Q fever in dairy cattle has been investigated in Latvia since 2012. In 2015, 10.7% of farms tested positive for the DNA of C. burnetii, its aetiological agent, in bulk tank milk. The presence of C. burnetii DNA and infectious bacteria in dairy products has been assessed in several countries, and because Latvian milk may contain them, parallel assessment in this country is recommended. Accordingly, the present study tested shop and farm retail dairy products from Latvia and included foreign products for comparison. Investigation was carried out of 187 samples of a diverse range of dairy products from 41 Latvian milk producers. Twenty-six comparable samples pooled from Estonia, France, Germany, Greece, Italy, Lithuania, the Netherlands, Poland and Spain were also included. The all-countries total number of fermented milk products was 160. Special attention was paid to products that could be more attractive to children because of their added chocolate, cacao, berry and fruit content. DNA was extracted and amplification of C. burnetii IS1111 was performed using a commercial PCR kit. Overall positivity was 60.56%. Domestic products were positive more often (60.96%) than foreign ones (57.69%). Only 26.67% of unpasteurised Latvian cow’s milk samples were positive whereas 76.47% of pasteurised equivalents and 63.13% of fermented milk products were. Sweetened and fruit-containing samples were 71.43% positive. The shedding of C. burnetii via milk should be monitored and only milk from healthy animals allowed for sale for direct human consumption without pasteurisation. Raw milk quality and the effectiveness of industrial heat treatment and pasteurisation methods in Latvia and other countries should be carefully assessed to ensure adequate consumer health protection.

Milk has been suggested to be a possible source of oestrogenically active compounds. In order to assess the health risk for milk consumers and ensure the safety of this staple part of the human diet, it is important to study the effect of xenooestrogen mixtures present in milk. This investigation used the available in vivo model to learn to what extent such compounds may be endocrine disruptors. The recommended immature golden hamster uterotrophic bioassay was chosen. A total of 132 animals were divided into nine groups of experimental animals and positive and negative control groups, each of 12 animals. The experimental females received ad libitum either one of five samples of raw cow’s milk from individual animals or one of four samples of pasteurised or ultra-high temperature treated cow’s milk as retail products. After 7 days, the animals were sacrificed and necropsied. Uterine weight increases were measured as the endpoint of oestrogenic activity in milk. The milk samples from individual cows and the retail milk samples did not show oestrogenic activity. However, in three groups, decreased uterine weights were observed. Considering that milk supplies are beneficial to health, contamination in this food should be avoided. There is a need for further animal experiments and epidemiological studies are warranted to evaluate any causative role of milk in human endocrinological disorders.

Enterococci are widespread, being part of the bacterial flora of humans and animals. The food chain can be therefore considered as the main route of transmission of antibiotic resistant bacteria between the animal and human populations. Milk in particular represents a source from which resistant bacteria can enter the human food chain. The aim of the study was to determine the occurrence and resistance to antimicrobial agents of Enterococcus spp. strains isolated from raw goat’s milk samples. A total of 207 goat’s milk samples were collected. Samples were cultivated on selective media and confirmed as E. faecium or E. faecalis and screened for selected resistance genes by PCR. Drug susceptibility determination was performed by microdilution on Sensititre EU Surveillance Enterococcus EUVENC Antimicrobial Susceptibility Testing (AST) Plates and Sensititre US National Antimicrobial Resistance Monitoring System Gram Positive CMV3AGPF AST Plates. Enterococcal strains totalling 196 were isolated, of which 40.8% were E. faecalis and 15.3% were E. faecium. All tested isolates were susceptible to linezolid, penicillin and tigecycline. For most other antimicrobials the prevalence of resistance was 0.5–6.6% while high prevalence of quinupristin/dalfopristin (51.5%), tetracycline (30%) and lincomycin (52%) resistance was observed. This study affords better knowledge concerning the safety of raw goat’s milk in terms of the enterococci possible to isolate from this foodstuff. It seems that enterococci in milk are still mostly susceptible to antimicrobials of major concern as multiply resisted drugs, such as gentamycin and vancomycin. However, the presence of multi-resistant strains in goat milk is cause for apprehension.

Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) are cytokines widely used in ex vivo monocyte differentiation experiments, vaccine formulations and disease treatment. The aim of this study was to produce recombinant bovine GM-CSF and IL-4 in an episomal expression system that conserves the postransductional modification of the native proteins and to use the products to differentiate bovine monocytes into dendritic cells. The recombinant proteins rGM-CSF and rIL-4 were expressed in PEAKrapid CRL-2828 human kidney cells, ATCC CRL-2828. The functional activity of the recombinant cytokines was monitored by registering morphological changes in bovine monocytes and assessing the expression of CD14 upon incubation with them. Both recombinant proteins were detected in the cell culture supernatant of transfected cells. Culture supernatants of transfected cells induced in bovine monocytes morphological changes that resemble macrophages or dendritic cells. In addition, bovine cells treated with rGM-CSF and rIL-4 showed reduced expression of the macrophage surface marker CD14 compared with untreated cells. This effect indicates the expected differentiation. The expression of the cytokines was stable after many successive cell passages and a freeze/thaw cycle. The semi-stable mammalian episomal expression system used in this study allowed us to easily produce functional bovine rGM-CSF and rIL-4 without the need for protein purification steps.

Proper conformational arrangement of the E2 molecules of bovine viral diarrhoea-mucosal disease virus (BVD-MDV) is crucial to obtain an effective recombinant vaccine candidate against the disease. In this study, we characterised a new molecule composed of two distinct sequences of the E2 glycoprotein of BVD-MDV and the Fc fragment of human immunoglobulin (BVDE2Fc). The chimaeric protein was expressed in mammalian cell lines of different species by adenoviral transduction and purified by immobilised metal-affinity chromatography. The N-glycans were profiled by HPLC, and the BVDE2Fc immunogenicity was assessed in male mice. The antigen-antibody reactions were evaluated by ELISA. The MDBK cell line was selected from among five for the final production of BVDE2Fc. After purification to over 90%, the N-glycan profile showed neutral and complex oligosaccharides. The mouse immunisation induced a strong humoral response, which produced antibodies able to attach to conformational epitopes on E2 molecules, while the Fc fragment barely contributed to the immune response. Additionally, BVDE2Fc attached to antibodies from bovine sera positive to distinct BVD-MDV subtypes, whereas the loss of BVDE2Fc structure during the deglycosylation process considerably diminished those interactions. These results demonstrate that the structure of E2 molecules arranged in tandem and attached to an Fc fragment could represent a viable design for future vaccine candidates against BVD-MD.

Because minipig skin is similar to human skin in anatomy and physiology, establishing an atopic dermatitis (AD) minipig model seems meaningful. We applied 1-fluoro-2,4-dinitrobenzene (DNFB) or ovalbumin onto the back skin of five Yucatan minipigs aged 8–10 months and 19 kg in median weight. Two minipigs with the same parameters served as controls. Both DNFB and ovalbumin mediated epithelial hyperplasia, spongiosis, and immune cell infiltration in the dermis, which is a typical histopathological feature of AD. Moreover, AD upregulated the Th1- and Th2-related cytokine expressions in DNFB- or in ovalbumin-treated skin. Notably, AD-induced minipigs exhibited greater cytokine serum concentrations. Histopathological finding and cytokine analysis revealed that DNFB or ovalbumin mediates AD. However, ovalbumin-treated minipig is a more reliable and precise AD model owing to the DNFB-induced severe skin damage. In summary, ovalbumin-treated skin shows similar AD as human in histopathological and molecular analysis.

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.

Bone repair in horses implies invasive surgeries and increased cost. Research on musculoskeletal disorders therapy in horses includes cell-based therapy with mesenchymal stromal cells (MSCs). Mesenchymal stromal cells can be obtained from bone marrow (BMMSCs). Unfortunately, BMMSCs have limited cell replication in vitro. The objective of this study was to develop a biologically immortalized equine stem cell line derived from bone marrow, with unlimited in-vitro proliferation and the ability to differentiate into bone cells. Equine BMMSCs were transfected and immortalized with human telomerase reverse transcriptase (hTERT) gene. Cell passages from equine immortal BMMSCs were characterized by the presence of stemness CD markers and expression of multi-potent differentiation genes (OCT-4, SOX2, and NANOG). Equine immortal BMMSCs were incubated in osteogenic medium and bone cell differentiation was determined by alkaline phosphatase and von Kossa staining, and osteogenic gene expression (osteocalcin, Runx2, and osterix). Telomerase activity was determined by telomeric repeat amplification technique. Results showed that equine immortal BMMSCs were able to replicate in-vitro up to passage 50 and maintain stem cell characteristics by the presence of CD90 and expression of multi-potent genes. Equine immortal BMMSCs were able to differentiate into bone cells, which was confirmed by the positive osteogenic staining and gene expression. Equine BMMSCs were successfully immortalized and maintained characteristics of stem cells and readily differentiated into osteogenic cells. Extending the life span of equine BMMSCs by transfection of the hTERT gene will revolutionize the clinical use of MSCs by making them available to orthopedic surgeons "off the shelf."

OBJECTIVE To evaluate whether cell-based and tissue-based immunofluorescent assays (IFAs) run in parallel could be used to detect glial fibrillary acidic protein (GFAP) autoantibodies in the CSF of dogs with meningoencephalitis of unknown origin (MUO) and other CNS disorders ANIMALS 15 CSF samples obtained from dogs with presumed MUO (n = 5), CNS disease other than MUO (5), and idiopathic epilepsy (5). PROCEDURES All CSF samples underwent parallel analysis with a cell-based IFA that targeted the α isoform of human GFAP and a tissue-based IFA that involved mouse brain cryosections. Descriptive data were generated. RESULTS Only 1 CSF sample yielded mildly positive results on the cell-based IFA; that sample was from 1 of the dogs with presumed MUO. The remaining 14 CSF samples tested negative on the cell-based IFA. All 15 CSF samples yielded negative results on the tissue-based IFA. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that concurrent use of a cell-based IFA designed to target the human GFAP-α isoform and a tissue-based IFA that involved mouse tissue cryosections was inadequate for detection of GFAP autoantibodies in canine CSF samples. Given that GFAP autoantibodies were likely present in the CSF samples analyzed, these findings suggested that epitopes differ substantially between canine and human GFAP and that canine GFAP autoantibody does not bind to mouse GFAP. Without a positive control, absence of GFAP autoantibody in this cohort cannot be ruled out. Further research is necessary to develop a noninvasive and sensitive method for diagnosis of MUO in dogs.

The objective of this preliminary study was to identify genomic regions that may predispose Gordon setters from the United Kingdom to familial protein-losing enteropathy (PLE) at a young age. A total of 106 related Gordon setters was used, including 6 affected dogs from an affected litter, 6 case controls from the same litter, 10 related/affected dogs, and 84 related/unaffected dogs. Genomic DNA was collected from each Gordon setter and extracted from buccal mucosal swabs. Genotyping of affected and unaffected dogs was carried out using the Canine Illumina HD SNP array and data generated were analyzed with PLINK software, using fixation index (Fst) and runs of homozygosity (ROH) methods. Pairwise Fst analyses between the affected and unaffected Gordon setter dogs identified various regions of differentiation on chromosomes 10, 18, 21, and 23 that contained several important genes. These regions revealed 5 candidate genes, including RARB, TTC7A, SOCS5, PIGF, and RHOD, that are associated with human inflammatory bowel disease (IBD) and could potentially be associated with PLE in Gordon setters. Run of homozygosity (ROH) analyses revealed additional unique regions on chromosomes 15 and 17. These regions contained genes SYT1, UCN, and FNDC that could also be potential candidates for PLE in Gordon setters. The biological functions of the identified genes provided initial insights into the pathophysiology of PLE. Further large-scale studies are warranted to investigate the possible causality of these genomic regions and any possible genetic markers that could be used in predicting susceptibility to PLE syndrome.