Ann M. Rashmir-Raven

Mutation in cyclophilin B that causes hyperelastosis cutis in american quarter horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin b-protein interactions and affects collagen folding

Background: Hyperelastosis cutis in horses is caused by a homozygous mutation in cyclophilin B (PPIB). Results: This mutation changes protein-protein interactions of CypB and delays folding of collagen. Conclusion: Mutant CypB has PPIase activity, but the synthesized collagen contains less post-translational modifications of lysine residues. Significance: CypB has other important functions besides its PPIase activity. The formation of these interactions is crucial for a correct biosynthesis of collagen. The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Inhibition of cyclooxygenase-2 expression and prostaglandin E2 production in chondrocytes by avocado soybean unsaponifiables and epigallocatechin gallate

OBJECTIVE To evaluate the anti-inflammatory effect of the combination of avocado soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in cytokine-activated equine chondrocytes. METHODS Production of type II collagen and aggrecan was verified by immunohistochemistry and Western blot. Chondrocytes were incubated with: (1) control media alone, (2) ASU (4 microg/ml; 8.3 microg/ml), (3) EGCG (4, 40, 400 ng/ml), or (4) the combination of ASU and EGCG for 24h. Cells were next incubated with control medium alone or with IL-1beta (10 ng/ml) and TNF-alpha (1 ng/ml). COX-2 gene expression by real-time PCR analysis and NF-kappaB nuclear translocation by immunohistochemistry were performed after 1h of incubation. PGE(2) production was determined by immunoassay after 24h of incubation. RESULTS Equine chondrocytes responded to cytokine activation by up-regulated gene expression of COX-2 and increased PGE(2) production. Activation was associated with NF-kappaB translocation. Individually, ASU and EGCG marginally inhibited COX-2 expression and PGE(2) production in activated chondrocytes. In contrast, the combination of ASU and EGCG reduced COX-2 expression close to non-activated control levels and significantly inhibited PGE(2) production. These reductions were statistically greater than those of ASU or EGCG alone. The inhibition of COX-2 expression and PGE(2) production was associated with inhibition of NF-kappaB translocation. CONCLUSION The present study demonstrates that the anti-inflammatory activity of ASU and EGCG is potentiated when used in combination. This combination may offer an attractive supplement or alternative to non-steroidal anti-inflammatory drugs (NSAIDs) in the management of osteoarthritis.

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts, propagation in cell culture, and description of ultrastructural and genetic characteristics

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.

Prevalence of Sarcocystis neurona sporocysts in opossums (Didelphis virginiana) from rural Mississippi

Sarcocystis species sporocysts were found in intestinal scrapings from 24 of 72 opossums (Didelphis virginiana) from rural Mississippi. The number of sporocysts in each opossum varied from a few ( < 100000) to 187 million. Sporocysts from 24 opossums were bioassayed for Sarcocystis neurona infections by feeding to gamma-interferon knockout (KO) mice. S. neurona was detected in the brains of KO mice fed sporocysts from 19 opossums by immunohistochemical staining with anti-S. neurona specific polyclonal rabbit serum, and by in vitro culture from the brains of KO mice fed sporocysts. The isolates of S. neurona from opossums were designated SN16-OP to SN34-OP. Merozoites from 17 of 19 isolates tested at the 25/396 locus were identical to previously described S. neurona isolates from horses. The high prevalence of S. neurona sparocysts in D. virginiana suggests that this opossum constitutes an ample reservoir of infection in the southern United States.

Ocular findings in Quarter Horses with hereditary equine regional dermal asthenia

OBJECTIVE To compare ocular structures of Quarter Horses homozygous for hereditary equine regional dermal asthenia (HERDA) with those of Quarter Horses not affected by HERDA (control horses) and to determine the frequency of new corneal ulcers for horses with and without HERDA during a 4-year period. DESIGN Cohort study of ocular structures and retrospective case series of horses with and without HERDA. ANIMALS The cohort portion of the study involved 10 Quarter Horses with HERDA and 10 Quarter Horses without HERDA; the retrospective case series involved 28 horses with HERDA and 291 horses without HERDA. PROCEDURES Ophthalmic examinations, Schirmer tear tests, tonometry, corneal pachymetry, histologic examinations, and scanning electron microscopy (SEM) were performed in cohorts of Quarter Horses with and without HERDA. Records were reviewed to determine the incidence of corneal ulcers in horses with and without HERDA during a 4-year period. RESULTS Corneal thickness of horses with HERDA was significantly less than that of control horses, but tear production of horses with HERDA was significantly greater than that of control horses. Results of SEM revealed zones of disorganized, haphazardly arranged collagen fibrils in corneas of horses with HERDA that were not evident in corneas of control horses. The incidence of corneal ulcers was significantly greater for horses with HERDA than for horses without HERDA during the 4-year period. CONCLUSIONS AND CLINICAL RELEVANCE Alterations in corneal thickness, arrangement of collagen fibers, and incidence of corneal ulcers indicated that abnormalities in horses with HERDA were not limited to the skin.

The effect of oxytocin on contractility of the equine oesophagus: a potential treatment for oesophageal obstruction.

This study was performed to determine the effect of administration of i.v. oxytocin on the contractility of the musculature associated with the equine oesophagus. Nine clinically normal horses were fitted with a nasogastric tube modified with inflatable latex cuffs. These cuffs were connected to piezoelectric pressure recording devices. Oxytocin in 3 different doses or saline controls were administered i.v. in a randomised block pattern. Systolic blood pressure, ECG, heart rate and nasogastric tube cuff pressures were then measured for 60 min. Administration of oxytocin i.v. at 0.11 and 0.22 iu/kg bwt, resulted in a short-term statistically significant relaxation of the musculature of the equine oesophagus. When oxytocin was administered at 0.11, 0.22 and 0.44 iu/kg bwt, no clinically significant cardiovascular changes were seen. In approximately 5% of the oxytocin administrations, signs of mild short-term abdominal discomfort were observed. In clinical cases of noncomplicated oesophageal obstruction, it is suggested that reduction in tone of oesophageal musculature may result in passage of oesophageal obstructions with reduced risk of oesophageal injury when compared to other traditional treatments.

Papillomatous pastern dermatitis with spirochetes and Pelodera strongyloides in a Tennessee Walking Horse

Papillomatous digital dermatitis is a common disease in cattle. The pastern dermatitis observed in a horse shared many of the gross characteristics of papillomatous digital dermatitis in cattle. Lesions included a mixture of proliferative and erosive changes, with a verrucose appearance in some areas. Microscopic similarities included pseudoepitheliomatous and papillomatous epidermal hyperplasia with hyperkeratosis, spongiosis of the epidermis, and intraepidermal spirochetes. The horse was also concurrently infected with Pelodera strongyloides. Papillomatous digital dermatitis in cattle is associated with poor husbandry practices. The environment of the affected horse was heavily contaminated with urine, manure, and other organic debris. Verrucous pododermatitis of horses may be the same as or similar to bovine papillomatous digital dermatitis, and these conditions have similar etiologies.

Tensile properties in collagen-rich tissues of Quarter Horses with hereditary equine regional dermal asthenia (HERDA).

REASONS FOR PERFORMING STUDY Hereditary equine regional dermal asthenia (HERDA) is an autosomal recessive disorder of Quarter Horses characterised by skin fragility. Horses with HERDA have a missense mutation in peptidyl-prolyl cis-trans isomerase B (PPIB), which encodes cyclophilin B and alters folding and post translational modifications of fibrillar collagen. OBJECTIVES The study aimed to test the hypothesis that tendons, ligaments and great vessels, which, like skin, are rich in fibrillar collagen, will also have abnormal biomechanical properties in horses with HERDA. STUDY DESIGN Ex vivo biomechanical study comparing horses with and without a diagnosis of HERDA. METHODS Forelimb suspensory ligament, superficial and deep digital flexor tendons; withers, forelimb and abdominal skin; the main pulmonary artery and the aortic arch were harvested from 6 horses with HERDA and 6 control horses without the HERDA allele. Tissues were distracted to failure. Tensile strength (TS), elastic modulus (EM) and energy to failure (ETF) were compared. RESULTS Horses with HERDA had significantly lower TS and EM in tendinoligamentous tissues and great vessels, respectively. The TS, EM and ETF were significantly lower in skin from horses with HERDA. Differences in TS and ETF were more extreme at the withers than at the forelimb or abdomen. CONCLUSIONS Tendinoligamentous tissue, great vessels and skin are significantly weaker in horses with HERDA than in horses lacking the PPIB mutation, substantiating that diverse tissues with high fibrillar collagen content are abnormal in HERDA and that the HERDA phenotype is not limited to the integument.

Risk factors associated with the presence of Sarcocystis neurona sporocysts in opossums (Didelphis virginiana)

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM) in horse in the Americas. The only known definitive host for this parasite in the United States is the opossum (Didelphis virginiana); however, despite the importance of the disease, the epidemiology of the parasite in the definitive host is poorly understood. To begin addressing these data gaps, potential risk factors were evaluated for their association with the presence of sporocysts of S. neurona in opossums live-trapped in March 1999 and November 1999 to May 2000. Sporocysts of S. neurona were found in 19 of the 72 animals examined. Potential risk factors evaluated were locality, trap date, age, gender, the presence of young in the pouch of females, and body condition score. Variables that were associated with the presence of S. neurona sporocysts were used in logistic regression analysis. Of the factors examined, season and body condition score were associated with increased odds of an animal harboring sporocysts.

Heritable equine regional dermal asthenia.

Hereditary equine regional dermal asthenia is a form of Ehlers-Danlos syndrome, and has an autosomal recessive mode of inheritance. Affected horses are typically born normal and develop lesions within the first 2 years of life. The most common symptoms of the disease include stretchy, loose skin that feels doughy or mushy. More severely affected horses experience spontaneous skin sloughing and extensive lacerations, hematomas, and seromas from minor trauma. Affected horses have a higher than expected incidence of corneal ulcers. DNA testing can normal, establish carrier and affected status. Palliative therapy is available, but no curative treatment exists.