Diclofenac was responsible for the decimation of Gyps vulture species on the Indian subcontinent during the 1980s and 1990s. Gyps vultures are extremely sensitive (the lethal dose 50 [LD50] ~ 0.1 mg/kg - 0.2 mg/kg), with toxicity appearing to be linked to metabolic deficiency, demonstrated by the long T1/2 (~12 h - 17 h). This is in striking comparison to the domestic chicken (Gallus gallus domesticus), in which the LD50 is ~10 mg/kg and the T1/2 is ~1 h. The phase 1 cytochrome P450 (CYP) 2C subfamily has been cited as a possible reason for metabolic deficiency. The aim of this study was to determine if CYP2C9 homolog pharmacogenomic differences amongst avian species is driving diclofenac toxicity in Gyps vultures. We exposed each of 10 CYP-inhibited test group chickens to a unique dose of diclofenac (as per the Organisation for Economic Co-operation and Development [OECD] toxicity testing guidelines) and compared the toxicity and pharmacokinetic results to control group birds that received no CYP inhibitor. Although no differences were noted in the LD50 values for each group (11.92 mg/kg in the CYP-inhibited test group and 11.58 mg/kg in the control group), the pharmacokinetic profile of the test group was suggestive of partial inhibition of CYP metabolism. Evaluation of the metabolite peaks produced also suggested partial metabolic inhibition in test group birds, as they produced lower amounts of metabolites for one of the three peaks demonstrated and had higher diclofenac exposure. This pilot study supports the hypothesis that CYP metabolism is varied amongst bird species and may explain the higher resilience to diclofenac in the chicken versus vultures.

Influenza A viruses (IAVs) are typically isolated and cultured by successive passages using 9- to 11-day-old embryonated chicken eggs (ECEs) and in 14-day old ECEs for virus mutational studies. Real-time reverse transcription-polymerase chain reaction tests (RT-PCRs) are commonly used for IAV diagnosis, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus. Efforts at isolating ostrich-origin IAVs from RT-PCR positive specimens using ECEs have often been unsuccessful, raising the possibility of a species bottleneck, whereby ostrich-adapted IAVs may not readily infect and replicate in ECEs, yet the capacity of an ostrich embryo to support the replication of influenza viruses has not been previously demonstrated. This study describes an optimised method for H5 and H7 subtype IAV isolation and propagation in 28-day old embryonated ostrich eggs (EOEs), the biological equivalent of 14-day old ECEs. The viability of EOEs transported from breeding sites could be maximised by pre-incubating the eggs for 12 to 14 days prior to long-distance transportation. This method applied to studies for ostrich-adapted virus isolation and in ovo studies will enable better understanding of the virus-host interaction in ostriches and the emergence of potentially zoonotic diseases.

International audience | Abstract OBJECTIVE To investigate the effects and duration of orally administered prednisolone on renal function evaluated by glomerular filtration rate (GFR) determination and creatinine (Cr) and symmetric dimethylarginine (SDMA) concentrations as well as on urinalysis, electrolytes, and hydric status in healthy dogs. ANIMALS 14 healthy Beagles. PROCEDURES In this prospective double-masked placebo-controlled study, dogs were randomized after baseline evaluation to receive a 7-day course of either prednisolone (1.5 to 2.0 mg/kg, PO, q 12 h) or a placebo. A repeated-measure design was performed, each dog participating in 4 successive sampling sessions. Clinical data, systolic blood pressure, CBC, and biochemical analyses including serum SDMA concentration, GFR determination, urine output quantification, and complete urinalysis were performed for all dogs the day before (D0) and at the end of steroid administration (D7) as well as 2 weeks (D21) and 4 weeks (D35) after the end of treatment. RESULTS At D7, when compared with baseline, GFR increased significantly in treated dogs, whereas creatinine and SDMA concentrations decreased significantly. GFR and Cr but not SDMA modifications persisted significantly at D21. None of the variables differed significantly from baseline at D35. The OR of presenting an albumin band on urine electrophoresis was 2.4 times as high in treated versus control dogs (OR, 36; 95% CI, 1.8 to 719.4; P = 0.02). CLINICAL RELEVANCE A short-term course of immune-suppressive prednisolone treatment in healthy dogs leads to a sustained but reversible renal hyperfiltration state. Modification in electrolytic variables can affect the clinical interpretation of blood work in such patients.

Hypoxia is a common pathological condition after spinal cord injury. Oestrogen-related receptor alpha (ERRα), as a key regulator of energy metabolism and mitochondrial functions, plays an important role in maintaining cell homeostasis. However, its role in hypoxic spinal microglia has not been fully elaborated. This study investigated the receptor’s activity when these cells are hypoxic and used as an in vitro model. In this study, microglia (BV2) were exposed to cobalt chloride as a hypoxic model, and the inverse agonist of ERRα, XCT790, and pyrido[1,2-α]-pyrimidin-4-one were used to regulate the expression of the receptor to explore the ERRα-related mechanisms involved in hypoxic spinal cord injury (SCI). ERRα promoted autophagy in BV2 cells and inhibited the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and the expression of anti-inflammatory factors under hypoxic conditions. It also promoted the expression of fibronectin type III domain containing protein 5 (FNDC5). When a hypoxic SCI occurs, ERRα may maintain the homeostasis of spinal cord nerve cells by regulating autophagy and the p38MAPK/nuclear factor-kappa B cell and FNDC5/brain-derived neurotrophic factor signalling pathways, which are beneficial to the recovery of these cells.

Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons. Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs’ normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively. The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts. The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

Domestic poultry is a natural reservoir of Campylobacter, the host–pathogen interaction being predominantly asymptomatic. This study investigated whether chickens remain asymptomatic partly because of lactic acid bacteria (LAB). Campylobacter spp. and LAB were isolated from the gut of poultry chickens using enrichment and screening assays and were identified via rDNA sequencing. The C. jejuni DC3 isolate was grown in different cell-free supernatants (CFS) generated from a priority LAB isolate. An in vivo challenge involving the C. jejuni and LAB isolates using a chicken model was performed to confirm the in vitro findings. Twelve presumptive LAB isolates had anti-C. jejuni activity based on cross-streak and agar plug assays, with Lactobacillus sakei L14 isolate exhibiting the highest activity. Inhibition by L. sakei L14 CFS of the growth of C. jejuni occurred in a dose-dependent manner. Campylobacter jejuni DC3 inhibition was most evident in CFS harvested at 72 h and produced by co-culture with the pathogen. Neutralisation of the CFS abrogated the observed inhibition. Co-infection with C. jejuni DC3 and L. sakei L14 in vivo, however, failed to inhibit C. jejuni colonisation in chickens. The results suggest that the anti-C. jejuni effect of L. sakei L14 in chickens may be due to mechanisms other than direct inhibition of growth.

Dogs with chronic kidney disease (CKD) may have alterations in the glomerular filtration barrier, including podocyte loss. Detection of podocyte mRNA in urine could be useful for assessing podocyturia in dogs with kidney disease. The objective of this study was to evaluate the presence of nephrin mRNA (NPHS1) and podocin mRNA (NPHS2) in urine sediments of dogs with naturally occurring CKD and healthy dogs. Twenty-four dogs, 14 with CKD and 10 as healthy controls, underwent clinical evaluation. The dogs with CKD were divided into two groups, according to the International Renal Interest Society criteria: stage 1 or 2 CKD (n = 5) and stage 3 or 4 CKD (n = 9). Urine was collected by catheterisation or free catch and RNA isolation from the urine sediments was optimised using glycogen as a co-precipitant. Detection of NPHS1 and NPHS2 in the sediment samples was performed using quantitative real-time PCR. Both types of mRNA were detected in samples from all groups, but the percentages of detection were higher in the group of dogs with stage 1 or 2 CKD and lower in the group of dogs with stage 3 or 4 disease. Physiological podocyturia was observed in healthy dogs, and the results suggest differential podocyturia in dogs with CKD, according to the stage of the disease, i.e. an increase in podocyturia in dogs at stage 1 or 2 and a reduction in podocyturia in dogs at stage 3 or 4.

Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan. The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated. The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified. A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

Multi-class and multi-residue analyses are very complex procedures because of the physico-chemical properties of veterinary drug residues and other contaminants. The purpose of the study was to develop an analytical method for the sensitive determination of 69 analytes in bovine milk by liquid chromatography electrospray ionisation–tandem mass spectrometry. Antimicrobial, anabolic hormone, lactone, β-agonist, mycotoxin and pesticide residues were analysed in 120 raw milk samples from different dairy farms in North Macedonia. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. The linear regression coefficients were higher than 0.99, the limits of detection ranged from 0.0036 to 47.94 μg/L, and the limits of quantification ranged from 0.053 to 59.43 μg/L. The decision limit values ranged from 0.062 to 211.32 μg/L and the detection capability from 0.080 to 233.71 μg/L. Average recoveries of the analytes spiked in raw milk were in the range of 70.83% to 109%, intra-day coefficient of variation (CV) values from 2.41% to 22.29%, and inter-day CV values from 3.48% to 23.91%. The method was successfully applied in the testing of bovine milk samples. In five samples residues were detected. They were sulfadimethoxine (in two samples), enrofloxacin, tetracycline and oxytetracycline and were at concentrations below the EU maximum residue limit. The method is useful for routine testing for this group of chemical hazards in bovine milk.

Enhancer of zeste homologue 2 (EZH2) is the human homologue of Drosophila zeste gene enhancer. The aim of this study was to determine the expression of EZH2 in canine mammary carcinomas (CMCs) and its relationship with clinicopathological features. The expression of EZH2 mRNA and protein in 53 CMC tissue and 8 normal mammary gland tissue samples was measured by quantitative real-time PCR and immunohistochemical staining assay, respectively. The relationship between EZH2 protein expression and clinicopathological features was analysed by χ2 test to further explore the clinical significance of EZH2 in CMCs. Compared with normal mammary gland tissues, EZH2 mRNA expressions were significantly increased in CMC tissues (P < 0.01). Moreover, normal mammary glands did not express the EZH2 protein but carcinomic glands did, and expression increased in CMCs with high histological grades, especially in histological grade II (P < 0.05). However, EZH2 expression was not related to age, tumour size, or metastasis (P > 0.05). The expression of EZH2 in one type of CMC was not significantly different from the expression in any other type (P > 0.05). EZH2 is highly expressed in CMCs, indicating that it can be used as a molecular marker for early diagnosis, prognosis, or therapy of CMCs.