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.

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.

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.

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.

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 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.

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.

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.

Paneth cells: histochemical and morphometric study in control and Solanum glaucophyllum intoxicated rabbits.

The intestinal epithelium has a critical roll in host defence. One specialised cell type involved in this function is the Paneth cell, which secretes many substances with antimicrobial properties in response to different stimuli. Under pathological conditions, changes in the Paneth cell number, morphology and location as well as in granule number, morphology and composition have been reported. In the normal animal, 1,25-dihydroxyvitamin D3 participates in the maintenance of mineral homeostasis, immunomodulation and cell proliferation and differentiation. Solanum glaucophyllum, a calcinogenic plant containing high levels of 1,25-dihydroxyvitamin D3, is responsible for a condition known as enzootic calcinosis in ruminants, characterised by loss of body condition and mineralization of soft tissues. Using and established rabbit model, this study analyses the changes that rabbit Paneth cells undergo during intoxication with S. glaucophyllum. Male New Zealand white rabbits were experimentally intoxicated with S. glaucophyllum for 15 or 30 days. Lectin, immunohistochemical and morphometric studies were carried out on Paneth cells from samples of jejunum. SBA, DBA and WGA lectins bound to Paneth cells-granules in both normal and intoxicated rabbits, with more heterogenity in the labelling of granules from intoxicated rabbits. Paneth cells in both groups were immunonegative for lysosyme. A time and dose-dependent increase in the size and number of Paneth cells was found in both intoxicated groups. We suggest that the changes described in these cells may be directly or indirectly induced by S. glaucophyllum intoxication.

Histological and lectin binding changes in the genital tract of mice infected with Tritrichomonas foetus.

An experimental murine model of bovine genital tritrichomonosis is described. Female mice were inoculated per vaginam with Tritrichomonas foetus and a sample of the study population was killed every 3 days up to 60 days post-infection. Microscopical changes in the reproductive organs were assessed and immunohistochemistry was used to detect T. foetus within these tissues. Lectin histochemistry was used to determine changes in the expression of carbohydrates within the reproductive mucosa. A range of microscopical changes were detected in the uterine endometrium by 10 days post-inoculation and these were associated with the presence of the protozoan. The endometrial changes included endometritis and ulceration, mucosal atrophy and glandular metaplasia, and were similar to those reported in naturally infected cows. Changes in lectin binding were recognized first in the vagina where there was increased binding of Ulex europaeus agglutinin-1 (UEA-1) which was maximal on day 16 post-inoculation. Within the uterus, there was increased binding of soy bean agglutinin (SBA) which was maximal on day 19 post-inoculation, and of peanut agglutinin (PNA) which was maximal on day 16 post-inoculation. These changes in carbohydrate expression parallel the infection kinetics, since they appeared first in the vagina and later in the uterus. The changes may reflect either a host reaction against the infection or the production of enzymes by T. foetus, which act to enhance adhesion and colonization of the genital organs by the organism. The kinetics and pathogenesis of this murine infection are similar to those of the natural bovine disease, suggesting that this model system may be valuable for further studies of this disease.

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.

Characterization of cytoskeletal proteins in follicular structures of cows with cystic ovarian disease.

The distribution of intermediate filaments (vimentin, cytokeratins, desmin) and microfilaments (alpha-smooth muscle actin and muscle specific actin) was studied immunohistochemically in bovine ovaries, with and without cystic ovarian disease. The immunohistochemically stained area (IHCSA), was quantified by image analysis, to evaluate the expression of these cytoskeletal proteins in the follicular wall of healthy antral, atretic, and cystic follicles. The granulosa cell layer of cystic follicles and atretic follicles had a significantly larger IHCSA for vimentin than did healthy antral follicles. Cytokeratins reacted lightly in the granulosa cells of antral follicles of normal ovaries, whereas granulosa cells of atretic and cystic follicles showed significantly higher IHCSA values. Immunohistochemical localization of desmin, muscle specific actin, and alpha-smooth muscle actin was restricted to the theca externa. This study supports earlier suggestions that strongly positive reactions with vimentin and cytokeratin antibodies observed in the granulosa cells of cystic follicles are due to the reorganization that occurs in the follicle during the process of cystic development, and are associated with changes in the expression of cytoskeletal proteins that are essential to proper cellular functioning.

Interaction of Tritrichomonas foetus with the reproductive tract of experimentally infected female BALB/c mice: ultrastructural evaluation.

The interaction of Tritrichomonas foetus with its host is a complex process that involves colonisation, attachment and persistence. The goal of the present study was to describe the interaction of T. foetus with the genital tract using a model of non-oestrogenised female BALB/c mice which had been intravaginally infected with a suspension of T. foetus during oestrus. Animals were sacrificed after 10 weeks and the uteri fixed and processed for light and electron microscopy. Ultrastructural analysis showed that the attached protozoa interacted with the mucosa through a somal projection. With an amorphous secretion at the protozoa-host cell interface. There was no direct contact between the protozoal plasma membrane and the epithelial cell membrane. Our results demonstrated the participation of an active phagocytosis and the destruction of T. foetus by eosinophils.

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.

Intoxication by Ipomoea sericophylla and Ipomoea riedelii in goats in the state of Paraíba, Northeastern Brazil.

A disease of the nervous system was observed in goats from two farms of the semiarid of the state of Paraíba, northeastern Brazil. Ipomoea sericophylla was found in one farm and I. riedelii in the other. Both plants were administered experimentally to five goats each. Both plants induced clinical signs similar to those observed in spontaneous cases. Two goats died spontaneously and five were euthanatized. Three goats recovered after the withdrawal of the plants. Histological examination showed that all goats that died spontaneously or were euthanized had diffuse vacuolation of neurons, macrophages of lymphatic tissues, and epithelial cells of pancreas, thyroid, renal tubules and liver. On electron microscopy of Purkinje cells, numerous dilated membrane bordered vacuoles were identified as lysosomes. On lectin-histochemical analysis, cerebellar cells gave positive reactions to Concanavalia ensiformis, Triticum vulgaris, and succinylated-T. vulgaris, which indicate the storage of alpha-D-mannose, alpha-D-glucose, beta-D-N-acetyl-glucosamine, and acetyl-neuraminic acid. The chemical analysis of I. sericophylla and I. riedelii showed 0.11 and 0.14% of swainsonine, respectively. The latter also contained calystegines B1, B2 and C1. It is concluded that I. sericophylla and I. riedelli cause a lysosomal storage disease.

Lectin-histochemistry: glycogenosis in cattle.

Ten out of 47 calves that were born in a small Brahman herd from southern Brazil developed progressive muscular weakness and tremors, lethargy and poor body condition. Necropsy was performed on three affected animals. The only gross lesion detected was paleness of the muscles of the trunk and limbs. Multiple cytoplasm vacuoles located in different tissues were the principal microscopic lesions. Vacuoles were particularly evident in skeletal muscles and myocardium. PAS-positive granules were numerous in skeletal muscle fibres and Purkinje fibres of the myocardium, but were also observed in the neurons of the brain and spinal cord, and in the vascular smooth muscle fibres from all the examined tissues. Pretreatment with diastase completely abolished the PAS reactivity. The vacuoles reacted strongly to Griffonia simplicifolia II and Concanavalia ensiformes lectins, whose biding pattern has been reported as useful for demonstration of glycogen. Examination of the electron micrographs revealed that glycogen was free within the cytoplasm or accumulated in membrane-bound granules of several tissues, especially in striated muscle, liver and neurons of the CNS. These findings were consistent with generalized glycogenosis.

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.

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.