Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes.

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

Induction of interleukin-8 and interleukin-12 in neonatal ovine lung following experimental inoculation of bovine respiratory syncytial virus.

This study aimed to determine the immunohistochemical expression of interleukin (IL)-1ß, tumour necrosis factor alpha (TNF)-α, interferon (IFN)-γ, IL-4, IL-6, IL-8, IL-10 and IL-12 and to measure the concentrations of these cytokines in lung tissue from lambs infected experimentally with bovine respiratory syncytial virus (BRSV). Lambs (n = 15) were inoculated at 2 days of age with 20 ml of viral inoculum (1.26 × 10(6) TCID50 per ml) or sterile medium (n = 15). Rectal temperature, pulse and respiratory rates were monitored daily in control and infected lambs. Lambs were killed and subject to necropsy examination at 1, 3, 5, 7 and 15 days post inoculation (dpi). There was a temporal association between pulmonary expression of these cytokines and lung pathology in BRSV-infected lambs. The cytokines IL-4 and IL-10 were not elevated, but there was a significant increase in IL-1ß, TNF-α, IFN-γ and IL-6 proteins and labelled cells, suggesting that these cytokines may play a role in the biological response to BRSV infection and contribute to the development of lung lesions. There was also a significant increase in the cytokine concentration and number of immunolabelled cells expressing IL-8 and IL-12 in infected lungs, suggesting that these cytokines might be used as therapeutic targets in the management of BRSV, in conjunction with measures to combat the causative pathogen and prophylactic methods aimed at preventing infection.

Widespread epithelioid angiosarcoma with ventricular wall involvement in a dog.

This paper reports on a canine angiosarcoma, presenting as an "undifferentiated metastasizing tumor". A 14-year-old female Cocker Spaniel was referred to the University of Extremadura Veterinary Clinic for clinical examination after suffering rapid deterioration, with chronic cough, anorexia and cachexia. One week after clinical examination, the dog died of right congestive heart failure and ventricular arrhythmia. Blood counts revealed lymphopenia and platelet depletion. The biochemistry profile was within normal limits, except for a drop in blood urea nitrogen. Cytological evaluation of liver and spleen biopsies revealed clustered anaplastic cells that lacked convincing tissue differentiation. Major findings at necropsy were enlarged spleen and multiple, beige to dark-red nodules ranging from 0.5 to 3 cm in diameter in the heart, lung, liver and spleen. At histological examination, multiple nests of anaplastic epithelioid cells were found in sections from all affected organs. Immunohistochemistry revealed widespread expression of CD31 and Factor VIII-related antigen. The neoplastic cells were negative for CD 18. The diagnosis of epithelioid angiosarcoma, localized in the myocardium, lung, liver and spleen was made. The primary site of the neoplasm could not be determined.

Dominant expression of interleukin-8 vs interleukin-1ß and tumour necrosis factor alpha in lungs of lambs experimentally infected with Mannheimia haemolytica.

AIMS: To quantify the number of cells infected with Mannheimia haemolytica and expressing interleukin (IL)-1ß, tumour necrosis factor alpha (TNFα) and IL-8 using immunohistochemistry, and to measure the immunoreactivity of cytokines in pulmonary tissue extracts using ELISA, in the lung of lambs experimentally infected with M. haemolytica, and to compare the patterns of expression of cytokines in airways at different times post-infection (p.i.). METHODS: Twenty 3-month-old lambs of both sexes were randomly assigned to two groups, viz infected (n=15), and uninfected controls (n=5). Each lamb in the infected group was inoculated with 1.5 x 10(9) cfu M. haemolytica in 5 mL sterile nutrient broth, control lambs were inoculated with 5 mL sterile nutrient broth and clinical signs were monitored. Infected and control animals were killed at 1, 3, 5, 7, and 15 days p.i. Histopathology and immunohistochemistry were conducted to determine the number of immunolabelled cells in pneumonic lungs, and study the pattern of expression of IL-1ß, TNFα and IL-8 in lung extracts using ELISA. RESULTS: Lesions in bronchi and bronchioles ranged from epithelial desquamation to bronchiolitis obliterans and necrosis. The alveoli had areas of seroproteinaceous fluid, fibrin and bacterial aggregates that evolved to foci of pyogranulomatous inflammation with clustered inflammatory cells, referred to as 'oat cells'. M. haemolytica antigen was observed in the cytoplasm of inflammatory cells. Labelling of IL-1ß, TNFα and IL-8 was observed in bronchial and bronchiolar epithelial cells, alveolar exudate, and in interstitial inflammatory infiltrate, with increased expression on 1 and 3 days p.i. for IL-1ß and TNFα, and 1, 3, and 5 days p.i. for IL-8. In lung tissue extracts, peak concentrations of IL-1ß (55 (SD 5) ng/mL), TNFα (92 (SD 6) pg/mL) and IL-8 (8 [SD 2] µg/mL) occurred at 3 days p.i. CONCLUSIONS: The results of this study suggested that the inflammatory cytokines IL-1ß, TNFα and IL-8 may play an important role in enhancing the biological response to M. haemolytica, and contribute to the development of lesions in the lung in pulmonary pasteurellosis in sheep. Given that the expression of IL-8 in lung was much greater than that of IL-1ß and TNFα, anti-cytokine agents directed at this mediator could be useful in the prevention and treatment of this disease.

Changes in concentrations of cortisol and melatonin in plasma, expression of synaptophysin, and ultrastructural properties of pinealocytes in goat kids in situations of stress due to early weaning: the effect of melatonin.

AIM: To analyse the changes in some histo-physiological parameters of the pineal gland of goat kids in situations of stress due to early weaning, and the effect of exogenous treatment with melatonin. METHODS: Twenty-four 6-day-old Verata goat kids were used; 12 suckled their dams throughout the study (non-weaned groups), and the other 12 were removed from their dams and fed a milk replacer (weaned groups). Six goat kids in each group were treated with melatonin, and the other six with double-distilled pyrogen-free water (Day 0). On Days 28-29, blood samples were collected at 0600, 1000, 1400, 1800, 2200, 0200 and 0600 hours, to determine concentrations of cortisol and melatonin in plasma. On Days 29 and 30, six animals per group (three at 1400 and three at 0200 hours, respectively) were subject to euthanasia and the weight of their pineal glands determined. The structural immunocytochemistry, morphometric analysis, ultrastructural analysis and immunotransmission electron microscopy of the pineal glands were established. RESULTS: Concentrations of cortisol in plasma were significantly higher in weaned than in non-weaned goat kids (p<0.05), and treatment with melatonin reduced the concentrations in weaned kids (p<0.05). Concentrations of melatonin in plasma showed a similar pattern in the four groups, with peak values at 0200 and troughs at 1400 hours. Mean concentrations of melatonin in plasma in weaned goat kids were significantly lower than those in the other groups (p<0.05). In weaned goat kids not treated with melatonin, the weight and volume of the pineal gland, and number of pinealocytes, were significantly lower when compared with those from non-weaned kids (p<0.05). Quantitative ultrastructural analysis of pinealocytes showed the relative volume of mitochondria, rough endoplasmic reticulum and Golgi complex was significantly lower in weaned than non-weaned goat kids (p<0.05); treatment with melatonin significantly increased these parameters in weaned kids. CONCLUSIONS: Taken together, these results indicate that treatment with melatonin in goat kids in situations of stress due to premature weaning could play an important role in the improvement of histo-physiological function of the pineal gland.

Histopathological and immunohistochemical findings in the lungs of pigs infected experimentally with Mycoplasma hyopneumoniae.

Pigs were infected intranasally with Mycoplasma hyopneumoniae and killed at intervals ranging from 7 to 35 days post-infection (dpi). Histopathological changes consisted of (1) exudates in airways and alveolar lumina, (2) peribronchial and peribronchiolar lymphoid hyperplasia, and (3) enlargement of alveolar septa. These changes were particularly marked from 7 to 28dpi, coinciding with significant increases in the expression, detected immunohistochemically, of cytokines (IL-1alpha, IL-1beta, IL-8, TNF-alpha and INF-gamma) and lymphoid markers (CD4+, CD8+, muramidase, IgG+, IgA+). Both the lesions and immunohistochemical signals declined in intensity beyond 35 days.

Morphometric and immunohistochemical study of the abomasum of red deer during prenatal development.

The red deer is well suited to scientific study, given its economic importance as an animal to be hunted, and because it has a rich genetic heritage. However, there has been little research into the prenatal development of the stomach of ruminants in general, and none for the red deer. For this reason, we undertook histological evaluation of the ontogenesis of the abomasum in red deer. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses from the initial stages of prenatal life until birth. The animals were divided for test purposes into five experimental groups: group I [1.4-3.6 cm crown-rump length (CRL); 30-60 days, 1-25% of gestation]; group II (4.5-7.2 cm CRL; 67-90 days, 25-35% of gestation); group III (8-19 cm CRL; 97-135 days, 35-50% of gestation); group IV (21-33 cm CRL; 142-191 days, 50-70% of gestation) group V (36-40 cm CRL; 205-235 days, 75-100% of gestation). In the organogenesis of the primitive gastric tube of red deer, differentiation of the abomasum took place at 67 days, forming a three-layered structure: the epithelial layer (pseudostratified), pluripotential blastemic tissue and serosa. The abomasal wall displayed the primitive folds of the abomasum and by 97 days abomasal peak areas were observed on the fold surface. At 135 days the abomasal surface showed a single mucous cylindrical epithelium, and gastric pits were observed in the spaces between abomasal areas. At the bottom of these pits the first outlines of glands could be observed. The histodifferentiation of the lamina propria-submucosa, tunica muscularis and serosa showed patterns similar to those described for the forestomach of red deer. The abomasum of red deer during prenatal life, especially from 67 days of gestation, was shown to be an active structure with full secretory capacity. Its histological development, its secretory capacity (as revealed by the presence of neutral mucopolysaccharides) and its neuroendocrine nature (as revealed by the presence of positive non-neuronal enolase cells and the neuropeptides vasoactive intestinal peptide and neuropeptide Y) were in line with the development of the rumen, reticulum and omasum. Gastrin-immunoreactive cells first appeared in the abomasum at 142 days, and the number of positive cells increased during development. As for the number of gastrin cells, plasma gastrin concentrations increased throughout prenatal life. However, its prenatal development was later than that of the abomasum in sheep, goat and cow.

Morphometric and immunohistochemical study of the omasum of red deer during prenatal development.

The red deer is an important study species because of its value in the national economy and because it provides a wealth of genetic material. To date, there has been little research into the prenatal development of the stomach of ruminants, and none of the red deer. We therefore performed a histological evaluation of the ontogenesis of the omasum in the red deer. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses of deer from the initial stages of prenatal life until birth. For test purposes, the animals were divided into five experimental groups: Group I (1.4-3.6 cm crown-rump length, CRL; 30-60 days, 1-25% of gestation); Group II (4.5-7.2 cm CRL; 67-90 days, 25-35% of gestation); Group III (8-19 cm CRL; 97-135 days, 35-50% of gestation); Group IV (21-33 cm CRL; 142-191 days, 50-70% of gestation); and Group V (36-40 cm CRL; 205-235 days, 75-100% of gestation). At 67 embryonic days, the omasum wall was differentiated, and comprised three layers: the epithelial layer, pluripotential blastemic tissue and serosa. The stratification of the epithelial layer was accompanied by changes in its structure, with the appearance of four laminae of different sizes; in order of appearance these were: primary at 67 days, secondary at 90 days, tertiary at 97 days and quaternary at 135 days. At around mid-gestation, lateral evaginations were formed from the stratum basale of the primary and secondary smaller laminae. These were the primitive corneum papillae. From 205 days, the corneum papillae were present in all four sizes of laminae. The histodifferentiation of the lamina propia-submucosa, tunica muscularis and serosa showed patterns of development similar to those reported for the rumen and reticulum of red deer. The omasum of red deer during prenatal life, especially from 67 days of gestation, was shown to be an active structure with full secretory capacity. Its histological development, its secretory capacity (detected by the presence of neutral mucopolysaccharides) and its neuroendocrine nature (detected by the presence of positive non-neuronal enolase cells and the neuropeptides vasoactive intestinal peptide and neuropeptide Y) were parallel to the development of the rumen and the reticulum. However, its prenatal development was later than that of the omasum in sheep, goat and cow.

Morphometric and immunohistochemical study of the reticulum of red deer during prenatal development.

Iberian red deer need to be conserved for their economic role and for their genetic importance as an important component of the ecosystem. Modifications currently being made to traditional management systems require a better understanding of the structure, function and development of their alimentary system. Here we describe a histomorphometric and immunohistochemical analysis of the stomach of 25 red deer embryos and fetuses from 30 days of gestation until birth (235 days). Differentiation of the reticular compartment from the primitive gastric tube begins at 67 days, forming a three-layered structure: epithelium, pluripotential blastemal tissue and serosa. The primitive reticular cells are initiated as small epithelial evaginations (primary ribs) at 117 days. At 142 days, lateral growths appear from the primary reticular ribs, forming the corneum papillae. The secondary reticular ribs form at 142 days as growths from the primary ribs. The uneven height of primary and secondary reticular ribs leads to the formation of cells of varying size. Growth of the reticular ribs involves the lamina propria but not the submucosa, so clear separation of these layers is maintained during histodifferentiation. Formation of the tunica muscularis from the pluripotential blastemal tissue begins at 67 days of intrauterine life, as two layers of longitudinally and circularly arranged myoblasts. Differentiation of the muscularis from the mucosa occurs at approximately 205 days, as longitudinal projections of the internal bundles of the tunica muscularis form the musculature of the primary ribs. The secretion of neutral and acid mucopolysaccharides by the reticular epithelial layer begins at 67 days, establishing the gradual adaptation of the mucosa to its protective function in postnatal life. Neuroendocrine (non-neuron enolase) and glial cells (glial fibrillary acidic protein and vimentin) were detected by immunohistochemistry, in a similar localization and intensity to that reported in the rumen. The neuropeptides vasoactive intestinal peptide and neuropeptide Y showed a positive immunoreaction in the reticular epithelium from 142 days of prenatal life, again earlier than reported for the rumen. In comparison with domestic ruminants, deer were shown to be less precocious with regard to development of gastric tube, in their capacity to secrete neutral mucopolysaccharides, and in their neuroendocrine nature, as determined by the detection of positive neuroendocrine and/or glial cells.

Morphometric and immunohistochemical study of the rumen of red deer during prenatal development.

Abstract A detailed study of the ontogenesis of deer stomach has not been undertaken to date, and our aim was to sequence several histological phenomena that occur during the ontogenesis of one of the gastric compartments, the rumen. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses of deer from the initial stages of prenatal life until birth. For the purposes of testing, the animals were divided into five experimental groups: group I, 1.4-3.6 cm crown-rump length, 30-60 days, 1-25% of gestation; group II, 4.5-7.2 cm crown-rump length, 67-90 days, 25-35% of gestation; group III, 8-19 cm crown-rump length, 97-135 days, 35-50% of gestation; group IV, 21-33 cm crown-rump length, 142-191 days, 45-70% of gestation; and group V, 36-40 cm crown-rump length, 205-235 days, 75-100% of gestation. The rumen of the primitive gastric tube was observed at approximately 60 days. At 67 days the rumen consisted of three layers: internal or mucosal, middle or muscular, and external or serosal layer. The stratification of the epithelial layer was accompanied by changes in its structure with the appearance of ruminal pillars and papillae. The outline of the ruminal papillae began to appear at 142 days of prenatal development as evaginations of the basal zone toward the ruminal lumen, pulling with it in its configuration the stratum basale, the lamina propria and the submucosa. From the pluripotential blastemic tissue at 60 days we witnessed the histodifferentiation of the primitive tunica muscularis, composed of two layers of myoblasts with a defined arrangement. It was also from the pluripotential blastemic tissue, at 97 days, that the lamina propria and the submucosa were differentiated. The serosa showed continuity in growth as well as differentiation, already detected in the undifferentiated outline phase. The tegumentary mucosa of deer rumen was shown without secretory capacity in the initial embryonic phases; neutral mucopolysaccharides appeared from 67 days. The presence of neuroendocrine cells (non-neuronal enolase) in the ruminal wall of deer during development was not detected until 97 days. The glial cells were detected at 142 days for glial fibrillary acidic protein and at 67 days for vimentin. The immunodetection of neuropeptides vasointestinal peptide and neuropeptide Y progressively increased with gestation period, starting from 97 days. In terms of the structure of the rumen of the primitive gastric tube, our observations revealed that the deer is less precocious than small and large domestic ruminants. Thus its secretory capacity, detected by the presence of neutral mucopolysaccharides, and its neuroendocrine nature, determined by the presence of positive non-neuronal enolase cells, were evident in more advanced stages of prenatal development than those detected in the sheep, goat and cow.

Ultrastructural and immunocytochemical characterization of interstitial cells in pre- and postnatal developing sheep pineal gland.

Pineal gland interstitial cells from 32 sheep embryos (from day 54 of gestation until birth) and 18 sheep (from 1 month to >2 years) were analysed using ultrastructural and immunohistochemical techniques. From day 98 of gestation and throughout postnatal development, a second cell type was observed in addition to pinealocytes; these cells displayed uniform ultrastructural features similar to those of CNS astrocytes. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis. Expression of phosphotungstic acid hematoxylin, glial fibrillary acidic protein and vimentin indicates that the second cell population in the developing ovine pineal gland is, in fact, a combination of glial-astrocyte cells at varying stages of maturity. Pineal interstitial cells started to show signs of functional activity evident in vascular tropism; such activity, evident from around day 98 of gestation, appeared to relate to the exchange of substances between the pineal parenchyma and blood vessels and, though it continued throughout postnatal development, was most evident in animals slaughtered between 9 months and 2 years of age (group II). Morphologically, functional activity in interstitial cells in this age-group was apparent in: 1, formation of specific contact sites between interstitial cells and nerve fibres in the perivascular space; and 2, the presence of numerous gap junctions between the bulbous endings of cytoplasmic processes.

In situ hybridization detection of bovine respiratory syncytial virus in the lung of experimentally infected lambs.

We studied the distribution of bovine respiratory syncytial virus (BRSV) RNA in lungs of experimentally inoculated lambs by in situ hybridization at different times postinoculation. The probe used for in situ hybridization was prepared by reverse transcription of BRSV RNA, followed by polymerase chain reaction (PCR) amplification of the cDNA. Twenty-five Merino lambs of both sexes with a live weight of 17 +/- 3 kg received an intratracheal inoculation of 20 ml saline solution containing 1.26 X 10(6) TCID50 BRSV (strain NMK7)/ml. Lambs were slaughtered 1, 3, 7, 11, and 15 days postinoculation (PID). Bronchial and bronchiolar epithelial cells were positive for BRSV nucleic acid by ISH at 1, 3, 7, and 11 PID. However, alveolar epithelial cells contained positive cells at 1, 3, and 7 PID. Cells containing viral RNA were detected from 1 to 11 PID in exudate within bronchial and bronchiolar lumina and from 3 to 7 PID in alveolar exudates. Positive hybridization signals were identified in interstitial mononuclear cells and in bronchi-associated lymphoid tissue from 3 to 11 PID. Mononuclear cells were located in peribronchiolar tissue and interalveolar septa. The highest signal intensity in positive cells was observed at 3 and 7 PID, coinciding with the most important histopathological findings.

A combined immunohistochemical and electron microscopic study of the second cell type in the developing sheep pineal gland.

Ultrastructural and immunohistochemical techniques were used to study the second cell type in sheep embryo pineal glands. Thirty-two embryos were studied from day 54 of development through birth. Specimens were arranged in four age groups, defined in terms of the most relevant histological features: Group 1 (54-67 days of prenatal development), Group 2 (71-92 days), Group 3 (98-113 days), and Group 4 (118-150 days). At 98 days, a second cell type was observed which differed from pinealoblasts and showed uniform ultrastructural characteristics similar to those of astrocytes in the central nervous system. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis: while all Type II cells stained positive to phosphotungstic acid hematoxylin, only 50% expressed glial fibrillary acidic protein. In the course of ovine intrauterine development, the vascular affinity of this second cell population, composed of glial-like or astrocytic cells at varying stages of maturity, leads to the formation of a limiting pineal barrier. This barrier may constitute the morphological expression of a hypothetical functional involvement in the exchange of substances between blood and pineal parenchyma.

[Comparative ontogenic analysis of the epithelium of the non-glandular stomach compartments of merino sheep]. / Estudio ontogenico comparativo de la lamina epitelial de los compartimentos gastricos no glandulares en ovinos merinos.

A total of 74 embryos and fetuses were used in a comparative analysis of the epithelium of the non-glandular stomach compartments of merino sheep during development. The mechanical protection showed by the tegumentary epithelium in the superficial layers of the rumen, reticulum and omasum is supported by a buffer system of neutral mucopolysaccharides secreted by the deeper strata. Neutral mucopolysaccharides first appeared in epithelial cells at 46 days of fetal life. Acid mucopolysaccharides, mucins, and mucoid compounds were not detected. Growth curves and formulas were constructed for the epithelial layers.

Pathological study of experimentally induced bovine respiratory syncytial viral infection in lambs.

An experimental model was designed to characterize lesions in the lung of lambs inoculated with bovine respiratory syncytial virus (BRSV). Twenty-five Merino lambs of both sexes, with a live weight of 17 +/- 3 kg, received an intratracheal inoculation of 20 ml saline solution containing 1.26 x 10(6) TCID50 BRSV (strain NMK-7) per ml. Lambs were slaughtered 1, 3, 7, 11 and 15 days postinoculation (DPI), and samples were taken for analysis using light-microscopic and immunohistochemical techniques. The results reflected the effect of the virus on airway epithelia. The presence of BRSV in ciliated bronchial and bronchiolar epithelial cells gave rise to cytopathological changes, including loss of cilia and cell necrosis; these changes might be expected to decrease the efficiency of mucociliary clearance, favouring the development of secondary bacterial bronchopneumonia. These results suggest a reduction in BRSV tropism for alveolar epithelia compared to bronchial and bronchiolar epithelia. Light-microscopic analysis revealed a narrowing of alveolar and airway lumina and a considerable interstitial inflammatory reaction.

Lesions in lambs experimentally infected with bovine respiratory syncytial virus.

An experimental model was designed to characterize lesions in the lung of lambs inoculated with bovine respiratory syncytial virus (BRSV). 25 Merino lambs of both sexes, with a live weight of 17 +/- 3 Kg, received an intratracheal inoculation of 20 ml saline solution containing 1.26 x 10(6) TCID50 BRSV (strain NMK-7) per ml. Lambs were slaughtered 1, 3, 7, 11 and 15 postinoculation days (PID), and histopathological, immunohistochemical and electron microscopic studies were performed. Results reflected a series of lesions, the most noteworthy of which were bronchiolitis obliterants with destruction of the mucociliary apparatus, the presence of syncytial cells in alveoli and a progressive interstitial reaction. BRSV antigen was detected in lung samples. These changes might be expected to decrease the efficiency of respiratory tract defence mechanisms, rendering the lung parenchyma susceptible to opportunist bacterial infection.

Spontaneous bovine respiratory syncytial virus infection in goats: pathological findings.

This paper presents the results of a study of eight cases of natural bovine respiratory syncytial virus (BRSV) infection in a herd of 50 Murciana kids from a intensive goat-farm in Spain. Clinical and pathological analysis confirmed the existence of natural BRSV infection in eight kids. In two of the eight animals there was also evidence of concurrent infection with P. haemolytica A.

The immunologic response to experimental infection with bovine respiratory syncytial virus in lambs.

An experimental model was designed to study the humoral immune response in lambs experimentally infected with bovine respiratory syncytial virus (BRSV), 25 Merino lambs of both sexes, with a live weight of 17 +/- 3 kg, received an intratracheal inoculation of 20 ml. saline solution containing 1.26 x 10(6) TCID50 BRSV (strain NMK-7) per ml. Blood samples for serological analysis were collected from the jugular vein of the animals on days 0, 1, 3, 5, 7, 9, 11 and 15 post inoculation. Serum proteins were determined by electrophoresis, and a qualitative and quantitative analysis of immunoglobulins (Ig) was carried out using immunoelectrophoresis (IE) and light nephelometry, respectively. Serum anti-BRSV antibody levels were tested by enzyme-linked immunoabsorbent assay (ELISA). Lambs were necropsied 1, 3, 7, 11 and 15 postinoculation days (p.i.d.), and samples were taken for immunohistochemical analysis. IgA, IgG and IgM were labelled on paraffin-embedded lung sections using an avidin-biotin-peroxidase complex. Results reflected an increase in gamma fractions in experimental animals with respect to controls. IE analysis of these sera revealed a polyclonal increase in immunoglobulins. Nephelometric examination showed an increase in serum IgA, IgG an IgM levels up to 7 p.i.d., coinciding closely with high titres for humoral virus antibodies. After 9 p.i.d., titres and immunoglobulin levels both fell. Immunoglobulin labelling in formol-fixed and paraffin-embedded lung tissue samples detected IgG and IgA, mainly in bronchial, bronchiolar and alveolar epithelia. IgM, however, was not detected in the lungs of infected animals.

Histomorphometric analysis of the omasum of sheep during development.

Histomorphometric and scanning electron microscopic analyses were performed on 74 embryos and fetuses and 20 sheep (early postnatal to adult age). Histologic differentiation of the omasum took place at 33 days of fetal life, with the appearance of first-order laminae. Second-, third-, and fourth-order laminae appeared at 39, 50, and 59 days, respectively. Neutral mucopolysaccharides first appeared in epithelial cells at 46 days of fetal life, decreasing quantitatively until birth, before subsequently stabilizing in postnatal life. Acid mucopolysaccharides, mucins, and mucoid compounds were not detected. Growth curves and formulas were constructed for each tissue layer. Initial tests involved multiplicative (y = axb), exponential (y = EXP [a + bx]), linear (y = a + bx), and polynomial models (y = a + bx + cx2 + dx3).

Histomorphometric analysis of the reticulum of the sheep during development.

Histomorphometric and scanning electron microscopic analysis were performed on 74 embryos and foetuses and on 20 sheep (early post-natal to adult age). Histodifferentiation of the reticulum took place at 33 days of foetal life. Reticular ribs were observed as evaginations of the epithelial stratum germinativum at 64 days. Neutral mucopolysaccharides first appeared in epithelial cells at 46 foetal days, thereafter to decrease gradually in number, subsequently stabilising in postnatal life. Acid mucopolysaccharides, mucins and mucoid compounds were not detected. Growth curves and formulae were constructed for each tissue layer. Initial test involved multiplicative (y = axb), linear (y = a+bx) and polynomial model (y = a+b+cx2+dx3).