Macrocyclic lactone toxicity due to abamectin in farm dogs without the ABCB1 gene mutation

Abstract

CASE HISTORY: A 5-year-old entire female Huntaway from a sheep and beef farm was one of four dogs that developed clinical signs including hypersalivation, depression, blindness and ataxia after the death of another dog. A 4-year-old female Huntaway farm dog from a second farm was observed to be sitting down more often than usual on the day after being fed part of a calf carcass that had been treated with an abamectin pour-on.

CLINICAL FINDINGS: The first dog was ataxic and depressed but did respond to sound. The second dog presented with an acute onset of blindness, mydriasis, absence of a menace response, hypersalivation, gait abnormalities (e.g. high stepping gait and ataxia), and depression. Other presenting signs included muscle tremors, dehydration and difficulty eating. No abnormalities were detected from routine haematology and biochemistry. Analysis of samples of plasma from both dogs revealed concentrations of abamectin of 0.149 mg/L and 0.260 mg/L for the first and second dogs, respectively. Buccal swabs taken from the first dog for DNA testing for the ABCB1 gene mutation, gave a negative result.

DIAGNOSIS: In addition to the presenting signs which suggested a toxicosis, both dogs had measurable concentrations of abamectin in plasma confirming exposure.

CLINICAL RELEVANCE: Farm dogs exposed to concentrated pour-on products containing abamectin have been poisoned and recover or die. The product labels do not carry any warnings as to the risk of poisoning to dogs. This paper discusses two incidents affecting six farm dogs, but the authors are aware of more toxicoses in farm dogs exposed to abamectin.     

Novel insertion mutation of ABCB1 gene in an ivermectin-sensitive Border Collie

P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an efflux pump for xenobiotics. In the Border Collie, a nonsense mutation caused by a 4-base pair deletion in the ABCB1 gene is associated with a premature stop to P-gp synthesis. In this study, we examined the full-length coding sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the aforementioned deletion mutation. The sequence was compared to the corresponding sequences of a wild-type Beagle and seven ivermectin-tolerant family members of the Border Collie. When compared to the wild-type Beagle sequence, that of the ivermectin-sensitive Border Collie was found to have one insertion mutation and eight single nucleotide polymorphisms (SNPs) in the coding sequence of the ABCB1 gene. While the eight SNPs were also found in the family members'' sequences, the insertion mutation was found only in the ivermectin-sensitive dog. These results suggest the possibility that the SNPs are species-specific features of the ABCB1 gene in Border Collies, and that the insertion mutation may be related to ivermectin intolerance.     

ABCB1-1Delta polymorphism can predict hematologic toxicity in dogs treated with vincristine.

BACKGROUND: Dogs that harbor the naturally occurring ABCB1-1Delta polymorphism experience increased susceptibility to avermectin-induced neurological toxicosis as a result of deficient P-glycoprotein function. Whether or not the ABCB1-1Delta polymorphism affects susceptibility to toxicity of other P-glycoprotein substrate drugs has not been studied. HYPOTHESIS: Dogs that possess the ABCB1-1Delta mutation are more likely to develop hematologic toxicity associated with vincristine than ABCB1 wild-type dogs. ANIMALS: Thirty-four dogs diagnosed with lymphoma were included in this study. METHODS: Cheek swab samples were obtained from dogs diagnosed with lymphoma that were to be treated with vincristine. DNA was extracted from cheek swabs and the ABCB1 genotype was determined. Hematologic adverse drug reactions were recorded for each dog and graded according to the Veterinary Comparative Oncology Group's criteria for adverse event reporting (Consensus Document). In order to avoid possible bias, ABCB1 genotype results for a particular patient were not disclosed to oncologists until an initial adverse event report had been submitted. RESULTS: Dogs heterozygous or homozygous for the ABCB1-1Delta mutation were significantly more likely to develop hematologic toxicity, specifically neutropenia (P= .0005) and thrombocytopenia (P= .0001), after treatment with vincristine than ABCB1 wild-type dogs. CONCLUSIONS AND CLINICAL IMPLICATIONS: At currently recommended dosages (0.5-0.7 mg/M(2)), vincristine is likely to cause hematologic toxicity in dogs with the ABCB1-1Delta mutation, resulting in treatment delays and unacceptable morbidity and mortality. Assessing the ABCB1-1Delta genotype before vincristine administration and decreasing the dosage may prevent toxicity and treatment delays resulting from neutropenia or thrombocytopenia.     

The ABCB1-1Δ mutation is not responsible for subchronic neurotoxicity seen in dogs of non-collie breeds following macrocyclic lactone treatment for generalized demodicosis

P-glycoprotein (P-gp), encoded by the multiple drug resistance gene ABCB1 (also known as MDR1 ), is an integral component of the blood brain barrier crucial in limiting drug uptake into the central nervous system. Altered expression or function of P-gp, as seen in dogs of the collie lineage homozygous for the nt228(del4) mutation of the ABCB1 gene ( ABCB1-1Δ ), can result in potentially fatal neurotoxicosis, especially following administration of systemic macrocyclic lactones (SML). Occasionally, dogs from unrelated breeds develop subchronic signs of neurotoxicity when receiving SML to treat generalized demodicosis. It is possible that these dogs are heterozygous carriers of the ABCB1-1Δ mutation, resulting in decreased P-gp activity and central neurotoxicosis. Cheek swabs were collected from 28 dogs with generalized demodicosis that had shown subchronic signs of neurotoxicity following daily oral administration of ivermectin or other SML. Ten of these animals received concurrent systemic treatment with other confirmed or putative P-gp substrates. After DNA extraction, the relevant portion of the ABCB1 gene was amplified by polymerase chain reaction, and sequenced. Twenty-seven dogs were homozygous normal while one dog was heterozygous for the ABCB1-1Δ mutation. Therefore, with the exception of one dog, the observed neurotoxicity could not be attributed to the ABCB1-1Δ mutation. Possible explanations for the adverse reactions observed include pharmacological interactions (administration of SML with other P-gp substrates or inhibitors), excessively high doses, polymorphisms in P-gp expression, uncharacterized mutations in the ABCB1 gene or in another gene, or phenomena unrelated to the SML–P-gp interaction.     

Identification of a nonsense mutation in feline ABCB1

The aim of this study was to sequence all exons of the ABCB1 (MDR1) gene in cats that had experienced adverse reactions to P‐glycoprotein substrate drugs (phenotyped cats). Eight phenotyped cats were included in the study consisting of eight cats that experienced central nervous system toxicosis after receiving ivermectin (n = 2), a combination product containing moxidectin and imidacloprid (n = 3), a combination product containing praziquantel and emodepside (n = 1) or selamectin (n = 2), and 1 cat that received the product containing praziquantel and emodepside but did not experience toxicity (n = 1). Fifteen exons contained polymorphisms and twelve exons showed no variation from the reference sequence. The most significant finding was a nonsense mutation (ABCB11930_1931del TC) in one of the ivermectin‐treated cats. This cat was homozygous for the deletion mutation. All of the other phenotyped cats were homozygous for the wild‐type allele. However, 14 missense mutations were identified in one or more phenotyped cats. ABCB11930_1931del TC was also identified in four nonphenotyped cats (one homozygous and three heterozygous for the mutant allele). Cats affected by ABCB11930_1931del TC would be expected to have a similar phenotype as dogs with the previously characterized ABCB1‐1Δ mutation.     

Breed distribution of the ABCB1-1Delta (multidrug sensitivity) polymorphism among dogs undergoing ABCB1 genotyping

OBJECTIVE: To evaluate the breed distribution of the ABCB1-1Delta polymorphism in a large number of dogs in North America, including dogs of several herding breeds in which this polymorphism has been detected and other breeds in which this polymorphism has not yet been identified. DESIGN: Cross-sectional study. ANIMALS: 5,368 dogs from which buccal swab samples were collected for purposes of ABCB1 genotyping. PROCEDURES: From May 1, 2004, to September 30, 2007, DNA specimens derived from buccal swab samples collected from 5,368 dogs underwent ABCB1 genotyping. These data were reviewed, and results for each dog were recorded in a spreadsheet, along with the dog's breed. The genotypes for each breed were tallied by use of a sorting function. RESULTS: The ABCB1-1Delta allele was identified in 9 breeds of dogs and in many mixed-breed dogs. Breeds that had the ABCB1-1Delta allele included Collie, Longhaired Whippet, Australian Shepherd (standard and miniature), Shetland Sheepdog, Old English Sheepdog, Border Collie, Silken Windhound, and German Shepherd Dog (a breed in which this mutation had not been detected previously). CONCLUSIONS AND CLINICAL RELEVANCE: The ABCB1-1Delta polymorphism is associated with increased susceptibility to many adverse drug reactions and with suppression of the hypothalamic-pituitary-adrenal axis and is present in many herding breeds of dog. Veterinarians should be familiar with the breeds that have the ABCB1-1Delta polymorphism to make appropriate pharmacologic choices for these patients.     

Biliary excretion of technetium-99m-sestamibi in wild-type dogs and in dogs with intrinsic (ABCB1-1Δ mutation) and extrinsic (ketoconazole treated) P-glycoprotein deficiency

P-glycoprotein (P-gp), the product of ABCB1 gene, is thought to play a role in the biliary excretion of a variety of drugs, but specific studies in dogs have not been performed. Because a number of endogenous (ABCB1 polymorphisms) and exogenous (pharmacological P-gp inhibition) factors can interfere with normal P-gp function, a better understanding of P-gp's role in biliary drug excretion is crucial in preventing adverse drug reactions and drug–drug interactions in dogs. The objectives of this study were to compare biliary excretion of technetium-99m-sestamibi (^99mTc-MIBI), a radio-labelled P-gp substrate, in wild-type dogs (ABCB1 wild/wild), and dogs with intrinsic and extrinsic deficiencies in P-gp function. Dogs with intrinsic P-gp deficiency included ABCB1 mut/mut dogs, and dogs with presumed intermediate P-gp phenotype (ABCB1 mut/wild). Dogs with extrinsic P-gp deficiency were considered to be ABCB1 wild/wild dogs treated with the P-gp inhibitor ketoconazole (5 mg/kg PO q12h × 9 doses). Results from this study indicate that ABCB1 mut/mut dogs have significantly decreased biliary excretion of ^99mTc-MIBI compared with ABCB1 wild/wild dogs. Treatment with ketoconazole significantly decreased biliary excretion of ^99mTc-MIBI in ABCB1 wild/wild dogs. P-gp appears to play an important role in the biliary excretion of ^99mTc-MIBI in dogs. It is likely that concurrent administration of a P-gp inhibitor such as ketoconazole will decrease P-gp-mediated biliary excretion of other substrate drugs as well.     

Novel genotyping assay for the nt230 (del4) ABCB1 gene mutation and its allele frequency in Border Collie dogs in Mexico

A 4-bp deletion in the ATP-binding cassette subfamily B member 1 (ABCB1) gene, also referred to as the multidrug resistance gene (MDR1), produces stop codons that cause premature termination of P-glycoprotein 1 (P-gp) synthesis. Dogs with the homozygous mutation do not express functional P-gp, which increases their sensitivity markedly to many common veterinary drugs. We detected the nt230 (del4) ABCB1 mutation in Border Collie dogs in western Mexico with a simple and affordable primer-introduced restriction analysis PCR (PIRA-PCR). PIRA-PCR clearly identified all genotypes in our sample of 104 dogs. Genotype frequencies were 0.952 (wild/wild), 0.029 (wild/mut) and 0.019 (mut/mut). Allele frequencies were 0.033 (mutant alleles) and 0.966 (wild-type alleles). In this small subset of the Mexican dog population, we found a higher prevalence of the nt230 (del4) MDR1/ABCB1 gene mutation than reported in other countries.     

Attenuation of an avian pathogenic Escherichia coli strain due to a mutation in the rpsL gene

The diseases caused by pathogenic Escherichia coli constitute a major economic loss to the poultry industry. The development of a live oral E. coli vaccine to prevent or reduce diseases in poultry had been the objective of our work. Four spontaneous streptomycin-dependent (str-dependent) mutants were generated from a virulent avian strain that contains a mutation in the fur region of the chromosome. Genetic analysis of the mutants indicated that the str-dependent phenotype was due to a base change of C --> T at base 272 in the rpsL gene. The mutants were tested for attenuation using the day-old chick model. Day-old birds, in groups of 20, were either challenged with 10(6) colony-forming units (CFU) of the str-dependent mutant, the parent strain (containing the fur mutation), or the wild-type strain without the fur mutation. The parent strain and the wild-type strain were highly virulent, and 80% or more of the birds died. None of the birds challenged with the str-dependent mutants died, indicating attenuation of the mutants. The protective effect of the mutant as a live vaccine against the challenge with 10(6) CFU of the wild-type strain EC317 was investigated. Vaccination by both aerosol (day 1) and oral (days 14 and 28) routes using 10(8) CFU of the str-dependent mutant (EC1598) had no effect on the occurrence of cellulitis in the birds. Two vaccinations given as aerosol on day 1 and given orally on day 14 also had no significant effect on the occurrence of systemic lesions. Three immunizations on days 1, 14, and 28 resulted in a significant reduction in the number of birds with systemic lesions. Antibody titers prior to challenge were not predictive of outcome of challenge.     

Polymorphisms in the ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies

Background: Variation in the ABCB1 gene is believed to play a role in drug resistance in epilepsy. Hypothesis/Objectives: Variation in the ABCB1 gene encoding the permeability‐glycoprotein could have an influence on phenobarbital (PB) resistance, which occurs with high frequency in idiopathic epileptic Border Collies (BCs). Animals: Two hundred and thirty‐six client‐owned BCs from Switzerland and Germany including 25 with idiopathic epilepsy, of which 13 were resistant to PB treatment. Methods: Prospective and retrospective case‐control study. Data were collected retrospectively regarding disease status, antiepileptic drug (AED) therapy, and drug responsiveness. The frequency of a known mutation in the ABCB1 gene (4 base‐pair deletion in the ABCB1 gene [c.296_299del]) was determined in all BCs. Additionally, the ABCB1 coding exons and flanking sequences were completely sequenced to search for additional variation in 41 BCs. Association analyses were performed in 2 case‐control studies: idiopathic epileptic and control BCs and PB‐responsive and resistant idiopathic epileptic BCs. Results: One of 236 BCs (0.4%) was heterozygous for the mutation in the ABCB1 gene (c.296_299del). A total of 23 variations were identified in the ABCB1 gene: 4 in exons and 19 in introns. The G‐allele of the c.‐6‐180T > G variation in intron 1 was significantly more frequent in epileptic BCs resistant to PB treatment than in epileptic BCs responsive to PB treatment (P_raw= .0025). Conclusions and Clinical Importance: A variation in intron 1 of the ABCB1 gene is associated with drug responsiveness in BCs. This might indicate that regulatory mutations affecting the expression level of ABCB1 could exist, which may influence the reaction of a dog to AEDs.     

Canine mdr1 gene mutation in Japan

Frequency of the 4-bp deletion mutant in canine mdr1 gene was examined in 193 dogs of eight breeds in Japan. The mutant allele was found in Collies, Australian Shepherds, and Shetland Sheepdogs, where its respective frequencies were 58.3%, 33.3%, and 1.2%. The MDR1 protein was detected on peripheral blood mononuclear cells (PBMC) from a MDR1/MDR1 dog, but not on PBMC from a mdr1-1Delta/mdr1-1Delta Collie. Rhodamine 123 was extruded from MDR1/MDR1 lymphocytes. That excretion was inhibited by a MDR1 inhibitor, verapamil. On the other hand, Rh123 excretion was not observed from lymphocytes derived from a mdr1-1Delta/mdr1-1Delta Collie. These results indicated that the mutant mdr1 allele also existed in Collie-breed dogs in Japan at high rates and that mdr1-1Delta /mdr1-1Delta dogs have no functional MDR1.     

Presence of Escherichia coli carrying the EAST1 toxin gene in farm animals

Enteroaggregative Escherichia coli (EAEC) heat-stable toxin 1 (EAST1) is a small toxin of proteic nature. The reference strain producing this toxin was originally detected in the stools of a diarrheic Chilean child. Today, we know that EAST1 is not solely associated with EAEC but also with many other diarrheic E. coli families. Some studies have established the role of EAST1 in human outbreaks of diarrhea. In addition, isolates from farm animals were shown, more recently, to carry the astA gene coding for EAST1. However, the relation between the presence of EAST1 and disease is not conclusive. In this article, the current state of the knowledge on the presence and probable role of EAST1 in farm animal diseases is reviewed and discussed.