Experimental infection of meadow voles (Microtus pennsylvanicus) with sheep scrapie

Meadow voles (Microtus pennsylvanicus) are permissive to chronic wasting disease (CWD) infection, but their susceptibility to other transmissible spongiform encephalopathies (TSEs) is poorly characterized. In this initial study, we intracerebrally challenged 6 meadow voles with 2 isolates of sheep scrapie. Three meadow voles acquired a TSE after the scrapie challenge and an extended incubation period. The glycoform profile of proteinase K-resistant prion protein (PrP^res) in scrapie-sick voles remained similar to the sheep inocula, but differed from that of voles clinically affected by CWD. Vacuolization patterns and disease-associated prion protein (PrP^Sc) deposition were generally similar in all scrapie-affected voles, except in the hippocampus, where PrP^Sc staining varied markedly among the animals. Our results demonstrate that meadow voles can acquire a TSE after intracerebral scrapie challenge and that this species could therefore prove useful for characterizing scrapie isolates.     

Immunohistochemical studies on the splenic lobe of the pancreas in young Japanese quails (Coturnix c. japonica)

The splenic lobe (Lobus splenicus) of the pancreas of young meat-type quails (Coturnix c. japonica) was examined by immunohistochemical and light microscopic methods. The endocrine cells are mainly grouped as alpha, beta and mixed islets. A large region consisting of alpha cells is located in the central region of the splenic lobe whereas numerous beta islets are detected in the periphery of the splenic lobe. Alpha islets are in the majority composed of toluidine blue positive A cells and a few toluidine blue negative D and / or avian pancreatic polypeptide (APP) endocrine cells. Beta islets contain only a few toluidine blue negative B and a few D cells. Immunohistochemical staining of the splenic lobe reveal in the centre of beta islets numerous insulin immunoreactive cells and scarcely in alpha islets, exocrine tissue and / or among acinar cells. Somatostatin immune-reactive cells form a circular layer in the periphery of beta islets whereas these cells are uniformly distributed throughout the alpha islet parenchyma and exocrine tissue. In conclusion, the morphology but also the endo- and exocrine functions of the splenic lobe of quails are similar to observations in other avian species such as chicken, duck, goose and pigeon.     

Immunohistochemical and ultrastructural study of endocrine cells from the pyloric region of the European mole (Talpa europaea)]

The distribution and the morphology of some endocrine cells (gastrin, somatostatin and 5-HT immunoreactive) in the pyloric region were studied in the Talpa europaea, an insectivore representing one of the most primitive living Eutherians. The immunohistochemical studies enabled us to identify and calculate the percentage of each cell type: the most numerous endocrine cells were gastrin immunoreactive; fairly numerous appeared somatostatin immunopositive; less numerous were 5-HT immunoreactive cells. While the ultrastructural observations let us describe four endocrine cell types: G cells producing gastrin, D cells containing somatostatin, EC cells of the gastric type producing 5-HT and D1 cells whose content is still unknown.     

Histopathological and immunohistochemical analysis of the endocrine and exocrine pancreas in twelve cattle with insulin-dependent diabetes mellitus (IDDM).

Histological and immunohistochemical studies were carried out on the pancreas of twelve cattle of insulin-dependent diabetes mellitus (IDDM). They showed clinical signs such as persistent hyperglycemia, glycosuria and decreased glucose tolerance, and some cases accompanied with or without ketonuria. Histopathologically, eight cattle were diagnosed as chronic IDDM, while others were acute IDDM. The most characteristic lesions of the pancreas in chronic IDDM showed a decrease in the size and number of pancreatic islets, interlobular and interacinar fibrosis, mild lymphocytic insulitis, and vacuolation of a few islets. Almost all cells in the atrophied islets had a small amount of ungranulated cytoplasm. Immunohistochemical examination revealed that the atrophied islet cells did not react to anti-insulin antibody, but occasionally reacted to anti-glucagon or somatostatin antibodies. A few solitary islets with mild lymphocytic infiltration, necrotic islets with occasional calcification, and atrophied islets with mild fibrosis were also observed. A few islets consisted of many islet cells with vacuolated cytoplasm including a small number of insulin-positive granules. Accumulation of glycogen granules was occasionally observed in these islets. Islet fibrosis was due to the proliferation of collagen fibers reactive to both anti-collagen type I and type III antibodies. In acute IDDM, the major islets consisted of the cells with vacuolated cytoplasm indicating the degranulation of islet cells. These islets contained many islet cells with shrunken cytoplasm and karyorrhectic nuclei. Lymphocytic infiltration was frequently observed in the islets which consisted of many islet cells having karyorrhectic nuclei and vacuolated and severely degranulated cytoplasm. Immunohistochemically, islet cells with vacuolated cytoplasm had a small amount of insulin-positive granules, suggesting severe degranulation of beta-cells. An increase in acinar islet-cells and proliferation of ductal epithelial cells showing insulin-immunoreactivity were observed. Bovine IgG-immunoreactive islet cells were frequently seen in the vacuolated islets. In summary, pathological observations suggested that beta-cells were being destroyed by an inflammatory process which selectively affected the pancreatic islets. Lymphocytic insulitis and anti-bovine immunoreactive islet cells were thought to be the most significant changes in determining the etiology and pathogenesis of bovine IDDM, and suggested their role in anti-islet autoimmunity in this form of diabetes.     

Immunohistochemical and ultrastructural investigation of granular cell tumours in dog, cat, and horse.

Six canine, one feline and one equine granular cell tumours (GCTs) were investigated electron microscopically and immunohistochemically. The tumours were tested for reactivity with monoclonal antibodies against vimentin and desmin and with polyclonal antibodies against cytokeratin, S-100 protein, glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE). All GCTs were characterized by their PAS positive cytoplasmic granules in light microscopy, which in electron microscopy appeared as lysosome-like granules. In each case two canine GCTs were stained by the antibody against cytokeratin, vimentin and S-100 protein. Cells of the equine GCT showed reactivity with the antiserum against S-100 protein. In the feline GCT no reactivity with any of the antibodies tested was observed. These differences of the immunohistochemical reactions of GCTs suggest a nonuniform histogenesis of GCTs in domestic animals. The reactivity of the tumour cells with the antiserum against NSE is discussed.     

Morphological, histological, and ultrastructural studies of the ovary of the cattle-tick Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae)

This study presents the morphology of the ovary, as well as the dynamics of the vitellogenesis process in oocytes of the cattle-tick Boophilus microplus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a lumen delimitated by a wall of small epithelial cells with rounded nuclei. In this tick species, the oocytes were classified into six stages varying from I to VI and according to: cytoplasm appearance and presence of the germ vesicle, yolk granules, and chorion. Oocytes of various sizes and at different developmental stages remain attached to the ovary through a cellular pedicel until completing stage V. Afterwards, they are liberated into the lumen and from there to the exterior. Some oocytes (classified as type VI) showed an atypical appearance indicating that some of the cellular components would be undergoing a degenerative process and/or reabsorption.     

Immunohistochemical and ultrastructural study of ito cells (fat-storing cells) in response to extrahepatic bile duct ligation in broiler chickens.

The Ito cell (fat-storing cell) lies in perisinusoidal space of liver and has a variety of functions. We investigated the immunohistochemistry and ultrastructure of Ito cells in normal and cholestatic livers of broiler chickens. Immunohistochemistry demonstrated that Ito cells expressed HHF35 muscle actin, vimentin, desmin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), chromogranin A and cytokeratins in normal livers. These cells were diffusely scattered throughout the lobules. Livers treated with extrahepatic bile duct ligation (BDL) showed cholestasis, fibrosis, proliferation of biliary ductules and Ito cells. The Ito cells were frequently found in fibrotic areas and were larger in size with more extensive immunoreactivity than those of normal livers. Ultrastructural study demonstrated that Ito cells were closely associated with the production of collagen fibers in BDL livers. These findings suggest that Ito cells actively react against hepatocytic injuries and play a major role in the hepatic fibrogenesis of cholestatic livers of chickens.     

Structural and ultrastructural changes in the lungs of cats Felis catus (Linnaeus, 1758) experimentally infected with D. immitis (Leidy, 1856)

Clinical signs are seldom observed in feline heartworm disease, and the pathophysiological changes in the lungs of infected animals remain undefined. The goal of this study was to evaluate the structural and ultrastructural changes in the lungs of cats experimentally infected with Dirofilaria immitis. Six healthy cats were each infected with two adult heartworms by intravenous transplantation (Receptor Group, RG). The control group consisted of two uninfected animals kept under the same conditions as the RG. At 42 days after transplantation, all cats were euthanized and necropsied for worm recovery and collection of lung samples for examination by light microscopy (LM) and transmission electron microscopy. By LM, lung sections from the six infected cats exhibited bronchial and bronchiolar lesions. Alterations in all tissues of the pulmonary arteries were observed in the infected animals. In conclusion, cats infected experimentally with D. immitis developed lesions in their lungs as a consequence of arterial disease and intense interstitial pneumonia.     

Molecular and ultrastructural studies of the fibrotic lesions of bovine focal proliferative fibrogranulomatous panniculitis (Lechiguana)

This work aimed to investigate some aspects related to the pathogenicity of Lechiguana, a bovine fibroproliferative lesion characterized by rapid collagen accumulation. Light and transmission electron microscopy and in situ hybridization studies were performed in order to elucidate the fibrogenic activity of this lesion. The characterization of fibroblastic plasticity in the lesion was done by immunohistochemical study for α-smooth-muscle cell actin. The ovoid-shaped cells presented positive reaction for α-smooth-muscle cell actin in their cytoplasm and, at the electron-microscopic level demonstrated basal lamina-like material adjacent to the external surface and collagen fibrils that corresponded to a cell population phenotypically similar to the myofibroblast. We also investigated α1 collagen type I mRNA at different times of evolution of Lechiguana lesions, using isotopic and non-isotopic in situ hybridization. The results strongly suggest the involvement of a myofibroblast-like cell population that expresses mRNA for type I collagen and is probably associated with the increase of collagen deposition.     

Microscopic and ultrastructural studies of necrotizing hepatopancreatitis in Pacific white shrimp (Penaeus vannamei) cultured in Texas.

Granulomatous hepatopancreatitis of unknown etiology has been considered an important disease of Texas shrimp mariculture since 1985. Samples of Pacific white shrimp (Penaeus vannamei) were collected during 1986, 1987, and 1990 from three farms and an experimental mariculture facility with histories of production loss and increased mortality rates. Histologic and ultrastructural examination of shrimp from the four sites demonstrated two morphologically distinct, Gram-negative, double-enveloped, intracytoplasmic bacteria in necrotic hepatopancreatic epithelium. The more numerous small, pleomorphic rod as well as the helical organism are both taxonomically unclassifiable. The helical organism lacked ultrastructural characteristics of previously described helical or spiral bacteria. The relationship between the two organisms is unknown, but the pleomorphic rod is thought to play a major role in the disease. The role of a bacterial agent(s) in subsequent disease episodes is suggested by the observation that the use of oxytetracycline-medicated feed resulted in increased production and survival. Hepatopancreatic tubular epithelial necrosis and shrimp mortality correlated directly with the extent of infection by the small pleomorphic rod. Individual discrete bacteria were identified microscopically by Steiner and Steiner's method. Three major developmental stages of the disease were characterized based on the extent and number of hepatopancreatic tubular epithelial cells containing bacteria, the degree of tubular interstitial inflammation, and the extent and chronicity of tubular necrosis. Additional studies are needed to clarify the roles of the different bacteria identified and the potential role of environmental factors on the disease process.     

Effect of pen size on behavioral, endocrine, and immune responses of water buffalo (Bubalus bubalis) calves.

Female water buffalo (Bubalus bubalis) calves (n = 28) aged 7 to 10 d were divided into four groups of seven animals each to examine the effects of space allowance (Group A: 2.6 indoor m2 + 2.0 outdoor m2/calf; Group B: 2.6 indoor m2/calf; Group C: 1.5 indoor m2/calf; Group D: 1.0 indoor m2/calf) on behavioral, endocrine, and immune variables for a period of 60 d. Animals were offered 7 L/d of a commercial acidified milk substitute. The calves averaged 45.9 kg initially and 92.4 kg finally. The behavior observations were conducted 7 d after grouping and fortnightly thereafter. At wk 4 and 8, the phytohemagglutinin (PHA) skin test was performed to induce aspecific delayed hypersensitivity. At wk. 1 and 3, calves were injected i.m. with keyhole limpet hemocyanin. Antibody titers were determined at weekly intervals for 7 wk. Calves in pens with greater space allowance (Groups A and B) were less active than Groups C and D (P<.001). The latter groups were also observed feeding more often at wk 7 (P<.01). Calves provided with an outdoor paddock spent less time standing than Groups C and D (P<.01), and lay with a greater number of outstretched legs (P<.001). Groups C and D showed a lower reaction to PHA in both skin tests than did Groups A and B (P<.001 and P<.05, respectively). Group A showed an antibody response consistently higher than groups B, C, and D (P<.01, P<.05, and P<.05, respectively). At the end of the experimental period, the calves were subjected to an isolation test lasting 10 min. Group D showed a longer duration of movement with respect to Groups A and B (P<.01); animals from Group C walked more than did Group A (P<.05). Cortisol concentration evaluated 0, 10, 45, 90, 150, and 225 min after separation from the group was higher in Groups C and D than in Groups A and B (P<.01). For all animals, the highest cortisol level was observed immediately after the isolation test (P<.001). Space restriction resulted in evidence of stress in the animals as shown by alterations in a number of physiological responses. However, the use of small groups of only seven animals per pen may have affected their reactions to space restriction. It is possible that using larger groups could change these conclusions.     

Blood Supply to the Abdominal and Pelvic Regions in the Mediterranean Pine Vole, Microtus duodecimcostatus (Rodentia, Arvicolidae)

The abdominal and pelvic blood supply of the Mediterranean pine vole ( Microtus duodecimcostatus ) is described. A total of 13 individuals (seven males, six females) captured in the field (Sanaüja, Spain) were analysed by injection of coloured latex solution and subsequent dissection. The arrangements observed in all specimens show some common traits in relation to the patterns established in other Arvicolidae studied ( Arvicola terrestris, Clethrionomys glareolus, Microtus H agrestis ). These shared characteristics are centred on the origin of the deep circumflex iliac and the medial femoral circumflex arteries. As a particular variation from other Arvicolidae, some specimens of M. duodecimcostatus exhibit a gastrolienal trunk in the coeliac axis. Furthermore, the origin of the prostatic or the vaginal artery from the pudendo-epigastric trunk observed in some specimens of M. duodecimcostatus has not previously been reported in other myomorph rodents.     

Hematologic, cytochemical, ultrastructural, and molecular findings of Hepatozoon-infected flat-headed cats (Prionailurus planiceps)

BACKGROUND: The flat-headed cat (Prionailurus planiceps) is a small wild cat of Southeast Asia and is considered extremely endangered. Little is known about the hematologic values, blood cell morphology, or hemoparasites of this species in relation to other Felidae. OBJECTIVES: The objective of this study was to report basic hematologic values and describe the light microscopic, cytochemical, and ultrastructural characteristics of blood cells in 2 wild-caught flat-headed cats. In addition, molecular analysis was done of a Hepatozoon organism found in the neutrophils of both cats. METHODS: Blood samples were collected into EDTA from the cephalic vein. A CBC, manual differential count, manual reticulocyte count, cytochemical stains (Sudan black B [SBB], alpha-naphthyl acetate esterase [ANAE], and beta-glucuronidase), and scanning and transmission electron microscopy were done using standard methods. RESULTS: HCT was slightly lower and reticulocyte counts and red cell distribution width were higher than the expected values for other species of cats. Hepatozoon organisms were found in the cytoplasm of neutrophils in both cats, but the number of infected neutrophils was very low (1%-2%). Neutrophils stained strongly positive for SBB, but were negative for ANAE and beta-glucuronidase. Hepatozoon-infected neutrophils were negative for SBB, but focally positive for ANAE and beta-glucuronidase. By transmission electron microscopy, gamonts of Hepatozoon sp were observed in neutrophils, and rarely free in plasma. Infected neutrophils had fewer specific granules and more mitochondria compared with noninfected neutrophils. PCR products of partial 18S rRNA revealed that the isolate of Hepatozoon in the flat-headed cats was closely related to that of the frog Hepatozoon sp. CONCLUSIONS: These results add to our understanding of hematologic values and blood cell morphology in Hepatozoon-infected flat-headed cats as well as the molecular analysis of the Hepatozoon organism, and may be useful for the health management and evaluation of hemoparasitic disease in this species.     

Morphological and Immunohistochemical Considerations on the Basal Ganglia in Pigeon (Columba livia)

Morphological and immunohistochemical studies carried out particularly around the level of the basal ganglia (BG) in birds, are reported and commented on. Our results showed, on paraffin embedded avian BG, both the histological features and the immunohistochemical findings on immunofluorescence distribution of some neuropeptides (especially Metenkephalin) in the striatal complex. By comparing our results of Metenkephalin immunoreactivity (Menkir) with the referred analogous ones of Substance P (SP) quoted in literature, we confirmed the strikingly similar labelling at the levels of the Lobus paraolfactorius (LPO) and Paleostriatum augmentatum (PA), in contrast with the very low immunoreactivity at the Paleostriatum primitivum (PP) levels. We were able also to demonstrate the strong MEnk-ir of the neurons of the Nucleus accumbens, Nucleus septalis and paraventricularis. All these findings are also in accord to the interpretation by many AA (Wynne and Gunturkun. 1995; Reinez et al., 1983). about the anatomical correspondence between the LPO-PA complex of birds and the caudate-putamen in mammals. Some MEnk +‘wooly like fibres’ described in our specimens in the PA (on the contrary reported in the Gpe segment of mammals) apparently escape these correspondence.