Ochratoxin A cytotoxicity on Madin–Darby canine kidney cells in the presence of alpha‐tocopherol: Effects on cell viability and tight junctions

Ochratoxin A (OTA) is a potent nephrotoxic fungi metabolite that affects animal and human health. At the cellular level, OTA is able to alter functions and viability by several mechanisms of action. Several strategies to counteract its toxicity have been studied. We investigated the role of α‐tocopherol in counteracting OTA oxidative damage in Madin–Darby canine kidney (MDCK) cells by pre‐incubating the cells for 3 hr with the antioxidant (1 nm , 10 μm ) and then adding OTA (0–1.2 μg/ml) for the following 24 hr. Cell viability, lactate dehydrogenase (LDH) release, TUNEL staining and occludin and Zo1 localization by immunofluorescence were determined. Here, 1 nm α‐tocopherol was shown to significantly reduce (p < .05) the cytotoxicity, LDH release and apoptotic rate induced by OTA. The presence of the antioxidant at the same concentration maintained the localization of occludin and Zo1 in the rim of the MDCK cells after the 24‐hr OTA exposure. These results indicate that a low concentration of α‐tocopherol could block OTA toxicity, supporting its defensive role in the cellular membrane.     

Inhibition of P‐glycoprotein by psychotherapeutic drugs in a canine cell model

Drug–drug interactions related to long‐term therapies are of increasing concern. Psychotherapeutic drugs, licensed for the use in dogs for the management of separation anxiety and other behavioural disorders, are examples of drugs used in long‐term therapies. In an in vitro system with canine P‐glycoprotein (P‐gp) expressing cell lines, three psychotherapeutic drugs with a different mode of action were tested for their ability to inhibit the canine multidrug transporter P‐gp. At 10 μm , the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant clomipramine inhibited P‐gp for 41% and 59%, respectively. In contrast, selegeline did not inhibit the function of the canine P‐gp.     

Resistance of Ovine,Caprine and Bovine Endothelial Cells to Clostridium perfringens Type D Epsilon Toxin In Vitro

Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD⁵⁰/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD⁵⁰/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD⁵⁰/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro.     

Molecular Cloning and Characterization of CD9 cDNA from Sheep and Cashmere Goat

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. Some CD9 cDNA have been cloned in mammals and certain fish genera in recent years, but goat and sheep counterparts of cattle, human and mouse have not been identified. To facilitate the studies, we cloned the cDNA encoding for CD9 of cashmere goat (Capra hircus) and sheep (Ovis aries), and expressed sheep CD9 in Escherichia coli cells. Structural analysis indicated for both goat and sheep that a 1123 bp cDNA spanned an open reading frame of 681 bp which predicted a protein of 226 amino acids with a typical TM4SF structure, including four highly conserved transmembrane domains, two extracellular domains and a CCG motif, which is a hallmark of the TM4SF. The predicted amino acid sequences were highly homologous to those of cattle, mouse and human CD9. Molecular phylogenetic analysis based on CD9 cDNA sequences indicated that goat and sheep CD9 were closely related to CD9 of cattle, which is in agreement with their morphological taxonomy.     

Sequence and structural analysis of the presumed downstream promoter of the canine mdr1 gene

The product of the canine mdr1 gene, P‐glycoprotein (P‐gp), plays an important role in chemotherapeutic drug resistance of several canine tumours. Increased expression of P‐gp by tumour cells is associated with the multidrug‐resistant phenotype. Because of its importance in cancer chemotherapy, a great deal is known about the regulation of mdr1 gene expression in human cancer patients and rodent cancer models. In contrast, there is no information regarding the regulation of P‐gp expression in dogs. Initial information regarding the regulation of mdr1 gene expression can be gained by evaluating the mdr1 promoter. The downstream promoter of the canine mdr1 gene was sequenced. Several regulatory elements were identified, including an AP‐1 site, AP‐2 site and SP‐1 site. The presumed canine mdr1 promoter was similar to that of other species; however, low overall sequence homology may suggest that aspects of P‐gp regulation are distinctive in dogs.     

Oral bioavailability of P‐glycoprotein substrate drugs do not differ between ABCB1‐1Δ and ABCB1 wild type dogs

Mealey, K.L., Waiting, D., Raunig, D.L., Schmidt, K.R., Nelson, F.R. Oral bioavailability of P‐glycoprotein substrate drugs do not differ between ABCB1‐1Δ and ABCB1 wild type dogs. J. vet. Pharmacol. Therap. 33 , 453–460. Previous studies have indicated that intestinal P‐glycoprotein (P‐gp) limits the oral bioavailability of substrate drugs and alters systemic pharmacokinetics. In this study, dogs lacking functional P‐gp were used to determine the contribution of P‐gp to the oral bioavailability and systemic pharmacokinetics of several P‐gp substrate drugs. The P‐gp substrates quinidine, loperamide, nelfinavir, cyclosporin and the control (non P‐gp substrate) drug diazepam were individually administered intravenously and per os to ABCB1‐1Δ dogs, which have a P‐gp null phenotype and ABCB1 wildtype dogs. ABCB1‐1Δ dogs have been shown to have greater brain penetration of P‐gp substrates, but limited information is available regarding oral bioavailability of P‐gp substrate drugs in this animal model. Plasma drug concentration vs. time curves were generated and pharmacokinetic parameters were calculated for each drug. There were no differences in oral bioavailability between ABCB1‐1Δ dogs and ABCB1 wildtype dogs for any of the drugs studied, suggesting that intestinal P‐gp does not significantly affect intestinal absorption of these particular substrate drugs in ABCB1‐1Δ dogs. However, small sample sizes and individual variability in CYP enzyme activity may have affected the power of the study to detect the impact of P‐gp on oral bioavailability.     

狐狸脑炎病毒的分离鉴定和流行病学调查

用狗肾传代细胞(MDCK)从3个养狐场的病死狐脏器中分得3株病毒,经电镜观察,理化学、生物学性状和血清学鉴定以及病毒结构蛋白分析,证明其为狐狸脑炎病毒。同时,对这3个发病狐场进行了流行病学调查,所获得的临床资料以及血清学、病毒学和细菌学检测结果进一步证实了这3个养狐场此次所发生的传染病是狐狸脑炎.     

Mitochondrial DNA diversity,origin, and phylogenic relationships of three Chinese large-fat-tailed sheep breeds

China is abundant of sheep genetic resources. A total of 55 sequences containing the Ovis aries mtDNA D-loop of three large-fat-tailed sheep breeds, named Lanzhou, Tong, and Han were retrieved from GenBank to investigate their genetic diversity, origin, and phylogenetic evolution. The results showed that the sheep breeds in our study proved to be extremely diverse, the average haplotype diversity and nucleotide diversity were 0.987 ± 0.006 and 0.03956 ± 0.00206, respectively. The 55 sequences gave 39 different haplotypes. Phylogenetic analyses revealed that there were three distinct mtDNA haplogroups: A, B, and C, in which haplogroup A was predominant and had experienced population expansion events. Clustering analysis showed that the large-fat-tailed sheep breeds clustered into one group and were closely related to the Mongolian sheep and then European mouflon sheep (Ovis musimon). The results contribute to the knowledge of Chinese sheep breeds and the plan of conservation programs on large-fat-tailed sheep.     

Isolation and identification of canine adenovirus type-2 from the upper respiratory tract of a dog

AIM: To report on the isolation and identification of canine adenovirus type-2 (CAV-2) from a greyhound dog with tracheitis/tonsillitis. METHODS: Virus isolation was performed with Madin and Darby canine kidney (MDCK) cell monolayers using standard virological techniques. The isolated virus was identified by haemagglutination inhibition and serum neutralisation tests. Viral DNA was extracted from infected MDCK cells and subjected to restriction endonuclease analysis using the endonuclease enzymes Bam HI, Bgl II, Eco RI and Hind III. RESULTS: A virus, designated 5 113-87, was isolated in MDCK cells yielding typical cytopathic effect. The virus could be neutralised with a CAV-2 specific reference antiserum and also showed some cross neutralisation with CAV-1 specific reference antiserum. The virus 5 113-87 had a high haemagglutination inhibition titre with CAV-2 antiserum using human group 0 red blood-cells and CAV-1 and CAV-2 reference antisera. This virus also had DNA restriction profiles identical to those of the reference CAV-2 (Toronto A26/61), whereas previously isolated strains of adenovirus from dogs in New Zealand had DNA restriction patterns identical to the prototype CAV-1 strain (Utrecht). CONCLUSION: The findings show that the virus 5 113-87 isolated from the upper respiratory tract of a dog in New Zealand is CAV-2.     

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5’LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

     

Establishment of a cell line for assessing drugs as canine P‐glycoprotein substrates: proof of principle

P‐glycoprotein (P‐gp), encoded by the ABCB1 (MDR1) gene, dramatically impacts drug disposition. P‐gp is expressed in the intestines, biliary canaliculi, renal tubules, and brain capillaries where it functions to efflux substrate drugs. In this capacity, P‐gp restricts oral absorption, enhances biliary and renal excretion, and inhibits central nervous system entry of substrate drugs. Many drugs commonly used in veterinary medicine are known substrates for canine P‐gp (vincristine, loperamide, ivermectin, others). Because these drugs have a narrow therapeutic index, defective P‐gp function can cause serious adverse drug reactions due to enhanced brain penetration and/or decreased clearance. P‐gp dysfunction in dogs can be intrinsic (dogs harboring ABCB1‐1Δ) or acquired (drug interactions between a P‐gp inhibitor and P‐gp substrate). New human drug candidates are required to undergo assessment for P‐gp interactions according to FDA and EMA regulations to avoid adverse drug reactions and drug–drug interactions. Similar information regarding canine P‐gp could prevent adverse drug reactions in dogs. Because differences in P‐gp substrates have been documented between species, one should not presume that human or murine P‐gp substrates are necessarily canine P‐gp substrates. Thus, our goal was to develop a cell line for assessing drugs as canine P‐gp substrates.     

Tissue Distribution of P‐Glycoprotein in Cats

Permeability glycoprotein (P‐gp) is a membrane‐bound efflux pump that exports various substances out of the cell. Variations in P‐gp expression play an important role in susceptibility to toxic substances, drug efficacy and disease risk. In the present study, the distribution of the MDR1‐gene product P‐gp was determined in normal tissues of domestic shorthair cats using immunohistochemistry. Two monoclonal antibodies C494 and C219 were used, recognizing a different epitope on the human P‐gp molecule. A consistent positive immunolabelling was obtained. The tissue distribution and cellular locations with antibody C494 were similar to those in man and dogs; with liver, colon, adrenal cortex and brain capillaries being consistently and intensely labelled. However, the immunolabelling in the kidney was in contradiction to man and dogs. The C219 antibody seems to react with a specific form of P‐gp, only expressed in feline tissues with a barrier function, i.e. endothelia of the brain, testes and ovaries, and intestinal epithelial cells in contact with the lumen.     

A comparative analysis of sphenoid bone between domestic sheep (ovis aries) and goat (capra hircus) using geometric morphometrics

The sphenoid bone forms the rostral part of the base of the neurocranium and is composed of two segments, the presphenoid [os praesphenoidale] and the basisphenoid [os basisphenoidale]. Rarely studied in osteology, we tested whether it can provide distinctive features between domestic sheep (Ovis aries L., 1758) and goat (Capra hircus L., 1758). For this goal, we studied a sample comprised by 53 dry modern skulls of adult sheep (n = 36) and goat (n = 17) subjects from a modern comparative collection by means of geometric morphometric techniques using a total of 26 anatomical points (2 saggital landmarks and 24 semilandmarks). Results showed that form (size + shape) differences appear between both species: sphenoid among sheep tends to be bigger, longer and wider than in goats, differences of width being mainly located on basisphenoid width.     

狐狸脑炎病毒的分离鉴定

通过ＭＤＣＫ传细胞从黑龙江某狐场疑似狐狸脑炎病狐的肝脏中分离到对本动物具有较强致病能力的强毒株,定名为ＦＥＶ－Ｈ。经系统鉴定,并与已知国内分离毒株狐狸脑炎病毒ＦＥＶ－８８０１,狐喉气管炎病毒ＦＡＶ－２比较,证实为狐狸脑炎病毒,属犬１型腺病毒（ＣＡＶ－１）。     

In vitro infection by Ehrlichia ruminantium of baby hamster kidney (BHK), Chinese hamster ovary (CHO-K1) and Madin Darby bovine kidney (MDBK) cells

The Welgevonden stock of Ehrlichia ruminantium, aetiological agent of heartwater, was propagated in baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO-K1) cells and Madin Darby bovine kidney (MDBK) cells. The cultures required supplementation of the medium with cycloheximide for reliable growth of E. ruminantium. Growth of the Welgevonden stock in BHK and CHO-K1 cells could lead to the development of suspension cultures suitable for the mass production of E. ruminantium for an inactivated elementary body vaccine.     

Cloning of the Genomes of Equine Herpesvirus Type 1 (EHV‐1) Strains KyA and RacL11 as Bacterial Artificial Chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV‐1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Escherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co‐transfection of viral DNA and recombinant plasmid pΔ71‐pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high‐molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full‐length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacL11 and pKyA, respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP‐expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full‐length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2‐negative progeny reconstituted from pRacL11 and pKyA (L11Δgp2 and KyAΔgp2) exhibited different growth properties. Whereas both L11Δgp2 and KyAΔgp2 extracellular titres were reduced by 15‐ to 32‐fold, plaque diameters were only markedly (50%) reduced in the case of KyAΔgp2.     

Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Expression of melatonin and its related synthase and membrane receptors in the oestrous corpus luteum and corpus luteum verum of sheep

Melatonin is an important factor involved in regulating reproduction; it is synthesized enzymatically by the sequential action of melatonin‐synthesizing enzymes, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT), and exerts its biological functions mainly through receptor‐mediated action. To evaluate the expression of melatonin, two melatonin‐synthesizing enzymes (HIOMT and AANAT), and membrane receptors (MT1 and MT2) in oestrous corpus luteum (CL) and CL verum of sheep (Ovis aries), we performed ELISA, qRT‐PCR, western blotting and immunohistochemistry. The quantitative results showed that melatonin, HIOMT and AANAT levels in the CL verum were significantly higher than those in oestrous CL (p < 0.05), whereas MT1 and MT2 exhibited no change between the oestrous CL and CL verum (p > 0.05); moreover, the localization results showed that HIOMT, AANAT, MT1 and MT2 were mainly expressed in large luteal cells (LLCs). In summary, the above results suggested that sheep CL has potential for the synthesis of melatonin; meanwhile, they also suggested that CL is one of the targets of melatonin. These results provide not only a basis for whether sheep CL can synthesize melatonin but also provide a reference for further study on the mechanism of melatonin in the CL.     

P‐glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity

The role of the transporter P‐glycoprotein (P‐gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo‐physiological features of the ruminant species, the constitutive expression of P‐gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real‐time quantitative PCR. P‐gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65‐fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6‐ and 120‐fold higher). The treatment with DEX did not elicit a significant effect on the P‐gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (P_eff S‐M = 3.99 × 10^?6 ± 2.07 × 10^?6) and DEX‐treated animals (P_eff S‐M = 6.00 × 10^?6 ± 2.5 × 10^?6). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.