Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis. Cell division cycle 42 (Cdc42) from sheep (Ovis aries) (OaCdc42) was cloned by rapid amplification of cDNA ends (RACE), and then tissue distribution and differential expression levels of OaCdc42 mRNA between infected and vaccinated sheep were analysed by RT-qPCR. The full-length cDNA of OaCdc42 was 1,609 bp containing an open reading frame (ORF) of 576 bp. OaCdc42 mRNAs were detected in the heart, liver, spleen, lung, kidneys, rumen, small intestine, skeletal muscles, and buffy coat, and the highest expression was detected in the small intestine. Compared to the control, the levels of OaCdc42 mRNA from sheep infected with Brucella melitensis or sheep vaccinated with Brucella suis S2 was significantly different (P < 0.01) after 40 and 30 days post-inoculation, respectively. However, the expression of OaCdc42 mRNA was significantly different between vaccinated and infected sheep (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, whereas no significant difference (P > 0.05) was noted 40 days post-inoculation. Moreover, the expression of OaCdc42 from both infected and vaccinated sheep showed irregularity. OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.

Objective: To investigate the relationship between runt-related transcription factor 2 (RUNX2) expression in canine nucleus pulposus (NP) cells and intervertebral disk aging in chondrodystrophoid dogs. Animals: 7 healthy Beagles (mean age, 35.6 months) and 11 Dachshunds with herniated disks (mean age, 61 months). Procedures: All dogs underwent MRI examination of the thoracic and lumbar vertebral column immediately before sample collection under general anesthesia. The disk center–to–CSF T2-weighted signal intensity ratio was determined for healthy Beagles. Samples of NP were obtained from nonherniated disks in healthy Beagles and from herniated disks during surgical treatment of hospitalized Dachshunds. Samples were evaluated for RUNX2 and matrix metalloproteinase 13 transcript expression via reverse transcriptase PCR assay; RUNX2 protein expression was evaluated via immunohistochemical analysis, and correlation between these variables and age of dogs was evaluated. A 3′ and 5′ rapid amplification of cDNA ends method was used to identify the RUNX2 coding region. Results: RUNX2 cDNA had > 97% conservation with the human cDNA sequence and approximately 95% conservation with the mouse cDNA sequence; RUNX2 and matrix metalloproteinase 13 mRNA expression and RUNX2 protein expression in NP cells were positively correlated with age. The disk center–to–CSF T2-weighted signal intensity ratio was negatively correlated with RUNX2 protein expression in the NP of healthy dogs. Conclusions and Clinical Relevance: Results indicated that RUNX2 mRNA and protein expression in the NP are enhanced in aging intervertebral disks in dogs.

Objective—To identify a causative mutation for dilated cardiomyopathy (DCM) in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes known to be associated with familial DCM in humans. Animals—5 Doberman Pinschers with DCM and congestive heart failure and 5 control mixed-breed dogs that were euthanized or died. Procedures—RNA was extracted from frozen ventricular myocardial samples from each dog, and first-strand cDNA was synthesized via reverse transcription, followed by PCR amplification with gene-specific primers. Ten cardiac genes were analyzed: cardiac actin, α-actinin, α-tropomyosin, β-myosin heavy chain, metavinculin, muscle LIM protein, myosinbinding protein C, tafazzin, titin-cap (telethonin), and troponin T. Sequences for DCM-affected and control dogs and the published canine genome were compared. Results—None of the coding sequences yielded a common causative mutation among all Doberman Pinscher samples. However, 3 variants were identified in the α-actinin gene in the DCM-affected Doberman Pinschers. One of these variants, identified in 2 of the 5 Doberman Pinschers, resulted in an amino acid change in the rod-forming triple coiled-coil domain. Conclusions and Clinical Relevance—Mutations in the coding regions of several genes associated with DCM in humans did not appear to consistently account for DCM in Doberman Pinschers. However, an α-actinin variant was detected in some Doberman Pinschers that may contribute to the development of DCM given its potential effect on the structure of this protein. Investigation of additional candidate gene coding and noncoding regions and further evaluation of the role of α-actinin in development of DCM in Doberman Pinschers are warranted.

Objective-To determine the effects of recombinant equine interleukin -1beta (reIL-1beta) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes. Sample Population-Articular cartilage from 9 young adult horses. Procedure-Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), polysulfated glycosaminoglycan, and hyaluronan, each at concentrations of 10 and 100 micrograms/ml and each with or without reIL-1beta. A commercial immunoassay was used to determine prostaglandin E2 (PGE2) concentrations in conditioned medium of similarly treated cells to quantify COX-2 activity. Results-Addition of reIL-1beta increased the expression of COX-2 in a dose-dependent manner, which was paralleled by an increased concentration of PGE2 in culture medium. Concentration of PGE2 in spent medium from reIL-1beta-treated chondrocytes was significantly reduced by DEX and PBZ; however, only DEX significantly reduced gene expression of COX-2. Conclusions and Clinical Relevance-Prostaglandin E2 is considered to be an important mediator in the pathophysiologic processes of arthritis, and cultured chondrocytes respond to interleukin-1 with enhanced expression and activity of COX-2. Palliative relief in affected horses is probably attributable, in part, to inhibition of PGE2 synthesis; however, analysis of these data suggests that of the 4 compounds tested, only DEX affects pretranslational regulation of the COX-2 gene in cultured equine chondrocytes.

Investigation of the structure of equine articular cartilage link protein (LP) from individuals ranging in age from 1 to 15 years identified 3 distinct isoforms having molecular weights of 46,000, 43,000, and 41,000. The relative amounts of each of the 3 isoforms altered with age. The largest form did not change with age; however, amounts of the Mr 43,000 and 41,000 forms increased with increasing age. The results suggested that an accumulation, in the extracellular matrix of cartilage, of these 2 smaller products may have arisen from proteolytic cleavage. The complete amino acid sequence of the protein core was determined from complementary DNA products prepared by polymerase chain reaction amplification of cartilage LP mRNA. The sequence had 96% similarity with human LP and with that of other species for which the primary structure has been determined. This high degree of sequence conservation and the isoform data indicate that extracellular processing of LP occurs by similar mechanisms in various species. At the transcription level, equine chondrocytes were found to express LP as 2 abundant mRNA of 5.0 and 3.0 kb, and a smaller mRNA of 1.5 kb. Processing of the LP mRNA in horses, thus, appears to be similar to that found in other species investigated, and although multiple transcripts are present, the coding region remains unaltered and only 1 protein product is made.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin β-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm2) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Objective-To examine the role of bovine viral diarrhea virus (BVDV) biotype on the establishment of fetal infection in cattle. Animals-30 mixed-breed pregnant cows. Procedure-Pregnant cows were inoculated oronasally with either i-VVNADL, originating from an infectious BVDV cDNA clone of the National Animal Disease Laboratory (NADL) isolate, or the parental virus stock, termed NADL-A. Results-All cows developed neutralizing antibodies to BVDV, and virus was commonly isolated from peripheral blood mononuclear cells or nasal swab specimens of NADL-A inoculated cows; however, virus was rarely isolated from specimens of i-VVNADL inoculated cows. i-VVNADL did not cause fetal infection, whereas all fetuses harvested from NADL-A inoculated cows at 6 weeks after inoculation had evidence of infection. Immunoblot analysis of fetal virus isolates revealed the absence of NS3, confirming a noncytopathic (NCP) biotype BVDV in the NADL-A stock. The sequence of the NCP contaminant (termed NADL-1102) and the i-VVNADL genome were virtually identical, with the exception of a 270 nucleotide-long insert in the i-VVNADL genome. Phylogenetic analyses revealed that NADL-1102 forms a monophyletic group with 6 other NADL genomes. Conclusions and Clinical Relevance-These data suggest that the contaminating NCP virus in the NADL-A stock was the ancestral NADL virus, which originally infected a bovine fetus and recombined to produce a cytopathic (CP) variant. Following oronasal infection of pregnant cows, viremia and transplacental transmission of CP BVDV to the fetus is rare, compared with the high occurrence of maternal viremia and fetal infection observed with NCP BVDV.

OBJECTIVE To evaluate the coding regions of ADAMTS17 for potential mutations in Chinese Shar-Pei with a diagnosis of primary open-angle glaucoma (POAG), primary lens luxation (PLL), or both. ANIMALS 63 Shar-Pei and 96 dogs of other breeds. PROCEDURES ADAMTS17 exon resequencing was performed on buccal mucosal DNA from 10 Shar-Pei with a diagnosis of POAG, PLL, or both (affected dogs). A candidate causal variant sequence was identified, and additional dogs (53 Shar-Pei [11 affected and 42 unaffected] and 95 dogs of other breeds) were genotyped for the variant sequence by amplified fragment length polymorphism analysis. Total RNA was extracted from ocular tissues of 1 affected Shar-Pei and 1 ophthalmologically normal Golden Retriever; ADAMTS17 cDNA was reverse transcribed and sequenced, and ADAMTS17 expression was evaluated by quantitative reverse-transcription PCR assay. RESULTS All affected Shar-Pei were homozygous for a 6-bp deletion in exon 22 of ADAMTS17 predicted to affect the resultant protein. All unaffected Shar-Pei were heterozygous or homozygous for the wild-type allele. The variant sequence was significantly associated with affected status (diagnosis of POAG, PLL, or both). All dogs of other breeds were homozygous for the wild-type allele. The cDNA sequencing confirmed presence of the expected variant mRNA sequence in ocular tissue from the affected dog only. Gene expression analysis revealed a 4.24-fold decrease in the expression of ADAMTS17 in ocular tissue from the affected dog. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that the phenotype (diagnosis of POAG, PLL, or both) is an autosomal recessive trait in Shar-Pei significantly associated with the identified mutation in ADAMTS17.

Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.