. (p)ppGpp atua também direta e indiretamente em vários outros processos celulares, como motilidade, resistência a antibióticos, virulência e persistência, indicando que (p)ppGpp é um regulador central que integra informação metabólica e respostas adaptativas. O presente trabalho buscou estudar a correlação da resposta estringente de B. subtilis com a carência de ácidos graxos e a busca por pequenas moléculas capazes de modular RelA (a principal proteína envolvida na síntese de (p)ppGpp) e impedir o acúmulo de (p)ppGpp. Para a indução da carência de ácidos graxos, foram utilizadas duas estratégias; uso da droga Cerulenina (inibidor de FabF) e mutantes condicionais no gene FabF. Observou-se que mutantes incapazes de ativar a resposta estringente (cepa ppGpp(0) ou RelAD264G) apresentaram grande perda de viabilidade celular durante a carência de ácidos graxos, ao passo que a cepa selvagem manteve sua viabilidade celular. A causa da morte se deu majoritariamente devido ao colapso do potencial de membrana. Apesar de não termos observado aumento de (p)ppGpp nas células selvagens durante a carência de ácidos graxos, observou-se uma redução da razão GTP/ATP, ao passo que na cepa ppGpp(0), a razão GTP/ATP aumentou, devido ao acúmulo de GTP. O uso da droga decoinina, capaz de reduzir os níveis intracelulares de GTP, resgatou parcialmente a viabilidade da cepa e impediu a perda do potencial de membrana, indicando que os níveis de GTP são importantes durante a carência de ácidos graxos em B. subtilis. Para a triagem de pequenas moléculas inibidoras do acúmulo de (p)ppGpp, foi utilizada uma biblioteca de 2320 diferentes compostos químicos, e buscou-se drogas capazes de reverter o fenótipo de crescimento lento de cepas de B. subtilis que acumulam (p)ppGpp (via mutação pontual; mutante RelAH77A e via tratamento com o indutor hidroxamato de arginina) em meio rico. A primeira etapa selecionou 40 moléculas capazes de resgatar o crescimento de células tratadas com arginina-hidroxamato, porém apenas uma, salicilanilida, foi capaz de também resgatar o crescimento da cepa RelAH77A. Todavia, apesar de ser capaz de acelerar o crescimento de B. subtilis esse efeito é limitado. Diversos análogos de salicilanilida foram testados, porém não apresentaram efeito superior a salicilanilida para a reversão do fenótipo de crescimento lento de B. subtilis. Em adição, a droga não foi capaz de aumentar a sensibilidade dos organismos a diversos antibióticos testados, e aparentemente é incapaz de alterar os níveis internos de (p)ppGpp, porém é capaz de causar alterações nos níveis de ATP. Logo, acredita-se que o efeito observado para o crescimento das células seja devido a efeitos indiretos, possivelmente envolvendo alteração de outros nucleotídeos fosforilados

In the environment, inside a host or other habitat, bacteria will always face adverse conditions, as for example exposure to antimicrobials or starvation. In situations like those, bacteria activate the stringent response, modulated by the alarmone (p)ppGpp. (p)ppGpp accumulation promotes inhibition of rRNA and tRNA transcription and suppression of translational process, at the same time that it activates several amino acid biosynthesis operons. It is known also that the stringent response it is related to other starvation stress in Escherichia coli, like lack of fatty acids, but there is no knowledge if the same occurs for Bacillus subtilis or other gram-positive bacteria. ppGpp acts directly and indirectly affecting several other cellular process, as motility, resistance to antibiotics, virulence and persistence, indicating that (p)ppGpp is a central regulator that integrates metabolic information and adaptive responses. This work aimed to study the correlation between the stringent response in B. subtilis with fatty acid starvation, and search for small moleculas capable of modulating RelA (the main enzyme responsible for ppGpp synthesis) and stop (p)ppGpp production. For fatty acid starvation induction, two strategies were used; use of the drug Cerulenin (inhibitor of the FabF protein) and conditional mutants of the FabF gene. We observed that mutants incapable of activating the stringent response (strains ppGpp(0) ou RelAD264G) presented great loss of viability during fatty acid starvation, whereas the wild-type strain keeps its viability. The main cause of death is due membrane rupture in some cells, but mainly due to membrane potential collapse. Although we did not observed increase of (p)ppGpp in wild-type strains during fatty acid starvation, we observed reduction in GTP/ATP ratios, a hallmark of (p)ppGpp production in gram-positive bacteria. In the strain ppGpp(0) GTP/ATP ratio increased, mainly due to GTP increase. Using the drug decoyinine, capable of reducing GTP levels, partially recued viability and protects cells of losing its membrane potential, indicating that GTP levels plays an important role during fatty acid starvation in B. subtilis. For the screening of small molecules capable of inhibit (p)ppGpp production, a library of 2320 different chemical compounds were used, and we looked for drugs capable of reverting the slow growth phenotype of B. subtilis strains with (p)ppGpp accumulation (using a mutant RelAH77A; and using a stringent response inductor, arginine hidroxamate). The first step selected for 40 molecules capable of rescuing the growth of cells treated with arginine hidroxamate, but only one drug, salicilanilyde could also rescue the growth of the strain RelAH77A. Although capable of rescuing growth of B. subtilis that accumulates (p)ppGpp, this rescue is limited. Several analogues of salicilanilyde were tested, but none were stronger than salicilanilyde itself in rescuing growth of slow growing strains of B. subtilis. In addition, the drug was not capable of increasing antibiotic sensibility and it is incapable of changing intracellular (p)ppGpp levels, but it does shifts ATP levels. Therefore, we believe that the observed effects of salicilanilyde is due indirect action, probably involving other phosphorylated nucleotides, rather than modifying (p)ppGpp levels

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.

In vivo assessment of closantel ovicidal activity in Fasciola hepatica eggs.

Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.

Anthelmintic closantel enhances bacterial killing of polymyxin B against multidrug-resistant Acinetobacter baumannii.

Polymyxins, an old class of antibiotics, are currently used as the last resort for the treatment of multidrug-resistant (MDR) Acinetobacter baumannii. However, recent pharmacokinetic and pharmacodynamic data indicate that monotherapy can lead to the development of resistance. Novel approaches are urgently needed to preserve and improve the efficacy of this last-line class of antibiotics. This study examined the antimicrobial activity of novel combination of polymyxin B with anthelmintic closantel against A. baumannii. Closantel monotherapy (16 mg l(-1)) was ineffective against most tested A. baumannii isolates. However, closantel at 4-16 mg l(-1) with a clinically achievable concentration of polymyxin B (2 mg l(-1)) successfully inhibited the development of polymyxin resistance in polymyxin-susceptible isolates, and provided synergistic killing against polymyxin-resistant isolates (MIC ⩾4 mg l(-1)). Our findings suggest that the combination of polymyxin B with closantel could be potentially useful for the treatment of MDR, including polymyxin-resistant, A. baumannii infections. The repositioning of non-antibiotic drugs to treat bacterial infections may significantly expedite discovery of new treatment options for bacterial 'superbugs'.

Does closantel in therapeutic doses display thyroid hormone-like activity in sheep?

The aim of this study was to define the thyroid hormone-like activity of closantel in sheep by measuring some blood parameters that are known to be influenced by thyroid hormones triiodothyronine (T3) and thyroxine (T4). Our hypothesis was that, if closantel possesses thyroid hormone-like activity, its use under in vivo conditions will result in changes similar to those in hyperthyroidism. The study was conducted in 20 Jezersko-Solchava breed sheep. Blood sampling was performed before and 10 days after routine anthelmintic treatment with closantel. Complete blood count, plasma cholesterol, triglycerides, protein, and albumin levels, as well as those of serum T3 and T4, were compared before and 10 days after closantel administration. This routine anthelmintic treatment of sheep with closantel did not significantly influence hematological parameters, thyroid hormone levels, or most of the biochemical parameters. No evidence was found for thyroid hormone-like activity of closantel in sheep. However, significantly (P < 0.01) elevated levels of plasma triglycerides were present 10 days after closantel administration.

First report of anthelmintic resistance in Haemonchus contortus in alpacas in Australia.

BACKGROUND: Parasitic nematodes can cause substantial clinical and subclinical problems in alpacas and anthelmintics are regularly used to control parasitic nematodes in alpacas. Although anthelmintic resistance has been reported in ruminants worldwide, very little is known about anthelmintic resistance in alpacas. The present study was carried out to confirm a suspected case of anthelmintic resistance in Haemonchus contortus in alpacas in Australia. METHODS: Post mortem examination of an alpaca was conducted to determine the cause of its death. To confirm a suspected case of macrocyclic lactone (ML) resistance in H. contortus in alpacas, a faecal egg count reduction test (FECRT) was performed using closantel (7.5 mg/kg) and ivermectin (0.2 mg/kg). Nematode species were identified by morphological and molecular methods. RESULTS: Post mortem examination of a 1-year-old female alpaca that had died following a brief period of lethargy, anorexia and recumbency revealed severe anaemia, hypoproteinaemia and gastric parasitism by adult Haemonchus contortus, despite recent abamectin (0.2 mg/kg) treatment. Based on these findings and the exclusive use of MLs in the herd over the preceding six years, ML resistance in parasitic nematodes of alpacas on this farm was suspected. FECRT revealed that the efficacy of closantel was 99% (95% CI 93-100), whereas that of ivermectin was 35% (95% CI 0-78), indicating that the treatment failure was likely due to the presence of ML-resistant nematodes. Larval culture of faecal samples collected following ivermectin treatment consisted of 99% H. contortus and 1% Cooperia oncophora, a result confirmed using a PCR assay. CONCLUSIONS: This study provides the first evidence of ML resistance in H. contortus in alpacas in Australia. Based on the extent of anthelmintic resistance in sheep gastrointestinal nematodes in Australia, veterinarians and alpaca owners should be encouraged to implement integrated parasite management strategies to improve nematode control in alpacas.