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.     

Frequency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany

MDR1 (ABCB1) P-glycoprotein exerts a protective function in the blood–brain barrier thereby limiting the entry of many drugs and other xenobiotics to the central nervous system. A nonsense mutation has been described for Collies and related dog breeds which abolishes this function and is associated with increased susceptibility to neurotoxic side effects of several drugs including ivermectin, moxidectin and loperamide. In order to evaluate the occurrence and frequency of this nt230 (del4) MDR1 mutation in Germany, we screened 1500 dogs. Frequency of the homozygous mutated genotype was highest for Collies (33.0%), followed by Australian Shepherd (6.9%) and Shetland Sheepdog (5.7%). Thirty-seven percent of the Wäller dogs and 12.5% of the Old English Sheepdogs were heterozygous for the mutant MDR1 (−) allele. Considering the predominant role of MDR1 P-glycoprotein in drug disposition and in particular for blood–brain barrier protection, MDR1 genotype-based breeding programs are recommended for improving the safety of drug therapy in these canine breeds.     

Development of a PCR-based diagnostic test detecting a nt230(del4) MDR1 mutation in dogs: verification in a moxidectin-sensitive Australian Shepherd

A subpopulation of dogs of the Collie and Australian Shepherd breeds show increased sensitivity to central nervous actions of ivermectin, doramectin, loperamide, and probably several other drugs. The molecular background for this greater sensitivity is a nonsense mutation in the MDR1 efflux pump, which is part of the functional blood-brain barrier and normally limits drug penetration into the brain. This report describes a rapid PCR-based method for detection of this nt230(del4) MDR1 mutation using a small amount of genomic DNA from blood cells. Thereby, homozygous intact, homozygous mutated, and heterozygous mutated MDR1 genotypes can be clearly differentiated by high resolution polyacrylamide gel electrophoresis. Using this diagnostic test two Collies and one Australian Shepherd were screened for the nt230(del4) MDR1 mutation. The Collies had no history of altered drug sensitivity and showed homozygous intact and heterozygous mutated MDR1 alleles, respectively. However, the Australian Shepherd developed clear signs of neurotoxicity including ataxia, crawling, acoustic and tactile hyperexcitability, and miosis after a single dose of moxidectin (400 microg/kg). For this dog two mutated MDR1 alleles were detected. This report describes for the first time moxidectin neurotoxicosis in a dog with a homozygous MDR1 mutation.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Development of a genotyping tool for a functionally relevant CYP2C19 allele (Phe100Asn,Ala103Val and Ile112Leu) in cynomolgus macaques

In cynomolgus macaques, which are widely used in drug metabolism studies, CYP2C19(formerly known as CYP2C75) is abundantly expressed in liver, metabolizes human CYP2Csubstrates and is thus an important drug-metabolizing enzyme. One of the cynomolgusCYP2C19 alleles (p.Phe100Asn, p.Ala103Val and p.Ile112Leu) results insubstantially reduced metabolic activity and thus is an important allele in drugmetabolism studies. For this allele, a genotyping tool was developed using allele-specificTaqMan probe. Genotyping 40 Cambodian cynomolgus macaques using this tool found 1homozygote, 17 heterozygotes and 22 wild type animals, and the result was confirmed bydirect-sequencing. Interestingly, this allele frequency was similar to that of Chinesecynomolgus macaques. The genotyping tool established is useful for drug metabolism studiesusing cynomolgus macaques.     

Safety of alternate-day treatment with TS-1TM (tegafur/gimeracil/oteracil) in tumor-bearing dogs: a pilot study

Tegafur is a prodrug of fluoropyrimidine 5-fluorouracil (5-FU), while TS-1^TM is an oral fixed-dose combination of three active drugs, tegafur, gimeracil, and oteracil. This pilot study evaluated the safety of tegafur/gimeracil/oteracil in the treatment of cancers in dogs. Tegafur/gimeracil/oteracil was administered orally at a mean dose of 1.1 mg/kg twice daily on alternate days, Monday-Wednesday-Friday, every week to 11 dogs with tumors. Partial response and stable disease were observed in one dog each, whereas six exhibited progressive disease. Three dogs were not assessed. Adverse events, the most serious being grade 2, were noted in seven dogs. Adverse events were acceptable, and the drug was effective in some dogs. Therefore, tegafur/gimeracil/oteracil may be useful for treating malignant solid tumors in canines.     

Evaluation of canine heat shock transcription factor 4 (HSF4) as a candidate gene for primary cataracts in the Dachshund and the Entlebucher Mountain dog

OBJECTIVE: Testing of the cataract-causing insertion/deletion mutation in the canine HSF4 gene for its linkage and association with primary cataracts (CAT) in Dachshunds and Entlebucher Mountain dogs. MATERIALS: Exon 9 with flanking intronic regions of the canine HSF4 gene was sequenced in 24 Dachshunds and 20 Entlebucher Mountain dogs. The HSF4 cDNA sequence of lens tissue was analyzed in a CAT-unaffected mixed-breed dog and in three CAT-affected dogs of different breeds, including a Wire-haired Dachshund, a Dachshund-mix and a German Shepherd dog. RESULTS: In all dogs investigated here, the previously reported CAT-causing mutation did not exist. We found a single nucleotide polymorphism (SNP) in intron 9, which was neither associated nor linked with the CAT phenotype in the two dog breeds. CONCLUSION: The CAT phenotype in the two dog breeds investigated here was not caused by the same mutation found to be associated with early-onset CAT in the Staffordshire Bull Terrier and Boston Terrier. The intronic SNP may be useful to test HSF4 for linkage with CAT in further dog breeds.     

Localization of uncoupling protein 1 (UCP-1) in the sebaceous gland of the dorsal region in the Sunda porcupine (Hystrix javanica)

Uncoupling protein 1 (UCP-1) was believed to be an exclusive protein found in the brown adipose tissue of small rodents and humans; however, recent studies show that the expression of UCP-1 protein has been found in the sebaceous glands of the mouse tail and human skin. There are a few reports about the presence of UCP-1 in the sebaceous glands of other rodents, such as the Sunda porcupine (Hystrix javanica), a wild spiny rodent commonly found in Indonesia with a large sebaceous gland. The aim of this study was to identify the presence of UCP-1 in the sebaceous glands on the skin of the Sunda porcupine. The skin from three regions (thoracodorsal, lumbosacral and apex caudal) of eight adult Sunda porcupines was used to detect UCP-1-immunopositive cells through immunohistochemistry. All three regions were found immunopositive to anti-UCP-1 antibody in the sebaceous gland of quill and hair follicles, and the epidermal layer in quill and hair follicles with various intensities. The result of immunohistochemistry revealed that the thoracodorsal and apex caudal region was the most intense immunoreaction followed by the lumbosacral region. These findings proved that the presence of UCP-1 was also identified in the sebaceous glands of other rodent (Hystrix javanica) and regions of the body, which has not been reported previously.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Feeding strategies for small-scale dairy systems based on perennial (<Emphasis Type="Italic">Lolium perenne</Emphasis>) or annual (<Emphasis Type="Italic">Lolium multiflorum</Emphasis>) ryegrass in the central highlands of Mexico

Small-scale dairying is an option for campesinos in Mexico. The costs of feeding are high and strategies based on quality forages are a priority. The performance, agronomic variables and feeding costs were evaluated for dairy cows continuously grazing perennial ryegrass–white clover for 9 h/day (PRG) or fed cut herbage from annual ryegrass for 8 weeks followed by 9 h/day for 6 weeks on a tethered rotational grazing pattern (ARG). All cows received 3 kg/day of an 18% crude protein (CP) concentrate. A 14-week split-plot on-farm experiment was designed with 10 cows from two participating farmers, and 1.5 ha per strategy. Milk yield was recorded weekly and milk composition, live weight and body condition score were recorded every 14 days. Net herbage accumulation was greater for ARG (8222 kg organic matter (OM)/ha) than for PRG (5915 kg OM/ha) (p < 0.05), with higher CP in PRG (p < 0.05). Milk yield was 19 kg/cow per day for PRG and 15.9 kg/cow per day for ARG (p > 0.05). Over 14 weeks, PRG produced 1422 kg more milk. There were no differences for live weight or condition score (p > 0.05), but linear regression shows a live weight gain of 0.200 kg/cow per day for PRG. Protein and fat content showed no differences (p > 0.05), but milk fat content in PRG was below standard. ARG had 60% higher costs, and margins were 38% higher in PRG. ARG has a place in rain-fed fields. The results provide viable options for improving these systems that may be suitable in their socio-economic context and their social and personal objectives.     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

A Serine12Stop mutation in PB1-F2 of the 2009 pandemic (H1N1) influenza A: a possible reason for its enhanced transmission and pathogenicity to humans

As the scientific community scrambles to define the ancestry and lineages of the eight segments of new pandemic H1N1 strain, we looked for unique genetic events in this virus''s genome to explain the newly found enhanced virulence and transmissibility among humans. Genome annotations of this virus identified a stop mutation replacing serine at codon 12 (S12Stop) of the PB1-F2 protein, a virulence factor in influenza A viruses. Here, we discuss the significance of this finding and how it may contribute to host specialization, explaining the virtual absence of the H1N1 influenza A virus strain in pig populations. This finding is expected to lead to a better understanding of the transmission and pathogenesis of the 2009 pandemic strain.     

The T allele at the g.1471620G>T in the EDG1 gene associated with high marbling in Japanese Black cattle is at a low frequency in breeds not selected for marbling

Our previous study detected a single nucleotide polymorphism (SNP), g.1471620G > T , in the 5' flanking region of the endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, which has been considered as a positional functional candidate for the gene responsible for marbling, and showed association of the g.1471620G > T SNP with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the g.1471620G > T SNP among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Short Horn, Holstein, and Brown Swiss breeds. The T allele at the g.1471620G > T SNP associated with high marbling was found at high frequency in Japanese Black breed that has been subjected to a strong selection for high marbling, while the allele was absent or at very low frequencies in the other breeds that have not been strongly selected for high marbling. Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequency of the T allele at the g.1471620G > T SNP in the EDG1 .