Macro-microscopic study on the toepad of ostrich (Struthio camelus)

The ostrich foot has four toepads, two on the 3rd digit, one on the 4th digit and one at metatarso-phalangeal joint. Previous studies have not detailed the histo-morphological structure of these toepads. In this study, we have described the macroscopic and microscopic structures of the toepad of ostrich (Struthio camelus). Numerous papillae with different direction, length and thickness have been observed grossly on the ventral surface of each toepad. Histological examinations have revealed that the epidermis of the ostrich toepad, similar to other digitigrades, consists of an outer stratum corneum and an inner stratum germinativum (which is subdivided into basal, intermediate and transitional layers). The stratum corneum has several layers of flattened horny cells. The nuclei of basal cells have several mitotic figures. The cytoplasm of the stratum germinativum cells has multiple lipid droplets and multigranular bodies (in transitional cells only). Scanning electron microscopic examination revealed presence of collagen fibers in mid and deep dermis of each toepad. These fibers run parallel and connect to each other by very thin fibrils which are branched, crossed with each other in an oblique direction. Such arrangement of these collagen fibers, thin fibrils and presence of digital cushion are likely to be responsible for the protection of the underlying soft tissues and absorption of concussion.     

Sites of persistence of feline calicivirus

Various tissues were collected from eight cats persistently infected with feline calicivirus (FCV) strain 255 to determine the sites of viral persistence. Tissues were tested by virus isolation and an immunohistochemical technique in which infected cells were detected in formalin-fixed, paraffin-embedded tissue sections using rabbit antiserum to FCV 255, a biotinylated second antibody and streptavidin-peroxidase. Virus was detected by one or both techniques in tonsillar tissues of each animal, and not in other samples. Infected cells were detected in samples from six of eight kittens, and in each animal were few in number, and were cells of the superficial tonsillar epithelium or the stratum germinativum of the adjacent fossa mucosa. Transmission electron microscopic examination of tissues from three of the cats revealed calicivirus-like particles in cells similar to those identified immunohistochemically. These results confirm that the tonsillar region is the major site of FCV persistence and indicate that virus replication during persistence is confined to the surface epithelium of the tonsil and adjacent fossa mucosa.     

Diagnosis of infectious canine hepatitis virus (CAV-1) infection in puppies with encephalopathy.

Nine weaned Labrador Retriever puppies from a litter of 11 were presented with signs of acute central nervous system (CNS) disease that included ataxia and blindness. All puppies died. Gross examination of tissues from 2 puppies revealed regionally diffuse hemorrhages in the brain stem and swollen hemorrhagic lymph nodes. Light microscopic examination of hematoxylin and eosin-stained tissues showed numerous large, basophilic intranuclear inclusion bodies within CNS vascular endothelium and occasionally in individual hepatocytes. Immunohistochemical staining of the tissue was positive using an antibody against canine adenovirus-1. Virus isolation for infectious canine hepatitis virus was achieved using inoculated cell cultures. Polymerase chain reaction amplification of DNA from cell culture material revealed shared homology with other mammalian adenoviruses.     

A Rapid Method for the Diagnosis of Equine Virus Abortion

Smears and imprints were made from the liver of 27 equine fetuses, believed to have aborted as a result of Equine Virus Abortion (EVA) infection. Several different fixatives and staining techniques were employed for the demonstration of typical intra-nuclear inclusion bodies in these preparations, and the following conclusions were reached. Methanol proved to be the best fixative and Pappenheim's panoptic method was the best staining technique, giving good contrast and definition of the inclusion bodies. Cytological methods provided a simple and rapid means of diagnosis, but histological sections provided evidence of lesions which was most useful when inclusion bodies were very difficult to find. However, cytological methods proved better than histological sections for the demonstration of EVA intranuclear inclusion bodies.     

Occurrence of intracytoplasmic inclusion bodies in the digestive epithelium of fallow deer (Dama dama L)

Intracytoplasmic inclusion bodies were observed in the digestive epithelium of fallow deer (Dama dama L) suffering from bovine virus diarrhea/mucosal disease. Similar inclusion bodies were also found in the ruminal epithelium of fallow deer subjected to overfeeding by supplementary food. Inclusion bodies were not found in the upper alimentary mucosa of clinically healthy deer but were frequently found when these tissues were subjected to autolysis. At electron microscopical studies the inclusion bodies were found to consist of granular protein-like material encircled by a single membrane. Such inclusion bodies may constitute a non-specific degenerative cell response which could be elicited by diverse factors including autolysis.     

Herpesvirus infection in tortoises (Malacochersus tornieri and Testudo horsfieldii)

Large numbers of pancake tortoises (Malacochersus tornieri) and Horsfield tortoises (Testudo horsfieldii) in three consignments imported into Japan died soon after arrival. Some tortoises in the first consignment were dead on arrival. Postmortem examination of two of the pancake tortoises and four of the Horsfield tortoises revealed necrotizing lesions of the oral mucosa in both species, primarily in the tongue. Eosinophilic to amphophilic inclusion bodies were visible in the nuclei of mucosal epithelial cells in the lesions. Similar inclusion bodies were observed in the liver, spleen, adrenal glands, stomach, lungs, kidneys, small and large intestines, pancreas, and cerebrum of the pancake tortoises and in the liver, spleen, and pancreas of the Horsfield tortoises. Electron microscopic examination of the cells containing inclusion bodies showed herpesvirus-like particles about 100 nm in diameter in the cytoplasm. Nested polymerase chain reaction analysis using a herpesvirus consensus primer method confirmed the presence of a characteristic herpesvirus base sequence in tissue from these lesions.     

Inclusion Body Hepatitis in Young Broiler Breeders Associated with a Serotype 2 Adenovirus in Ontario,Canada

In July 2001 in southwestern Ontario, a young broiler breeder pullet flock experienced 2.1% mortality from 10 to 14 d of age. Mortality prior to and after this period was normal. Clinical signs included depression, lateral recumbency, and death with feed present in the crop. Necropsy lesions included friable, pale yellow-white livers scattered with hemorrhages. Histological examination revealed necrotizing hepatitis with large basophilic intranuclear inclusion bodies in the hepatocytes compatible with inclusion body hepatitis (IBH). An adenovirus from livers was isolated in chicken hepatoma cells. Virus neutralization and DNA analysis based on restriction fragment length polymorphism performed on this isolate were consistent with group I fowl adenovirus serotype 2. The origin of the adenovirus involved in this outbreak of IBH could not be established with certainty.     

Hepatic intranuclear inclusion bodies in a cockatoo bird (Cacatua sulphurea)

One hundred and nineteen cockatoos (Cacatua sulphurea) were kept at the Animal Quarantine Station in Denpasar, Indonesia. These birds were intended to be exported overseas. Nine birds were found dead in a period of 17 days. Necropsies were performed on three, and tissues were taken for microbiological and histopathological examination. Examinations revealed that two birds had both Newcastle disease and aspergillosis, and one bird was found to contain hepatic intranuclear inclusion bodies suggestive of Pacheco's parrot disease. Newcastle disease and aspergillosis in cockatoos have frequently been diagnosed in our laboratory. However, the finding of hepatic intranuclear inclusion bodies in a cockatoo is considered to be the first in Indonesia.     

Herpesvirus-associated papillomas in koi carp (Cyprinus carpio).

From January through November 1994, 32% (7/22) of koi carp (Cyprinus carpio) maintained in indoor aquariums developed proliferative cutaneous lesions that consisted of single to multiple 2-10-mm whitish to pink fleshy masses usually associated with fin rays. Although scaleless koi were more commonly affected (3/6) than were normally scaled koi (4/16), the difference in incidence rates was not significant (chi2 text, P > 0.05). Lesions typically resolved spontaneously in 1-3 wk, occasionally persisted for >3 mo, and recurred in several fish after 2-5 mo. Fish were otherwise asymptomatic. Wet mount preparations from lesions were densely cellular and consisted of hyperplastic epidermal cells of normal morphology without parasites or inflammatory cells. Histologically, biopsies were consistent with papillomas and were characterized by a marked benign epidermal hyperplasia without inclusion bodies or inflammatory infiltrate. Transmission electron microscopic examination revealed intranuclear and intracytoplasmic herpesvirus virions. Virus isolation attempts were unsuccessful.     

Intranuclear inclusions in cells infected with Newcastle disease virus.

Cells infected by Newcastle Disease Virus were observed to contain both intracytoplasmic and intranuclear inclusion bodies. Ultrastructurally, they consisted of twisted strands of about 18-20 nm diameter resembling nucleocapsids. The presence of these inclusions was detected irrespective of host cell or pathogenicity of the virus. In immunofluorescence and immunogold labelling experiments, these structures were tagged by an anti-P protein monoclonal antibody. In summary, we show that intracytoplasmic and intranuclear inclusion bodies, hitherto used as a taxonomic characteristic for the genus Morbillivirus of the Paramyxoviridae, also occur in a member of the genus Rubulavirus.     

Avian polyomavirus infection of a fledgling budgerigar (Melopsittacus undulatus) and differential diagnoses of viral inclusions in psittacine birds--case report and mini-review

A two-week-old budgerigar (Melopsittacus undulatus) of an outdoor aviary died suddenly and was submitted for determination the cause of illness and death. Macroscopically, the sparsely feathered animal was in a poor body condition. Histopathological examination revealed in various mesenchymal and epithelial tissues, numerous up to 15 microm in diameter large intranuclear, amphophilic to basophilic inclusion bodies with a clearing of the centre. Additionally, a feather dysplasia and retention hyperkeratosis of feather follicles was found. Ultrastructurally, viral particles of approximately 35 nm in diameter were detected in the feather follicle epithelium. A PCR for Avian Polyomavirus on fresh skin samples was negative whereas on formalin-fixed kidney samples with a high amount of viral inclusion bodies yielded a positive result. In addition, viral inclusion body diseases, like Avian Poxvirus, Psittacine Beak and Feather disease virus, Avian Adenovirus, Psittacine Herpesvirus and papillomavirus of psittacines are summarized and compared in the present article.     

Detection of fowlpox virus DNA by in situ hybridization using a biotinylated probe

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.     

Intranuclear Inclusions in Cells Infected with Newcastle Disease Virus

Cells infected by Newcastle Disease Virus were observed to contain both intracytoplasmic and intranuclear inclusion bodies. Ultrastructurally, they consisted of twisted strands of about 18–20 nm diameter resembling nucleocapsids. The presence of these inclusions was detected irrespective of host cell or pathogenicity of the virus. In immunofluorescence and immunogold labelling experiments, these structures were tagged by an anti-P protein monoclonal antibody. In summary, we show that intracytoplasmic and intranuclear inclusion bodies, hitherto used as a taxonomic characteristic for the genus Morbillivirus of the Paramyxoviridae, also occur in a member of the genus Rubulavirus.     

Fecal microbiome of growing pigs fed a cereal based diet including chicory(Cichorium intybus L.) or ribwort(Plantago lanceolata L.) forage

Background: The purpose of this study was to investigate how inclusion of chicory forage or ribwort forage in a cereal-based diet influenced the fecal microbial community(microbiome) in newly weaned(35 days of age) piglets.The piglets were fed a cereal-based diet without(B) and with inclusion(80 and 160 g/kg air-dry forage) of vegetative shoots of chicory(C) and leaves of ribwort(R) forage in a 35-day growth trial. Fecal samples were collected at the start(D0), 17(D17) and 35(D35) days after weaning and profiles of the microbial consortia were generated using terminal restriction fragment length polymorphism(T-RFLP). 454-FLX pyrosequencing of 16 S r RNA gene amplicons was used to analyze the microbial composition in a subset of the samples already analyzed with T-RFLP.Results: The microbial clustering pattern was primarily dependent on age of the pigs, but diet effects could also be observed. Lactobacilli and enterobacteria were more abundant at D0, whereas the genera Streptococcus, Treponema,Clostridium, Clostridiaceae1 and Coprococcus were present in higher abundances at D35. Pigs fed ribwort had an increased abundance of sequences classified as Treponema and a reduction in lactobacilli. However, the abundance of Prevotellaceae increased with age in on both the chicory and the ribwort diet. Moreover, there were significant correlations between the abundance of Bacteroides and the digested amount of galactose, uronic acids and total non-starch polysaccharides, and between the abundance of Bacteroidales and the digested amount of xylose.Conclusion: This study demonstrated that both chicory and ribwort inclusion in the diet of newly weaned pigs influenced the composition of the fecal microbiota and that digestion of specific dietary components was correlated with species composition of the microbiota. Moreover, this study showed that the gut will be exposed to a dramatic shift in the microbial community structure several weeks after weaning.     

Isolation and characterization of an antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion body disease virus (IBDV: Retroviridae)

The relationship between a retroviral infection and the development of nonviral intracytoplasmic inclusion bodies was studied in a Boa constrictor model. Twelve juvenile age- and size-matched inclusion body disease (IBD)-negative boas were randomly divided into three groups. Each group was inoculated intraperitoneally with 1 ml of an IBD virus (IBDV)-infected liver homogenate or 1 ml of normal boa liver homogenate (sham-inoculated control) or was left untreated. All boas were monitored for development of IBD by daily examination and serial liver biopsy over 1 year. The 4 IBDV-inoculated boas became IBDV and inclusion positive by 10 weeks postinoculation. The average size and density of inclusion bodies increased with the duration of infection. Ultrastructurally, inclusion bodies <2 microm in diameter consisted of intracytoplasmic aggregates of granular electron-dense material that were not membrane limited. Larger inclusions (3-6 microm in diameter) were characterized as membrane-bound aggregates of amorphous to granular electron-dense material admixed with membranelike fragments. The sham-inoculated and untreated control snakes did not become inclusion or IBDV positive. Direct comparison of the protein electrophoretograms of IBDV-infected and normal boa tissues demonstrated a prominent 68-kd protein band unique to infected inclusion-positive tissues. Monoclonal antibodies directed against the 68-kd protein band specifically labeled inclusion bodies. The results of this study demonstrate that IBD inclusions represent an intracytoplasmic accumulation of an antigenically distinct IBDV-associated protein.     

An outbreak of adenoviral infection in inland bearded dragons (Pogona vitticeps) coinfected with dependovirus and coccidial protozoa (Isospora sp.).

Thirty of 200 (15%) hatchling inland bearded dragons were found dead after a short period (48 hours) of weakness and lethargy. The most common clinical signs were head tilt and circling. Six bearded dragons with neurological signs were euthanized, and postmortem examination revealed no gross abnormalities. Microscopically, severe, randomly distributed hepatocellular necrosis with large basophilic intranuclear inclusion bodies in numerous hepatocytes was noted. Small-intestinal enterocytes contained intracytoplasmic coccidial protozoa (Isospora sp.) and occasional enterocytes had basophilic intranuclear inclusion bodies. Transmission electron microscopy revealed both 80- and 20-nm-diameter viral particles, which were consistent with adenoviruses and dependoviruses, respectively. Adenoviral outbreaks in groups of animals are uncommon. An adverse synergistic effect of the coccidiosis with the adenoviral infection may have played a critical role in the high morbidity and mortality in this case.     

Adenoviral fibrinous enteritis in calves

Two calves coming from different herds showed at post-mortem examination a fibrinous enteritis with mucosal necrosis particularly in the ileal-colonic tract. Histologically, amphophilic intranuclear inclusion bodies were the main feature. They were located in the endothelial cells of the intestine and mesenteric lymph cells. Ultramicroscopic examination of the inclusion bodies revealed the presence of an adenovirus. In one case the virus was isolated on cell cultures and further classified as bovine adenovirus type 4.     

Inclusion body disease in two captive Australian pythons (Morelia spilota variegata and Morelia spilota spilota)

Two captive Australian pythons, one carpet and one diamond python, presented with signs of central nervous system dysfunction. The carpet python was agitated. Its head was tilting and it was incoordinated and had convulsions. It was treated with antibiotics and anthelmintics but was eventually euthanased after failing to respond to therapy. The diamond python had flaccid paralysis of the caudal half. It was not treated and became disoriented and died. Hepatocytes from both pythons contained irregular 2 to 10 m m eosinophilic intracytoplasmic inclusion bodies. The brain of the diamond python was not available for examination. Occasional neurones in the carpet python brain contained similar inclusion bodies and other changes suggestive of viral infection. The clinical signs and histopathological findings in both pythons were consistent with boid inclusion body disease.     

Herpes simplex infection in a juvenile orangutan (Pongo pygmaeus pygmaeus).

A juvenile orangutan (Pongo pygmaeus pygmaeus) died after 8 days of diarrhea and vomiting. Necropsy showed petechial hemorrhages in the skin, the myocardium, and the peritoneal membranes. The lungs were hyperemic and edematous, and the liver and spleen were enlarged. Histologic changes consisted of interstitial pneumonia, hepatitis, and splenic hyperplasia. Numerous eosinophilic intranuclear inclusion bodies were visible in pulmonary epithelial cells, hepatocytes, and splenic endothelial cells. Electron microscopic examination revealed herpesvirus in hepatocyte nuclei. Polymerase chain reaction of liver tissue demonstrated the presence of a herpes simplex virus-1.