Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10-11, and the PI among siblings (PIsib) was 1.5 x 10-5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.

Copper-associated hepatitis in a young Dalmatian dog in Japan.

A male 25-month-old Dalmatian dog attended our veterinary hospital because of anorexia and high circulating liver enzyme activities. Abdominal computed tomography showed a slightly small liver with rounded edges, and laparoscopic examination showed that the liver was yellowish. Histopathological examination revealed multifocal necrosis of hepatocytes and severe chronic hepatitis. Rhodanine staining showed severe copper accumulation in hepatocytes and a quantitative analysis of the copper content of the liver showed substantial accumulation (10.3 mg/g dry mass), suggesting a diagnosis of copper-associated hepatitis. Previously reported canine mutation in the COMMD1, the gene responsible for the copper-associated hepatitis in the Bedlington terrier, was not identified. To our knowledge, this is the first report of copper-associated hepatitis in a Dalmatian in Japan.

Bilateral microphthalmos with cyst in a neonatal foal.

A 1-day-old miniature horse filly was presented to the University of Florida Veterinary Ophthalmology service for evaluation of multiple ocular anomalies which were present from birth. Protruding from the right orbit was a large, fluctuant, red, dry and variably ulcerated mass. A globe could not be appreciated clinically in the left orbit. Ocular ultrasound of both orbits was performed. This revealed a lobular, hypo-echoic structure filling the right orbit, with multiple hyperechoic septations; normal ocular structures were not identified. Ultrasound of the left orbit revealed a microphthalmic eye, with a well-defined, hyperechoic structure in the vitreous that was thought to be the lens. Due to irreversible blindness, the foal was humanely euthanized. Histopathology and immunohistochemistry of the orbital contents revealed bilateral microphthalmos with cyst, a congenital defect rarely reported in the veterinary literature.

Histologic evaluation of the diversity of epidermal laminae in hooves of horses without clinical signs of laminitis.

OBJECTIVE: To evaluate the histologic diversity of epidermal laminae in hooves from horses without clinical signs of laminitis. SAMPLE POPULATION: Formalin-fixed samples of stratum internum obtained from the mid region of the dorsal aspect of the hoof wall from the forelimbs of 35Thoroughbred cadavers (including foals [n = 9], yearlings [5], 2 year olds [6], racing horses [5], and mares [10]). PROCEDURES: Paraffin-embedded laminar tissues were stained with H&E for the evaluation of architectural variety of primary epidermal laminae (PEL) and secondary epidermal laminae (SEL). For detection of cytokeratin (CK) expression in epidermal laminae, immunohisto-chemical staining was performed by use of anti-CK14 and anti-CK8.12 antibodies. RESULTS: The morphology of the PEL, SEL, and tips of PEL was classified into 3, 5, and 3 patterns, respectively. Differences in the predominant type of SEL depended on their location with respect to the laminar interface. In SEL attached to the sides of PEL, the basal cells were immunoreactive to CK14 and CK8.12, which was interpreted as a normal pattern. In some SEL at the tips of PEL, the suprabasal cells expressed CK14, CK8.12, or both, which constituted a hyperplastic pattern. CONCLUSIONS AND CLINICAL RELEVANCE: The histologic diversity of epidermal laminae from hooves of Thoroughbreds was attributable to the combined morphology of PEL and SEL. Detection of hyperplastic changes in the laminar interface does not justify a diagnosis of laminitis because such changes can develop independent of clinical disease. The classification system used here should aid investigators in making a more accurate histologic evaluation of laminae.

Immunohistochemical evaluation of canine ovarian cysts.

To clarify the immunohistochemical characteristics of canine ovarian cysts, 109 canine ovarian cysts (57 cysts of subsurface epithelial structures: SES, 26 graafian follicle cysts, 12 cystic rete ovarii and 14 cysts difficult to classify morphologically) were examined regarding their lining cells immunohistochemically using antibodies against placental alkaline phosphatase (PLAP), S100, inhibin alpha, desmin and AE1/AE3. Both cysts of SES and cystic rete ovarii had a positive immunoreaction to desmin and AE1/AE3, whereas all cysts all but graafian follicle cysts were negative for inhibin alpha. PLAP-positive immunoreaction was observed only in cysts of SES. Graafian follicle cysts had a positive immunoreaction to inhibin alpha, but were negative for PLAP, desmin and AE1/AE3. Fourteen cysts were difficult to classify morphologically because these cysts had single-squamous lining cells and lacked other morphological characteristics. However, these unclassified cysts were immunohistochemically divided into two groups, including positive and negative cysts, by the reactivity of PLAP. The PLAP-positive cysts were considered large cysts of SES. These results suggest that PLAP was a useful marker for classification of cysts of SES, although cysts originating from SES are not always positive for this antigen.

Immunohistochemical evaluation of canine ovarian tumors.

Canine ovarian tumors (epithelial tumor, sex-cord stromal tumor, germ cell tumor) classifying into 9 histological types were examined immunohistochemically using placental alkaline phosphatase (PLAP), cytokeratin7 (CK7), desmin, S100, AE1/AE3, inhibin alpha, vimentin, and alfa feto-protein (AFP). The papillary and tubular types observed in epithelial tumors were immunoreactive for desmin and AE1/AE3. The papillary type was also immunoreactive for PLAP and CK7. The solid type, nest type, cord type, palisade type, cystic type and spindle type, which were observed in sex-cord stromal tumors, showed a positive immunoreaction for S100 but little or no positive immunoreaction for inhibin alpha with an exception of positive result in the palisade type. Most of the sex-cord stromal tumors were AE1/AE3-positive except for the palisade type. In the cobblestone type observed in germ cell tumors, only vimentin and AFP were positive. The present study elucidated the detailed histological and immunohistochemical characteristics of canine ovarian tumors.

Expression of monocarboxylate transporter 1 (MCT1) in the dog intestine.

In this study, the expression and distribution of monocarboxyolate transporter 1 (MCT1) along the intestines (duodenum, jejunum, ileum, cecum, colon and rectum) of dogs were investigated at both the mRNA and protein levels. The expression of MCT1 protein and its distribution were confirmed by Western blotting and immunohistochemical staining using the antibody for MCT1. We identified mRNA coding for MCT1 and a 43-kDa band of MCT1 protein in all regions from the duodenum to the rectum. Immunoreactive staining for MCT1 was also observed in epithelial cells throughout the intestines. MCT1 immunoreactivity was greater in the large intestine than in the small intestine. MCT1 protein was predominantly expressed on the basolateral membranes along intestinal epithelial cells, suggesting that MCT1 may play an important role in lactate efflux and transport of short-chain fatty acids (SCFAs) to the bloodstream across the basolateral membranes of the dog intestine.