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 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.

OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram. PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti–human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed. RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C. CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

Objective: To determine whether increased gene expression of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) in laminae of horses with starch gruel–induced laminitis was accompanied by increased enzyme activity and substrate degradation. Sample: Laminae from the forelimb hooves of 8 healthy horses and 17 horses with starch gruel–induced laminitis (6 at onset of fever, 6 at onset of Obel grade 1 lameness, and 5 at onset of Obel grade 3 lameness). Procedures: Gene expression was determined by use of cDNA and real-time quantitative PCR assay. Protein expression and processing were determined via SDS-PAGE and quantitative western blotting. Protein distribution and abundance were determined via quantitative immunofluorescent staining. Results: ADAMTS-4 gene expression was increased and that of versican decreased in laminitic laminae, compared with expression in healthy laminae. Catalytically active ADAMTS-4 also was increased in the tissue, as were ADAMTS-4–cleavage fragments of versican. Immunofluorescent analyses indicated that versican was depleted from the basal epithelia of laminae of horses at onset of Obel grade 3 lameness, compared with results for healthy laminae, and this was accompanied by regional separation of basal epithelial cells from the basement membrane. Aggrecan gene and protein expression were not significantly affected. Conclusions and Clinical Relevance: Changes in gene and protein expression of ADAMTS-4 and versican in the basal epithelium of laminitic laminae indicated a fundamental change in the physiology of basal epithelial cells. This was accompanied by and may have caused detachment of these cells from the basement membrane.

Objective: To determine whether a novel optimized plasmid carrying the porcine growth hormone–releasing hormone (GHRH) wild-type cDNA administered at a lower dose was as effective at eliciting physiologic responses as a commercial GHRH plasmid approved for use in Australia. Animals: 134 gilts. Procedures: Estrus was synchronized and gilts were bred. Pregnant gilts were assigned to 2 treatment groups (40 gilts/group) or 1 untreated control group (24 gilts). Gilts in one of the treatment groups received the commercial GHRH plasmid, whereas gilts in the other treatment group received a novel optimized GHRH plasmid; both plasmids were administered IM in the right hind limb, which was followed by electroporation. Sow and litter performance were monitored for the 3 gestations after treatment. Results: A significant increase in insulin-like growth factor-I concentrations, decrease in perinatal mortality rate, increase in the number of pigs born alive, and increase in the weight and number of pigs weaned were detected for both groups receiving the GHRH-expressing plasmids, compared with values for the control group. Additionally, there was a significant decrease in sow attrition in GHRH-treated females, compared with attrition in the control group, during the 3 gestations after treatment. Conclusions and Clinical Relevance: Both of the GHRH plasmids provided significant benefits for sow performance and baby pig survivability for pregnant and lactating sows and their offspring during the 3 gestations after treatment, compared with results for untreated control gilts. Use of a novel optimized plasmid reduced the effective plasmid dose in these large mammals.