S‐100 Protein‐Positive Mast Cells in Canine Paranal Sinus (Sinus paranalis)

The aim of this study was to establish the immunocytochemical expression of S‐100 protein in mast cells, localized in the wall of dog's paranal sinus. Control serial sections were used for immunocytochemical detection of tryptase‐positive mast cells. It was observed that S‐100‐positive cells have the same morphology and localization as the tryptase‐positive mast cells, which indicated that S‐100‐positive cells are most probably mast cells with abilities as dendritic cells. In conclusion, for the first time, the current study gave evidence that mast cells in this organ possess one more function, such as dendritic cells.     

Bone Morphogenetic Protein‐6 (BMP‐6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre‐antral Follicles Cultured In Vitro

BMP‐6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP‐6) and recombinant follicle‐stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP‐6 (Experiment 2). Secondary follicles were cultured in αMEM⁺ alone (control medium) or supplemented with BMP‐6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP‐6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP‐6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non‐cultured control and after in vitro culture (MEM and 1 ng/ml BMP‐6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP‐6 at 1 ng/ml treatment. In conclusion, the low BMP‐6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.     

DNA Immunization Against Very Virulent Infectious Bursal Disease Virus with VP2‐4‐3 Gene and Chicken IL‐6 Gene

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2‐4‐3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin‐6 (ChIL‐6) genes were constructed and designated as pALTER‐MAX‐VP2‐4‐3 and pALTER‐MAX‐ChIL‐6 respectively. Several DNA vaccination experiments were performed: 1‐week‐old chickens were intramuscularly injected with only plasmid pcDNA3‐VP2, pALTER‐MAX‐VP2‐4‐3 or mixture with pALTER‐MAX‐ChIL‐6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER‐MAX‐VP2‐4‐3 and pALTER‐MAX‐ChIL‐6 three times conferred protection for 90% of chickens. Enzyme‐linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER‐MAX‐ChIL‐6 were higher than those immunized simply with plasmid pcDNA3‐VP2 or pALTER‐MAX‐VP2‐4‐3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT‐PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2‐4‐3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL‐6 plasmid significantly increased the protection after challenge with the very virulent strain.     

Limited Genetic Diversity and Gene Expression Differences between Egg‐ and Non‐Egg‐Related Salmonella Enteritidis Strains

Salmonella Enteritidis strains of egg‐ and non‐egg‐related origin were characterized molecularly to find markers correlated with the egg‐contaminating property of Salmonella Enteritidis. Isolates were examined by random amplified polymorphic DNA (RAPD), plasmid profiling and phage typing. Furthermore, the presence of 30 virulence genes was tested by PCR. In genetic fingerprinting and gene content, only small differences between the strains were found and no correlation was observed with the origin (egg‐related versus non‐egg‐related). A major RADP group was present in both egg‐ and non‐egg‐related strains, but other smaller RAPD groups were present as well in both categories of strains. Phage types PT4 and PT21 were predominant. Differential mRNA expression levels of fimA and agfA under conditions of growth simulating the conditions during egg formation were determined by real‐time RT‐PCR. Although differences in fimA and agfA expression levels were observed between the strains, these could not be correlated with the origin of the strains (egg‐related versus non‐egg‐related). The highest expression levels of agfA and fimA were only found in two non‐egg‐related strains, which seemed to be correlated with the presence of a 93 kb plasmid instead of the 60 kb virulence plasmid. Our results seem to indicate only a limited role for at least type I fimbriae (encoded by fim operon) in egg contamination by Salmonella Enteritidis.     

Pregnancy‐Induced ISG‐15 and MX‐1 Gene Expression is Detected in the Liver of Holstein–Friesian Heifers During Late Peri‐Implantation Period

The bovine embryonic signal interferon‐τ (IFN‐τ) produced by the trophoblast is known to pass through the uterine fluid towards the endometrium and further into the maternal blood, where IFN‐τ induces specific expression of interferon‐stimulated gene expression (ISG), for example in peripheral leucocytes. In sheep, it was shown experimentally by administration of IFN‐τ that ISG is also detectable in the liver. The objective was to test whether ISG can be detected in liver biopsy specimens from Holstein–Friesian heifers during early pregnancy. Liver biopsies were taken on day 18 from pregnant and non‐pregnant heifers (n = 19), and the interferon‐stimulated protein 15 kDa (ISG‐15) and myxovirus‐resistance protein‐1 (MX‐1) gene expression was detected. The expression of both MX‐1 (p: 24.33 ± 7.40 vs np: 9.00 ± 4.02) and ISG‐15 (p: 43.73 ± 23.22 vs 7.83 ± 3.63) was higher in pregnant compared to non‐pregnant heifers (p < 0.05). In conclusion, pregnancy induced ISG‐15 and MX‐1 gene expression in the liver already at day 18 in cattle.     

Acceleration of Second and Fourth Metatarsal Fracture Healing with Recombinant Human Bone Morphogenetic Protein‐2/Calcium Phosphate Cement in Horses

Objective— To compare the efficacy of recombinant human bone morphogenetic protein‐2 (rhBMP‐2)/calcium phosphate (CP) to autogenous cancellous bone graft (CBG) and to no treatment on bone healing, in surgically induced osteotomies and ostectomies of the accessory metatarsal bones in an equine model. Study Design— Experimental. Animals— Adult horses (n=9). Methods— Segmental ostectomies of the second metatarsal bone (MT2) and osteotomies of the fourth metatarsal bone (MT4) were performed bilaterally in 9 horses. There were a total of 35 defects (1 MT4 was previously fractured) created and supplemented randomly either with no treatment (untreated control), rhBMP‐2/CP cement, or matrix (CPC or CPM), or CBG. Radiography was performed every 2 weeks until study endpoint at 12 weeks. After euthanasia, bone healing was evaluated using radiography, mechanical testing, and histology. Data were analyzed with ANOVA followed by the Duncan's Multiple Range Test or nonparametric analyses. Results— At 12 weeks, radiographic scores for union were significantly greater for the rhBMP‐2 (P<.0001) and CBG (P=.004) groups compared with the untreated control group, for both MT2 ostectomies and MT4 osteotomies. The rhBMP‐2 treated MT2 had greater maximum torque to failure in torsion than CBG and control limbs at 12 weeks (P=.011). Histologic analysis demonstrated increased bone formation and more mature bone at the ostectomy site for MT2 in the rhBMP‐2 and CBG groups compared with the untreated control group. Conclusion— Injection of rhBMP‐2/CP into surgically induced ostectomies and osteotomies of the accessory metatarsal bones might accelerate early bone healing in the horse. Clinical Relevance— RhBMP‐2/CP may be as effective if not superior to CBG as an adjuvant treatment to accelerate healing of bone defects.     

Large pituitary adenoma in an 8‐year‐old Arabian stallion

An 8‐year‐old Arabian stallion weighing 361 kg presented to Louisiana State University Veterinary Teaching Hospital with a 3‐month history of weight loss, exercise intolerance, long hair coat and recent history of seizures and aimless wandering in the pasture. An initial presumptive diagnosis of pituitary pars intermedia dysfunction (PPID) was made based on clinical signs. The initial examination revealed weight loss and loss of body condition (BCS 3/9), hypertrichosis, muscle wasting and reluctance to move when prompted. A neurological examination revealed dull mentation with no evidence of proprioceptive deficits in the limbs. Mild hyperglycaemia and a stress leucogram were noted on initial biochemical panel and haematology, respectively. Plasma adrenocorticotrophic hormone (ACTH) concentrations before and after thyrotropin releasing hormone (TRH) stimulation were markedly increased. Rapid slice computed tomography (CT) scan of the head before and after contrast revealed a large mass in the region of the pituitary gland suggestive of macroadenoma causing PPID. Prior to imaging, treatment consisted of supportive nursing care. Due to size of the pituitary gland (measuring 4.6 × 4.6 × 3.8 cm) and the presence of seizure‐like activity and dull mentation, the stallion was subjected to euthanasia. A necropsy was not performed. Pituitary macroadenomas in horses affected with PPID, who show neurological signs such as seizure‐like activity, dull mentation and aimless wandering, might have a poor prognosis and treatment with pergolide mesylate might not reduce pituitary gland size or relieve clinical signs. A CT scan is indicated in horses with neurological signs suspected of PPID to further evaluate pituitary gland size and surrounding structures and rule out other causes to better assess prognosis.     

Pre‐ and Post‐Partum Mild Underfeeding Influences Gene Expression in the Reproductive Tract of Cyclic Dairy Cows

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post‐partum. This study investigated the effects of pre‐ and post‐partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post‐partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post‐partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi‐ and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post‐partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post‐partum patterns of IGF‐1, insulin, β‐hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid‐related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post‐partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post‐partum dairy cows.     

Plant‐frugivore interactions in an intact tropical forest in north‐east Thailand

Fleshy‐fruited plants in tropical forests largely rely on vertebrate frugivores to disperse their seeds. Although this plant–animal interaction is typically considered a diffuse mutualism, it is fundamental as it provides the template on which tropical forest communities are structured. We applied a mutualistic network approach to investigate the relationship between small‐fruited fleshy plant species and the fruit‐eating bird community in an intact evergreen forest in northeast Thailand. A minimum of 53 bird species consumed fruits of 136 plant species. Plant‐avian frugivore networks were highly asymmetrical, with observed networks filling 30% of all potential links. Whereas some of the missing links in the present study might be due to undersampling, forbidden links can be attributed to size constraints, accessibility and phenological uncoupling, and although the majority of missing links were unknown (58.2%), many were probably due to a given bird species being either rare or only a very occasional fruit eater. The most common frugivores were bulbuls, barbets and fairy‐bluebirds, which were responsible for the majority of fruit removal from small fleshy fruited species in our system. Migratory birds seemed to be a minor component of the plant‐frugivore networks, accounting for only 3% of feeding visits to fruiting trees; they filled 2% of the overall potential networks. The majority of interactions were generalized unspecific; however, Saurauia roxburghii Wall. appeared to be dependent on flowerpeckers for dispersal, while Thick‐billed Pigeons were only seen to eat figs.     

A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function

Objectives

Glutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.

Methods

Banked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.

Results

Canine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in V_max for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.

Conclusions

An Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.     

Reversed patent ductus arteriosus and multiple congenital malformations in an 8‐day‐old Arabo‐Friesian foal

An 8‐day‐old Arabo‐Friesian filly was presented with signs of severe dyspnoea, tachypnoea, coughing and cyanotic mucous membranes. On auscultation, a bilateral grade V/VI continuous heart murmur and heart rate of 155 beats/min (sinus tachycardia) were detected. Lung ultrasonography revealed pronounced comet tail artefacts indicating lung oedema. Echocardiography showed right ventricular hypertrophy, a 1.2 cm muscular ventricular septal defect with a left‐to‐right shunt, a stenotic bicuspid pulmonary valve and severe mitral and tricuspid valve regurgitation. Tricuspid regurgitation peak velocity indicated a right ventricular systolic pressure of 119 mmHg. The pulmonary artery was severely dilated and a 1 cm diameter patent ductus arteriosus was found. Colour flow Doppler showed systolic ductal flow reversal with right‐to‐left shunting through the ductus. Arterial partial oxygen pressure and saturation were lower in the metatarsal artery (25 mmHg, saturation 52.6%) than in the carotid artery (31 mmHg, saturation 64.3%). Due to the poor prognosis, the foal was subjected to euthanasia and necropsy confirmed the ultrasonographic findings. Patent ductus arteriosus is a rare condition and occurs most frequently in combination with tetralogy and pentalogy of Fallot. A genetic basis for congenital cardiac disease, especially for ventricular septal defects, in Arabians and for aortic rupture and aorto‐pulmonary fistulation in Friesians has been reported. Whether cross‐breeding leads to an increased prevalence is unknown. This is the first case report with echocardiographic visualisation of reversed ductal flow in a neonatal Arabo‐Friesian foal.