Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Immunological and pathological investigations in equine experimental uveitis

Background

The pathogenic mechanism of equine recurrent uveitis (ERU) is still poorly defined and many variations between experimental animal models and spontaneous disease exist.

Objectives

The aim of our study was to investigate if Th17 cell-mediated response plays role in the pathogenesis of the used experimental model in horses and to reveal its pathological findings.

Methods

Experimental uveitis was induced in 6 healthy horses. The concentrations of retinal autoantigen CRALBP and IL-17 were measured using ELISA in aqueous humor and vitreous body of the 12 inflamed eyes as well as in 12 control non-inflamed eyes taken from 6 horses in slaughter house. After centrifugation of the two eye media, smears were prepared and cytological investigation was performed. Tissue specimens were taken from all eye globes and were submitted to histopathological investigation.

Results

CRALBP and IL-17 concentrations were significantly elevated in eye media of horses with experimental uveitis in comparison with controls. Cytological and histopathological findings corresponded to the changes characteristic of chronic immune-mediated inflammation with mononuclear cell infiltration of uvea, choroid, retina, and eye media as well as severe retinal destruction.

Conclusions

Our study demonstrated the involvement of the retinal autoantigen CRALBP as well as IL-17 in the pathogenesis of experimental uveitis in horses. These findings suggests that this experimental uveitis in horses may serve as a suitable animal model for investigation of IL-17- mediated immune response during spontaneous autoimmune uveitis in horses as well as in humans.

     

Neutrophil myeloperoxidase measurements in plasma, laminar tissue, and skin of horses given black walnut extract

OBJECTIVE: To compare measurements of myeloperoxidase (MPO) in plasma, laminar tissues, and skin obtained from control horses and horses given black walnut heartwood extract (BWHE). ANIMALS: 22 healthy 5- to 15-year-old horses. PROCEDURES: Horses were randomly assigned to 4 groups as follows: a control group given water (n = 5) and 3 experimental groups given BWHE (17) via nasogastric intubation. Experimental groups consisted of 5, 6, and 6 horses that received BWHE and were euthanatized at 1.5, 3, and 12 hours after intubation, respectively. Control horses were euthanatized at 12 hours after intubation. Plasma samples were obtained hourly for all horses. Laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia. Plasma and tissue MPO concentrations were determined via an ELISA; tissue MPO activity was measured by use of specific immunologic extraction followed by enzymatic detection. RESULTS: Tissues and plasma of horses receiving BWHE contained significantly higher concentrations of MPO beginning at hour 3. Laminar tissue and skin from horses in experimental groups contained significantly higher MPO activity than tissues from control horses. Concentrations and activities of MPO in skin and laminar tissues were similar over time. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWHE administration causes increases in MPO concentration and activity in laminar tissue and skin and the time of increased MPO concentration correlates with emigration of WBCs from the vasculature. These findings support the hypothesis that activation of peripheral WBCs is an early step in the pathogenesis of acute laminitis.     

Clinical features and heritability of hypoadrenocorticism in Nova Scotia Duck Tolling Retrievers: 25 cases (1994-2006)

OBJECTIVE: To evaluate the clinical features and heritability of naturally occurring hypoadrenocorticism in Nova Scotia Duck Tolling Retrievers (NSDTRs). DESIGN: Retrospective case series. ANIMALS: 25 NSDTRs with hypoadrenocorticism. PROCEDURES: Questionnaires completed by owners of NSDTRs with hypoadrenocorticism and medical records from veterinarians were reviewed for information regarding diagnosis, age at diagnosis, concurrent diseases, age at death, and cause of death. Pedigrees were analyzed for heritability and mode of inheritance of hypoadrenocorticism (including complex segregation analysis of pedigrees of 1,515 dogs). RESULTS: On the basis of results of ACTH stimulation testing, hypoadrenocorticism was diagnosed in 16 female and 9 male NSDTRs (including 6 full siblings). Median age at diagnosis was 2.6 years; the diagnosis was made prior to 2 years of age in 11 dogs. Seventeen dogs had hyponatremia, hyperkalemia, or both, and serum electrolyte concentrations were within reference ranges for 8 dogs at the time of diagnosis. Median survival time after diagnosis for 4 dogs that died or were euthanized as a result of medical causes was 1.6 years. Heritability was calculated at 0.98 with no sex effect, and complex segregation analysis fit a major gene model with an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: In NSDTRs, hypoadrenocorticism was diagnosed at an earlier age, compared with published reports of age at diagnosis among the general dog population. Among the study dogs, 32% had no serum electrolyte abnormalities at the time of diagnosis, and the disease appeared to have an autosomal recessive mode of inheritance in the breed.     

Cerebral Blastomyces dermatitidis infection in a cat

CASE DESCRIPTION: An 8-year-old domestic shorthair cat was evaluated because of signs of depression, circling, and visual deficits. CLINICAL FINDINGS: The cat had no cutaneous lesions, and results of an ophthalmologic examination and thoracic radiography were within reference limits. Computed tomography of the brain revealed a mass lesion involving the right parietal, temporal, and occipital lobes; the mass was in broad-based contact with the skull and smoothly marginated and had strong homogenous enhancement after contrast agent administration. During craniectomy, samples of the mass were collected for cytologic and histopathologic evaluations and microbial culture. A diagnosis of Blastomyces dermatitidis-associated meningoencephalitis with secondary pyogranulomatous inflammation was made. TREATMENT AND OUTCOME: Amphotericin B (0.25 mg/kg [0.11 mg/lb], IV) was administered on alternate days (cumulative dose, 1.75 mg/kg [0.8 mg/lb]). To minimize the risk of nephrotoxicosis, assessments of serum biochemical variables (urea nitrogen and creatinine concentrations) and urinalyses were performed at intervals. The third dose of amphotericin B was postponed 48 hours because the cat became azotemic. The cat subsequently received fluconazole (10 mg/kg [4.5 mg/lb], PO, q 12 h) for 5.5 months. Six months after discontinuation of that treatment, the cat appeared healthy and had no signs of relapse. CLINICAL RELEVANCE: Brain infection with B dermatitidis is typically associated with widespread disseminated disease. The cat of this report had no evidence of systemic disease. Blastomycosis of the CNS should be considered as a differential diagnosis for brain lesions in cats from areas in which B dermatitidis is endemic.     

Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.     

Evaluation of stocking density and subtherapeutic chlortetracycline on Salmonella enterica subsp. enterica shedding in growing swine

The objective of this research was to determine the effect of stocking density and inclusion of subtherapeutic chlortetracycline in the diet on Salmonella fecal prevalence and antimicrobial resistance in growing swine. A 2 x 2 factorial design was employed on a privately owned commercial swine farm. Four finisher rooms were included in the study. Two of the rooms received 50 g/tonnes of chlortetracycline in the ration, two rooms received no antimicrobials in the feed. In each room, alternate pens were assigned to either high stocking density (0.60 m2/pig) or low stocking density (0.74 m2/pig). Pigs were placed in the finisher rooms at 10 weeks of age and followed for 6 weeks. Individual fecal samples were collected from the floors of each pen and cultured once weekly. Antimicrobial resistance phenotypes were determined. Data were analyzed using multilevel, multivariable logistic regression. Pigs fed chlortetracycline were at increased odds (OR 6.88, 95% CI 2.77-17.12) to shed Salmonellae. No other associations between treatments (CTC and stocking density) and Salmonella prevalence or reduced susceptibility to antimicrobials were identified. Variance in the odds of a fecal sample to be positive was distributed mostly at the lowest level, the individual fecal sample. The increased risk of shedding associated with inclusion of subtherapeutic chlortetracycline in swine diets is discordant with previous results by our group, suggesting farm or strain specific factors may impact this association. Understanding this risk may provide a potential intervention for controlling Salmonella pre-harvest.