Effect of Newcastle disease virus level of infection on embryonic length, embryonic death, and protein profile changes.

BACKGROUND AND AIM: Newcastle disease virus (NDV) is an obligate intracellular parasite. Virus can only live on living cells. The embryonated chicken eggs (ECEs) are one of the growth media of virus that is a cheap, easy to do, and accurate for showing patterns of virus change in the host. Higher virus titers indicate the higher number of viruses and more virulent to infect host. This research aimed to investigate the effect of different level of NDV titer infection in ECEs on protein profile, embryonic length, mortality, and pathological change. MATERIALS AND METHODS: The study used a completely randomized design of six treatments and seven replications. The treatments were different level of NDV titer infection in allantoic fluid (AF) of 9-11 days ECEs, i.e., P1=20, P2=26, P3=27, P4=28, P5=29, and P6=210 hemagglutination unit (HAU). All samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were analyzed using one-way ANOVA with p=0.05 for length of the embryo and descriptive analysis for embryo mortality, pathology change, and protein band. RESULTS: The result showed that protein profile of NDV-infected ECEs of all different levels is more complex than protein profile of no NDV-infected ECEs. NDV infected of all different levels showed longer size embryo, higher mortality embryo at the first 2 days, and higher occurrence of hemorrhagic in all part of bodies of embryo than those of no NDV infected. CONCLUSION: It was concluded that NDV infection of all different level decreased health conditions of chicken embryo of ECEs of 9-11 days old. Different level of NDV infection of ECEs of 9-11 days old showed no significantly different embryo profiles. However, all of the NDV-infected embryos were shorter, death on the 2nd day, and suffered more hemorrhage on all body surfaces than uninfected NDV embryos.

Prevalence of enterotoxigenic Escherichia coli (ETEC) in hospitalized acute diarrhea patients in Denpasar, Bali, Indonesia.

The relationship between enterotoxigenic Escherichia coli (ETEC) and hospitalized patients with acute diarrhea was examined in a study conducted in two hospitals from June 2000 to May 2001 in Denpasar, Bali, Indonesia. A total of 489 hospitalized patients with acute diarrhea were enrolled, and their rectal swabs were screened for enteric bacterial pathogens. Toxins, colonization factor antigens (CFAs), in vitro antimicrobial susceptibility and seasonal distribution patterns associated with ETEC were ascertained. The diagnosis of ETEC infection and CFAs association were performed with GM-1 ELISA and Dot blot immunoassays. Enterotoxigenic Escherichia coli was isolated from the rectal swabs of 14.9% of the patients. The distribution of toxins among the ETEC strains found was ST in 51 (69.9%), while LT and ST/LT were found in 28.8% and 1.3% respectively. The highest isolation rate for ETEC was found among children between the ages of 1 and 15 years. Colonization factor antigens were identified in 28.8% of the ETEC strains. A high prevalence of CFA was found among the rectal swabs of patients with ST isolates. High frequency of resistance to ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, tetracycline and cephalothin was displayed among the ETEC strains. All ETEC strains were susceptible to norfloxacin, ciprofloxacin and nalidixic acid. The results of this study document the prevalence of ETEC in hospitalized patients with acute diarrhea in Denpasar, Bali, Indonesia. Data generated in this study depicts the prevalence of ETEC diarrhea and CFA types among diarrhea patients in the tourist city of Denpasar, Bali, Indonesia.

Genetic diversity of Salmonella enterica serovar Paratyphi A from different geographical regions in Asia.

AIMS: Subtyping of Salmonella Paratyphi A isolates from India, Pakistan, Indonesia and Malaysia was carried out by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these isolates from different endemic countries. METHODS AND RESULTS: A total of 39 human isolates of Salmonella Paratyphi A from Pakistan, India, Indonesia and Malaysia were studied using PFGE analysis following digestion of chromosomal DNA with XbaI. Seven isolates from Pakistan were resistant to ampicillin, tetracycline and cotrimoxazole. It was noted that Salmonella Paratyphi A isolates obtained from outbreaks in India had limited genetic diversity and probably belonged to closely related clones. Significant genetic homogeneity was observed among antimicrobial-resistant isolates from Pakistan and antimicrobial-sensitive isolates from Pakistan and Indonesia, respectively. CONCLUSIONS: PFGE was a useful subtyping technique to differentiate Salmonella Paratyphi A from different endemic countries. However, it fails to differentiate the antimicrobial-resistant and -sensitive strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing and comparing strains of Salmonella Paratyphi A. Our study suggests that a limited number of clones are responsible for paratyphoid fever in these countries.

Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.

Norwalk-like viruses (NLVs), rotavirus and adenovirus are reportedly responsible from 4 to 42% of non-bacterial acute sporadic gastroenteritis. The incidence of NLVs, adenovirus and rotavirus infections in Indonesia is unclear. A total of 402 symptomatic cases from Indonesian patients with acute gastroenteritis and 102 asymptomatic controls that tested negative for bacteria and parasites were screened for the presence of NLVs, rotavirus and adenovirus using the reverse transcriptase-polymerase chain reaction (RT-PCR), Rotaclone kits and Adenoclone kits. Specific prototype probes were used to ascertain which NLV prototypes were present in the area. NLVs were detected in 45/218 (21%), rotavirus was detected in 170/402 (42%) and adenovirus was detected in 11/273 (4%) samples examined. Genetic analysis of the RT-PCR products using specific prototype probes for NLVs indicated that the prototypes were 42% Taunton agent and 58% Hawaii/Snow Mountain agent. Comparative data on patients showed that the incidence of rotavirus infections was two times greater than the NLVs infections, and that adenovirus infections were the least prevalent. All of the control samples tested were negative for NLVs and adenoviruses, however 8/70 (11%) of the samples were positive for rotaviruses. The high incidence of enteric viral-related infections is a threat among acute diarrheic patients in Jakarta, Indonesia.

Characterization of Norwalk-like virus associated with gastroenteritis in Indonesia.

Norwalk Virus and Norwalk-like viruses (NLVs) are reportedly responsible for 2.5-4.0% of nonbacterial acute gastroenteritis (NBAG) worldwide. To help clarify the impact of NLVs on NBAG in Indonesia, stool specimens from 102 patients, 74 with NBAG and 28 with BAG, were screened for the presence of NLVs, using a reverse transcription-polymerase chain reaction (RT-PCR) assay. The specimens were subtyped using prototype-specific oligonucleotide probes and were sequenced and compared with published NLV sequences. Of the 102 specimens examined, 31 (30%) were found to be positive for NLVs. Type-specific probe analysis of the RT-PCR products indicated that 31 isolates hybridized to UK1 (Taunton agent) and UK3/4 (Hawaii agent/Snow Mountain agent) prototype strains. The results of this study indicate that prototype strains of NV or NLVs co-circulate in Indonesia and contribute to the overall level of acute gastroenteritis throughout the region.

Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritis.

Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis.

Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia.

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem among children and adults in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The significance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs has previously been shown to induce protective immunity against diarrhea in animal models. The aim of this study was to determine toxin-associated CFAs of ETEC isolated from a diarrheal disease case-control study in Jakarta, Indonesia. Thirteen hundred and twenty-three diarrheic and control patients with lactose-fermenting colonies were screened by ganglioside GM1-enzyme-linked immunosorbent assay (GM1-ELISA) for heat-labile (LT) and heat-stable (ST) toxins. Two hundred and forty-six (19%) ETEC isolates identified by GM1-ELISA for the LT/ST toxins were screened for CFAs by Dot blot assay using monoclonal antibodies against CFA/I, II, and IV and against the putative colonization antigens (PCF) PCFO159, PCFO166, CS7, and CS17. Of the 246 ETEC isolates, 177 (72%) elaborated ST, 56 (23%) produced LT, while 13 (5%) elicited both the ST and LT toxins. CFA testing of the 246 ETEC isolates showed that 21 (8%) expressed CFA/I, 3 (1%) exhibited CFA/II, 14 (6%) elaborated CFA/IV, while 7 (3%) expressed PCFO159 and PCFO159 plus CS5. No CFAs or PCFs could be associated with 201 (82%) of the ETEC strains. This report documents the types of CFAs associated with ETEC strains in Jakarta, Indonesia. These data may help current research efforts on the development of CFA-based vaccines for humans against ETEC and provide additional information for future ETEC vaccine trials in Southeast Asia.

Vibrio parahaemolyticus associated with cholera-like diarrhea among patients in North Jakarta, Indonesia.

A diarrhea study was conducted in North Jakarta, Indonesia from December 1996 through December 1997. Vibrio parahaemolyticus was isolated from 333 (6.1%) of 5442 rectal swab samples collected from patients with cholera-like diarrhea. Vibrio cholerae O1 was isolated from 545 (10.0%) and V. cholerae non-O1 from 183 samples (3.4%), respectively. Patients positive for V. parahaemolyticus were mostly adults between 20 and 40 years of age, with males constituting 62%. A majority (65%) of these patients demonstrated watery diarrhea with a frequency of fewer than 10 episodes per 24 hour. A large number of the patients had abdominal pain (83%) and vomiting (76%) and were non-febrile (90%). The highest isolation rate (9.6%) of V. parahaemolyticus was found during the dry season (June, July) and the lowest (4.5%) in the rainy season (December, January, February). All of the V. parahaemolyticus isolates were hemolytic on human blood agar (positive Kanagawa) but none was urease positive. Disk diffusion antibiotic susceptibility tests performed on the isolates demonstrated resistance to ampicillin (98%), cephalothin (24%), kanamycin (15%), colistin (97%), neomycin (2%) and ceftriaxone (0.3%). All isolates (100%) were sensitive to chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin.

Shigella spp. surveillance in Indonesia: the emergence or reemergence of S. dysenteriae.

From June 1998 through November 1999, Shigella spp. were isolated in 5% of samples from 3,848 children and adults with severe diarrheal illness in hospitals throughout Indonesia. S. dysenteriae has reemerged in Bali, Kalimantan, and Batam and was detected in Jakarta after a hiatus of 15 years.

Cholera in Indonesia in 1993-1999.

Cholera-specific surveillance in Indonesia was initiated to identify the introduction of the newly recognized Vibrio cholerae non-O1, O139 serotype. Findings from seven years (1993-1999) of surveillance efforts also yielded regional profiles of the importance of cholera in both epidemic and sporadic diarrheal disease occurrence throughout the archipelago. A two-fold surveillance strategy was pursued involving 1) outbreak investigations, and 2) hospital-based case recognition. Rectal swabs were transported to Jakarta for culture and isolates were characterized by serotypic identification. Outbreak findings showed that V. cholerae O1, Ogawa serotype, was the predominant etiology in all 17 instances of investigated epidemic transmission. Monitoring of eight hospitals representing seven provinces provided 6,882 specimens, of which 9% were culture positive for V. cholerae: 589 (9%) for O1 and 20 (< 1%) for non-O1 strains. Proportional representation of V. cholerae O1 among cases of sporadic diarrheal illness was variable, ranging from 13% in Jakarta to < 1% in Batam. Overall, 98% of V. cholerae O1 cases were the Ogawa serotype. There was no instance of non-O1, O139 serotype introduction in either epidemic or sporadic disease form. Anti-microbial drug susceptibility was consistently demonstrated, both temporally and spatially, except against colistin. Evidence is provided that epidemic and sporadic cholera occurrence in western Indonesia is associated with periods of low rainfall. Conversely, in the more eastern portion of the country, heavy rainfall may have contributed to epidemic cholera transmission.

In vitro antibiotic susceptibility of Neisseria gonorrhoeae in Jakarta, Indonesia.

Antibiotic susceptibilities were determined for 122 Neisseria gonorrheae isolates obtained from 400 sex workers in Jakarta, Indonesia, and susceptibilities to ciprofloxacin, cefuroxime, cefoxitin, cefotaxime, ceftriaxone, chloramphenicol, and spectinomycin were found. All isolates were resistant to tetracycline. A number of the isolates demonstrated decreased susceptibilities to erythromycin (MIC >/= 1.0 microg/ml), thiamphenicol (MIC >/= 1.0 microg/ml), kanamycin (MIC >/= 16.0 microg/ml), penicillin (MIC >/= 2.0 microg/ml), gentamicin (MIC >/= 16.0 microg/ml), and norfloxacin (MIC = 0.5 microg/ml). These data showed that certain antibiotics previously used in the treatment of gonorrhea are no longer effective.

Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area.

A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine-attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single-dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied.

Norwalk-like virus and bacterial pathogens associated with cases of gastroenteritis onboard a US Navy ship.

Acute gastroenteritis is a potential cause of substantial morbidity in U.S. military personnel during deployment. This study investigated the microbial causes of diarrhea in U.S. troops on exercises in Southeast Asia aboard the U.S.S. Germantown from March through May 1996. A total of 49 (7%) patients with diarrhea reported to sick call during a 3-month deployment involving 721 personnel. Diarrheal samples from 49 patients were subjected to bacterial and parasitologic examination, but sufficient samples from only 47 of 49 were available for analysis of the presence of Norwalk-like virus (NLV). Of the 49 diarrhea cases, 10 (20.4%) appeared to be due to bacterial etiology alone, 10 (20.4%) due to bacteria and the prototype Taunton agent (TNA), 11 (22.4%) due to TNA only, and 4 (8.0%) due to parasites. Norwalk-like virus RNA was present in 21 (45%) of 47 stool samples from the diarrhea cases, 10 with bacterial etiologies and 11 without bacterial or parasitic etiologies. No pathogen was detected in 14 (29%) of the cases. Four of the controls showed the presence of parasitic organisms. Of the 11 cases in which enterotoxigenic Escherichia coli was isolated, 8 were positive for colonization factor antigen (CFA/IV), and 3 were CFA-negative. The bacterial pathogens tested were all susceptible to gentamicin, and furadantin, but were resistant to ceftriaxone and norfloxacin, including 75% of the Campylobacter spp. These data support the view that the major cause of diarrhea for troops deployed in this geographic area is most likely NLVs.

An outbreak of El Tor cholera associated with a tribal funeral in Irian Jaya, Indonesia.

An outbreak of El Tor biotype cholera occurring in a rural village in Irian Jaya, Indonesia was evaluated for risk factors associated with death from cholera. Among those dying in the village during the epidemic, a significant association between membership in one of the five tribal groups in the village complex was associated with an elevated risk of suffering a cholera death (odds ratio = 5.9). Interviews with members of the decedents' families revealed a very strong association (odds ratio = 11.6) between risk of cholera death and having attended the two day funeral of a woman who died of a cholera-like illness a few days prior to an outbreak of cholera-like diarrheal disease in the village complex. Recent flooding may have contributed to the creation of an environment conducive to cholera transmission.

Comparison of direct-plating and enrichment methods for isolation of Vibrio cholerae from diarrhea patients.

A direct-plating method on thiosulfate citrate bile salts sucrose agar (DIR-TCBS) in conjunction with enrichment in alkaline peptone water (APW) incubated for both 6 h and 24 h followed by subculture onto TCBS (APW6h-TCBS and APW24h-TCBS, respectively) was performed on 16,034 rectal swab samples for isolating Vibrio cholerae. A total of 2,932 (18.3%) rectal swab samples were positive for V. cholerae O1 biotype El Tor, with the Ogawa serotype constituting 99.2% of the isolates. There were no significant differences in V. cholerae O1 isolation rates between the three culture systems nor between the combinations of any two systems. However, direct plating plus enrichment demonstrated a significantly higher V. cholerae O1 isolation rate than DIR-TCBS alone (P < 0.02). Conversely, enrichment procedure, alone or in combination with DIR-TCBS, yielded significantly more (P < 0.0001) V. cholerae non-O1 isolates than DIR-TCBS alone. The length of incubation time of the enrichment broth, 6 h, offers no significant advantages over 24 h for the isolation of V. cholerae O1 and non-O1. A 24-h enrichment broth incubation period has the practical advantage of being easy to integrate into a normal laboratory workday, whereas 6-h broth enrichment, although more commonly recommended, requires that arrangements be made for after-hours subculture.

Enterotoxigenic Escherichia coli diarrhea among young children in Jakarta, Indonesia.

The incidence of diarrhea and enterotoxigenic Escherichia coli (ETEC) infection was evaluated in children six months to five years of age from an urban community in Jakarta, Indonesia. From January through May 1994, 408 children were monitored in their homes for diarrheal disease. Thirty-six percent (148 of 408) of the study children had at least one episode of diarrhea during the study period. Twenty-nine (19.6%) of the 148 children with diarrhea had ETEC isolated from a rectal swab sample at least once during the surveillance period; five children had ETEC isolated from two distinct episodes of diarrhea, giving a total of 34 episodes of ETEC positive diarrhea in the study group. Ten of 34 episodes were associated with heat-labile toxin, 15 of 34 with heat-stable toxin, and seven of 34 with both toxins. The mean age of children with diarrhea (1.7 years), whether ETEC positive or negative, was significantly lower than those who did not have diarrhea (2.4 years) during the study period; 82% of the children with ETEC were less than two years of age. This study demonstrates a high incidence of ETEC diarrhea among young children in Jakarta, and suggests this site would be suitable for ETEC vaccine efficacy trials.

PIP: During a 4-month period in 1994, 408 children 6 months to 5 years of age (mean, 2.4 years) from a densely populated slum section (Kapuk) of West Jakarta, Indonesia, were monitored in their homes for diarrheal disease. Many homes in this community lack running water or toilet facilities. Overall, 148 (36%) of these children had at least one diarrhea episode during the study period. 29 children (19.6%) with diarrhea had enterotoxigenic Escherichia coli (ETEC) isolated from a rectal swab sample at least once during the surveillance period and five children had ETEC isolated from two distinct diarrhea episodes, for a total of 34 episodes of ETEC-positive diarrhea. 10 of the 34 episodes were associated with heat-labile toxin, 15 with heat-stable toxin, and 7 with both toxins. Annualized rates of diarrhea and ETEC infections were estimated at 2.2 and 0.3 per child, respectively. The rate of children with diarrhea declined steadily with increasing age: 52% at 6-11 months, 48% at 12-23 months, 28% at 24-35 months, 30% at 36-47 months, and 12% at 48-60 months. 82% of children with ETEC were under 2 years of age. The high incidence of ETEC diarrhea recorded in this study suggests the feasibility of ETEC vaccine efficacy trials in this population.

Simple differentiation of Vibrio cholerae O139 from V. cholerae O1 and non-O1, non-O139 by modified CAMP test.

Strong positive CAMP reactions were demonstrated by 121 Vibrio cholerae O139 and 504 El Tor isolates, and weak positive CAMP reactions were shown by 235 non-O1, non O139 isolates when these isolates were tested by a modified CAMP technique. Thirty-five classical biotype V. cholerae O1 isolates included in the tests were all CAMP negative.

Flagellar serotypes of Salmonella typhi in Indonesia: relationships among motility, invasiveness, and clinical illness.

While the H1-d flagellar serotype of Salmonella typhi has been found worldwide, the H1-j serotype occurs only in Indonesia. A cross-sectional survey in Indonesia compared epidemiologic, clinical, and pathogenetic characteristics of these two serotypes. S. typhi isolates were collected from patients with acute typhoid fever in four Indonesian cities. Flagellar serotype was determined by polymerase chain reaction amplification of the fliC locus of the flg gene. Of 321 isolates, 51 (15.9%) were H1-j. Patients with H1-j infection were older than those with H1-d (P < .001). Among 30 patients with known clinical outcomes, H1-j infection was associated with milder clinical illness than H1-d (P = .06). In vitro, H1-j isolates were both less motile on semi-solid agar plates (P = .004) and less invasive of HEp-2 cells (P = .002) than H1-d isolates. The association of decreased severity of illness with decreased motility and invasiveness suggests that flagellar properties are a component of S. typhi's virulence.

Persistent, recurring diarrhea in a colony of orangutans (Pongo pygmaeus) caused by multiple strains of Campylobacter spp.

A colony of 10 orangutans (Pongo pygmaeus) experienced persistent, recurring diarrhea caused by multiple infections with Campylobacter jejuni and C. coli. Infections appeared to have occurred through several mechanisms, including fecal-oral transmission between orangutans, and possibly transmission by houseflies contaminated with the organisms from nearby chicken feces. Among the 14 fecal and environmental C. jejuni isolates, 4 different antibiotic susceptibility profiles were detected; there were also 4 different profiles among the 8 isolates of C. coli. In 5 orangutans, there were back-to-back infections by different strains of C. jejuni, suggesting that a single C. jejuni infection may not confer protective immunity against heterologous strains circulating in the same vicinity. Transmission was effectively interrupted by environmental modifications and a 7-day course of oral erythromycin.

Modified CAMP test for biogrouping Vibrio cholerae O1 strains and distinguishing them from strains of V. cholerae non-O1.

A modified CAMP test was used to identify 973 Vibrio cholerae isolates by phenotype. Eltor and non-O1 strains were CAMP positive; classical strains were CAMP negative. Sausage-shaped zones of hemolysis of eltor strains were easily distinguished from narrower bands of non-O1 isolates. For O1 isolates, there was 100% agreement between the CAMP test and inhibition by polymyxin B.