Molecular sexing of Jungle crow (Corvus macrorhynchos japonensis) and Carrion crow (Corvus corone corone) using a feather

A molecular sexing method using the intron‐size difference between chromo‐helicase‐DNA binding protein (CHD1) genes on the Z and W sex chromosomes (CHD1Z and CHD1W, respectively) was developed for wild Jungle crows and wild Carrion crows. The polymerase chain reaction product sizes were 661 bp for CHD1Z and 465 bp for CHD1W in both the Jungle and the Carrion crows. Each male crow sample produced a single fragment (CHD1Z), whereas female samples produced two fragments (CHD1Z and CHD1W), which differed by 200 bp in crows. In the present study, sexing live Jungle and Carrion crows was performed by applying the method to a feather for molecular sexing.     

Characterization of Campylobacter jejuni in large-billed crows (Corvus macrorhynchos) in Tochigi prefecture,Japan

As free-living crows are a potential source of Campylobacter infections in broilers and cattle, we characterized Campylobacter spp. isolated from crows using multilocus sequence typing and antimicrobial susceptibility testing. We obtained 82 samples from 27 birds captured at seven different times using a trap set in Tochigi prefecture, Japan. Campylobacter jejuni was isolated from 55 (67.1%) of the 82 samples and classified into 29 sequence types, of which 21 were novel. Tetracycline and streptomycin resistance rates were 18.2% and 3.6%, respectively. These results show that most types of C. jejuni infecting crows differ from those isolated from humans, broilers, and cattle. Thus, the importance of free-living crows as reservoirs of Campylobacter infections in broilers and cattle may be limited.     

Gross morphological features of the air sacs of the hooded crow (Corvus cornix)

Air sacs are considered to be one of the controlling factors of bird behaviour and habits in addition to their roles in ventilation, regulating body temperature, swimming and flight. As a scavenger and an omnivorous flight bird, air sacs of the hooded crow were the focus of this study. Eight healthy, adult hooded crows were used to examine the morphological characteristics of the air sacs, which were examined grossly and with latex and cast preparations. In general, the morphological overview of the hooded crow air sacs is similar to other avian species. We observed nine air sacs; four paired sacs (cervical, cranial thoracic, caudal thoracic and abdominal air sacs) and one unpaired sac; the clavicular air sac. The cervical air sac communicated to the lung through the medioventral bronchus and had three diverticula; intermuscular, subscapular and subcutaneous. The clavicular air sac communicated with lung through the medioventral bronchus and had subscapular, axillary, humeral, subpectoral and sternal diverticula. The cranial and caudal thoracic air sacs were communicated with lung through the lateroventral bronchi and the both sacs did not have any diverticula. The abdominal air sacs were posterior to the caudal thoracic air sacs. The left abdominal sac was the largest air sac. The right and left abdominal sacs gave off branches to diverticula that pneumatized synsacrum. The abdominal air sacs gave off femoral diverticula behind the hip joint as well as perirenal diverticula.     

Combined infection by avian poxvirus and Collyriclum faba in an American crow (Corvus brachyrhynchos).

An adult American crow (Corvus brachyrhynchos) from Virginia, USA, was diagnosed with combined infection of avian poxvirus and the skin fluke Collyriclum faba. The flukes and viral inclusions were combined in a large (4 x 4 cm) multilobulated proliferative mass on the ventrum just cranial to the cloaca. The flukes were identified using light microscopy of organisms obtained by antemortem wedge biopsy. Intraepithelial cytoplasmic inclusions consistent with poxvirus infection were seen on histopathologic examination of the mass.     

Gross morphological,histological and scanning electron specifications of the oropharyngeal cavity of the hooded crow (Corvus cornix pallescens)

The present study was carried out on the oropharyngeal cavity of the hooded crow to investigate the gross and microscopic structures via gross anatomy, light microscopy and scanning electron microscopy (SEM). The gross anatomy clarified the elongated triangular shape of the oropharyngeal cavity with a non-protruding tongue with a bifid apex. The lingual body contained median groove rostrally and separated caudally from the root by a transverse papillary crest. The laryngeal mound located posterior to the lingual root, contained midline laryngeal cleft and bounded caudally by a transverse row of pharyngeal papillae. The palate contained choanal cleft rostrally and infundibular slit caudally in addition to five palatine ridges. By light microscopy, the dorsal lingual epithelium was highly keratinised stratified squamous with a lingual nail in the most rostral part of the apex. Then, the thickness of the keratin layer decreased caudally, while in the ventral surface, the lining epithelium became non-keratinised. The entoglossum supported the lingual body and root, but not extended to the apex. The lining epithelium of the palate was also keratinised stratified squamous and became none-keratinised at the oral side of the choanal cleft. There were numerous lobules of polystomatic salivary glands in the lingual root and the palate. SEM revealed the arrangement of different types of papillae covering both the floor and the roof of the oropharynx besides numerous openings of salivary glands in the lingual root, laryngeal mound and the palate. These findings reflect the functional relationship of the oropharyngeal cavity of the hooded crow during feeding.     

Seasonal changes in the histochemical properties of the olfactory epithelium and vomeronasal organ in the Japanese striped snake, Elaphe quadrivirgata

Seasonal changes in the histochemical properties of the vomeronasal and olfactory epithelia of the Japanese striped snake were examined in four seasons, viz. the reproductive, pre-hibernating, hibernating and post-hibernating seasons. In the vomeronasal and olfactory supporting cells, secretory granules were much more abundant in the hibernating season than in the other seasons. In the vomeronasal and olfactory receptor cells, the lipofuscin granules were much fewer in the post-hibernating season than in the other seasons. In histochemical studies with 21 lectins, several lectins stained the vomeronasal and olfactory epithelia (receptor cells, supporting cells and free border) more weakly in the hibernating season than in the reproductive season. However, all lectins stained both epithelia in the hibernating season after sialic acid removal in a similar manner as in the reproductive season after sialic acid removal. These lectin histochemical studies indicate that sialic acid residues in the vomeronasal and olfactory epithelia are more numerous in the hibernating season than in the reproductive season. The results suggest that during hibernation, the vomeronasal and olfactory receptor cells possibly undergo rapid cell turnover, and that during this time, the vomeronasal and olfactory epithelia are securely protected from pathogens by an innate immune defence system.     

Lectin histochemical study on the olfactory bulb of the newt, Cynops pyrrhogaster

The function and/or morphological features of the vomeronasal olfactory system remain unclear in aquatic animals, although the system appeared first in urodeles based on phylogenic data. We examined the lectin binding patterns in the olfactory bulb of a semi-aquatic urodele, the Japanese red-bellied newt, Cynops pyrrhogaster, using 22 different lectins. Eleven of the lectins showed specific binding to the nerve fibres and glomeruli in the olfactory bulb. Among these, Wheat germ agglutinin, pokeweed and peanut agglutinin preferentially bound the main olfactory bulb, reflecting variation in the expression of glycoconjugates between the main and accessory olfactory bulbs. By contrast, the types of lectins bound to the Cynops olfactory bulb were considerably different from those reported in other urodele families. These results suggest a histochemical distinction between the main and accessory olfactory bulbs, and that glycoconjugate expression may differ significantly among urodele families.     

DNA vaccination of the American crow (Corvus brachyrhynchos) provides partial protection against lethal challenge with West Nile virus

The New York 1999 strain of West Nile virus (WNV) is nearly 100% fatal in the American crow (Corvus brachyrhynchos). We evaluated four WNV vaccine formulations in American crows, including intramuscular (i.m.) DNA vaccine, i.m. DNA vaccine with adjuvant, orally administered microencapsulated DNA vaccine, and i.m. killed vaccine. Neutralizing antibodies developed in approximately 80% of crows that received the DNA vaccine i.m. (with or without adjuvant), and in 44% that received the killed vaccine. However, no crows that received the oral microencapsulated DNA vaccine or the placebo developed WNV antibodies. All crows were challenged 10 wk after initial vaccination. No unvaccinated crows survived challenge, and survival rates were 44% (i.m. DNA vaccine), 60% (i.m. DNA vaccine with adjuvant), 0% (oral microencapsulated DNA vaccine), and 11% (killed vaccine). Peak viremia titers in the birds that survived were significantly lower as compared to titers in birds that died. Parenteral administration of a WNV DNA vaccine was associated with reduced mortality but did not provide sterile immunity.     

Pathology of fatal highly pathogenic H5N1 avian influenza virus infection in large-billed crows (Corvus macrorhynchos) during the 2004 outbreak in Japan

Highly pathogenic H5N1 avian influenza viruses were isolated in 9 large-billed crows that died in Kyoto and Osaka prefectures in Japan from March to April in 2004. We studied 3 of the 9 crows using standard histologic methods, immunohistochemistry, and virus isolation. The most prominent lesions were gross patchy areas of reddish discoloration in the pancreas. The consistent histologic lesions included severe multifocal necrotizing pancreatitis, focal degeneration and necrosis of neuron and glial cells in the central nervous system, and focal degeneration of cardiac myocytes. All of these tissues contained immunohistochemically positive influenza viral antigens. The virus was isolated from the brain, lung, heart, liver, spleen, and kidney of the crows examined. Thus we concluded that highly pathogenic avian influenza virus was associated with clinical disease, severe pathologic changes, and death in the 3 crows.     

Ultrastructural and enzyme histochemical aspects of amyloidosis in the bovine renal medulla.

Electron micrsocopic studies of bovine kidneys with amyloidosis showed macrophage-like cells in the interstitial amyloid that were either disseminated or in small groups. They had rather uniform protracted vacuoles filled with parallel fibrils. Similar vacuoles were also found in some reticular interstitial cells. In some samples electron-dense vacuoles and vacuoles containing fibrils were seen. Protracted lysosomal enzyme activity was seen in macrophage-like cells and reticular interstitial cells; these areas of activity showed green birefringence with Congo red. The vacuoles were thought to be lysosomes, and the parallel amyloid fibrils could be a consequence of intracellular formation or of phagocytosis.     

Prevalence of thermophilic campylobacters in crows (Corvus levaillantii, Corvus corone) and serogroups of the isolates

A total of 500 fecal droppings of crows collected from a seashore of an ocean bay and from a cemetery on a hill surrounded by a forest were examined for thermophilic campylobacters, and the Skirrow's biovars and Penner's serogroups of the isolates were determined. The organisms were isolated from 169 (62.6%) of 270 seashore crow samples and 106 (46.1%) of 230 cemetery crow samples. During the investigation period from May 1986 to April 1987, the monthly isolation rate of thermophilic campylobacters in the seashore crow varied from 32.0 to 85.0%. C. jejuni, C. coli, and C. laridis were isolated from 150, 21 and 14 samples, respectively. In case of the cemetery crow, the monthly isolation rate varied from 20.0 to 75.0%, and C. jejuni, C. coli, and C. laridis were detected from 80, 12 and 16 samples, respectively. Among 192 strains of C. jejuni selected from 98 seashore and 57 cemetery crow samples, 106 (93.0%) of 114 seashore crow strains and 69 (88.5%) of 78 cemetery crow strains were identified as Skirrow's biovar I. Of 192 strains of C. jejuni serogrouped, 169 strains were classified into 20 serogroups. The Penner's serogroup 2, one of common serogroups among poultry and human isolates in Japan, was the most predominant in crow strains.     

Variety in histochemical characteristics of the olfactory receptor cells in a flatfish, barfin flounder (Verasper moseri)

Variety in histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP9.5) and calretinin, and by lectin histochemistry with Phaseolus vulgaris leucoagglutinin (PHA-L) in the olfactory epithelium (OE) of the barfin flounder (Verasper moseri). PGP 9.5 immunoreactivity was observed in the ORC situated in the upper three fourths of the OE. Calretinin immunoreactivity was observed in the ORC which seemed to be immunonegative for PGP 9.5. These cells were located in the upper two thirds of the OE. PHA-L staining was observed in small subsets of the ORC. PGP 9.5 and calretinin immunoreactivities and PHA-L staining were also observed in the crypt cells unique to the fish OE. These findings suggest the different properties of olfactory perception among fish ORC.     

Ultrastructural alterations of the thyroid follicular cells in domestic fowls following neonatal olfactory bulbectomy

1. In order to investigate if ultrastructural alterations of thyroid follicular cells are induced by removal of the olfactory bulb at hatching, chicks were olfactory bulbectomised within 24 h after hatching and the thyroid follicular cells were examined at 5 months of age by electron microscopy. 2. Olfactory bulbectomised chickens showed a well-developed Golgi area, a highly-developed rough endoplasmic reticulum, many mitochondria and a paucity of secretory granules and lysosomes in thyroid follicular cells. 3. These hypertrophic ultrastructural changes in follicular cells of operated birds indicated that the thyroids glands were stimulated by neonatal olfactory bulbectomy. 4. This finding may suggest an establishment of the olfactory bulbothyroid glandular system during the early days of life in the chicken.     

Ultrastructural histochemical evaluation of growth plate cartilage matrix from healthy and osteochondritic swine

A contributing factor to the lack of understanding the cause of osteochondritic syndromes has been incomplete knowledge of the morphology of lesions in subclinical stages of the disease. In osteochondritic growth plate cartilage from growing swine, the morphology of the pericellular matrix surrounding hypertrophic zone chondrocytes is abnormal and is characteristic of a matrix in which the ordered interactions of matrix macromolecules with each other and with the plasma membrane have been altered. In the present study, ultrastructural histochemical techniques were used to analyze the nature of macromolecular interactions in the pericellular matrix in normal growth plate cartilage, and selective enzyme digestions of normal growth plate cartilage were used to simulate the morphology found in osteochondritic lesions. Results showed that a pericellular macromolecular material which was both ferrocyanide positive and trypsin sensitive was essential for stabilizing the cell membrane/pericellular interface in normal growth plates. The highly variable morphology of this same material in osteochondritic lesions was simulated by hyaluronidase digestion. Since similar pericellular matrix abnormalities have not been described in other diseases of growth plate cartilage, they may represent a matrix abnormality unique to the vascularization failure of osteochondritic syndromes. Our ability to simulate the ultrastructural morphology of subclinical osteochondritic lesions enhances the potential for understanding the macromolecular changes found in the pericellular matrix of osteochondritic cartilage. Based on these results, a new hypothesis is presented for the early sequence of events in the pathogenesis of osteochondrosis.     

Differential expression of histochemical characteristics in the developing olfactory receptor cells in a flatfish, barfin flounder (Verasper moseri)

Differentiation of the histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5) and calretinin (CR) and lectin histochemistry for Phaseolus vulgaris agglutinin-L (PHA-L) in the developing olfactory epithelium (OE) of the barfin flounder. PGP 9.5 immunoreactivity was diffuse and CR immunoreactivity was restricted at day 7, but these immunoreactivities became intense in the OE toward day 91. Crypt cells were first identified at day 56. PHA-L staining was faint at day 28, but became intense toward day 91. These findings suggest that PGP 9.5-immunopositive cells, CR-immunopositive cells, crypt cells and PHA-L-reactive cells differentiate independently in the developing OE and constitute subsets of the ORC in the OE.     

Histomorphology of the stomach, proventriculus and ventriculus of the red jungle fowl

The cranial chamber (proventriculus) and caudal chamber (ventriculus) of the stomach of the Red jungle fowl (Gallus gallus spadiceus) were examined by means of light microscopy. Both chambers presented folds of the tunica mucosa lined by a simple prismatic epithelium that was positive for neutral mucin. Simple tubular glands occupied the lamina propria of both chambers; in the ventriculus of older birds, they showed a coiled base. These ventricular glands were lined by simple cuboidal cells represented by the chief cells and a few large basal cells. The luminal and tubular koilin rodlets and folds of the ventriculus were positive to periodic acid Schiff (PAS) stain. The proventricular glands were situated between the inner and outer layers of the lamina muscularis mucosae. Cells lining the tubulo-alveolar units of the proventricular glands showed a dentate appearance. Vacuoles were not observed, and the cells were negative for Alcian-PAS stain. The tunica submucosa was very thin in the proventricular wall. In the ventriculus, it was not separated from the lamina propria owing to the absence of any lamina muscularis mucosae. The tunica muscularis of the proventriculus was formed by a thick inner layer of circular smooth muscle fibres and a thin outer layer of longitudinal fibres. In addition to these layers, oblique muscle fibres formed the most internal layer of the tunica muscularis in the ventriculus.     

Growth factors and their receptors in the olfactory system.

The olfactory epithelium is unique in the mammalian nervous system as it is a site of continual neurogenesis. Constant turnover of primary sensory neurons in the periphery results in continuous remodeling of neuronal circuits and synapses in the olfactory bulb throughout life. Most of the specific mechanisms and factors that control and modulate this process are not known. Recent studies suggest that growth factors, and their receptors, may play a crucial role in the development and continuous regeneration of olfactory neurons, i.e. particularly in neuronal proliferation, neurite outgrowth, fasciculation and synapse formation of the olfactory system. The potential role of the following factors and their receptors in different species are reviewed: Nerve growth factor (NGF); insulin-like growth factors (IGFs); fibroblast growth factors (FGFs); epidermal growth factor (EGF); transforming growth factor alpha (TGF alpha); amphiregulin (AR) and transforming growth factors beta (TGFs beta).     

West Nile virus antibody prevalence in American crows (Corvus brachyrhynchos) and fish crows (Corvus ossifragus) in Georgia, USA

Crows have been the centerpiece of avian West Nile virus (WNV) surveillance and research in North America. This work has demonstrated variation in susceptibility to WNV infection between American (Cor vus brachyrhynchos) andFish Crows (Corvus ossifragus). The higher WNV-associated mortality rate in American Crows compared with Fish Crows suggests that WNV antibody prevalence would be greater in the Fish Crow population. The objectives of this study were to 1) determine whether Fish Crows had higher WNV antibody prevalencethan American Crows, 2 ) determine th e persistence o f antibodies to WNV in naturally infected Fish Crows, and 3) develop a technique to distinguish Fish Crows from American Crows on the basis of sequence analysis and restriction enzyme digestion of a mitochondrial DNA fragment. West Nile virus antibody prevalence was 16.5% (n = 97) in Fish Crows and 5.7% in American Crows (n = 53) collected from Georgia between 2004 and 2006. Antibodies persisted at high titers for 12 mo in Fish Crows. This is the first report of WNV antibody persistence in a crow species. A polymerase chain reaction technique paired with restriction enzyme digestion easily distinguished American Crows from Fish Crows on the basis of a mitochondrial DNA fragment.     

Phylogenic aspects of the amphibian dual olfactory system

The phylogenic significance of the subdivision of dual olfactory system is reviewed mainly on the basis of our findings by electron microscopy and lectin histochemistry in the three amphibian species. The dual olfactory system is present in common in these species and consists of the projection from the olfactory epithelium (OE) to the main olfactory bulb (MOB) and that from the vomeronasal epithelium (VNE) to the accessory olfactory bulb (AOB). The phylogenic significance of subdivisions in the dual olfactory system in the amphibian must differently be interpreted. The subdivision of the MOB into its dorsal region (D-MOB) and ventral region (V-MOB) in Xenopus laevis must be attributed to the primitive features in their olfactory receptors. The middle cavity epithelium lining the middle cavity of this frog possesses both ciliated sensory cells and microvillous sensory cells, reminding the OE in fish. The subdivision of the AOB into the rostral (R-AOB) and caudal part (C-AOB) in Bufo japonicus formosus must be regarded as an advanced characteristic. The lack of subdivisions in both MOB and AOB in Cynops pyrrhogaster may reflect their phylogenic primitiveness. Since our lectin histochemistry to detect glycoconjugates expressed in the olfactory pathway reveals the subdivisions in the dual olfactory system in the amphibian, the glycoconjugates may deeply participate in the organization and function of olfactory pathways in phylogeny.