Masaru Furuya

Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.

Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum

Background: Apicomplexan parasites, including Cryptosporidium, possess organelles associated with host cell invasion. Results: A chicken-derived monoclonal antibody inhibited host cell invasion by C. parvum. The corresponding antigen, which localizes to the apical region of the parasite, was identified as elongation factor-1α (EF-1α). Conclusion: EF-1α mediates cryptosporidial cytoskeletal complex critical for host cell invasion. Significance: The findings suggest that cryptosporidial EF-1α could serve as a novel anti-infective target antigen. The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Morphological changes and viability of Cryptosporidium parvum sporozoites after excystation in cell-free culture media.

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0-3 h (84.3% for banana-shaped and 49.2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan.

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.

Effect of low pH on the morphology and viability of Cryptosporidium andersoni sporozoites and histopathology in the stomachs of infected mice

The genus Cryptosporidium includes many common parasites infecting animals and humans, and is a major cause of diarrheal illness worldwide. The biology of gastric Cryptosporidium spp., including replication in the stomach, has not been well documented. This study evaluated the viability of Cryptosporidium andersoni sporozoites in gastric environments after excystation and examined the endogenous development and histopathological changes in the stomachs of infected mice, using a novel type of C. andersoni. Sporozoites were affected by low pH (61.6% viability after 3h at pH2.0). Electron microscopy revealed developmental parasites on the gastric foveolae but not on the surface of the gastric mucosa. Histopathological examinations at 1, 2, 4 and 12 weeks p.i. uncovered three different lesions. The gastric mucosa of foveolae filled with parasites was extended and the amount of neutral mucopolysaccharide at the mucosal surface was decreased with the first type of lesion. The gastric mucosa was atrophied, some gastric glands were disrupted and the amount of acid mucopolysaccharide at the mucosal surface was increased with the second type. Finally, the gastric mucosa was slightly extended and goblet cells were present in the gastric mucosa, indicating intestinal metaplasia, in the third type. No parasites were detected in these areas with increased acidic mucin and indications of metaplasia. The results suggest that C. andersoni parasites could not survive in acidic environments for a long period before invading host cells and preferentially develop in neutral sites of the gastric mucosa, resulting in histopathological changes and chronic shedding of oocysts.

Gicerin, an Ig-superfamily cell adhesion molecule, promotes the invasive and metastatic activities of a mouse fibroblast cell line

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and plays an important role during development through its adhesive properties. Gicerin has two isoforms that differ in their cytoplasmic domains; s‐gicerin is the shorter and l‐gicerin the longer form of the protein. Gicerin is over‐expressed in some sporadic tumors as well as in developing tissues. To provide direct evidence that gicerin has the potential to participate in malignant aspects of tumor cell behavior, a gicerin cDNA was introduced into L‐929 cells, an endogenous gicerin‐negative mouse fibroblast and subsequently analyzed for changes in their invasive and metastatic potential by implantation into nude mice and chick embryos. Compared with parental cells, both gicerin isoform transfectants showed an enhanced cell growth and invaded deeply into surrounding tissues from implanted sites in both animal models. Furthermore, l‐gicerin transfectants markedly enhanced metastasis to the lung. These findings suggest that gicerin promotes the tumor growth and invasion, and the isoform bearing the longer cytoplasmic domain may play a role in metastasis. J. Cell. Physiol. 197: 103–109, 2003© 2003 Wiley‐Liss, Inc.

Gicerin, a cell adhesion molecule, promotes the metastasis of lymphoma cells of the chicken

Gicerin is an immunoglobulin superfamily cell adhesion molecule purified from chicken gizzards. This molecule displays an adhesive interaction with a laminin-like protein as well as with gicerin itself. Gicerin appears in embryonic tissues and plays a role in chick development through its cell adhesive properties. An increase in gicerin expression is found in some sporadic tumors of the chicken. To elucidate the possible role of gicerin in tumor progression in chickens, we introduced gicerin cDNA into an endogenous gicerin negative lymphoma MDCC-MSB1 cell line, and subsequently analyzed them for changes in their metastatic potentials. After intravenous implantation of the gicerin transfectants into chickens, the metastatic potential to the lung, liver and kidney was enhanced compared with parental MDCC-MSB1 cells. Self-aggregation activity was increased in gicerin transfectants. In addition, adhesive and migratory activities of the gicerin transfectants to the gicerin ligands were enhanced in vitro. These findings indicate that gicerin can contribute to the malignancy and metastatic properties of lymphoma.

Molecular cloning and analysis of the mouse gicerin gene.

Gicerin is a cell adhesion molecule, which has five immunoglobulin-like loop structures in an extracellular domain followed by a single transmembrane domain and a short cytoplasmic tail. We have reported that gicerin participates in neurite extension and structural organization of the nervous system, and its expression in the nervous system is high during the development and dramatically decreased after birth. To elucidate the mechanism how the expression of gicerin is regulated, we performed a genomic cloning of a mouse gicerin. A fragment of 16 kbp genomic clone contained 8 kbp gicerin gene composed of 16 exons with 6 kbp upstream region. Genomic cloning revealed that two isoforms of gicerin were generated by an alternative splicing of exon 15 results in cytoplasmic domains composed of either 63 or 21 amino acids. As for an expressional regulation of gicerin, we found that the mRNA content of gicerin in PC12 cells was regulated by cAMP. Quantitative-PCR analysis revealed that forskolin induced four-fold increase of gicerin mRNA. To characterize the involvement of its promoter region, we examined the promoter activity in PC12 cells by a luciferase-reporter assay. We found that a CRE site located at 60 bp upstream of gicerin gene was responsible for the increase of its mRNA induced by forskolin.

Identification of novel tumour-associated antigens in canine mammary gland tumour.

UNLABELLED Canine mammary gland tumour (MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour-associated antigens (TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini-arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT-dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs. CLINICAL RELEVANCE HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT.

The detection of toxigenic Corynebacterium ulcerans from cats with nasal inflammation in Japan.

Corynebacterium ulcerans (toxigenic C. ulcerans) produces the diphtheria toxin, which causes pharyngeal and cutaneous diphtheria-like disease in people, and this bacterium is commonly detected in dogs and cats that are reared at home. It is considered dangerous when a carrier animal becomes the source of infection in people. To investigate the carrier situation of toxigenic C. ulcerans of cats bred in Japan, bacteria were isolated from 37 cats with a primary complaint of rhinitis in 16 veterinary hospitals in Osaka. Toxigenic C. ulcerans was detected in two of the cats. By drug sensitivity testing, the detected bacterium was sensitive to all investigated drugs, except clindamycin. It appears necessary to create awareness regarding toxigenic C. ulcerans infection in pet owners because this bacterium is believed to be the causative organism for rhinitis in cats.