P‐33 Poxvirus infection in two cats

A 3‐year‐old Burmese cat was presented with a history of nonresolving crusted and papular lesions of the face and prior treatment with prednisolone. Skin biopsies revealed typical pox lesions with hyperplasia and ulceration of the epidermis and eosinophilic cytoplasmic inclusion bodies in the epidermal cells. In the upper dermis there was prominent diffuse mast cell infiltration and mild neutrophilic and eosinophilic inflammation. Rare cytoplasmic inclusion bodies were also present in swollen endothelial cells of dermal venules, which showed no other degenerative changes. Histological diagnosis was confirmed by electron microscopic evidence of pox virus particles in inclusion bodies of epidermal cells. The lesions resolved within 6 weeks with systemic antibiotic therapy and supportive care. A 2‐year‐old domestic short‐haired cat was presented with multiple disseminated papular and ulcerative pox lesions with central eschar over the entire body. Histologically, large epidermal inclusion bodies (up to 6 μm in diameter) were present. Widespread haemorrhage and vascular wall necrosis was visible in the dermis and subcutis. Some subcutaneous vessels showed neutrophilic vasculitis. In addition to diffuse dermal neutrophilic and eosinophilic inflammation, a lymphohistiocytic panniculitis was also present. The cat died as a result of massive haemorrhage and lymphedema, despite supportive care. Funding: Self‐funded.     

Prevalence of Dirofilaria immitis and Dipetalonema reconditum in greyhounds

Blood samples from 331 greyhounds in the Hunter Valley and nearby coastal areas of New South Wales were examined for microfilariae using a filtration technique. Species were identified by histochemical staining; 10.9% of the greyhounds were infected with Dirofilaria immitis and 3.6% with Dipetalonema reconditum. The prevalence of infection of both species was significantly greater in summer than in winter (p = less than 0.05). Infection with D. immitis was correlated with differences in age, sex, bodyweight and coat colour, and a reported lack of stamina and the presence of a cough. No significant association was found. Diethylcarbamazine citrate was used for prophylaxis in 8.8% of all the greyhounds examined.     

Effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells.

Uptake, transfer to rough endoplasmic reticulum, and intracellular growth of Brucella abortus were studied in Vero cells treated with endocytic and metabolic inhibitors. Infection of Vero cells was suppressed when inhibitors of energy metabolism (iodoacetate, dinitrophenol), receptor-mediated endocytosis (monodansylcadaverine, amantadine, methylamine), or endosomal acidification (chloroquine, ammonium chloride, monensin) were added to the inoculum. Inhibition was not observed when these drugs were added after the inoculation period. Infection of Vero cells by B abortus was inhibited by dibutyryl-cyclic adenosine monophosphate and Vibrio cholerae enterotoxin, but was stimulated by dibutyryl-cyclic guanosine monophosphate and escherichia coli heat-stable enterotoxin a. Uptake of B abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B abortus and noninvasive E coli was similar to that of viable brucellae. Intracellular growth of B abortus was not affected by cycloheximide. Results indicate that: B abortus may be internalized by a receptor-mediated phagocytic process; transfer of B abortus from phagosomes to rough endoplasmic reticulum may require endosomal acidification; and replication of B abortus within the rough endoplasmic reticulum may not depend on protein synthesis by the host cell.     

Confirmation of rinderpest in experimentally and naturally infected cattle using microtitre techniques

Summary Virulent rinderpest virus was detected by immunoperoxidase staining of microtitre bovine kidney cell cultures within 24 to 48 hours of inoculation with prescapular lymph node and spleen homogenates from experimentally infected steers. Rinderpest virus specific cytopathic effects were evident from 48 hours in microtitre plates and from 72 hours in rolled tube cultures. Nasal and ocular secretions collected from cattle naturally infected with rinderpest and inoculated into bovine kidney cell cultures did not readily yield cytopathic virus in both tubes and microtitre plates, but immunoperoxidase staining of microtitre cultures on the fourth day of inoculation detected replication of virus in cultures inoculated with ocular and nasal secretions from seven of 17 cattle tested.

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Resumen Se detectó el virus virulente de mediante la tinción con inmunoperoxidasa de cultivos de células de ri?ón bovino en bandejas de microtitulación, después de la inoculación de estos con suspensiones homogenizadas de ganglios linfáticos preescapulares y de bazo provenientes de novillos infectados experimentalmente. El efecto citopático del virus de la peste bovina fue evidente desde las 48 horas en bandejas de microtitulación y desde las 72 horas en tubos de cultivo giratorios. Secreciones oculares y nasales colectadas de ganado infectado en forma natural con la peste bovina e inoculadas en cultivos de células de ri?ón bovino, no mostraron efecto citopático fácilmente en tubos giratorios o bandejas de microtitulación, pero la tinción de las bandejas con inmunoperoxidasa reveló replicación del virus a partir del cuarto día de inoculación con secreciones oculares y nasales en siete de los 17 animales examinados.

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Résumé Un virus bovipestique virulent a été décelé par le test de coloration à l'immunoperoxydase de cellules rénales bovines en culture dans des plaques de microtitrage et infectées 48 heures plus t?t avec des homogénats de ganglions lymphatiques et de rate provenant de bouvillons infectés expérimentalement. Les effets cytopathogènes du virus étaient évidents au bout de 48 h dans les plaques de microtitrage et 72 h dans les tubes en rollers. Les sécrétions nasales et oculaires prélevées sur du bétail infecté naturellement par la peste bovine et inoculées sur des cellules rénales bovines n'ont pas toujours montré d'effet cythopathogène aussi bien dans les tubes que dans les plaques de microtitrage. Cependant, la coloration à la peroxydase au jour 4 après l'inoculation a permis de déceler la présence de virus dans 7 cas sur 17.

     

Deep Freezing of Boar Semen Packaged in Plastic Bags and Straws

For practical reasons, a large volume (i.e. 5 ml) of frozen boar semen per insemination dose is desirable, but successful freezing has not been achieved, since optimal cooling rates have not yet been established. Post-thaw motility and the acrosome intep'ty of semen from four boars frozen with a programmable freezin machine, in mini-(0.25 ml), maxi-(5 ml) plastic straws and in 10 × 5 cm PVC- or Teflon FEP-plastic bags (0.35 – 0.12 mm thick, 5 ml) was studied. The freezing of the semen was monitored using thermocouples placed in the straws and the bags. The freezing curve started from +5°C, at a rate of −3°C/min, to – 6°C, it was held for 1 min at −6°C, and was followed by further drop to −100°C at a rate of −20°C/min, with subsequent storage in LN₂. The bags had a much shorter freezing point plnteau, compared to the maxi-straws. Post-thaw sperm motility was significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. The freezing procedure did not cause major acrosomal damages, significantly more normal apical ridges being present in the bags and mini-straws than in the maxi-straws. This in vitro evaluation indicates that the freezing method employed is satisfactory for freezing large volumes of boar semen into plastic bags .     

Distributions of extracellular matrix and carbonic anhydrase-III during bovine palatine ridge development.

The present study describes histological alterations and immunohistochemical distributions of extracellular matrices (ECMs) and the carbonic anhydrase isozyme-III (CA-III) during the period of bovine palatine ridge formation. Morphogenesis of bovine palatine ridges was preceded by epidermal placodes and the mesenchymal condensation (MC). During the early stages of less than 44 cm crown rump length (CRL), fibronectin (FN) was distributed densely in the MC. Strong reactions against type I collagen (C-1) were detected outer to the FN positive site. In the stages of more than 44 cm CRL, FN and C-1 were distributed diffusely in subepithelial mesenchyme. Laminin (LN) and type IV collagen were distributed in the epithelial and endothelial basement membranes (BMs) in all of the stages examined, except in the stage of 7 cm CRL, where LN was not detected only in the BM just beneath the epidermal placode. CA-III was detected in basal epithelial cells except for palatine ridge rudiments in the stages of more than 21 cm CRL. It is suggested that the expressions of LN and CA-III might play a role in the spatial determination of rudiments of bovine fetal palatine ridges.     

Effect of short-term calf removal at three stages of a follicular wave on fate of a dominant follicle in postpartum beef cows.

The hypothesis that ovulation in response to short-term (48 h) calf removal (CR) is dependent on the developmental stage of the dominant follicle was tested in two studies. The objective of Exp. 1 was to characterize the fate of a dominant follicle following 48-h CR on d 2, 4, or 8 of a postpartum follicular wave. Ovaries of 61 beef cows were examined daily by transrectal ultrasonography starting at d 20 to 21 postpartum. Treatments were no CR (n = 14) and CR on d 2 (n = 12), 4 (n = 16), or 8 (n = 10) of first detected follicular wave. Percentage of cows that ovulated a dominant follicle following treatment was not different among groups (P = 0.62). Maximum size of dominant follicles was larger in cows that ovulated (P = 0.002) than in cows that did not ovulate. The objectives of Exp. 2 were 1) to determine whether a follicular wave could be synchronized in anestrous cows following injection of 1 mg of estradiol benzoate (EB) and 200 mg of progesterone (P4; EB + P4); 2) to characterize the fate of dominant follicles following 48-h CR at three stages of a synchronized follicular wave; and 3) to determine whether estrous cycles of normal length followed ovulation in cows pretreated with EB + P4. Ovaries of 50 anestrous beef cows were examined daily as in Exp. 1. Treatments were sesame oil (SO) injected (i.m.) on d 25 postpartum and no CR (n = 9); EB + P4 and no CR (n = 9); EB + P4 and CR on 6 (n = 12), 8 (n = 9), or 12 (n = 11) d after injection. The EB and P4 injections were given on d 25 postpartum. Variability in day of emergence of subsequent follicular waves was lower in cows receiving EB + P4 than in SO-injected cows (P < 0.05). The percentage of cows that ovulated was not different (P = 0.16), but CR increased the percentage of cows that ovulated when groups that received EB + P4 were compared to the EB + P4 group that did not have CR (53.1 vs 11.1%, respectively; P < 0.05). Maximum diameter of dominant follicles was larger (P = 0.05) in ovulatory follicles. The luteal phase was longer (P < 0.03) in cows receiving EB + P4 injection (10.6 +/- 1.2 d) than in cows receiving SO (4.4 +/- 2.2 d). In summary, the maximum size of ovulatory follicles was greater than that of nonovulatory follicles, the ovulatory response of postpartum anestrous cows was maintained through d 8 of a follicular wave, synchronization of follicular waves was accomplished in postpartum cows using EB + P4, and the subsequent luteal phase length was increased in animals that were administered EB + P4.     

A novel HPLC method for glutathione-insulin transhydrogenase assay using fluorescence labeled insulin

The activity of rat liver glutathione-insulin transhydrogenase (GIT) was measured by HPLC. The degradation of fluorescein isothiocyanate-I (FITC-I)-labeled insulin is separated into several peaks, which are bound different amount of FITC-I. We selected mono-fluorescein-thiocarbamylated insulin to estimate the decrease of insulin content and it became possible to assay GIT activity. This novel method was time-saving and simple, and this system could utilize instead of previous method.     

Changes in the concentration of mRNAs for the inhibin subunits in ovarian follicles after administration of gonadotropins to progestin treated ewes.

RNA was extracted from single or small groups of ovine ovarian follicles after treatment of ewes with FSH and/or LH. The content of mRNA for the alpha-inhibin and beta A-inhibin subunits was analyzed by hybridization with specific cDNA probes. All ewes were treated with progestin vaginal pessaries to suppress spontaneous preovulatory follicle maturation and ewes were given three intramuscular injections of gonadotropins at 8-hr intervals starting 24 hr prior to collection of ovaries. In experiment I, both Schering-FSH and NIDDK-oFSH-17 (oFSH) significantly increased alpha- and beta A-inhibin mRNA per ewe in 2-5 mm follicles and tended to increase alpha- and beta A-inhibin mRNA in large (greater than 5 mm) follicles. In experiment II, oFSH and NIDDK-oLH-25 (oLH) were administered in a 2X2 factorial arrangement. Separate administration of oFSH or oLH increased (P less than .05) the alpha-inhibin mRNA concentration in large follicles. alpha-inhibin mRNA concentration in 4-5 mm follicles was also increased by oFSH but was decreased by oLH. Concomitant treatment with oFSH and oLH did not change alpha-inhibin mRNA concentrations from those measured in oFSH treated ewes. In experiment II, beta A mRNA concentrations followed a pattern similar to that of alpha A mRNA, but the differences were not statistically significant. We conclude that, in the ewe, exogenous FSH increases the concentration of inhibin mRNA in the whole follicle. The ability of exogenous oLH to alter expression of the inhibin subunit genes may depend upon the stage of follicle maturation.     

In situ hybridization analysis of ovarian prostaglandin endoperoxide synthase mRNA throughout the periovulatory period of the ewe.

Cellular alterations in level of expression of mRNA encoding for prostaglandin endoperoxide synthase were quantified within ovarian tissues of sheep obtained before, during and after induction of the preovulatory surge of LH and ovulation with LHRH. This was accomplished by isotopic in situ hybridization using a selective cRNA probe to ovine prostaglandin endoperoxide synthase mRNA. A significant elevation in mRNA was detected within the theca interna of the preovulatory follicle at 8, 16 and 24 hr following administration of LHRH. Very close to the time of ovulation (ie., at 24 hr post-LHRH) a marked rise in mRNA was observed in association with epithelial cells covering the apical surface of the follicle. Ovarian cyclooxygenase metabolites of arachidonic acid produced during the ovulatory process in the ewe originate within the thecal layer and germinal epithelium of the follicle destined to ovulate.