Masaaki Satoh

Characteristics of a Novel Type of Bovine Cryptosporidium andersoni

ABSTRACT We isolated oocysts that resemble Cryptosporidium andersoni from cattle grazing on a farm in Japan. The partial sequences of genes from the isolate were coincident with published sequences of genes of C. andersoni. Since the isolate was able to infect SCID mice, the isolate appears to be a novel type of C. andersoni.

Morphological and Immunohistochemical Features of Cryptosporidium andersoni in Cattle

Light and electron microscopic features and immunohistochemical features of Cryptosporidium andersoni (C. andersoni) and host reaction in the mucosa were studied. Although the affected cattle demonstrated no apparent clinical signs, a severe infection of C. andersoni was observed in the abomasum. C. andersoni were round in shape, measured 6-8 μm in size and were mainly observed to be freely located in the gastric pits, being attached in occasional cases to the surface of the abomasum epithelium. Frequent inflammatory cells had infiltrated the lamina propria of the affected mucosa, and frequent mitotic figures were observed in epithelial cells at the dilated isthmus. To access the cell kinetics, the number of epithelial cells infected with C. andersoni were counted and compared with noninfected cattle. The number of gastric pit cells in infected cattle was significantly higher than that in the controls. The number of proliferative cells determined by the Ki-67 antigen in C. andersoni infected cattle was also significantly higher than that in the controls. Transmission electron microscopy and scanning electron microscopy revealed that the morphology of the C. andersoni organism was common to those of other Cryptosporidium spp. immunohistochemically, several commercial antibodies against Cryptosporidium spp. showed positive reactions at the wall of these oocysts or parasitophorous vacuoles. This report is possibly the first to discuss the prominent hyperplasia of the abomasum mucosa, as well as morphologic features of C. andersoni in cattle.

Gene analysis of Cryptosporidium parvum HNJ-1 strain isolated in Japan.

We analyzed genetically Cryptosporidium parvum HNJ-1 strain, which is the Japanese reference strain isolated from human in Japan. DNA sequences of genes for thrombospondin-related adhesive protein of Cryptosporidium-1 and Cryptosporidium-2 (TRAP-C1, TRAP-C2), heat shock protein 70 (HSP70), oocyst wall protein (COWP), beta-tubulin, alpha-tubulin, polythreonine-region (Poly-T), elongation factor 1 alpha (EF-1α), and 18S rRNA of this strain were determined. They showed high rate of homology to published sequences of genotype 2 strains, which were considered to be infective to both humans and animals. However, HNJ-1 had synonymous and non-synonymous substitutions in the nucleotide sequence of TRAP-C1 and beta-tubulin among HNJ-1 and published sequences of genotype 2 strains. These results implied that HNJ-1 strain was an unique subpopulation of genotype 2 strain of C. parvum.

Characterization of Cryptosporidium canis isolated in Japan

Oocysts that morphologically resemble Cryptosporidium canis oocysts were isolated from a stray dog captured in the northeastern part of the main island of Japan. The DNA sequence of the 18S rRNA gene of the isolate showed high homology to the published sequence of C. canis that was isolated in USA by Fayer et al., J Parasitol, 87:1415–1422, (2001). The isolate phylogenetically belonged to the C. canis cluster; however, its DNA sequence showed two base substitutions. This suggests the genetic diversity of C. canis.

CGRP induces [Ca2+]i rise and glycoconjugate secretion in feline tracheal submucosal gland

Submucosal glands were isolated from feline trachea. The intracellular Ca2+ concentration ([Ca2+]i) of the acinar cells of isolated glands was measured using the fluorescent dye Fura-2. Calcitonin gene-related peptide (CGRP) at 10(-8) to 10(-5) M produced a significant and sustained rise in the [Ca2+]i of isolated glands, reaching a maximal response of 127% of the prior baseline level but did not alter the intracellular adenosine 3, 5-cyclic monophosphate ([cAMP]i). In a Ca(2+)-free solution, CGRP produced no significant alteration in [Ca++]i. Glycoconjugate secretion from isolated glands was stimulated by CGRP in a dose dependent fashion, reaching a maximal response of 167% of control at 10(-6) M but was without effect in tracheal explants. Further, CGRP did not produce any significant increase in glycoconjugate secretion in the Ca(2+)-free medium. These findings indicate that CGRP stimulates glycoconjugate secretion from airway submucosal glands by inducing Ca2+ influx from the extracellular solution.

Discrimination of Cryptosporidium species by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was used for the discrimination of three species and one genotype of the protozoan parasite Cryptosporidium: the C. parvum, C. andersoni, C. muris, and C. muris Japanese field mouse genotype. A set of primers specific for the 18S rRNA gene of Cryptosporidium was used in the DGGE; consequently, the four strains showed different banding patterns. This is a potentially convenient and precise method for the discrimination of Cryptosporidium spp.

Tumor necrosis factor attenuates β agonist-evoked Cl− secretion in canine tracheal epithelium

We examined the effect of human recombinant TNF alpha on the potential difference (PD) and short circuit current (SCC) of canine tracheal epithelium using an Ussing chamber. Luminal or submucosal TNF (2 to 200 U/ml) produced no significant alterations in the basal PD or SCC values. Pretreatment with luminal TNF significantly reduced isoproterenol (ISOP, 10(-6) M)-evoked increases in SCC and PD to 57% and 66% of that with ISOP alone, respectively, with a significant decrease in conductance (G) to 87% of that with ISOP alone in a dose-dependent fashion, from 10 to 200 U/ml. Even after ISOP (10(-6) M)-evoked PD and SCC had reached a plateau, TNF produced significant decreases in PD and SCC up to 79% and 83% of that with ISOP alone, respectively, in a dose-dependent fashion, from 50 to 200 U/ml. Amiloride did not alter the inhibitory action of TNF on ISOP-evoked SCC and PD values. Antiserum against TNF abolished the inhibitory action of TNF on ISOP-evoked response. In contrast, submucosal TNF did not alter PD, SCC or G. These findings indicate that TNF attenuates beta agonist-evoked increases in chloride secretion across airway epithelium.

An expectorant, stepronin, reduces airway secretion in vitro

Stepronin (SPN) is clinically used as an expectorant, and thenoic acid (TA) is its metabolite. We examined the effects of these drugs on the bioelectric parameters [potential difference (PD), short circuit current (SCC), conductance (G)] of the posterior epithelial membrane of canine trachea and on those of the mucus glycoprotein secretion from feline tracheal isolated glands. PD and SCC were obtained using an Ussing chamber and G was calculated as the ratio SCC/PD. Neither SPN nor TA significantly altered the baseline values of PD and SCC. However, in the mucosal solution, both SPN and TA significantly inhibited PD and SCC evoked by isoproterenol (ISOP), whereas G remained unchanged. Amirolide did not alter the inhibitory action of SPN and TA. Mucus glycoprotein secretion from isolated glands was estimated by measuring trichloride acetic acid-precipitable [3H]-glycoconjugates. SPN and TA significantly reduced mucus glycoprotein secretion. Further, when stimulated by methacholine, these agents significantly inhibited mucus glycoprotein secretion from isolated glands. These findings suggest that SPN inhibits airway secretion in vitro by both decreasing Cl- secretion from epithelial cells and mucus glycoprotein secretion from submucosal glands.