African swine fever virus (ASFV) causes one of the most dangerous diseases of pigs and wild boar – African swine fever (ASF). Since its second introduction into Europe (in 2007), the disease has been spreading consistently, and now ASF-free European countries are at risk. Complex interactions between the host’s immune system and the virus have long prevented the development of a safe vaccine against ASF. This study analysed the possibility of neutralisation of the ASFV in vitro by sera collected from ASF-survivor animals. Two pig and three wild boar serum samples were collected from previously selected potential ASF survivors. All sera presented high antibody titres (>5 log₁₀/mL). Primary alveolar macrophages were cultured in growth medium containing 10% and 20% concentrations of selected sera and infected with a haemadsorbing ASFV strain (Pol18_28298_O111, genotype II). The progress of infection was investigated under a light microscope by observing the cytopathic effect (CPE) and the haemadsorption phenomenon. Growth kinetics were investigated using a real-time PCR assay. Haemadsorption inhibition was detected in the presence of almost all selected sera; however, the inhibition of virus replication in vitro was excluded. In all samples, a CPE and decreasing quantification cycle values of the viral DNA were found. Anti-ASFV antibodies alone are not able to inhibit virus replication. Interactions between the humoral and cellular immune response which effectively combat the disease are implicated in an ASF-survivor’s organism.

OBJECTIVE To investigate the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. SAMPLE 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculoskeletal disease. PROCEDURES Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or 5 mg/mL) medium for 6 or 24 hours. Explant metabolic activity and percentage of dead cells were assessed with a resazurin-based assay and live-dead cell staining, respectively, at each time point. Drug effects were assessed relative to findings for the respective control group. RESULTS Application of TA (at both concentrations) did not significantly change the cell viability of FC-DDFT explants. For FC-NB explants, TA at 6 mg/mL significantly reduced the metabolic activity and increased the percentage of dead cells at both time points. With either MPA concentration, FC-DDFT and FC-NB explants had reduced metabolic activity and an increased percentage of dead cells at 24 hours, whereas only MPA at 5 mg/mL was cytotoxic at the 6-hour time point. CONCLUSIONS AND CLINICAL RELEVANCE In ex vivo explants, TA was less cytotoxic to equine FC-DDFT and FC-NB cells, compared with MPA. Further work is warranted to characterize the drugs' transcriptional and translational effects as well as investigate their cytotoxicity at lower concentrations.

OBJECTIVE To compare pregnancy-associated glycoprotein 1 (PAG1) concentrations in maternal (jugular vein) and fetal (uterine vein) circulations and amniotic fluid samples between pregnant ewes that were and were not experimentally infected with bovine viral diarrhea virus (BVDV). ANIMALS 11 healthy pregnant yearling ewes. PROCEDURES Before study initiation, all ewes were naïve to BVDV and confirmed pregnant by transabdominal ultrasonography at approximately 60 days of gestation. At 65 days of gestation, ewes were intranasally inoculated with a noncytopathic BVDV type 1b strain (concentration, 107 TCID50/mL; 2 mL/nostril; n = 6) or an equal volume of BVDV-free viral culture medium (control; 5). A blood sample was collected for measurement of PAG1 concentration before inoculation. At 80 days of gestation, each ewe was anesthetized and underwent an ovariohysterectomy. While sheep were anesthetized, blood samples from the jugular and uterine veins and an amniotic fluid sample were collected for measurement of PAG1 concentration. Fetal tissues underwent real-time PCR analysis for BVDV RNA, and placental specimens underwent histologic evaluation and immunohistochemical staining for BVDV antigen. RESULTS At 80 days of gestation, BVDV RNA in fetal tissues and mild placentitis were detected in 5 of 6 BVDV-inoculated ewes. Mean PAG1 concentrations in the maternal and fetal circulations of BVDV-inoculated ewes were significantly less than those in control ewes. Mean amniotic fluid PAG1 concentration did not differ significantly between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE Concentration of PAG1 in the maternal circulation may be a useful biomarker for determining placental health in sheep after viral infection of the reproductive tract.

OBJECTIVE To use a biopolymer delivery system to investigate the ability of interleukin (IL)-4 to recruit neutrophils into subcutaneous tissues of equids. ANIMALS 16 horses and 2 ponies. PROCEDURES Animals were assigned to 3 experiments (6/experiment). Effects of recombinant equine (Req) IL-4 (100, 250, or 500 ng/site) versus a positive control (ReqIL-8; 100 ng, 250 ng, or 1 μg/site) and a negative control (Dulbecco PBSS or culture medium) on neutrophil chemotaxis were assessed after SC injection into the neck with an injectable biopolymer used as the vehicle. Tissue samples including the biopolymer plug were collected by biopsy at various time points from 3 hours to 7 days after injection. Neutrophil infiltration was evaluated by histologic scoring (experiments 1, 2, and 3) or flow cytometry (experiment 3). RESULTS Histologic neutrophil infiltration scores did not differ significantly among treatments at most evaluated time points. On flow cytometric analysis, log-transformed neutrophil counts in biopsy specimens were significantly greater for the ReqIL-8 treatment (1 μg/site) than the negative control treatment at 3 but not 6 hours after injection; results did not differ between ReqIL-4 and control treatments at either time point. Negative control treatments induced an inflammatory response in most equids in all experiments. CONCLUSIONS AND CLINICAL RELEVANCE Flow cytometry was a more reliable method to estimate neutrophil migration than histologic score analysis. The ReqIL-4 treatment did not induce a detectable neutrophil response, compared with the negative control treatment in this study. Evidence of inflammation in negative control samples suggested the biopolymer is not a suitable vehicle for use in equids.

Septic arthritis is an important disease in horses, necessitating aggressive and prolonged therapy. In order to guide therapy, reliable methods of detecting the eradication of infection are needed. Therefore, the objective of this study was to investigate detection of eradication of infection in an experimental model of equine septic arthritis using standard diagnostic techniques. For this purpose, 17 adult horses were assigned to 3 experimental groups. The middle carpal joint of each horse was injected with Escherichia coli (Septic group, n = 8), lipopolysaccharide (LPS) (LPS group, n = 6), or sterile saline (Control group, n = 3) at day 0. Contralateral joints were not injected. Standard therapy was applied to all joints except non-injected joints in the Control group at day 1. Sequential samples of synovial fluid (SF) were collected for bacterial culture using 3 culture media [Columbia blood agar (CBA), brain heart infusion broth (BHI), and Signal blood culture medium] and for cytological evaluation [percentage neutrophils (PN), total nucleated cell count (TNCC), and total protein (TP)]. Escherichia coli-specific polymerase chain reaction (PCR) was carried out to detect E. coli DNA in synovial fluid. Culture and PCR were positive for E. coli in all joints injected with E. coli at day 1 and 1 joint was positive on BHI at day 4. Based on the results of bacterial culture, PCR, and TNCC, the elimination of infection in our experimental model occurred by day 4 post-infection in 6 out of 7 cases. Total protein (TP) and PN remained elevated at clinical threshold used for diagnosis of septic arthritis until day 14. In our experimental model of E. coli-induced arthritis, we conclude that TP and PN may not be good indicators for detecting the eradication of bacterial infection caused by E. coli from infected and subsequently treated joints.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease, a chronic contagious enteritis of ruminants that causes major economic losses. Several studies, most involving large free-stall herds, have found environmental sampling to be a suitable method for detecting MAP-infected herds. In eastern Canada, where small tie-stall herds are predominant, certain conditions and management practices may influence the survival and transmission of MAP and recovery (isolation). Our objective was to estimate the performance of a standardized environmental and targeted pooled sampling technique for the detection of MAP-infected tie-stall dairy herds. Twenty-four farms (19 MAP-infected and 5 non-infected) were enrolled, but only 20 were visited twice in the same year, to collect 7 environmental samples and 2 pooled samples (sick cows and cows with poor body condition). Concurrent individual sampling of all adult cows in the herds was also carried out. Isolation of MAP was achieved using the MGIT Para TB culture media and the BACTEC 960 detection system. Overall, MAP was isolated in 7% of the environmental cultures. The sensitivity of the environmental culture was 44% [95% confidence interval (CI): 20% to 70%] when combining results from 2 different herd visits and 32% (95% CI: 13% to 57%) when results from only 1 random herd visit were used. The best sampling strategy was to combine samples from the manure pit, gutter, sick cows, and cows with poor body condition. The standardized environmental sampling technique and the targeted pooled samples presented in this study is an alternative sampling strategy to costly individual cultures for detecting MAP-infected tie-stall dairies. Repeated samplings may improve the detection of MAP-infected herds.

Objective-To develop a method for experimental induction of equine rhinitis A virus (ERAV) infection in equids and to determine the clinical characteristics of such infection. Animals-8 ponies (age, 8 to 12 months) seronegative for antibodies against ERAV. Procedures-Nebulization was used to administer ERAV (strain ERAV/ON/05; n = 4 ponies) or cell culture medium (control ponies; 4) into airways of ponies; 4 previously ERAV-inoculated ponies were reinoculated 1 year later. Physical examinations and pulmonary function testing were performed at various times for 21 days after ERAV or mock inoculation. Various types of samples were obtained for virus isolation, blood samples were obtained for serologic testing, and clinical scores were determined for various variables. Results-ERAV-inoculated ponies developed respiratory tract disease characterized by pyrexia, nasal discharge, adventitious lung sounds, and enlarged mandibular lymph nodes. Additionally, these animals had purulent mucus in lower airways up to the last evaluation time 21 days after inoculation (detected endoscopically). The virus was isolated from various samples obtained from lower and upper airways of ERAV-inoculated ponies up to 7 days after exposure; this time corresponded with an increase in serum titers of neutralizing antibodies against ERAV. None of the ponies developed clinical signs of disease after reinoculation 1 year later. Conclusions and Clinical Relevance-Results of this study indicated ERAV induced respiratory tract disease in seronegative ponies. However, ponies with neutralizing antibodies against ERAV did not develop clinical signs of disease when reinoculated with the virus. Therefore, immunization of ponies against ERAV could prevent respiratory tract disease attributable to that virus in such animals.

Objective—To determine the differentiation of canine adipose tissue–derived mesenchymal stem cells (ASCs) into endothelial progenitor cells (EPCs). Animals—3 healthy adult Beagles. Procedures—Canine ASCs were isolated and cultured from adipose tissue, and endothelial differentiation was induced by culturing ASCs in differentiation medium. Morphological and immunophenotypic changes were monitored. Expression of endothelial-specific markers was analyzed by conventional and real-time RT-PCR assay. The in vitro and in vivo functional characteristics of the endothelial-like cells induced from canine ASCs were evaluated by use of an in vitro solubilized basement membrane tube assay, low-density lipoprotein uptake assay, and in vivo solubilized basement membrane plug assay. Results—After differentiation culture, the cells developed morphological changes, expressed EPC markers such as CD34 and vascular endothelial growth factor receptor 2, and revealed functional characteristics in vitro. Furthermore, the induced cells allowed vessel formation in solubilized basement membrane plugs in vivo. Conclusion and Clinical Relevance—Results indicated that canine ASCs developed EPC characteristics when stimulated by differentiation medium with growth factors. Thus, differentiated canine ASC-EPCs may have the potential to provide vascularization for constructs used in regenerative medicine strategies.

Objective-To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis Sample-Femoral head cartilage from 16 dogs undergoing total hip replacement Procedures-Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and nducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis. Results-Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups. Conclusions and Clinical Relevance-Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.

Objective: To determine the effects of 2 conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12) on synthesis of prostaglandin (PG) E2 and F2α and expression of prostaglandin H synthase-2 (PGHS-2) of adult and fetal bovine endometrial epithelial cells in vitro. Sample: Primary cultures of endometrial epithelial cells obtained from 4 adult cows and 4 fetal bovine carcasses. Procedures: Cells were exposed to 0, 50, 100, or 200μM cis-9, trans-11 or trans-10, cis-12 CLA isomers for 24 hours. Culture media collected before and after 6 hours of stimulation of cells with phorbol 12-myristate 13-acetate were assayed to detect PGE2 and PGF2α via ELISA. After stimulation, cells were collected for western blot analysis to quantify PGHS-2. Results: Concentrations of PGF2α and PGE2 were significantly lower in culture media of adult and fetal endometrial epithelial cells exposed to any concentration of either CLA than they were in media of cells not exposed to CLAs. The trans-10, cis-12 CLA isomer seemed to decrease PG production more markedly than did the cis-9, trans-11 CLA isomer. Most concentrations of both CLAs significantly reduced culture media PGE2:PGF2α concentration ratios of cells. Exposure of cells to CLAs did not affect expression of PGHS-2 protein. Conclusions and Clinical Relevance: Results of this study indicated CLAs significantly decreased PGF2α and PGE2 concentrations and PGE2:PGF2α concentration ratios for cultures of adult and fetal endometrial epithelial cells with no apparent effect on PGHS-2 expression. Similar effects in cows could have effects on maternal recognition of pregnancy and immune function.