Katsuya Kitoh

Dissemination of extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow.

Toxoplasma gondii is an intracellular parasite. It has been thought that T. gondii can disseminate throughout the body by circulation of tachyzoite-infected leukocytes (intracellular parasite) in the blood flow. However, a small number of parasites exist as free extracellular tachyzoites in the blood flow (extracellular parasite). It is still controversial whether the extracellular parasites in the blood flow disseminate into the peripheral tissues. In this study, we evaluated the dissemination efficiency of the extracellular and intracellular parasites in the blood flow using GFP-expressing transgenic parasite (PLK/GFP) and DsRed Express-expressing transgenic parasite (PLK/RED). When PLK/GFP and PLK/RED tachyzoites were injected, as intracellular and extracellular forms respectively, at the same time into the tail vein of a mouse, many disseminated green fluorescent PLK/GFP tachyzoites were observed in the lung, the spleen, the liver and the brain. However, only a few red fluorescent PLK/RED tachyzoites were detected in these organs. When PLK/GFP and PLK/RED tachyzoites were injected in the opposite manner, that is, as extracellular and intracellular forms respectively, the majority of tachyzoites in these tissues were PLK/RED tachyzoites. Collectively, these results indicate that intracellular tachyzoites mainly disseminate throughout the body and that extracellular tachyzoites hardly contribute to parasite dissemination.

Pathological changes of renal tubular dysplasia in Japanese Black cattle

Pathological studies were conducted on 91 Japanese Black cattle with a hereditary disease which induced growth retardation, long hooves and renal failure. In calves one to two months old, no gross abnormalities were observed in the kidneys, but microscopical examinations revealed immature epithelia which were arranged irregularly and not attached to the basement membranes in some proximal tubules. In animals three to 36 months old, the kidneys had shrunk perceptibly and had grey-white radial streaks; microscopically they showed severe interstitial fibrosis with round-cell infiltration in the outer zone of the medulla and cortex, and reductions in the numbers of glomeruli and tubules. In the fibrotic areas there were immature epithelia with an irregular arrangement, and the basement membrane of the tubules was thickened. It was concluded that renal tubular dysplasia was the primary lesion of the disease, and that interstitial fibrosis and reductions in the numbers of nephrons were secondary lesions.

Clinical features of renal tubular dysplasia, a new hereditary disease in Japanese Black cattle

A new hereditary disease characterised by renal failure, poor growth and long hooves in Japanese Black cattle (wagyu) has been recognised in a region of central Japan since 1990. The number of calves affected has increased gradually, with the incidence reaching 17 of 485 (3.51 per cent) in 1995. Almost all the calves were slightly undersized at birth, and repeatedly had diarrhoea during the neonatal period. They began to show signs of growth retardation with proportional body and elongation of the hooves from about two to five months of age, but they had an almost normal or only slightly decreased appetite. The concentrations of urea nitrogen, creatinine and inorganic phosphorus in serum were high, and the affected calves excreted diluted urine frequently. Among 25 cases, the urine of 21 contained occult blood, 24 contained protein and two contained glucose. In 29 calves observed for 30 to 130 days, the course of the disease varied; in 21 of them it remained unchanged, six became gradually worse and two became severely debilitated and died. The disease was diagnosed as renal tubular dysplasia by histopathological examination.

Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins.

To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7.0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

Homozygosity mapping of the locus responsible for renal tubular dysplasia of cattle on bovine Chromosome 1

Abstract. Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation with an autosomal recessive trait. In the present study, we mapped the locus responsible for the disease (RTD) by linkage analysis with an inbred paternal half-sib pedigree obtained from commercial herds. By analyzing segregation of microsatellite markers in the half-sibs, significant linkage was observed between the RTD locus and markers on bovine Chromosome (Chr) 1 with the highest lod score of 11.4. Homozygosity mapping with the inbred pedigree further defined the localization of the RTD locus in a 4-cM region between microsatellite markers BMS4003 and INRA119. Mapping of the RTD locus on bovine Chr 1 will facilitate cloning and characterization of the gene responsible for this disease.

Up-regulation of hyaluronan receptors in Toxoplasma gondii-infected monocytic cells.

The apicomplexan, obligate intracellular parasite Toxoplasma gondii orally infects humans and animals. The parasites cross the intestinal epithelium, invade leukocytes in the general circulation and then disseminate into the peripheral organs. The mechanism of extravasation of the infected leukocytes, however, remains poorly understood. It is known that adhesion of leukocytes to extracellular matrix (ECM) is an important factor in extravasation, and CD44 and ICAM-1 on the leukocyte surface are known receptors for hyaluronan (HA), an ECM component. In this study, we demonstrated up-regulation of CD44 and ICAM-1 expression on the surface of T. gondii-infected human monocytic THP-1 cells and fresh isolated human monocyte. T. gondii-infected THP-1 cells adhered more efficiently to immobilized HA than did non-infected cells. T. gondii-infected monocytes in the general circulation might preferentially adhere to the ECM and migrate out from blood vessels, so transporting parasites into the peripheral organs.

Toxoplasma gondii tachyzoite-infected peripheral blood mononuclear cells are enriched in mouse lungs and liver.

The intracellular parasite Toxoplasma gondii is thought to disseminate throughout the host by circulation of tachyzoite-infected leukocytes in the blood, and adherence and migration of such leukocytes into solid tissues. However, it is unclear whether T. gondii-infected leukocytes can migrate to solid organs via the general circulation. In this study, we developed a real-time quantitative PCR (qRT-PCR) method to determine the rate of infection of peripheral blood mononuclear cells (PBMCs) flowing into and remaining within solid organs in mice. A transgenic T. gondii parasite line derived from the PLK strain that expresses DsRed Express, and transgenic green fluorescent protein-positive PBMCs, were used for these experiments. Tachyzoite-infected PBMCs were injected into mouse tail veins and qRT-PCR was used to measure the infection rates of the PBMCs remaining in the lungs, liver, spleen and brain. We found that the PBMCs in the lungs and liver had statistically higher infection rates than that of the original inoculum; this difference was statistically significant. However, the PBMC infection rate in the spleen showed no such enhancement. These results show that tachyzoite-infected PBMCs in the general circulation remain in the lungs and liver more effectively than non-infected PBMCs.

Inheritance of renal tubular dysplasia in Japanese Black cattle

Correspondence to Dr Kitagawa A RENAL disease combined with growth retardation and overgrown hooves has been observed in Japanese Black beef cattle in central Japan since 1990 (Ohba and others 2001). The incidence rate was reported to be 3-51 per cent among newborn animals in 1995. The onset of clinical signs is at approximately three months of age or later, and the disease is chronic. The meat of affected cattle is condemned because of uraemia, causing large economic losses. The main lesion is found in the kidneys (Sasaki and others 2001). The primary lesion is a dysplasia of epithelial cells at the renal proximal tubules in younger calves, followed by interstitial fibrosis with a reduction in the numbers of glomeruli and tubules in older cattle; therefore, the disease was named renal tubular dysplasia (RTD). Epidemiological and clinical investigations (Ohba and others 2001) have suggested that the disease is caused by consanguineous mating. In order to determine genetic factors of the disease, pedigree and medical records were collected for more than 1000 cattle which were related to affected animals. This short communication reports the results of the genetic analysis of the pedigrees of cattle affected with RTD. Between June 1992 and October 1996, a total of 78 Japanese Black cattle (45 males and 33 females) were diagnosed by histopathology to be suffering from RTD in Gifu Prefecture. The pedigree and medical records of the affected cattle and their relatives were collected. Records of 38 pedigrees covering 1109 cattle, including the 78 affected calves, were used for analyses. The coefficient of inbreeding was calculated by the tabular method (Tier 1990), using the records of 17 sires and pedigrees of 15 animals with RTD, covering 89 cattle. The level of statistical significance was set at P<0-05. Values were expressed as means (se). In analysing the difference in the numbers of male and female affected cattle, 78 calves (45 males and 33 female) affected with RTD and 246 of their siblings were used. To analyse how well the simple autosomal recessive genetic hypothesis for inheritance fitted, the chi-squared test was used on the records of 30 cattle affected with RTD and 91 of their siblings. To confirm whether the RTD trait was inherited as a simple autosomal recessiveness, the chi-squared test was also used for the mating results. To calculate a gene frequency, 3465 cattle, including 72 calves with RTD, were chosen, and it was assumed that the population was in Hardy-Weinberg equilibrium. The chi-squared test was also used to analyse whether the observed number of newborns affected with RTD agreed with the expected number of offspring produced by mating between carriers of RTD in the carrier sires and their offspring assumed to be in the HardyWeinberg equilibrium. The calculation of significance followed a two-sample t test with Welchs correction. Fig 1 shows the pedigrees of the 78 cattle with RTD. Most affected calves were born from matings between sire B and its half-sisters (mating pattern III, 70 per cent) or sire A and its granddaughters (mating pattern II, 15 per cent). The calves of sire A showed excellent marbling, a desirable characteristic. All the affected calves had a common ancestor, sire C. From examining beef medical records for the 78 cattle affected with RTD, it was found that the parents had no clinical problems during their lifetimes, or physical or laboratory abnormalities, such as uraemia, associated with RTD. The average inbreeding coefficients of RTD (n=66) and normal calves Number of I I I I I I

Renal reabsorption of magnesium and calcium by cattle with renal tubular dysplasia

The concentrations of magnesium and calcium in the serum and urine and their rates of clearance were determined in cattle with renal tubular dysplasia, an autosomal recessive hereditary disease associated with a deletion of the paracellin-1 gene in Japanese Black cattle. There were no significant differences in the serum or urine magnesium concentrations between normal cattle and cattle which were heterozygous or homozygous for the condition. Serum calcium concentrations tended to be lower in the homozygous cattle, and the serum creatinine and urea nitrogen concentrations were significantly higher in the homozygous cattle. The ratio of magnesium:creatinine and the fractional excretion of magnesium were higher in cattle with the disease than in normal cattle. There were no significant differences in urine calcium concentration, the calcium:creatinine ratio, and fractional excretion of calcium between normal cattle and cattle which were homozygous or heterozygous for the condition. The creatinine clearance was significantly lower in the homozygous cattle than in normal cattle. The clearance, excretion rate, reabsorption rate and reabsorption rate:clearance ratio of magnesium in cattle with renal tubular dysplasia were significantly lower than in normal cattle. The clearance rate and reabsorption rate of calcium were also significantly lower in the affected cattle, but the excretion rate and reabsorption rate:clearance of calcium were not different between the normal cattle and the cattle homozygous for the condition. In cattle with the condition the rate of reabsorption of magnesium by the kidneys was low, but the rate of reabsorption of calcium was normal.

Relaxing and Contracting Activities of Heartworm Extract on Isolated Canine Abdominal Aorta

Effect of adult heartworm (HW) crude extract on isolated canine abdominal aortic strips precontracted with noradrenaline was examined by recording isometric changes in tension. HW extract caused contraction of the aortic strip at a low concentration (LC) and its relaxation at a high concentration (HC). In aortic strips without endothelium, LC extract elicited a contraction similar to that in the strips with endothelium, whereas HC extract failed to produce any relaxation but instead produced a contraction. The relaxing effect of HC extract was blocked after treatment with 300 µM NG-nitro-l-arginine methyl ester hydrochloride, with reversal by additional treatment with 3 mM l-arginine. It was also markedly reduced or abolished after treatment with 3 µM oxyhemoglobin or 1 µM methylene blue. Fractionation of HW extract by high-performance liquid chromatography revealed that the relaxing and contracting activities are due to different substances in the extract. The results indicate that HW extract contains 2 different vasoactive substances, 1 causing contraction of canine abdominal aorta via a direct action on the smooth muscle, and the other its relaxation indirectly by releasing nitric oxide from endothelial cells. These vasoactive substances might play a role in HW extract-induced shock in dogs, and in the pathogenesis of HW infection.