Ecological studies on reovirus pollution of rivers in Toyama Prefecture

Reoviruses (reos) were isolated from river water in various areas of Toyama Prefecture. The frequency of reo isolation was higher in the river water, the basin of which has a larger human population. The degree of river contamination with reo paralleled that with the Escherichia coli group of organisms, and reos were frequently isolated from sewage, too. The high antibody-positive (greater than or equal to 1:8 or greater than or equal to 1:10) rates against reos in humans and other animals tested (swine, cattle, and field rodents) indicated their wide-spread infection with reos. These results suggested that the major source of reos present in the river water may be the excretion by humans and other animals, especially the former. Survival experiments in which reos were added into the filtered or centrifuged river water and kept at various temperatures, revealed that reos survived for more than 3 years at 5 C. In the field experiment where reos suspended in cellophane tubes were kept in an agricultural water stream in winter (water temperatures below 10 C), they survived for 6 months until the water temperature rose above 20 C in summer.     

Epidemiological studies on the background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture. I. Epidemiology of infection with Rickettsia tsutsugamuchi among field rodents in endemic and nonendemic areas

In order to clarify the epidemiological background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture, Japan, since 1978, comparative surveys have been carried out between endemic and nonendemic areas. Rickettsia tsutsugamushi (Rt) was isolated at a rate of about 36% (158/439) from field rodents in the endemic area while it was not isolated from any of 280 in nonendemic areas. In all of six stations in the endemic area, a significantly high proportion of rodents were found to be Rt carriers. However, no Rt was isolated from rodents captured from July to September. The organism was isolated from rodents captured in the other months, especially in a high proportion in November when infestation of rodents with Leptotrombidium pallidum was at its peak. When the rodents were examined by indirect immunofluorescence staining, the rate of anti-Rt antibody-positive animals was about 55% (157/287) and about 17% (62/368) in endemic and nonendemic areas, respectively. Larvae of mites collected from the rodents were found to belong to four genera and 11 species. Among them L. pallidum was the only mite that had been known to be a vector of Rt. L. pallidum was found most frequently and in abundance from rodents in the endemic area, whereas it was present in very small numbers in rodents in nonendemic areas. The infestation of rodents with L. pallidum showed a seasonal variation, i.e. two peaks per year, in spring and autumn, and the number of mites detected was markedly greater in November than in spring. Rt was isolated from L. pallidum on rodents captured in the endemic area.     

Virologic and serologic studies on an outbreak of echovirus type 11 infection in a hospital maternity unit.

An outbreak of echovirus type 11 (E-11) infection occurred among newborn babies in a hospital maternity unit in the summer of 1971. The results of studies are as follows: 1) Forty-one of 188 infants developed febrile illness with stomatitis during one and a half months from July to September. E-11 was isolated from stool specimens of 14 infants and two throat swabs. Antibody response to the virus was shown in all the 19 cases examined. Some of their mothers were suffering from subclinical infection. 2) The isolates were identified as a variant of E-11 which is not neutralized with antiserum against prototype E-11. Antiserum against the current virus neutralized both current and prototype viruses. 3) Sucrose gradient centrifugation of sera from infants revealed that the neutralizing antibody activity resided more predominantly in 19S than in 7S fractions. These antibodies reacted more specifically with the current strain than with the prototype Gregory strain.     

Ecology of Non-O 1 Vibrio cholerae in Toyama Prefecture

The ecology of non-O 1 Vibrio cholerae and Vibrio mimicus as causes of cholera-like diarrhea or seafood-associated gastroenteritis has been investigated in Toyama Prefecture since 1980. The relationship between biological or serological characteristics of the isolates and their enteropathogenicity is discussed. Overall isolation rates from river water, sea water, and fish were 24.0, 59.5, and 33.7%, respectively, the isolation frequency being, in general, extremely high in the summer season, although the organisms were detected all year around in the case of sea water. Most isolates from river water were unable to grow on plates of TCBS agar to which colistin was added at a concentration of 1 μg/ml (CL-TCBS). These strains quickly fermented cellobiose. O-51 and O-70 were the two most frequently detected serogroups among them and they did not show enteropathogenicity in the rabbit ileal loop (RIL) test. On the other hand, almost all isolates from sea water and fish as well as those from human diarrhea cases were able to grow on CL-TCBS, but were unable to ferment cellobiose quickly. O-36, O-10, O-6, O-8, O-39, and O-26 were the dominant serogroups of these isolates, and some of them showed enteropathogenicity in the RIL test. Six out of 98 isolates from river water, 14 out of 116 from sea water, and 19 out of 112 from fish were classified as Vibrio mimicus. All of these strains were able to grow on CL-TCBS and quickly fermented mannose but not cellobiose. I-41 was the most common serogroup among them and some of these strains showed enteropathogenicity in the RIL test. Production of a cholera-like enterotoxin among the isolates in Toyama Prefecture, if any, seemed to be poor.     

Ecological Studies on Reovirus Pollution of Rivers in Toyama Prefecture. II. Molecular Epidemiological Study of Reoviruses Isolated from River Water

In order to clarify the source of reovirus pollution in river water, comparative surveys have been carried out between reovirus isolates from river water and those from sewage, human or animal, by making use of the analysis of genomic RNA-migration pattern of reovirus in polyacrylamide gel electrophoresis (electropherotype). The strains of reovirus serotype 1 and 2 isolated from river water were classified into 3 and 9 electropherotypes, respectively, and 8 out of these 12 types were also found among strains isolated from sewage or human. When the monthly distribution of the river isolates classified by electropherotypes was compared with that of the sewage isolates, there were cases in which strains of the same electropherotype were simultaneously isolated from both sources. The electropherotypes of 3 isolates from pig and field rodents were different from those of the other isolates. The electropherotype of an oyster isolate coincided with that of some of the isolates from humans and river water. These results indicate that the major sources of reoviruses polluting river water may be the human excretion.     

Epidemiological studies on the background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture. II. Anti-Rickettsia tsutsugamushi antibody-positive rate in inhabitants of endemic and nonendemic areas

With a view to clarifying the actual state of inapparent infection of tsutsugamushi diseases, inhibitants of endemic and nonendemic areas were screened for anti-Rickettsia tsutsugamushi antibody (anti-Rt antibody) by the indirect immunofluorescence test. The anti-Rt antibody-positive rate in the inhabitants of the endemic area (about 50%) was statistically significantly higher than that in the nonendemic area (14.7%). The antibody titer in the inhabitants of the endemic area was 10-160, and the number of inhibitants showing a high antibody titer was 2-4 times larger than that of the nonendemic area. A total of 257 volunteers in the endemic area were analyzed for the changes in anti-Rt antibody titer over 1.5-2 years on an individual basis. An increase in the antibody titer was found in 20 inhabitants. There was no difference in the anti-Rt antibody-positive rate between male and female in either the endemic or the nonendemic area. The positive rate was also compared as to the distribution by 10 years of age. In the endemic area, there were no significant differences in the positive rate between any pair of 10-year age groups from 30s to 60s, whereas in the nonendemic area, the positive rate in the teen-age group was significantly lower than those in the age groups of 20 years or older. In Yamada district, the numbers of serum samples obtained from each age group were about the same, and the distribution of the positive rates showed a normal distribution. The nurse students having their homes in Toyama Prefecture were plotted on the map as for their anti-Rt antibody and geographical distribution. The results showed that many of them having homes in the endemic area were positive for the antibody, while some antibody-positives were scattered all over Toyama Prefecture.     

Epidemiological studies on caprine arthritis-encephalitis virus infection in Jordan

To investigate the seroprevalence of, and risk factors for, caprine arthritis-encephalitis virus (CAEV) infection in different breeds of goats in Jordan, sera from 1100 goats from three different geographical regions in Jordan were analyzed. Prevalence of antibodies to CAEV was determined using a competitive ELISA test. A semi-structured questionnaire was used to collect information on herd health and management. Questionnaire data were tested in a multivariable logistic regression model to elucidate risk factors associated with CAEV seropositivity. In addition, the incidence of CAEV antibodies was investigated in six goat herds located in the northern part of Jordan. Out of the 69 goat herds investigated, 16 (23.2%) had antibodies against CAEV. Individual goat true seroprevalence to CAEV was 8.9%. The highest CAEV seroprevalence was observed in goats older than 3-years and younger than 6-years of age. The seroprevalence of CAEV in goats was significantly higher (P < 0.05) in the northern part of Jordan than that in central or southern parts of Jordan. The multivariable logistic regression model identified large herd size (OD = 2.0; 95% CI: 1.1, 2.7), addition of new animals to the herd (OD = 1.3; 95% CI: 0.3, 1.6) and contact with other goat herds (OD = 1.1; 95% CI: 0.9, 2.0) as risk factors for CAEV seropositivity. The incidence of CAEV seropositivity in the six herds monitored in the northern part of Jordan ranged from 2.4 to 5.3%.     

Studies on the enteropathogenic mechanism of non-O 1 Vibrio cholerae isolated from the environment and fish in Toyama Prefecture

Enteropathogenic mechanisms of non-O 1 Vibrio cholerae were investigated using strains from the environment and those from fish in Toyama Prefecture. None of the 93 non-O 1 V. cholerae strains produced a detectable level of choleratoxin-like-enterotoxin (CT-like-enterotoxin) in Syncase medium, while 23 strains showed a distinct fluid accumulation in the rabbit ileal loop test (RIL). These RIL-positive strains neither produced CT-like-enterotoxin in vitro in the other four kinds of media which are considered suitable for CT production, nor in vivo in the ligated ileal loop. Approximately one-third of RIL-positive strains produced a fluid accumulating factor (FAF) which was not neutralized with anti-CT serum. FAF of a representative strain (Strain 79-9-2) was inactivated by heating at 100 C for 10 min, and has a molecular weight within the range of 50,000 to 100,000 daltons. Most accumulated fluids in RIL after inoculation with whole cultures of RIL-positive strains contained both hemolytic and cytotoxic principles. Desquamation of epithelial cells, inflammatory edema, neutrophile infiltration, loss of goblet cells and frequent hemorrhages were observed in sections of ligated ileal loop inoculated with whole cultures or concentrated culture filtrates of CT-like-enterotoxin-negative but RIL-positive strains. In contrast, neither desquamation of epithelial cells nor hemorrhage was observed in sections after inoculation with those of a CT-like-enterotoxin positive strain (Strain E 8498). These results indicated that most RIL-positive non-O 1 V. cholerae strains from the environment and fish isolated in Toyama Prefecture produce little CT-like-enterotoxin, but some of them produce FAF with cytotoxic activities.     

Epidemiological studies on jute diseases

Summary Hyphae of M. phaseoli failed to grow on unsterilized natural soil and were completely lysed within 4 days of exposure. Germination of sclerotia in natural soil was inhibited indicating soil fungistasis. Lysis of mycelium and inhibition of germination of sclerotia could be annulled by addition of various organic nutrients and fertilizers to natural soil or by autoclaving the soil. Germination of dormant sclerotia in natural soil was stimulated by root-exudates of host and non-host plants. Population of sclerotia buried in unsterilized natural soil gradually declined and after 15 months only 35 per cent of the initial number could be recovered; more than 80 per cent of these germinated when nutrients were added. Data suggest poor saprophytic ability of M. phaseoli in mycelial form and the involvement of dormant sclerotia in the survival of the organism in soil.     

Epidemiological studies of HCMV infection in children by restriction analysis of long-PCR products

Sequences of MIE, pol, gB gene longer fragments (2.0-2.6 kb) were amplified in two stage PCR (nested PCR) from 46 PEG-urine extracts. Restriction of particular amplicons with selected endonucleases identified 19 distinct HCMV genotypes: 8 for MIE, 6 for gB and 5 for pol. MIE profiling revealed highest differentiation power. Our data showed the correlation among MIE, pol, and gB genotypes and clinical disease, indicating that genotypes may contribute the course of HCMV infection. For newborns with symptomatic infection, the highest level of variability among three genes analysed was obtained in comparison to samples of newborns with asymptomatic infection. Long PCR technique coupled with restriction analysis may be successfully applied in HCMV genotypes differentiation in samples taken directly from HCMV infected individuals, thus may be useful in prognosis of clinical infection.     

Molecular epidemiology of echoviruses 11 and 13, based on an environmental surveillance conducted in Toyama Prefecture, 2002-2003

Nineteen echovirus 11 (E11) and 12 E13 isolates were isolated from three rivers in Toyama Prefecture, Japan, during an environmental surveillance conducted from April 2002 to March 2003. The nucleotide sequences of E13 isolates were closely related to those from patients with aseptic meningitis, with less than 1.3% divergence in the VP1 region of the viral capsid gene, and belonged to the same clade responsible for a worldwide outbreak that started in 2000. In contrast, E11 isolates were clustered into three genomic groups and were not closely related to echovirus strains isolated from patients. These results suggest that the combination of both virus isolation from environmental sources and phylogenetic analysis could be complementary assessment approaches to trace prevalent and minor circulating enteroviruses in the human population.