A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

Simple and rapid determination of triclabendazole and its metabolites in bovine and goat muscle tissue.

Triclabendazole (TCB) is widely used for prevention and treatment of parasitic infections in animals. Improper use can result in drug residues in animal tissues and cause health problems to humans through consumption. A simple and reliable analytical method for the determination of TCB and its metabolites in bovine and goat muscle using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Analytes were extracted using acetonitrile and purified using enhanced matrix removal cartridge. Chromatographic separation was carried out on a BEH Shield RP18 column. The analytes were detected in positive-mode electrospray ionization mass spectrometry using multiple reaction monitoring. Average recoveries of 96.1%-105.6% with coefficients of variation of 1.9%-8.4% were obtained at fortification levels of 0.5, 2.5, 25, and 50 µg/kg for TCB and 5.0, 25, 250, and 500 µg/kg for its metabolites (triclabendazole sulfoxide, triclabendazole sulfone, and keto-TCB). A good linear regression was obtained with the mixed standard solutions in the range of 0.05-20 µg/L for TCB and 0.5-200 µg/L for its metabolites. The limit of quantification and limit of detection ranged from 0.05 to 0.75 µg/kg and from 0.1 to 1.5 µg/kg, respectively. Moreover, this method was successfully applied to 33 real samples.

Veterinary deworming agent-induced toxic optic neuropathy: a case report.

BACKGROUND: Veterinary antiparasitic drugs are widely used in countries and regions in which parasitic diseases are endemic, which leads to the risk of accidental ingestion and poisoning in humans.  CASE PRESENTATION: A 40-year-old male patient with a history of cirrhosis sought medical attention on November 25, 2021, due to progressive vision loss. He had previously taken triclabendazole and bithionol and was diagnosed with toxic optic neuropathy on examination. Steroid, neurotonic, and high-pressure oxygen therapy were ineffective. CONCLUSIONS: Triclabendazole and bithionol have potential risk of optic neurotoxicity and should be considered for enhanced supervision and warning labels.

In vitro and in vivo studies on a group of chalcones find promising results as potential drugs against fascioliasis.

About a third of the world population is infected by helminth parasites implicated in foodborne trematodiasis. Fascioliasis is a worldwide disease caused by trematodes of the genus Fasciola spp. It generates huge economic losses to the agri-food industry and is currently considered an emerging zoonosis by the World Health Organization (WHO). The only available treatment relies on anthelmintic drugs, being triclabendazole (TCBZ) the drug of choice to control human infections. The emergence of TCBZ resistance in several countries and the lack of an effective vaccine to prevent infection highlights the need to develop new drugs to control this parasitosis. We have previously identified a group of benzochalcones as inhibitors of cathepsins, which have fasciolicidal activity in vitro and are potential new drugs for the control of fascioliasis. We selected the four most active compounds of this group to perform further preclinical studies. The compound's stability was determined against a liver microsomal enzyme fraction, obtaining half-lives of 34-169 min and low intrinsic clearance values (<13 µL/min/mg), as desirable for potential new drugs. None of the compounds were mutagenic or genotoxic and no in vitro cytotoxic effects were seen. Compounds C31 and C34 showed the highest selectivity index against liver fluke cathepsins when compared to human cathepsin L. They were selected for in vivo efficacy studies observing a protective effect, similar to TCBZ, in a mouse model of infection. Our findings strongly encourage us to continue the drug development pipeline for these molecules.

Triclabendazole and clofazimine reduce replication and spermine uptake in vitro in Toxoplasma gondii.

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii. Although this infection is generally asymptomatic in immunocompetent individuals, it can cause serious clinical manifestations in newborns with congenital infection or in immunocompromised patients. As current treatments are not always well tolerated, there is an urgent need to find new drugs against human toxoplasmosis. Drug repurposing has gained considerable momentum in the last decade and is a particularly attractive approach for the search of therapeutic alternatives to treat rare and neglected diseases. Thus, in this study, we investigated the antiproliferative effect of several repurposed drugs. Of these, clofazimine and triclabendazole displayed a higher selectivity against T. gondii, affecting its replication. Furthermore, both compounds inhibited spermine incorporation into the parasite, which is necessary for the formation of other polyamines. The data reported here indicate that clofazimine and triclabendazole could be used for the treatment of human toxoplasmosis and confirms that drug repurposing is an excellent strategy to find new therapeutic targets of intervention.

The Fasciola hepatica egg development and hatching test (EDHT) using commercial drugs: a simple protocol for tackling anthelminthic resistance and promoting cattle productivity.

Fasciolosis is an important zoonotic disease caused by the trematode Fasciola hepatica, and it causes great losses in bovine production. The anthelmintic resistance is a major problem in the control of fasciolosis. In this study, the F. hepatica egg development and hatching test (EDHT) was used for the evaluation of the ovicidal activity of commercial drugs, commonly used for treating infected cattle, which reflects F. hepatica anthelminthic resistance in infected bovines, according to recent literature. Bile samples from F. hepatica naturally parasitized cattle were obtained from slaughterhouses in the cities of Lages and Otacílio Costa, Santa Catarina State, Brazil. The bile was washed, the eggs were recovered, quantified, and distributed in universal collectors, with a minimum of 1,000 eggs per vial. Four commercial drugs were used in this study, containing albendazole sulfoxide (ABDZ), closantel (CSTL), nitroxynil (NTXL), and triclabendazole with fenbendazole (TBZF). The drugs were diluted according to the manufacturer instructions. All drugs, and the respective control, were tested in triplicates, with the quantity of recovered eggs determining the number of drugs to be tested. The vials were incubated for 28 days at 27 °C, and the eggs were classified according to their degree of development under a stereomicroscope. In total, 121 egg samples were analyzed. Two samples were identified as resistant to TBZF. Undetermined resistance/susceptibility has been found in two isolates treated with ABDZ, one treated with NTXL and six treated with TBZF. CSTL did not present ovicidal activity and cannot be used in EDHT. This is the first time that commercial drugs were used in F. hepatica EDHT.

[A case report:the progress of fascioliasis from hepatic phase to biliary phase].

This is a case report of fascioliasis that progressed from the hepatic to the biliary phases over 2 years. A woman in her late 60s ate Zingiber mioga from the field, which was followed by abdominal pain that occurred 1 month later. Although CT and MRI studies revealed an increase in blood eosinophils as well as multiple hepatic nodules, they vanished quickly. After 2 years, an MRCP study revealed multiple flat lesions, which were diagnosed as adult fascioliasis. Definitive diagnosis was provided by enzyme-labeled antibody method using fasciola-specific antigen. Triclabendazole was administered once to complete the treatment.

Efficacy of flukicides on Fasciola gigantica, a food-borne zoonotic helminth affecting livestock in Bangladesh.

Fasciola gigantica, the causative agent of tropical fasciolosis, is a food-borne zoonotic trematode that affects around 80% livestock of Bangladesh. Triclabendazole (TCBZ), nitroxynil (NTON) and oxyclozanide (OCZN) are frequently used against fascioliasis; however, the current status of potency of these flukicides was unknown. In this study, in vitro efficacy of TCBZ, NTON and OCZN at various concentrations on F. gigantica has been evaluated by relative motility (RM), morphological distortions of apical cone through an inverted microscope, architectural and ultra-structural changes through histopathological and scanning electron microscopy (SEM). It is observed that TCBZ, NTON and OCZN at higher concentrations significantly (P < 0.05) reduced RM of the flukes compared to untreated control. NTON at 150 µg mL−1 was the most potent to reduce the motility within 4 h whereas TCBZ and OCZN were much delayed. Histopathological changes showed swollen, extensive cracking, numerous vacuoles and splitting of the tegument surrounding the spines; spine dislodged from its socket in treated flukes compared to untreated worms. Histopathological changes were more conspicuous at higher doses of TCBZ, NTON and OCZN. SEM has shown the disruption of the apical cone, apart from swelling of the tegument on the ventral surface corrugation and disruption of the ventral apical cone. All these changes indicate that NTON is the most potent in killing flukes in vitro among the tested flukicides and suggest the presence of TCBZ-resistant fluke populations in Bangladesh. It is imperative to explore the in vivo effects of these flukicides and subsequently their molecular mechanisms.

Liver function markers and haematological dynamics during acute and chronic phases of experimental Fasciola hepatica infection in cattle treated with triclabendazole.

Fasciola hepatica, a worldwide-distributed liver fluke, is one of the causative agents of fasciolosis, a zoonotic disease that affects livestock and humans. In livestock, fasciolosis causes huge economic losses worldwide, reducing animal fertility, milk production, weight gain and condemnation of livers. In spite of the availability of drugs, such as triclabendazole (TCZ), for the treatment of fasciolosis, they do not necessarily prevent liver damage or parasite reinfection and can eventually increase parasite resistance. The aim of this research was to relate the hepatic function, haematological parameters, leukocyte counts in circulation and parasite egg shedding during F. hepatica acute and chronic phases of infection in cattle as well as to determine how these parameters change with TCZ-treatment of chronically infected cattle. Our results show that increased levels of serum aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were detected in early stages of the experimental infection. Moreover, high circulating eosinophil count and plateletcrit levels were correlated with fluke number in livers from infected cattle. On the other hand, although TCZ-treatment in the chronic phase of infection reduced parasite burden and damage in the liver, it was not able to completely avoid them. In conclusion, our work sheds light into the physiopathological mechanisms induced during fluke infection in cattle, revealing the complexity of the host response to the infection, together with the effects of TCZ-treatment in chronically infected animals.

Triclabendazole Treatment Failure for Fasciola hepatica Infection among Preschool and School-Age Children, Cusco, Peru1.

We conducted a retrospective cohort study of children who had chronic fascioliasis in the highlands of Peru to determine triclabendazole treatment efficacy. Children passing Fasciola eggs in stool were offered directly observed triclabendazole treatment (>1 doses of 10 mg/kg). Parasitologic cure was evaluated by using microscopy of stool 1-4 months after each treatment. A total of 146 children who had chronic fascioliasis participated in the study; 53% were female, and the mean ± SD age was 10.4 ± 3.1 years. After the first treatment, 55% of the children achieved parasitologic cure. Cure rates decreased after the second (38%), third (30%), and fourth (23%) treatments; 17 children (11.6%) did not achieve cure after 4 treatments. Higher baseline egg counts and lower socioeconomic status were associated with triclabendazole treatment failure. Decreased triclabendazole efficacy in disease-endemic communities threatens control efforts. Further research on triclabendazole resistance and new drugs to overcome it are urgently needed.

Acute fasciolosis in an alpaca: a case report.

BACKGROUND: The popularity of new world camelids, particularly alpacas, is growing rapidly in Ireland, presenting a clinical challenge to veterinary practitioners who may not have worked with these species previously. To the authors' knowledge, the clinical course of a case of acute fasciolosis in an alpaca has not previously been reported, and fasciolosis has not been reported at all in alpacas in Ireland, making this case report a valuable addition to the current literature. CASE PRESENTATION: A three-year-old male castrated huacaya alpaca was admitted to UCD Veterinary Hospital with a two-day history of colic and tenesmus. He had been treated with albendazole, dexamethasone and potentiated amoxycillin by the referring veterinary practitioner with no response. On initial clinical exam, sensitivity to abdominal palpation was the only abnormality. However, the alpaca proceeded to show abnormal lying positions, tenesmus and reduced faecal output over the next 24 h. A general blood panel demonstrated moderate anaemia, marked hyperglobulinaemia and moderately increased hepatocellular and hepatobiliary enzyme activity. Abdominal radiography revealed enlargement of the first forestomach compartment without evidence of gastrointestinal obstruction or peritonitis. An abdominal ultrasound exam revealed an elongated, heterogenous mass in the caudoventral abdomen that appeared to be contiguous with the liver. FNA of this mass revealed that it was in fact a liver lobe with biliary stasis and inflammation. Faecal sedimentation demonstrated Fasciola hepatica eggs. In spite of treatment with triclabendazole and supportive treatment including blood transfusion, the alpaca's condition continued to deteriorate and he was euthanised. On post-mortem exam, acute fasciolosis was diagnosed. CONCLUSIONS: The clinical presentation and course of a case of acute fasciolosis in an individual alpaca is described, including the results of a range of diagnostic tests that were carried out. The final diagnosis is supported by a description of post-mortem findings. This information will serve as a resource for veterinary practitioners involved in the diagnosis and treatment of similar cases.

The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica.

Fasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

[Hepatic tumor or giant liver fascciola?: case report]. / ¿Tumor hepático o Fasciola hepática gigante?: reporte de caso.

Hepatic Fasciola is a frequent disease in the Peruvian highlands. We present a case of hepatic Fasciola from the Andean zone of La Libertad, with symptoms of several months of evolution with pain in the right hypochondrium, jaundice and coluria. An abdominal CT scan was performed, demonstrating a liver mass probable neoplasm, and was referred to the Institute of Neoplastic Diseases. Laboratory tests were completed finding eosinophilia and variable increases in liver function tests. Fascioliasis was presented as a differential diagnosis and a Western Bloot examination was performed confirming its diagnosis. Treatment with 2 cycles of Triclabendazole was started, with a favorable clinical evolution.

Pilot Evaluation of Two Fasciola hepatica Biomarkers for Supporting Triclabendazole (TCBZ) Efficacy Diagnostics.

Fasciola hepatica, the causative agent of fasciolosis, is a global threat to public health, animal welfare, agricultural productivity, and food security. In the ongoing absence of a commercial vaccine, independent emergences of anthelmintic-resistant parasite populations worldwide are threatening the sustainability of the few flukicides presently available, and particularly triclabendazole (TCBZ) as the drug of choice. Consequently, prognoses for future fasciolosis control and sustained TCBZ application necessitate improvements in diagnostic tools to identify anthelmintic efficacy. Previously, we have shown that proteomic fingerprinting of F. hepatica excretory/secretory (ES) products offered new biomarkers associated with in vitro TCBZ-sulfoxide (SO) recovery or death. In the current paper, two of these biomarkers (calreticulin (CRT) and triose phosphate isomerase (TPI)) were recombinantly expressed and evaluated to measure TCBZ efficacy via a novel approach to decipher fluke molecular phenotypes independently of molecular parasite resistance mechanism(s), which are still not fully characterised or understood. Our findings confirmed the immunoreactivity and diagnostic potential of the present target antigens by sera from TCBZ-susceptible (TCBZ-S) and TCBZ-resistant (TCBZ-R) F. hepatica experimentally infected sheep.

Screening the Pathogen Box for Identification of New Chemical Agents with Anti-Fasciola hepatica Activity.

Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry. For decades, triclabendazole has been the unique anti-Fasciola drug that can effectively treat this disease. However, triclabendazole resistance in fascioliasis has more recently been reported around the world, and thus, the discovery of novel drugs is an urgent need. The aim of this study was to investigate the fasciocidal properties of 400 compounds contained in the Pathogen Box. The first stage of the screening was carried out by measuring the fasciocidal activity on metacercariae at a concentration of 33 µM each compound (the standard dose). Subsequently, the activities of the most active compounds (n = 33) at their 50% inhibitory concentration (IC50) values against metacercariae were assayed, and the results showed that 13 compounds had IC50s of ≤10 µM. The second stage queried the activities of these compounds at 33 µM against adult flukes, with seven of the compounds producing high mortality rates of >50%. Four hit compounds were selected on the basis of their predicted nontoxic properties, and the IC50 values obtained for adult worms were <10 µM; thus, these compounds represented the best fasciocidal compounds tested here. A cytotoxicity assay on four types of cell lines demonstrated that three compounds were nontoxic at their most active concentration. In conclusion, three hit compounds identified in this proof-of-concept study are potential candidates in the discovery of new fasciocidal drugs. Further studies are warranted.

Pharmacologic interaction between oxfendazole and triclabendazole: In vitro biotransformation and systemic exposure in sheep.

The aim of the current work was to evaluate a potential pharmacokinetic interaction between the flukicide triclabendazole (TCBZ) and the broad-spectrum benzimidazole (BZD) anthelmintic oxfendazole (OFZ) in sheep. To this end, both an in vitro assay in microsomal fractions and an in vivo trial in lambs parasitized with Haemonchus contortus resistant to OFZ and its reduced derivative fenbendazole (FBZ) were carried out. Sheep microsomal fractions were incubated together with OFZ, FBZ, TCBZ, or a combination of either FBZ and TCBZ or OFZ and TCBZ. OFZ production was significantly diminished upon coincubation of FBZ and TCBZ, whereas neither FBZ nor OFZ affected the S-oxidation of TCBZ towards its sulfoxide and sulfone metabolites. For the in vivo trial, lambs were treated with OFZ (Vermox® oral drench at a single dose of 5 mg/kg PO), TCBZ (Fasinex® oral drench at a single dose of 12 mg/kg PO) or both compounds at a single dose of 5 (Vermox®) and 12 mg/kg (Fasinex®) PO. Blood samples were taken to quantify drug and metabolite concentrations, and pharmacokinetic parameters were calculated by means of non-compartmental analysis. Results showed that the pharmacokinetic parameters of active molecules and metabolites were not significantly altered upon coadministration. The sole exception was the increase in the mean residence time (MRT) of OFZ and FBZ sulfone upon coadministration, with no significant changes in the remaining pharmacokinetic parameters. This research is a further contribution to the study of metabolic drug-drug interactions that may affect anthelmintic efficacies in ruminants.

Improving the Dissolution of Triclabendazole from Stable Crystalline Solid Dispersions Formulated for Oral Delivery.

Triclabendazole belongs to the class II/IV of the Biopharmaceuticals Classification System, and its low aqueous solubility represents a major drawback during the development of effective dosage forms. Therefore, the goal of this study was to elucidate whether polymeric solid dispersions would represent a suitable approach to overcome such disadvantage. Due to the lack of information on triclabendazole release, four different dissolution media were evaluated to analyze drug dissolution rate. The polymeric solid dispersions were characterized by X-ray diffraction and Fourier transform infrared spectroscopy. The selected final formulations were further stored for 24 months, and their physical stability was evaluated by means of X-ray diffraction and drug dissolution assays. Drug solubility studies indicated that poloxamer 407 (P407) solubilized a higher amount of drug than polyethylene glycol 6000. Drug-to-carrier ratio, nature of the selected carriers, and the type of dissolution media were important factors for increasing dissolution. By infrared spectroscopy, there were no specific interactions between the drug and polymers. The physicochemical characterization of the systems showed a detectable evidence of drug amorphization by increasing the carrier ratio. Micromeritic studies indicated that raw triclabendazole, physical mixtures, and reference formulation showed poor flow properties, in contrast to the triclabendazole:P407 solid dispersion sample. Both the crystalline properties and dissolution rate of selected samples were very similar after 24 months at room temperature. Thus, considering physical stability and dissolution studies, the development of the solid dispersion is a very suitable methodology to improve triclabendazole dissolution and, potentially, its biopharmaceutical performance.

Recent developments in the epidemiology, diagnosis, and treatment of Fasciola infection.

PURPOSE OF REVIEW: This review aims at describing the latest research in Fasciola epidemiology, diagnosis, treatment, and control in endemic countries. RECENT FINDINGS: The geographic distribution and range of reservoirs for Fasciola hepatica continues to expand. The impact of fascioliasis goes beyond human disease to affect food security and income in developed and developing countries. Promising serologic and molecular methods to diagnose fascioliasis have been described, but are not widely available. Triclabendazole remains the only highly active medication to treat human and livestock infected with juvenile and adult forms of Fasciola spp. Efforts to control fascioliasis may be hindered by the emergence of resistance to triclabendazole among livestock and subsequently in humans. SUMMARY: Increased awareness and surveillance are likely to uncover the real distribution and burden of fascioliasis in human. Research into new drugs or adjuvants to tackle the emerging resistance to triclabendazole is imperative to treat and control Fasciola infection.

Assessment of the pharmacological interactions between the nematodicidal fenbendazole and the flukicidal triclabendazole: In vitro studies with bovine liver microsomes and slices.

Parasitic diseases have a significant impact on livestock production. Nematodicidal drugs, such as fenbendazole (FBZ) or its oxidized metabolite oxfendazole (OFZ), can be used along with the trematodicidal triclabendazole (TCBZ), to broaden the spectrum of anthelmintic activity. However, co-exposure to these compounds could lead to drug-drug (D-D) interactions and eventually alter the clinical profile of each active principle. The aim of this study was to assess the presence of such interactions by means of two in vitro models, namely bovine liver microsomal fractions and bovine precision-cut liver slices (PCLSs). To this end, an in vitro assessment involving incubation of FBZ and TCBZ or a combination of FBZ and TCBZ was carried out. Results with microsomal fractions showed a 78.4% reduction (p = .002) in the rate of OFZ production upon co-incubation, whereas the sulfoxide metabolite of TCBZ (TCBZSO) exhibited a decreasing tendency. With PCLS, OFZ accumulation in the incubation medium increased 1.8-fold upon co-incubation, whereas TCBZSO accumulation decreased by 28%. The accumulation of FBZ and OFZ in the liver tissue increased upon 2-hr co-incubation, from 2.1 ± 1.5 to 18.2 ± 6.1 (p = .0009) and from 0.4 ± 0.1 to 1.3 ± 0.3 nmol (p = .0005), respectively. These results confirm the presence of D-D interactions between FBZ and TCBZ. Further studies are needed to determine the extent of involvement of drug-metabolizing enzymes and membrane transporters in interactions between compounds largely used in livestock production systems.