Compartmentalisation: an example of a national official assurance system.

In South Africa's experience, compartmentalisation has been extremely effective in maintaining an animal subpopulation of a specific health status, based on segregation and biosecurity management. Compartmentalisation promotes animal disease control and underwrites a national official assurance system that provides zoosanitary trade guarantees for animals and animal products. South Africa started to develop the concept of compartmentalisation during the 1950s for the pig sector, due to African swine fever being endemic in wildlife in the north of the country, where certain biosecurity measures were used to protect domestic pigs. Compartmentalisation protocols were formalised and officially published from 2001 as voluntary systems, first for the pig industry and later for the poultry (chicken and ostrich) industry. South Africa uses five basic principles for animal health compartmentalisation, namely, segregation, biosecurity, record-keeping, surveillance and official inspection. These are applied in line with the World Organisation for Animal Health Terrestrial Animal Health Code guidelines. Industry and the farmer bear the costs of compartmentalisation and, in turn, obtain not only animal disease protection but also trade advantages due to health assurances. Compartmentalisation ensures optimal use of government resources as the expenses for animal disease control are shared with private industries. International acceptance and standardisation of compartments is advocated as a measure to provide assurances for disease freedom, based on risk-specific mitigation measures.

D'après l'expérience de l'Afrique du Sud en matière de compartimentation, celleci s'est révélée extrêmement efficace pour préserver le statut sanitaire de souspopulations animales données grâce à l'application de mesures de ségrégation et de gestion de la biosécurité. La compartimentation favorise le contrôle des maladies animales et participe à un système officiel national d'assurance visant à apporter des garanties sanitaires aux échanges d'animaux et de produits d'origine animale. L'Afrique du Sud a commencé à expérimenter le concept de compartimentation dans les années 1950 en l'appliquant au secteur porcin qui se trouvait menacé par la peste porcine africaine présente à l'état endémique dans la faune sauvage des régions du nord du pays, à travers des mesures de biosécurité spécifiques mises en place pour protéger les porcs domestiques. Des protocoles de compartimentation ont été officiellement adoptés et publiés à partir de 2001 sous forme de systèmes basés sur le volontariat, destinés en premier lieu au secteur porcin puis au secteur avicole (élevages de poulets et d'autruches). La compartimentation appliquée en Afrique du Sud fait appel à cinq principes fondamentaux, à savoir la ségrégation, la biosécurité, la tenue de registres d'élevage, la surveillance et l'inspection officielle. Ces principes sont mis en oeuvre conformément aux lignes directrices du Code sanitaire pour les animaux terrestres de l'Organisation mondiale de la santé animale. Les coûts de la compartimentation sont pris en charge par le secteur privé et les éleveurs, qui à leur tour en retirent non seulement une protection contre les maladies animales mais aussi des avantages compétitifs grâce aux garanties sanitaires qu'ils sont en mesure d'apporter à leurs partenaires commerciaux. La compartimentation garantit une utilisation optimale des ressources publiques car les dépenses liées au contrôle des maladies animales sont partagées avec le secteur privé. Les auteurs plaident en faveur d'une reconnaissance et normalisation internationales des compartiments en tant que méthode permettant de garantir le statut indemne de maladie sur la base de mesures spécifiques d'atténuation des risques.

Su propia experiencia lleva a Sudáfrica a concluir que la compartimentación ha resultado sumamente eficaz para preservar el estado sanitario de determinadas subpoblaciones animales con medidas de segregación y seguridad biológica. La compartimentación facilita el control de enfermedades animales y trae aparejado un sistema nacional de garantía oficial por el cual los animales y productos de origen animal destinados al comercio se acompañan de una garantía zoosanitaria. Sudáfrica empezó a desarrollar el concepto de compartimentación en el decenio de 1950 para aplicarlo al sector porcino, dado que la peste porcina africana era endémica en la fauna salvaje del norte del país, donde se aplicaron ciertas medidas de seguridad biológica para proteger a los cerdos domésticos. En 2001 se formalizaron y publicaron oficialmente protocolos de compartimentación con carácter de sistema voluntario, primero para el sector porcino y algo después para el de producción avícola (pollos y avestruces). Sudáfrica aplica cinco principios básicos para la compartimentación zoosanitaria, a saber, segregación, seguridad biológica, mantenimiento de registros, vigilancia e inspección oficial, siguiendo las pautas marcadas por la Organización Mundial de Sanidad Animal en su Código Sanitario para los Animales Terrestres. Los costos de la compartimentación recaen en industriales y productores, quienes a cambio salen ganando no solo con la protección contra enfermedades animales, sino también con las ventajas comerciales que les procuran las garantías sanitarias. La compartimentación asegura un uso óptimo de los recursos del Gobierno, toda vez que este comparte con el sector privado los gastos de la lucha zoosanitaria. Los autores proponen que a escala internacional se acepte y se estandarice el uso de compartimentos como medida de garantía de la ausencia de enfermedades, partiendo para ello de medidas de mitigación adaptadas específicamente a cada tipo de riesgo.

First comprehensive low-density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families.

Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F(2) generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F(2)s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated from each embryo and from blood samples of the parents and grandparents. The families have been genotyped with 353 equine microsatellites and 6 biallelic markers, and 42 linkage groups were formed. In addition, the physical location of 85 of the markers is known, and this has allowed 37 linkage groups to be anchored to the physical map. The inclusion of dams in the genotyping analysis has allowed the generation of a genetic map of the X chromosome. Markers have been assigned to all 31 autosomes and the X chromosome. The average interval between markers on the map is 10.5 cM, and the linkage groups collectively span 1780 cM. The results demonstrate the benefits for horse linkage mapping studies of genotyping on these unique full-sibling families, which comprise relatively few individuals, by the generation of a comprehensive low-density map of the horse genome.

Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences.

A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 microl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at -20 degrees C.

Development of equine endometrial glands from fetal life to ovarian cyclicity.

Branched tubular endometrial glands are key features of the uterine lining in mares. The epithelium in the neck regions of the glands is similar to that lining the lumen of the uterus, whereas the epithelium in the deeper, coiled portions of the glands secretes protein rich histotrophe or 'uterine milk' during dioestrus and pregnancy. The present study was performed to characterize endometrial histology in perinatal and peripubertal fillies and to investigate the influences of specific steroid treatments on endometrial gland development in the prepubertal period. Uteri obtained from equine fetuses (n=5) between days 150 and 320 of gestation, and from foals (n=5) during the first 2 months of life, were compared histologically with endometrial biopsies taken from yearling fillies (n = 8) around the onset of ovarian cyclicity. In addition, biopsies were obtained from two prepubertal fillies that were treated for sequential periods with injections of progesterone or oestradiol benzoate each day to mimic the peripheral hormone changes associated with puberty. At day 250 of gestation the luminal epithelium was starting to invaginate into the underlying stroma and, by day 300 of gestation, the first cross-sections of very simple tubular gland structures were visible. These tubular gland structures developed into clusters of rudimentary gland cross-sections during the early postnatal period but, thereafter, gland development was arrested until the epithelial invaginations became more obvious during the first oestrus at the onset of puberty. However, final differentiation and maturation of the secretory portions of the glands occurred only after the first ovulation. Furthermore, in prepubertal fillies this maturation process could not be elicited by treatment with progesterone only and an initial period of oestrogen priming was essential to induce full differentiation of the endometrial glands.

Immunolocalization of a novel protein (P19) in the endometrium of fertile and subfertile mares.

One of the major progesterone-dependent endometrial proteins in the mare is a novel 19 kDa lipocalin (P19). This protein is secreted by the endometrial glands and is readily detectable in uterine secretions during the luteal phase of the oestrous cycle and early pregnancy. The function of P19 is unknown, but since most lipocalins act as carriers of small hydrophobic molecules, it probably transports a maternal factor to the conceptus during pregnancy. In this study, a high titre antiserum raised against recombinant-derived P19 was used to detect by immunohistochemistry the protein in endometrial biopsies from normal healthy mares and mares with endometrosis. Immunoreactive P19 was undetectable in prepubertal fillies and in anoestrous mares and was barely detectable in oestrous mares. However, it was present in large amounts during dioestrus and very early pregnancy, and in moderate amounts after day 20 of gestation. In six subfertile mares with endometrosis, an abnormal pattern was found at all stages of the reproductive cycle, especially in the 'gland nests'. These showed either an absence of P19 or an accumulation of the protein due to a lack of secretion, thus indicating asynchronous glandular activity. These results identified a high proportion of abnormal, asynchronous endometrial glands in a group of subfertile mares and indicated that abnormal secretion of P19 may be one of the reasons for the reduced fertility rates observed in aged mares with endometrosis.

Cell proliferation patterns in the equine endometrium throughout the non-pregnant reproductive cycle.

Immunohistochemical detection of the proliferation marker Ki-67 antigen was used to monitor mitotic activity in the endometrium of mares. The monoclonal antibody MIB1 was validated for use on equine tissues by demonstrating its reaction with activated peripheral blood lymphocytes, and endometrial biopsies were recovered from 26 non-pregnant mares at selected stages during the reproductive cycle. The proportion of positively stained nuclei was counted in five random areas on each histological section to determine the percentage and type of proliferating cells. Multiplication rates in the types of cell found in the superficial strata, comprising the luminal epithelium, the epithelium of the gland necks and the stromal cells of the stratum compactum, were greatest during oestrus, presumably under the influence of oestrogens secreted by the growing ovarian follicles. In contrast, the mitotic activity in the cells of the deeper secretory portions of the endometrial glands was restricted to a brief phase between day 3 and day 7 of dioestrus, most likely as a delayed response to the decreasing oestrogen concentrations after ovulation. Some of the degenerate glands in subfertile mares did not follow this pattern of increased epithelial proliferation at that stage. After day 7 of dioestrus, the proliferation rates of cells in the endometrium decreased to basal values and remained low for as long as progesterone concentrations remained evaluated, even during prolonged dioestrus. The technique enabled characterization of normal cell proliferation patterns in the endometrium of mares and it will be a useful tool in the future for monitoring the endometrial responses of reproductively healthy and subfertile mares.

Factors controlling epidermal growth factor (EGF) gene expression in the endometrium of the mare.

Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non-pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial pathology (endometriosis) or because of incompatible extraspecific embryo transfers (donkey-in-horse pregnancies). Only a few samples showed weak EGF expression during the late luteal phase of the oestrous cycle. During normal pregnancy, the previously observed dramatic increase of expression after day 40 of gestation was confirmed. Although aged mares suffering from endometriosis and mares carrying an extraspecific donkey conceptus showed the same increase of EGF mRNA in normal glands, this was virtually absent from gland cross-sections compromised due to inflammatory or fibrotic changes. Administration of various doses and combinations of progesterone and oestrogen for < 35 days yielded negative or only weakly positive hybridization results, whereas progesterone alone for > or = 40 days upregulated EGF expression strongly irrespective of additional treatment with oestrogen. This is the first experimental evidence that EGF expression in the endometrium can be induced by progesterone alone. The requirement for prolonged progesterone priming is of considerable interest in the context of the unusually late stage of gestation at which placental attachment commences in equids.

Cell proliferation patterns during development of the equine placenta.

Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.

Expression of epidermal growth factor and its receptor in equine placental tissues.

Northern blot and in situ hybridization techniques have demonstrated a marked increase in mRNA encoding epidermal growth factor (EGF) in the endometrium of mares, coincident with the start of interdigitation between the allantochorion and endometrium during placentation. In the present study, the unusually high EGF expression in the epithelium of the endometrial glands was shown to be maintained until at least day 250 of gestation (term = 320-340 days) in mares carrying normal horse conceptuses. However, in mares carrying failing donkey-in-horse pregnancies created by embryo transfer, EGF expression was severely retarded in those areas of the endometrium that were heavily infiltrated with lymphocytes and which showed a failure of placental development. Specific receptors for EGF were also detected in tissue homogenates from pregnant mares using 125I-labelled human EGF. Binding was high in the fetal membranes (allantochorion), both before implantation (days 30-34) and in the fully developed placenta (days 150-250), and was equivalent to the level of binding to homogenates of adult liver and kidney. Binding was much reduced in endometrial homogenates before implantation and from non-pregnant mares but increased after implantation to reach values equivalent to those exhibited by the fetal membranes. Scatchard analysis of displacement curves indicated a single class of high-affinity binding sites in the fetal membranes and pregnant endometrium sampled at day 150 of pregnancy and chemical cross-linking of the receptor-125I-labelled EGF complexes in fetal membranes revealed two radiolabelled bands of 170 kDa and 150 kDa. A large excess of insulin-like growth factor I (IGF-I) failed to displace any labelled EGF from the tissue homogenates. The marked and sustained upregulation of endometrial EGF expression during pregnancy in mares, and the presence of EGF receptors in the fetal allantochorion and maternal endometrium, suggest a possible role for EGF in the marked growth of these two tissues during placentation in equids.

Semen quality after thawing: correlation with fertility and fresh semen quality in dogs.

Fifty-four semen samples from five dogs were evaluated both, fresh and after thawing. Some of these semen samples were mixed with autologous prostatic fluid after thawing and used to inseminate each of nine bitches 4-7 times intravaginally. All bitches conceived and the mean number (+/- SD) of conceptuses, number of corpora lutea and ratio between conceptuses and corpora lutea (implantation rate) were 5.7 +/- 2.8, 9.4 +/- 1.1 and 0.63 +/- 0.34, respectively. The mean incidence of normal sperm morphology and progressively motile spermatozoa for all semen samples were 71.5 +/- 13.5% and 74.4 +/- 7.1%, respectively, in fresh semen and 52.0 +/- 18.5% and 53.4 +/- 12.6% in frozen-thawed semen. Extension rate was 1:3 for all semen samples and the mean sperm concentration after thawing was 12.08 +/- 6.66 x 10(7) ml-1. The only semen quality variables after thawing that were correlated with implantation rate were the number of spermatozoa inseminated on day-2 and number of progressively motile spermatozoa inseminated on day-2 (where day 0 is the day of onset of dioestrus as determined by cytology) (Spearman's rank correlation coefficient > 0.7, n = 9, P < 0.05). This study suggests that it is essential that frozen-thawed semen is inseminated on day-2 and that an insemination dose of 10-11 x 10(7) progressively motile frozen-thawed spermatozoa is adequate to achieve a mean implantation rate of 75% or higher. The incidence of either proximal or distal cytoplasmic droplets in fresh semen was negatively correlated with motility after thawing in three of five dogs (Spearman's rank correlation < -0.5, n = 6-17, P < 0.05). Neither the percentage spermatozoa with normal morphology in fresh semen nor the percentage progressively motile spermatozoa in fresh semen nor the concentration of spermatozoa after thawing were correlated with motility after thawing. Fresh semen quality, with the exception of the incidence of retained cytoplasmic droplets, has little value in predicting the progressive motility after thawing in frozen dog semen.

The effects of metergoline combined with PGF2alpha treatment on luteal function and gestation in pregnant bitches.

This study was designed to determine the nature of the antiluteotrophic effect of metergoline on pregnant bitches, the occurrence of clinically evident side-effects, and the efficacy of PGF(2alpha) in initiating abortion after a course of metergoline therapy. Starting on Days 18 to 20 after the onset of diestrus, 8 adult pregnant beagle bitches were treated twice daily with 0.4 to 0.5 mg/kg po metergoline for 5 d. After receiving no treatment for the next 5 d, metergoline administration was repeated for a further 3 d, followed by twice-daily intramuscular injections of dinoprost tromethamine at 250 mug/kg. There was an overall trend for the plasma progesterone concentration to decrease during the first and second course of metergoline therapy and to rise during the intervening period of no treatment. None of the bitches aborted during or after the first 5 d of metergoline administration. No side-effects were evident during the metergoline therapy. After pretreatment with metergoline a mean of 4.8 injections of PGF(2alpha) was necessary to ensure complete abortion. All abortions were rapid and uneventful, except for the usual side-effects associated with PGF(2alpha). The plasma progesterone concentration at the onset of PGF(2alpha) treatment was positively correlated to the number of PGF(2alpha) injections needed to complete the abortion process. The plasma progesterone concentration was < 8 nmol/l for several days as a result of metergoline therapy in 6 of the 8 bitches. Only 1 bitch, however, aborted before PGF(2alpha) therapy was initiated. In the other 7 bitches PGF(2alpha) appeared to be necessary for abortion. The results suggest that the effect of metergoline has to be considered incompletely luteolytic at the doses used in this study. Even prolonged suppression of luteal function in early to mid-gestation, however, did not cause abortion without the previously documented luteolytic and/or ecbolic effects of PGF(2alpha). The average interestrus interval of cycles treated with metergoline and PGF(2alpha) was shorter (mean 182.2 d, SD 7.9 d, n = 6) than expected for this group of bitches (mean 211.4 d, SD 31.5 d, n = 7).

Success with intravaginal insemination of frozen-thawed dog semen--a retrospective study.

This retrospective study reports on the fertility of bitches (n = 40) that were inseminated into the fornix vaginae with frozen-thawed sperm from 9 sperm donors. In most bitches, the inseminations were repeated daily although, in 5 bitches, 1-2 d were skipped between some inseminations. All semen had been frozen by the same method and all prostatic fluid had been frozen at -18 degrees C prior to use. Sixteen Beagle bitches (Group 1) were inseminated with frozen-thawed semen to which 3-5 ml prostatic fluid had been added post-thaw. Ten German Shepherds and 4 other bitches of similar size (Group 2) were inseminated after adding 6-10 ml prostatic fluid to the frozen-thawed semen. Ten different German Shepherd bitches (Group 3) were inseminated without using prostatic fluid. The volumes of individual insemination doses were 3.5-6 ml, 7-12 ml and 1-3 ml for Groups 1, 2 and 3 respectively. The number of progressively motile sperm per insemination varied from 9 x 10(6) to 300 x 10(6). Sixteen of 16, 13 of 14 and 6 of 10 bitches conceived in Groups 1, 2 and 3 respectively. Mean litter sizes were 4; 5.6 and 4 in Groups 1, 2 and 3 respectively. The pregnancy rate of Groups 1 and 2 combined was higher than that of Group 3 (P < 0.01). The mean number of foetuses per bitch bred was 5.2 (SD 3.2) for Group 2, which was higher than the 2.4 (SD 2.8) of Group 3 (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)