Association between Degree of Anaplasia and Degree of Inflammation with the Expression of COX-2 in Feline Injection Site Sarcomas.

Feline injection site sarcomas (FISSs) are mesenchymal neoplasms that develop at the sites of delivery of vaccines or other injectable products. Vaccine adjuvants can trigger an intense and persistent inflammatory response that may lead to neoplastic transformation. The proinflammatory role of cyclo-oxygenase (COX)-2 is well known and its overexpression has prognostic value in multiple neoplastic processes. One hundred and seventeen FISSs were evaluated for the degree of inflammation and anaplasia. Immunohistochemistry was used to determine the expression of COX-2 in these sarcomas. There was a significant association between the degree of inflammation and the expression of COX-2 by neoplastic cells. COX-2 expression was lower in tumours with higher degrees of anaplasia. These findings may be useful in predicting the sensitivity of FISSs to treatment with COX-2 inhibitors. The potential therapeutic use of such agents could then be restricted to tumours with lower degrees of anaplasia.

Clinico-pathological findings in natural cases of "mascadera" in goats.

"Mascadera" is a chronic emaciating neuropathy affecting goats; it produces significant economic losses in many regions and its cause is unknown. Here, the histological lesions found in 15 animals naturally affected by the disease are described. Complete necropsy was performed and tissue samples were collected for histopathological study. Severe atrophy of the masseter and buccinator muscles and tongue was observed, as well as vacuolar degeneration of neurons in the nuclei of the trigeminal, facial, and glossopharyngeal nerves. No relevant lesions were observed in other tissues. These findings and the clinical signs are consistent with those observed by other authors in animals spontaneously and experimentally intoxicated with Prosopis juliflora. The disease may be due to consumption of a similar species present in our country that is still unknown. Further research on the etiology and pathogenesis of this disease is needed to establish appropriate prevention guidelines.

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Spontaneous photosensitization by Heterophyllaea pustulata Hook. f. (Rubiaceae), in sheep from Northwestern Argentina.

Heterophyllaea pustulata Hook. f. (Rubiaceae) is a phototoxic plant. It grows in the Andean area of northwest of Argentina, and it causes significant economic losses in the livestock. This plant induces dermal lesions by photosensitization probably due to its content of photosensitizing anthraquinones. This paper describes an outbreak of poisoning in Corriedale sheepfold, which had an incidence of 49%. Ear skin biopsies and blood samples were collected of six affected animals. Liver enzymes remained within the reference limits. Histopathologically, a deep necrotizing dermatitis was identified in all samples. H. pustulata was identified in the areas of grazing. Anthraquinone concentration in leaves was 0.84% p/p, expressed as rubiadin. All findings allow us to conclude that the diagnosis is a primary photosensitization. Huge regional economic losses could be attributed to H. pustulata poisoning, although its toxicity has been little studied.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

Efficacy and productive performance of moxidectin in feedlot calves infected with nematodes resistant to ivermectin.

Anthelmintic resistance (AR) of gastrointestinal nematodes to macrocyclic lactones is an increasingly common worldwide phenomenon limiting cattle production. This has motivated the search for alternatives, such as new active compounds, added drug synergisms, different doses, and alternate administration routes. The aim of this study was the assessment of moxidectin (MXD) performance in feedlot calves with a history of AR to ivermectin (IVM). Crossbred female calves aged 6-7 months and weighing 163kg (SD=34kg) were divided into 3 groups of 35 animals each. They were assigned to the following antiparasitic treatment groups: IVM group (0.2mg/kg IVM); MXD group (0.2mg/kg MXD), and ricobendazole+levamisole (RBZ+LEV) group (7.5mg/kg RBZ+8mg/kg LEV). On days 0, 26, and 47, fecal samples were taken and the weight of each animal was registered. Anthelmintic efficacy (by fecal egg count reduction), total weight gain (TWG) and average daily weight gain (AWG) were compared between the groups. A mixed SAS procedure was used for statistical analysis. Fecal egg count reduction 26 days post-treatment (PT) was calculated at 28% for the IVM group, 85% for the MXD group, and 99% for the RBZ+LEV group. AWGs (Standard Error) of 1.095g (56), 1.264g (49), and 1.340g (52) were registered for the IVM, MXD, and RBZ+LEV groups, respectively (p<0.05). Coprocultures revealed that MXD more effectively reduced Haemonchus spp. and Cooperia spp. egg counts than IVM. This resulted in higher AWGs and TWGs for this group; similar results were seen for the RBZ+LEV group as well. In this study, animals treated with MXD gained about 160 more g/day than animals treated with IVM. This represents a gain of 16 USD per animal over the 47 day trial.

Immunohistochemical expression of growth factors in the follicular wall of normal and cystic ovaries of sows.

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF-I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF-I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF-II was very similar to pattern noted in IGF-I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF-II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.

The effect of anthelmintic resistance on the productivity in feedlot cattle.

The aim of this study was to evaluate, in a commercial feedlot, the effect of different anthelmintic drugs on the productivity of naturally infected calves from a cow-calf operation, where resistance to ivermectin (IVM) has been previously detected. The study began with the random selection of 80 calves whose weight was 132 ± 12 kg. Four groups were made: IVM, ricobendazole (RBZ), ricobendazole plus levamisol (RBZ + LEV) and a control group (CG) without treatment. On days 0, 21, 42, 70, 98 and 126, manual collection of fecal matter and individual weight were registered. Mixed SAS procedure was used for statistical analysis. The percentages of fecal egg count reduction test (FECRT) calculated 21 days post treatment (PT) were 18%, 96% and 100% for the IVM, RBZ and RBZ + LEV groups, respectively. Body weight (± SEM) at the end of the trial was 266 kg (± 0.9), 269 kg (± 1.1), 276 kg (± 1.3), 280 kg (± 1.9) for CG, IVM, RBZ and RBZ + LEV groups, respectively. The effect on live weight was highly significant (p < 0001). After 126 days of fattening, the deleterious effect of the combination of Cooperia and Haemonchus in the IVM group on body weight was evident. Undetected animals carrying anthelmintic resistant (AR) worms entering the feedlot, could cause major productivity losses.

Protective effects of intranasal immunization with recombinant glycoprotein d in pregnant BALB/c mice challenged with different strains of equine herpesvirus 1.

Equine herpesvirus (EHV)-1 induces respiratory infection, neurological disorders and abortion in horses. Most of the currently available attenuated or inactivated vaccines against this infection are administered intramuscularly and only provide partial protection against the respiratory disease. The present study examines the effect of intranasal immunization with purified EHV-1 recombinant glycoprotein D (gD) in BALB/c mice followed by challenge with three different EHV-1 strains during early to mid-pregnancy. The induced viral infection was evaluated by virus isolation, DNA detection by polymerase chain reaction, histopathology and immunohistochemical localization of antigen in the lung, placenta and uterus. Non-immunized mice showed clinical signs of infection, positive virus isolation from lungs and uteri, and abortion induced by one of the virus strains. Endometrial lesions developed in some of these animals that have been described previously only in horses. Immunized mice and their offspring had no viral infection or typical lesions. Intranasally administered gD therefore induced partial or complete protection against three different EHV-1 strains in BALB/c mice.

Intermediate filament proteins expression and carbohydrate moieties in trophoblast and decidual cells of mature cat placenta.

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Lectin-binding pattern in ovarian structures of rats with experimental polycystic ovaries.

Numerous experimental models in different species have been developed for the study of polycystic ovarian syndrome. In this study, we used a model of induction of polycystic ovaries (PO) in rats by exposure to constant light to study the distribution and variations of glycosylated residues present in the different ovarian structures. Seven biotinylated lectins were used (Con-A, WGA, DBA, SBA, PNA, RCA and UEA-I) on tissue sections, and detection was performed using the streptavidin/peroxidase method. In tissue sections was observed an increase in affinity for Con-A in the granulosa and theca interna of growing follicles and cysts in animals with PO in relation to the control group. Follicular cysts showed higher affinity for WGA and RCA-I which growing follicles in the same group, and there was a decrease in affinity for PNA in the cysts in relation to the growth of follicles in both groups. Atretic follicles in both groups showed greater labelling with lectins PNA, SBA and RCA-I in relation to healthy follicles. It could also be noted that the zona pellucida of cystic follicles lost the affinity for the lectin Con-A. There was no staining on follicles in any category with the lectins DBA and UEA-I, although it was staining in the corpus luteum (control group) and in the mesothelium and interstitial glands of both groups with DBA. These observations probably reflect changes in the glycosaminoglycans present in the different ovarian compartments or in the glycosylation of cellular components essential for proper follicular dynamics.

Effects of different anesthetics in the murine model of EHV-1 infection.

Mice are commonly used as an experimental model to investigate the Equid herpesvirus 1 (EHV-1) infection. This model easily reproduces the disease, and the clinical signs are more or less similar to those observed in the horse, the natural host. During natural infection, the acute course of respiratory infection is mandatory for the development of adaptive immune response. Since interactions between EHV-1 and anesthetics are possible, the study investigated whether the early events of murine pulmonary immune response could be affected by different anesthetics. Therefore, mice were experimentally infected with a unique EHV-1 strain under the effects of ether, ketamine/xylazine, or isoflurane. Clinical signs and histopathological lesions in the lungs were described, and the cell death and proliferation rates of sham-inoculated or infected animals were quantified using immunohistochemistry. Clinical signs were more severe in animals anesthetized with ether. Qualitative differences in the recruited inflammatory cells were observed following application of anesthesia. The level of infection between the infected groups was not statistically significant. However, lungs from ketamine/xylazine-anesthetized animals showed the highest cell death rates, whereas those from isoflurane-anesthetized animals showed the highest proliferation rates. It has been emphasized that anesthetics alone or their interactions with EHV-1 modify the response against the infection. An appropriate selection of the anesthetic during experimental studies is relevant to minimize wrong conclusions.

Lectin binding pattern in the uterus of pregnant mice infected with Tritrichomonas foetus.

Bovine genital tritrichomonosis is caused by the protozoon Tritrichomonas foetus and leads to embryonic death and abortion. The complexity of handling bovine experimental systems has led to the development of alternative models. The infection has been reproduced in pregnant BALB/c mice. In the pathogenesis of the disease, adhesion of the protozoon to host cell surface glycoproteins is important. Labelling with soya bean agglutinin (SBA) and peanut agglutinin (PNA) lectins increases in the luminal and glandular uterine epithelium of non-pregnant infected mice. The aim of the present study was to determine whether these changes also occur in pregnant infected BALB/c mice. Female BALB/c mice were inoculated intravaginally with T. foetus and, 15 ± 3 days post infection, were paired with males overnight. Infected and control mice were sacrificed 6, 8 and 10 days later. Samples of uterus were labelled with a panel of biotinylated lectins. Infected mice showed increased binding of PNA and SBA. There was also increased binding of concanavalin (Con-A) by luminal epithelium and Ricinus communis agglutinin (RCA-1) by glandular epithelium at day 6 post coitum. These changes may be due to the production of enzymes by T. foetus, which could act to enhance adhesion and colonization and thus favour infection.

Effects of Solanum glaucophyllum toxicity on cell proliferation and apoptosis in the small and large intestine of rabbits.

Vitamin D regulates mineral homeostases and enterocyte proliferation and differentiation. Hypervitaminosis D generates changes in cell proliferation, differentiation and apoptosis in several organs. We analysed morphometric parameters and proliferative and apoptotic indices in the intestinal epithelium of rabbits with hypervitaminosis D induced by the chronic treatment with the calcinogenic plant Solanum glaucophyllum. Rabbits were treated for 15 or 30 days. A group was treated for 15 days and led to possible recovery for 30 days. Another group was nutritionally restricted for 30 days. Morphological, morphometric, proliferative and apoptotic changes were found in the treated animals. Mild atrophy and reduced proliferation was found in the jejunum and ileum. Apoptosis increased in the crypts of the ileum and in the superficial epithelium and crypts of the rectum. Most of the alterations were partially recovered. The possible involvement in these changes of the hypervitaminosis D-like state induced by S. glaucophyllum is discussed.

Histopathological, immunohistochemical, lectinhistochemical and molecular findings in spontaneous bovine abortions by Campylobacter fetus.

Bovine campylobacteriosis (BC) is a venereal disease caused by Campylobacter fetus characterized by temporary infertility with mild endometritis, early embryonic death and occasional abortions. The objectives of this study were to describe and identify C. fetus in spontaneous bovine abortion on the basis of histopathological, immunohistochemical, lectinhistochemical and polymerase chain reaction (PCR) techniques. The most frequent foetal lesion was neutrophilic bronchopneumonia and interstitial pneumonia. Other commonly observed lesions included non-suppurative interstitial enteritis, hepatitis, pericarditis, myositis, myocarditis, and meningitis. In this study, C. fetus fetus was phenotypically classified in all bovine foetuses from lungs and abomasal fluids. Immunohistochemistry (IHC) staining revealed positive stained Campylobacter organisms with typical morphology. Lectin binding patterns not showed great differences between the infected and the non-infected groups. The most important changes were a minor peanut agglutinin (PNA) and DBA binding in the alveolar cells of the lungs and DBA globet cells in some of the C. fetus-positive foetuses. Individual variations in each lectin binding pattern complicate the evaluation of the lectins results. All foetuses positive to IHC were positive by PCR. Better efficiency of PCR was obtained from abomasal fluids than from lung tissues. The association of culture and phenotypic techniques with histopathology, IHC and PCR allowed a better characterization and description of BC.

Calbindin D28k expression and the absence of apoptosis in the cerebellum of Solanum bonariense L-intoxicated bovines.

Solanum bonariense intoxication is characterized by cerebellar neuronal vacuolation, degeneration, and necrosis. Cerebellar Purkinje cells seem especially susceptible, but more research is needed to determine the pathogenesis of neuronal necrosis and the mechanism of Purkinje cell susceptibility. Calbindin D28k (CbD28k) is highly expressed in Purkinje cells and has been used as a marker for normal and degenerative Purkinje cells. The goal of this study was to describe S bonariense-induced disease by ascertaining Purkinje cell-specific degenerative changes using CbD28k expression and to correlate this with apoptosis in Purkinje cells, as determined using TUNEL (transferase-mediated dUTP-biotin nick end-labeling) and ultrastructural changes. In all cases, an increase in both dose and duration of S bonariense intoxication resulted in a decrease in the number of Purkinje cells. CbD28k immunohistochemistry was an excellent marker for Purkinje cells because immunoreactivity did not change in normal or degenerative tissues. This finding suggests that excessive calcium excitatory stimulation does not induce rapid neuronal degeneration and death. As found in previous studies, TUNEL tests and electron microscopy suggest that Purkinje cell degeneration and death are not occurring via an apoptotic process. These findings suggest that S bonariense poisoning induces progressive Purkinje cell death that is not mediated by excitotoxicity or apoptotic activation.

Glycoconjugate histochemistry in the small and large intestine of normal and Solanum glaucophyllum-intoxicated rabbits.

Vitamin D participates in mineral homeostasis, immunomodulation, cell growth and differentiation. The leaves of Solanum glaucophyllum contain high levels of 1,25-dihydroxyvitamin D3 as glycoside derivatives and their chronic ingestion generates a hypervitaminosis D-like state. We analyzed changes on carbohydrate expression as a cell differentiation indicator on samples of the small and large intestine of S. glaucophyllum-intoxicated rabbits, using conventional and lectin histochemistry. Male New Zealand white rabbits were intoxicated with S. glaucophyllum during two or four weeks and killed the day after. A group of animals ("possibly recovered group") were intoxicated during 15 days and killed at day 45 of the beginning of the experiment. We found changes in the lectin binding pattern in the small and large intestine of the intoxicated rabbits. Some of these changes were reverted in the possibly recovered group. Vitamin D could be a new regulator factor of the intestinal glycosylation process.

The guinea pig as an animal model for Ipomoea carnea induced alpha-mannosidosis.

The toxic effects of Ipomoea carnea subsp. fistulosa were evaluated in guinea pigs by administration of dry leaves during 45 days. Swainsonine and calystegines B(1), B(2) and C(1) were isolated and quantified. Clinical signs included emaciated and loss of body weight. Histological evaluation demonstrates numerous vacuoles in the cytoplasm of pancreas, liver and renal cells. Vacuolation was also evident in neurons of brain stem, mainly pontine nuclei. Neuronal lectin binding pattern showed a strong positive reaction to Con-A (Concanavalia ensiformis), WGA (Triticum vulgaris), sWGA (succinylated T. vulgaris) and LCA (Lens culinary). This result is coincident with the lectin histochemistry staining pattern of the vacuoles described in CNS of ruminants. We conclude that I. carnea subsp. fistulosa induces an intralysosomal accumulation of mannose-containing oligosaccharides in guinea pigs, which makes it a valuable animal model for the reproduction of induced alpha-mannosidosis.

Lectin-binding sites on the normal and pathologic uterus of sows.

The lectin-binding pattern was compared in the normal and pathological uterus of sows during the ovarian cycle. The following biotinylated lectins were used: Con A, DBA, SBA, PNA, RCA-I, UEA-I and WGA. Glycoconjugate labelling showed differences between phases of ovarian cycle and presence of morphologic lesions. Cystic endometrial hyperplasia increased the RCA-I reaction in the apical region of the glandular epithelium. There was higher intensity of labelling of WGA in the glandular epithelium in uteri with endometritis. In addition, increased Con A binding in the glandular epithelium and mild reduction of UEA-I reactivity in the glycocalyx of the glandular epithelium were detected in the cases of endometritis. The results of this study show that morphologic alterations modify the sugar pattern in the porcine uterus. These modifications in glycoconjugates may be one of the reasons for decreased fertility in sows.

Heat shock protein 70 and sex steroid receptors in the follicular structures of induced ovarian cysts.

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERalpha than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERalpha in the treated group. An increase in total ERalpha protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERbeta in animals with COD, and the total protein expression of ERbeta was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.