Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

Effects of oxytocin,lipopolysaccharide (LPS), and polyunsaturated fatty acids on prostaglandin secretion and gene expression in equine endometrial explant cultures

Increased secretion of prostaglandin F₂α (PGF₂α) within the uterus because of uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence PG production in many species, although the effects on the mare remain unknown. The present study aimed to determine fatty acid uptake in equine endometrial explants and evaluate their influence on PG secretion and expression of enzymes involved in PG synthesis in vitro. Equine endometrial explants were treated with 100 μM arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid and then challenged with oxytocin (250 nM) or lipopolysaccharide (LPS; 1 μg/mL). Production of PGF₂α and PG E₂ (PGE₂) was measured, and mRNA expression of enzymes involved in PG synthesis was determined with quantitative real-time PCR. Media concentrations of PGF₂α and PGE₂ were higher (P < 0.0001) from endometrial explants challenged with oxytocin or LPS compared with controls despite which fatty acid was added. Only DHA lowered (P < 0.0001) media concentrations of PGF₂α and PGE₂ from explants. Endometrial explants stimulated with oxytocin had increased expression of PG-endoperoxide synthase 1 (PTGS1; P < 0.02), PG-endoperoxide synthase 2 (PTGS2; P < 0.001), PG F₂α synthase (PGFS; P < 0.01), PG E₂ synthase (PGES; P < 0.01), and phospholipase A₂ (PLA₂; P < 0.005) compared with controls and regardless of fatty acid treatment; whereas stimulation with LPS increased expression of PTGS2 (P < 0.004), PGFS (P < 0.03), PGES (P < 0.01), and PLA₂ (P < 0.01) compared with controls and regardless of fatty acid treatment. Treatment with PUFAs, specifically DHA, can influence PG secretion in vitro through mechanisms other than enzyme expression.     

GLUT4 but not GLUT1 expression decreases early in the development of feline obesity

The increase in obesity in people and pets has been phenomenal. As in man, obesity in pets is a risk factor for many diseases including diabetes mellitus. Recently, tissue-specific regulation of glucose metabolism in fat and muscle tissue has been identified as an important factor for insulin sensitivity and it has been hypothesized that glucose uptake into tissues is altered in obesity causing insulin resistance. The purpose of this study was to determine the expression of the glucose transporter proteins GLUT4 and GLUT1 in muscle and fat from lean and obese cats. Seventeen domestic felines were tested in the lean state and again after a 6-month period of ad libitum food intake which led to a significant increase in weight (P<0.0001). Obese cats showed a significantly higher area under the curve (AUC) for glucose, AUC for insulin and a significant decrease in glucose percentage disappearance per min (K-value) (P=0.013, 0.018 and 0.017, respectively) during an intravenous glucose tolerance test, but no change in baseline glucose or glycosylated hemoglobin concentrations. GLUT4 expression was decreased in biopsies of both muscle (P=0.002) and fat (P=0.001) in the obese animals. GLUT4 in muscle and fat significantly and negatively correlated with the insulin AUC (r²=0.36, P=0.004 and r²=0.18, P=0.040, respectively). GLUT1 expression showed no significant change in the obese cats in either tissue. It is concluded that the changes in GLUT4 are early derangements in obesity and occur before glucose intolerance is clinically evident.     

First recovery of A/equine/Fontainebleau/1/79 influenza viruses in Italy

In Italy epizootics of equine influenza often occur, but no virus isolation has been reported since 1971. This paper describes the antigenic and biochemical characterization of two equine influenza viruses isolated in Italy from 1985 to 1989. The virus isolates were shown to differ antigenically from earlier strains of the same subtype, A/equine/Miami/1/63 (H3N8). Monoclonal antibody analysis showed that the haemagglutinins of these strains were serologically indistinguishable from A/equine/Fontainebleau/1/79, a variant of A/equine/Miami, never isolated in Italy before. One of the two virus isolates was obtained from a horse immunized with a bivalent inactivated influenza vaccine, not containing A/equine/Fontainebleau/79 antigens.

The vaccine failure underlines the importance of antigenic relatedness between currently circulating viruses and vaccine strains. Therefore, to improve the protection afforded by equine immunization, the vaccine composition should be decided according to the results of a virological surveillance activity, systematically conducted among horses.     

Evaluation of the chemoprophylactic efficacy of 10% long acting injectable moxidectin against gastrointestinal nematode infections in calves in Belgium

The chemoprophylactic efficacy of a single dose of the 10% long acting (LA) injectable formulation of moxidectin on nematode infections in calves, was evaluated. Two similar groups of 11 female, first grazing season Holstein calves were turned out in early May on separate plots of a single, naturally infected pasture. Until 56 days post-treatment (pt), the percentage reduction in faecal egg output was 100%, remaining above 90% during the entire trial, except for day 126 pt. More than 90% of the larvae in the treated group were identified as Cooperia until 140 days after treatment and more than 70% during the rest of the trial, whereas in the control group Cooperia was the most abundant species until day 84 pt and Ostertagia from 126 days pt onwards. The reduction in faecal egg output in the treated group was reflected in the mean pepsinogen levels being below the pathogenic threshold at the end of the grazing season (1.8 units of tysrosine (U tyr)) and the absence of diarrhoea during the second half of the grazing season. In the control group pepsinogen levels remained high (mean: 5.5 U tyr) and prolonged diarrhoea occurred in the second half of the grazing season. Furthermore, the weight gain for the treated group at the end of the grazing season was 41.9 kg higher than for the control group. At necropsy, the reduction in O. ostertagi worm burden in the treated group was 97.5% compared to the control group, while the reduction in C. oncophora worm burden was 57%. An additional benefit of the long acting parasitological control, was reduced pasture contamination.     

Comparison of the disease incidences of Finnish Ayrshire and Finnish Black and White dairy cows

Finnish Ayrshire and Finnish Black and White cows were compared regarding the incidences of early and late mastitis, parturient paresis, ketosis, ovarian disorders, metritis and the risk of having a test-day somatic-cell count >200,000 cells ml⁻¹ at any of the first three monthly test days in lactation. In a retrospective cohort study 101,793 cows from 5844 tie stalls and 11,811 cows from 437 loose-housing systems from all over Finland were followed from calving in 2000 until the end of lactation. The observed incidences of those cows were then analysed using generalised linear mixed models. Finnish Black and White cows had higher incidences of all diseases except ovarian disorders. Although the differences were statistically significant in all models except metritis and early mastitis in loose-housing systems, they were, in our view, only important on the national level (for the breeding organisations), and of little importance for the farmers.     

The expression of intermediate filaments in canine mammary glands and their tumors

Monoclonal antibodies specific for different types of intermediate filaments (cytokeratin, vimentin, desmin and neurofilaments) were used to study the histogenesis of canine mammary glands and 57 canine mammary tumors by immunocytochemistry. The intra- and interlobular duct epithelium, acinar, and intralobular myoepithelial cells stained positively for cytokeratin. Peripheral ductal and acinar cells, as well as interstitial cells, stained positively for vimentin. A similar staining pattern was seen in adenomas, complex adenomas, benign mixed tumors, ductular carcinomas, and one myoepithelioma-like tumor. Additionally, cytokeratin positive cells were scattered interstitially in one single adenoma, most complex adenomas, some benign mixed tumors, complex carcinomas, and in the malignant mixed tumors. All stromal cells stained positively for vimentin. The fibrosarcomas were positive only for vimentin, while the following expressed both desmin and cytokeratin: epithelial-like cells in one adenoma, three complex adenomas, the myoepithelioma-like tumor, the single comedo carcinoma, two complex carcinomas, the single lobular carcinoma, one malignant mixed tumor, and three osteosarcomas. Epithelial-like cells in one adenoma, six complex adenomas, two benign mixed tumors, two complex carcinomas, the lobular carcinoma, and the malignant schwannoma stained for neurofilaments. Three tumors, one adenoma, one complex adenoma, and the lobular carcinoma expressed both desmin and neurofilaments in addition to cytokeratin and vimentin. The results show the expression of different types of intermediate filaments and indicate that there might be a stem cell origin in most of the canine mammary tumors.     

The Effect of Cysteine-Rich Secretory Protein-3 and Lactoferrin on Endometrial Cytokine mRNA Expression After Breeding in the Horse

The equine uterus undergoes a transient innate immune response after breeding, also known as mating-induced endometritis. The deposition of spermatozoa triggers the expression of pro-inflammatory cytokines, which results in the migration of polymorphonuclear neutrophils (PMNs) into the endometrium and the uterine lumen. Select seminal plasma proteins, specifically cysteine-rich secretory protein 3 (CRISP-3) and lactoferrin, have been shown to affect the activity of the PMNs, either by suppressing (CRISP-3) or promoting (lactoferrin) the phagocytosis of spermatozoa based on their viability in vitro. Conjointly, many components of inseminate, including seminal plasma, bacteria, and spermatozoa itself, have shown to have an effect on the expression of endometrial cytokines after breeding. The objective of this study was to determine if select proteins affect the mRNA expression of endometrial cytokines after insemination. Six mares were bred during four consecutive estrous cycles with treatments in randomized order of: 1mg/mL CRISP-3, 150 ug/mL lactoferrin, seminal plasma, or Lactated Ringer’s Solution (LRS) to a total volume of 10 mL combined with 1×10⁹ progressively motile spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis. No treatment effects were found for the mRNA expression of IL-1β, IL-6, IL-8, IL-10, TNFα, and IFNγ, while lactoferrin significantly suppressed the mRNA expression of IL-1RN when compared to LRS. In conclusion, the seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on the expression of select endometrial cytokines at 6 hours post breeding.     

Identification and quantification of amines in the equine caecum

Acute laminitis has been associated with the release of compounds, as yet unidentified, produced by hindgut fermentation which affect blood flow to the digit. The objectives of this study were to identify amine compounds in equine caecal and colonic contents, some of which are known to have vasoactive properties. In addition, the concentrations of amines in caecal contents of horses fed either grass or hay diets were compared. Fifteen amines were identified in equine hindgut contents in concentrations greater than 1 microM. The caecal concentrations of phenylethylamine, isoamylamine, cadaverine, diaminoheptane and spermidine were significantly higher in horses on spring/summer grass compared with those on winter grass or hay. These data show that many amines are present in the equine hindgut, some of which may have the potential to cause peripheral vasoconstriction if released into the circulation from the gastrointestinal tract.     

Immunohistochemical expression of progesterone receptors, growth hormone and insulin growth factor-I in feline fibroadenomatous change

The immunohistochemical expression, tissue-specific and cell-specific distribution patterns of progesterone receptors (PR), growth hormone (GH) and insulin growth factor-I (IGF-I) have been studied in 22 cases of feline fibroadenomatous change (FFAC). PR and GH were detected in all cases and were distributed homogeneously throughout the lesion, while IGF-I was detected in 77% of the cases at the site of ductal budding. The simultaneous expression of PR, GH and IGF-I was detected in epithelial cells in 14 of 22 cases while PR and GH expression only was detected in epithelial cells in 11 cases. Cases that expressed GH and IGF-I without PR expression in the stroma were the most numerous. Double immunohistochemical staining showed the co-localisation of PR and GH in a subset of ductal epithelial cells located between basal/myoepithelial and luminal cells (probably undifferentiated stem cells). These results suggest that ligand-activated progesterone receptors may induce the local synthesis of GH which in turn may exert its proliferative action directly and also indirectly through the production of other growth factors, such as IGF-I, in an autocrine/paracrine manner.     

Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.     

Morphological, immunohistochemical, and ultrastructural findings in an embryonal rhabdomyosarcoma of a newborn thoroughbred foal

This study reports the rare case of a rhabdomyosarcoma in the shoulder of a newborn Thoroughbred foal. Histomorphologic, immunohistochemical, and ultrastructural investigations revealed an embryonal rhabdomyosarcoma with various differences: small round undifferentiated cells, rhabdomyoblast-like neoplastic cells, and spindle-shaped cells. Desmin expression appears in 30% of small round cells and in as many as 90% of spindle-shaped cells. Vimentin co-expression occurs in 90% of small round and spindle-shaped neoplastic cells but only in few (30%) rhabdomyoblast-like cells. Myoglobin was detected in 50% of the rhabdomyoblast-like cells and in 30% of spindle-shaped cells but not in small round neoplastic cells. This is the first time that rhabdomyosarcoma markers Myf-4 and Myo D1 were found in horses. Myf-4 and Myo D1 were expressed in about half of the neoplastic cells in immediately fixated specimens. Ultrastructural sarcoma-like packets and formations of primitive Z-bands, as well as a discontinuous basal lamina, were found and served to confirm our diagnosis.

Introduction

Malignant neoplasia of striated muscles in domestic animals represents uncommon findings.[1] Only single cases of rhabdomyosarcoma in horses of varying ages (2-19 years) and races (Quarter horses, Appaloosas, Thoroughbreds, ponies) were described. [2, 3, 4, 5, 6 and 7]In human medicine, histopathologically different partly overlapping types of rhabdomyosarcoma are distinguished, reflecting stages of differentiation and the morphology of the neoplastic cells[8 and 9]: (1) embryonal rhabdomyosarcoma (subtypes: spindle cell, small cell) and its botryoid variant; (2) alveolar (synonymous with juvenile) rhabdomyosarcoma; and (3) pleomorphic (synonymous with adult) rhabdomyosarcoma.In veterinary pathology, rhabdomyoma, rhabdomyosarcoma, and “embryonal rhabdomyosarcoma of the urinary bladder in dogs” are actually distinguished histopathologically.[10] But in older references the nomenclature of human medicine is used.In horses, alveolar rhabdomyosarcoma occur in the extremities.[2 and 5] Botryoid rhabdomyosarcomas were described in the equine uterus [4] and the urinary bladder. [7] Pleomorphic rhabdomyosarcomas are found in the tongue [6] and inside the thorax. [3]Differential diagnoses include fibrosarcoma, melanoma, anaplastic lymphosarcoma, and anaplastic carcinoma. Immunohistochemical[10, 11 and 12] and ultrastructural [13 and 14] investigations are helpful in confirming a diagnosis of rhabdomyosarcoma.Myo D1, Myf-3, and Myf-4 belong to a group of myogenic regulatory proteins whose expression determines commitment and differentiation of primitive mesenchymal cells into skeletal muscle.[15] Myf-3 protein, the human homologue of the murine Myo D1 gene product, is a factor capable of myogenic determination, whereas Myf-4, the human homologue of myogenin, is required downstream as a differentiating factor. [15]This paper reports morphologic, immunohistochemical, and ultrastructural findings of an unusual case of embryonal rhabdomyosarcoma in the right shoulder of a newborn foal.

Case history

A newborn Thoroughbred foal showed a swelling in the right shoulder. This thickening was growing fast and at the age of 3 months a carneous mass of 5 kg was removed surgically by the local veterinarian. A distal dehiscence of the suture appeared and the swelling recurred within the following 3 months.

Clinical findings

When the foal was presented to the veterinary clinic at the age of 6 months, it showed a good general condition and no evidence of lameness. The right shoulder laterally showed a thick firm swelling of 20 cm. (Fig 1). At the caudodistal edge, a 10 cm deep fistula was found and sonographic examination showed multiple cysts within the tissue. Cytologic examination of the cyst fluid revealed numerous neutrophils and some adipocytes. Additional clinical findings revealed mild blood leukocytosis.     

Evaluation of the long-term oral consequences of equine exodontia in 50 horses

The aims of this study were to objectively evaluate and quantify the process of post-extraction cheek teeth (CT) dental drift in horses, and to report on associated disorders of CT wear and long-term periodontal health. Fifty horses that had CT oral extraction because of apical infection were prospectively re-examined and a full oral examination, including measurements of some dental parameters, was performed.Narrowing of the extraction space was noted in all cases with complete closure occurring in 18% of horses. The rate of dental drift was calculated as 15.7% of extraction space/year (range 4–50%) and was not associated with the age at extraction (P = 0.78) or frequency of dental care since extraction (P = 0.48). There was a significant negative relationship between the rate of dental drift and the duration of time since extraction (P = 0.008). Overgrowths were present on the opposite CT row in 98% of horses, including opposite the extracted CT and on the Triadan 06s and 11s. No significant difference was noted in either the number of diastemata (P = 0.9) or periodontal disease score (P = 0.8) between the extraction and the contralateral cheek tooth rows.     

Capability of the nematode-trapping fungus Duddingtonia flagrans to reduce infective larvae of gastrointestinal nematodes in goat feces in the southeastern United States: dose titration and dose time interval studies

Infection with gastrointestinal nematodes, particularly Haemonchus contortus, is a major constraint to goat production in the southeastern United States. Non-anthelmintic control alternatives are needed due to increasing resistance of these nematodes to available anthelmintics. Two studies were completed in Central Georgia in August 1999, and April–May 2000, using Spanish does naturally infected with Haemonchus contortus, Trichostongylus colubriformis, and Cooperia spp. to evaluate effectiveness of nematode-trapping fungi as a biological control agent. In the first experiment, five levels of Duddingtonia flagrans spores were mixed with a complete diet and fed once daily to the does (three per treatment) in metabolism crates. The treatment concentrations were (1) 5×10⁵, (2) 2.5×10⁵, (3) 10⁵, and (4) 5×10⁴ spores per kilogram body weight (BW), and (5) no spores. Fungal spores were fed for the first 7 days of the 14-day trial, and fecal samples were collected daily from individual animals for analysis of fecal egg count and establishment of fecal cultures. Efficacy of the fungus at reducing development of infective larvae (L3) in the fecal cultures was evaluated. The mean reduction in L3 from day 2 of the treatment period until the day after treatment stopped (days 2–8) was 93.6, 80.2, 84.1, and 60.8% for animals given the highest to lowest spore doses, respectively. Within 3–6 days after termination of fungal spore feedings, reduction in L3 development was no longer apparent in any of the treated animals. In a second experiment, effectiveness of 2.5×10⁵ spores of D. flagrans per kilogram BW fed to does every day, every second day, and every third day was evaluated. Reduction in L3 development by daily feeding was less in the second experiment than in the first experiment. Daily fungal spore feeding provided more consistent larval reduction than intermittant feeding (every second or third day). When fed daily under controlled conditions, D. flagrans was effective in significantly reducing development of L3 and appears to be an effective tool for biocontrol of parasitic nematodes in goats.     

Immunohistochemical study of intermediate filaments and neuroendocrine marker expression in leydig cells of laboratory rodents

The aims of this study were to detect the expression of intermediate filaments and to verify the existence of marker substances for neuronal and neuroendocrine cells within the interstitial Leydig cells of laboratory rodent's testes, such as it has been described in other species. Adult male rats, mice, gerbils, Syrian hamsters and guinea-pigs were used and the localization of the different markers was achieved by the streptoavidin-peroxidase immunohistochemical method. The present study demonstrates in all rodents studied a similar pattern of localization in Leydig cells of intermediate filaments (vimentin, cytokeratin, neurofilament 200 kD and glial fibrillary acidic protein) and other marker substances (S-100, CgA, substance P and neurone-specific enolase), which are typical of neuroendocrine (APUD cells or paraneurones) and glial cells. The expression of these substances, related to neurotransmitters or neurohomones and other proteins characteristic of neuroendocrine cells, could suggest that it is a neural crest derived cell. Although this study provides more evidences about the immunoexpression of neuronal and glial markers in Leydig cells, this fact cannot be related directly to their embryological origin, because the current data support the hypothesis of a mesenchymal origin of the Leydig cells.     

Microbiological and serological study of leptospirosis in horses at slaughter: first isolations

A bacteriological survey of kidneys from 145 abattoir horses was performed, which resulted in the isolation of two Leptospira strains. The isolates were serologically typed as belonging to serogroups Australis and Pomona, and REA identified them as L. interrogans serovar Bratislava and L. kirschneri serovar Tsaratsovo, respectively. These are the first Leptospira isolates obtained from horses in Portugal and the Bratislava strain is the first serogroup Australis strain to be isolated in this country. The 145 horses were also serologically tested for leptospiral antibodies, and 37% had MAT titres #10878;1:10.