Tomoyuki Shibahara

Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome.

The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan, was investigated in specific-pathogen-free (SPF) chickens. One-day-old to 15-mo-old SPF chickens were inoculated intramuscularly, orally, and intranasally with liver homogenates from HPS chickens or isolated serotype 4 adenovirus. There were no clinical signs before death. The mortality rate in all groups of 1-day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates of 7-mo-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25%, respectively. In 15-mo-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%. Gross lesions were hydropericardium and swelling and congestion of the liver with occasional petechial hemorrhages. Histologically, the liver had diffuse or multifocal hepatic necrosis and hemorrhage with intranuclear inclusion bodies noted within hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at 1 day old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, cecum, kidney, and lung of the chicks inoculated at 1 day old. Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS. This study indicates that HPS adenovirus is able to reproduce HPS lesions and mortality in SPF chicks and even adult chickens and that it is a highly pathogenic strain.

Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks.

Ten 250-day-old broiler breeders, seven 16-day-old broiler chicks, and three 25-day-old broiler chicks suffering from hydropericardium syndrome (HPS) in Japan were examined histologically, immunohistochemically, and ultrastructurally. Clinically, the chickens died suddenly without apparent signs. The mortality rates were 6.4%, 20.2%, and 26.1%, respectively. The common characteristic histologic lesion was necrosis of hepatocytes, accompanied by intranuclear inclusions of hepatocytes and hemorrhages. In the spleen, there were activation of macrophages in splenic sinus and ellipsoids and erythrophagocytosis in the splenic sinus. The interlobular interstitium of the lung showed marked edema. The air and blood capillary areas of parabronchi included many macrophages with yellow pigments. With immunoperoxidase staining, intranuclear inclusion bodies within degenerating hepatocytes stained positively for group I adenovirus antigen. Ultrastructurally, numerous viral particles (65-70 nm in diameter) were demonstrated in the intranuclear inclusions of hepatocytes. Group I adenovirus (serotype 4) was isolated from liver samples of adult broiler breeders and broiler chicks with HPS. This study suggests that HPS may be caused by group I adenovirus.

Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector

Erysipelothrix rhusiopathiae Koganei 65-0.15 strain, the live swine erysipelas vaccine for subcutaneous injection, has been shown to colonize the tonsils of pigs after oral inoculation. We thus evaluated the possible use of the strain as a vector for oral vaccination against mycoplasmal pneumonia of swine. Recombinant E. rhusiopathiae strains expressing the C-terminal domain of the P97 adhesin of Mycoplasma hyopneumoniae were constructed and examined for vaccine efficacy in mice and pigs. Mice subcutaneously inoculated with the recombinant strains were protected from challenge exposure to a virulent E. rhusiopathiae. Administration of milk replacer containing recombinant E. rhusiopathiae expressing the M. hyopneumoniae protein protected pigs from death after exposure to E. rhusiopathiae and significantly reduced the severity of pneumonic lung lesions caused by infection with M. hyopneumoniae.

Experimental West Nile Virus Infection in Aigamo Ducks, A Cross between Wild Ducks (Anas platyrhynchos) and Domestic Ducks (Anas platyrhynchos var. domesticus)

Abstract Four 2-wk-old and four 4-wk-old aigamo ducks, a cross between wild and domestic ducks (Anas platyrhynchos and Anas platyrhynchos var. domesticus, respectively), were infected with the NY99 strain of West Nile virus (WNV) to investigate WNVs pathogenicity in aigamo ducks and the possibility that they could transmit WNV. In the group of infected 2-wk-old aigamo ducks (2w-infection group), all of the ducks ate and drank less and showed decreased activity, some showed ataxia, and one died. Meanwhile, the group of infected 4 wk olds (4w-infection group) showed no clinical signs during the experimental period. Viremia was observed in all of the ducks in both age groups. Peak viral titers in the three surviving members of the 2w-infection group were 103.7–105.3 plaque-forming units (PFU)/ml serum; the peak was 107.1 PFU/ml serum in the 2w duck that died from the infection. Peak viral titers in the 4w-infection group were 104.1–104.9 PFU/ml serum. Viral shedding in the oral and/or cloacal cavity was observed in all four members of the 2w-infection group and in three of the four members of the 4w-infection group. These results suggest that WNV-infected aigamo ducks can transmit WNV. Although aigamo ducks are reared in East Asia, where WNV is an exotic pathogen, the virus could be introduced and spread there in the future; thus it is important to take precautions against an introduction, and measures to prevent infection to aigamo duck operations should be prepared.

Isolation of parapoxvirus from a cow treated with interferon-γ

A virus was isolated from peripheral blood leukocytes of a cow which was kept in an isolated pen after it was injected with recombinant bovine interferon-gamma. The virus was identified as a member of genus Parapoxvirus in the family Poxviridae on the basis of electron microscopic observations and serological tests. Parapoxvirus has seldom been isolated other than from papular lesions, the characteristic sign of parapoxvirus infection. This is the first report of parapoxvirus isolation from the peripheral blood of a cow without any clinical signs. These results show that parapoxviruses are capable of causing persistent infection in cattle without clinical signs and can be activated by stress factors that induce modification of immune reactions. Relationships between the isolated virus and other parapoxviruses isolated previously from cattle in Japan were investigated and discussed.

Comparative Pathology of Heart and Liver Lesions of Broiler Chickens That Died of Ascites, Heart Failure, and Others

Pathologic changes of 120 dead broilers from a flock of 1000 birds were compared. Macroscopically, the changes were divided into three pathologic types: ascites (retention of ascitic fluid), heart failure (right heart dilation and/or hydropericardium without retention of ascitic fluid), and others (without ascites or heart failure). The rates of ascites, heart failure, and others were 55.8% (67/120), 33.3% (40/120), and 10.8% (13/120), respectively. Histologically, cardiac histologic changes (myocardial degeneration with calcification and epicardial fibrosis) were seen in 40 of 67 (59.7%) ascites cases. Hepatic histologic changes (hepatocytic degeneration/necrosis and hepatic capsule fibrosis) were seen in 64 of 67 (95.5%) ascites cases. Cardiac histologic changes were seen in 24 of 40 heart failure cases (60.0%). Hepatic histologic changes were seen in 36 of 40 (90.0%) heart failure cases. Six of 13 others had hepatic histologic changes, but the remaining seven birds had no significant histologic changes. Ascites and heart failure cases had almost the same percentages of hepatic and cardiac histologic changes. This study indicates that two pathologic types, ascites and heart failure, may be closely related conditions and that hepatic histologic changes may be common and fundamental lesions in the pathogenesis of these pathologic types.

Ultrastructural characteristics and molecular identification of Entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity

Protozoan parasites of the genus Entamoeba infect many classes of vertebrates and are primarily classified based on morphological criteria. To date, only a few species have been proven to cause disease. Here, we examined the pathology of infected pigs with hemorrhage and detected Entamoeba parasites. Isolates were characterized genetically and ultrastructurally to identify the species. Histopathologically, bleeding and thrombus formation were seen only in the large intestine mucosa, where a large number of trophozoites or some Entamoeba cysts were observed around breakdowns in the lamina propria. No screw-shaped bacteria were detected in the lesions, and no pathogenic bacteria such as Brachyspira spp. were detected in fecal cultures. Interestingly, electron microscopy revealed that the parasites possessed mitochondrial organelles, unlike other Entamoeba spp. The isolates were identified as Entamoeba suis by PCR analysis and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. In phylogenetic analyses based on the actin gene, the E. suis isolate formed a cluster with Entamoeba histolytica and Entamoeba invadens, as well as with other parasites of the Amoebidae. Whether the pathogenicity of the E. suis isolate is affected by the severity of infection or host health status remains unclear; however, our results suggest that E. suis could cause or exacerbate clinical symptoms such as hemorrhagic colitis or diarrhea.

Needle-free jet injection of small doses of Japanese encephalitis DNA and inactivated vaccine mixture induces neutralizing antibodies in miniature pigs and protects against fetal death and mummification in pregnant sows

Japanese encephalitis (JE) virus causes abortion and stillbirth in swine, and encephalitis in humans and horses. We have previously reported that immunogenicity of a DNA vaccine against JE was synergistically enhanced in mice by co-immunization with a commercial inactivated JE vaccine (JEVAX) under a needle-free injection system. Here, we found that this immunization strategy was also effective in miniature pigs. Because of the synergism, miniature pigs immunized twice with a mixture of 10 μg of DNA and a 1/100 dose of JEVAX developed a high neutralizing antibody titer (1:190 at 90% plaque reduction assay). Even using 1 μg of DNA, 3 of 4 pigs developed neutralizing antibodies. Following challenge, all miniature pigs with detectable neutralizing antibodies were protected against viremia. Pregnant sows inoculated with 10 or 1 μg of DNA mixed with JEVAX (1/100 dose) developed antibody titers of 1:40-1:320. Following challenge, fetal death and mummification were protected against in DNA/JEVAX-immunized sows.

Genetic identification of Entamoeba polecki subtype 3 from pigs in Japan and characterisation of its pathogenic role in ulcerative colitis

To date, three Entamoeba spp. (E. suis, zoonotic E. polecki and E. histolytica) have been identified in pigs, but their pathogenicity and molecular classification have not been fully determined. Examination and pathological analysis of pigs (n=3) with diarrhoea was conducted and revealed the presence of Entamoeba organisms. We performed a genetic analysis of the isolate using the small-subunit ribosomal RNA (SSU rRNA) gene region to identify the species. A severe ulcerative colitis was observed histopathologically with inflammatory cells, including macrophages and neutrophils, infiltrating the mucous membranes of the cecum and colon. Many Entamoeba trophozoites were found at the erosion site or at ulcerative lesions. Pathogenic viruses or bacteria were not detected. The SSU rRNA sequence of the Entamoeba isolate was found to be completely homologous to that of E. polecki subtype 3.

Systemic Streptococcus dysgalactiae subspecies equisimilis infection in a Yorkshire pig with severe disseminated suppurative meningoencephalomyelitis.

ABSTRACT An 18-week-old female breeding Yorkshire pig displayed symptoms of astasia and subsequently died. Histologically, severe disseminated suppurative meningoencephalomyelitis was detected, as were numerous myocardial microabscesses. Gram-positive cocci were detected in these suppurative lesions, and these cocci reacted with an antibody against Streptococcus C group species. Gram-positive cocci were isolated from the liver, spleen, kidney, heart, lungs, pleural abscess and articular fluid of the right tarsal joint. The isolates were β-hemolytic, categorized into Lancefield group C and were identified as Streptococcus dysgalactiae subspecies equisimilis by analysis of the 16S ribosomal DNA sequence. This is the first report of systemic S. equisimilis infection in a pig with severe disseminated suppurative meningoencephalomyelitis.