The temporal dynamics of humoral immunity to Rickettsia typhi infection in murine typhus patients.

OBJECTIVES: This study examined individuals with Rickettsia typhi infection in the Lao People's Democratic Republic (Lao PDR) to (a) investigate humoral immune dynamics; (b) determine the differences in reference diagnostic results and recommend appropriate cut-offs; (c) determine differences in immune response after different antibiotic treatments; and (d) determine appropriate diagnostic cut-off parameters for indirect immunofluorescence assay (IFA). METHODS: Sequential serum samples from 90 non-pregnant, adults were collected at seven time-points (days 0, 7, 14, 28, 90, 180 and 365) as part of a clinical antibiotic treatment trial. Samples were tested using IFA to determine IgM and IgG antibody reciprocal end-point titres against R. typhi and PCR. RESULTS: For all 90 individuals, reciprocal R. typhi IgM and IgG antibody titres ranged from <400 to ≥3200. The median half-life of R. typhi IgM was 126 days (interquartile range 36-204 days) and IgG was 177 days (interquartile range 134-355 days). Overall median patient titres for R. typhi IgM and IgG were significantly different (p < 0.0001) and at each temporal sample collection point (range p < 0.0001 to p 0.0411). Using Bayesian latent class model analysis, the optimal diagnostic cut-off reciprocal IFA titer on patient admission for IgM was 800 (78.6%, 95% CI 71.6%-85.2% sensitivity; 89.9%, 95% CI 62.5%-100% specificity), and for IFA IgG 1600 (77.3%; 95% CI 68.2%-87.6% sensitivity; 99%, 95% CI 95%-100% specificity). CONCLUSIONS: This study suggests suitable diagnostic cut-offs for local diagnostic laboratories and other endemic settings and highlights antibody persistence following acute infection. Further studies are required to validate and define cut-offs in other geographically diverse locations.

Differential patterns of endothelial and leucocyte activation in 'typhus-like' illnesses in Laos and Thailand.

Scrub typhus is responsible for a large proportion of undifferentiated fevers in south-east Asia. The cellular tropism and pathophysiology of the causative agent, Orientia tsutsugamushi, remain poorly understood. We measured endothelial and leucocyte activation by soluble cell adhesion molecule enzyme-linked immunosorbent assays in 242 Lao and Thai patients with scrub or murine typhus, leptospirosis, dengue, typhoid and uncomplicated falciparum malaria on admission to hospital. Soluble E-selectin (sE-selectin) levels were lowest in dengue, sL-selectin highest in scrub typhus with a high sE-selectin to sL-selectin ratio in leptospirosis patients. In scrub typhus patients elevated sL-selectin levels correlated with the duration of skin rash (P = 0.03) and the presence of eschar (P = 0.03), elevated white blood cell (WBC) count (P = 0.007), elevated lymphocyte (P = 0.007) and neutrophil counts (P = 0.015) and elevated levels of sE-selectin correlated with the duration of illness before admission (P = 0.03), the presence of lymphadenopathy (P = 0.033) and eschar (P = 0.03), elevated WBC (P = 0.005) and neutrophil counts (P = 0.0003). In comparison, soluble selectin levels in murine typhus patients correlated only with elevated WBC counts (P = 0.03 for sE-selectin and sL-selectin). Soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1 levels were not associated significantly with any clinical parameters in scrub or murine typhus patients. The data presented suggest mononuclear cell activation in scrub typhus. As adhesion molecules direct leucocyte migration and induce inflammatory and immune responses, this may represent O. tsutsugamushi tropism during early dissemination, or local immune activation within the eschar.

Foot and mouth disease in the Lao People's Democratic Republic: II. Seroprevalence estimates, using structured surveillance and surveys of abattoirs.

An examination of the seroprevalence of foot and mouth disease (FMD) virus was conducted in the Lao People's Democratic Republic (Lao PDR) from 1996 to 2005, using structured surveillance and abattoir-based studies. Under structured surveillance, seropositivity ranged from 65.7% (Vientiane Capital, 1996) to 3% (Houaphan, 2005) for cattle and buffalo; and from 2.8% (Vientiane Capital, 1998) to 0% in separate studies of pigs. In each study, species composition was significantly associated with seroprevalence rates. For abattoir surveys, the majority of samples (60.5%) came from Vientiane Capital (33.0%), Savannakhet (14.0%) and Champasak (13.5%) provinces. The overall proportion of animals testing positive for the presence of antibodies against the FMD virus was 18.7% (ranging from 50.8% in Vientiane Province to 1% in Phongsali). Generally, antibodies against serotype O were the most prevalent. Cattle and buffalo that tested as seropositive were significantly older than the seronegative animals (p < 0.00005). The overall proportional seropositivity was significantly different for different species, as was the case with the antibodies against serotypes O, A and Asia 1. Some 22% of cattle, 55% of buffalo and 23% of pigs demonstrated seropositivity but this varied significantly between provinces.

Foot and mouth disease in the Lao People's Democratic Republic: I. A review of recent outbreaks and lessons from control programmes.

Foot and mouth disease (FMD) causes sporadic disease outbreaks in the Lao People's Democratic Republic (Lao PDR). As the Lao PDR is a major thoroughfare for transboundary animal movements, regular FMD outbreaks occur, causing economic hardship for farmers and their families. In this review of the recent history of FMD in the Lao PDR between 1997 and 2006, the authors examine the virological and epidemiological aspects of the disease and appropriate control measures, including the distribution of outbreaks, causative serotypes and the molecular epidemiology of the viruses, as well as large-scale vaccination programmes. The dominant serotype, type O, was reported every year from 1998 to 2005. The majority of outbreaks occurred in Vientiane Capital (n = 42; 28%) and the highest number of outbreaks were reported in cattle (n = 94; 61%); followed by buffalo (n = 41; 27%) and pigs (n = 18; 12%). All type A outbreaks occurred in cattle. Type Asia 1 outbreaks were reported in the central provinces around Vientiane Capital between 1996 and 1998.

Characterisation of a novel lyssavirus isolated from Pteropid bats in Australia.

A novel lyssavirus isolated from Pteropid bats in Australia (Australian Bat Lyssavirus, ABLV) has been characterised using gene sequence analyses, electron microscopy and a panel of monoclonal antibodies. Electron microscopic examination of Pteropid bat and mouse brain material as well as virus isolated from tissue culture medium, showed the presence of bullet-shaped rhabdovirus particles and structures characteristic of lyssavirus. Analysis using nucleocapsid (N) specific monoclonal antibodies, showed a strong relationship between this new lyssavirus and serotype 1 rabies. The nucleotide sequence of the prototype strain of ABLV was determined from the initiator methionine codon for the nucleocapsid protein (N protein) to the amino terminus of the polymerase gene (L protein), a distance of 5344 nucleotides. Comparisons of the deduced N, phosphoprotein (P), matrix protein (M), and glycoprotein (G) proteins showed that ABLV was more closely related to serotype 1 classic rabies viruses than to other members of the Lyssavirus genus. The percent relatedness of the ABLV proteins when compared to the cognate proteins of PV (Pasteur vaccine strain) rabies was 92, 75, 87 and 75% for the N, P, M and G proteins, respectively. Phylogenetic studies of N protein sequences showed clearly that ABLV is an unrecognised member of the Lyssavirus genus and represents a new genotype, genotype 7.

Serotype identification of Australian bluetongue viruses using a rapid fluorescence inhibition test.

Rapid serotyping of bluetongue virus (BTV) isolates is required to facilitate the choice of an appropriate serotype-specific vaccine in a disease situation or to improve surveillance of BTV serotype prevalence. This communication describes the development and validation of a bluetongue virus fluorescent inhibition test (BTV FIT) as a rapid method to serotype Australian BTV isolates. The BTV FIT uses virus neutralisation principles similar to those used in the rabies rapid fluorescent focus inhibition test. The BTV FIT has the ability to provide an accurate serotype identification within 24 h thereby abbreviating the serotyping process by 3-4 days relative to conventional virus neutralisation assays and making the BTV FIT comparable time-wise with the polymerase chain reaction technique. The development of the BTV FIT is described using BTV reference viruses which have been isolated in Australia, and validation of the assay by assessment of five Australian BTV isolates of unknown serotype by comparison with the plaque inhibition method. The use of the BTV FIT readily facilitated rapid and accurate serotype identification of Australian BTV reference viruses and five unknown BTV isolates with results indicating full agreement with the plaque inhibition method.

Rapid identification of Australian bunyavirus isolates belonging to the Simbu serogroup using indirect ELISA formats.

The Bunyavirus genus, belonging to the Bunyaviridae family, is comprised of a large group of antigenically and geographically disparate arthropod-borne viruses of medical and veterinary significance. In Australia, viruses belonging to the Simbu serogroup of the Bunyavirus genus, Akabane, Tinaroo, Peaton, Aino, Douglas, Thimiri and Facey's Paddock have been isolated. In this communication we describe two indirect ELISAs, referred to as the Simbu serogroup ELISA (SG-ELISA), and the Simbu typing ELISA (ST-ELISA), for the identification of these Simbu serogroup viruses. Infected cell lysate antigens prepared from Simbu serogroup virus isolates were assessed in the SG-ELISA for reactivity with a mouse monoclonal antibody (4H9/B11/F1). The monoclonal antibody reacted strongly with all Australian members of Simbu serogroup reference viruses and is proposed for use as a serogrouping reagent for Simbu viruses. Furthermore, the ST-ELISA enabled specific identification of viruses from within this group by recognition of characteristic reaction patterns between infected cell lysate antigens and a panel of polyclonal antisera raised to Simbu serogroup viruses.

Identification of epizootic haemorrhagic disease of deer virus serotypes using a fluorescence inhibition test.

A fluorescence inhibition test (FIT) is described for serotyping rapidly isolates of epizootic haemorrhagic disease of deer virus (EHDV). The test used a serogroup-reactive monoclonal antibody in a immunofluorescence procedure to detect virus which resisted neutralisation by antisera to any of the eight known EHDV serotypes. The EHDV FIT provided an accurate serotype identification procedure for all eight reference serotypes and, in comparison with the plaque inhibition assay, abbreviated the serotyping process by three to four days.

A rapid indirect ELISA for the serogrouping of Australian orbiviruses.

This communication describes the development and evaluation of a simple and rapid method for the classification of Australian orbiviruses into one of seven established serogroups (i.e. bluetongue, epizootic haemorrhagic disease of deer, Palyam, Eubenangee, Corriparta, Wallal, Warrego) or an 'ungrouped' category. The Australian orbivirus serogrouping ELISA (SG-ELISA) utilised a sodium deoxycholate-treated cell lysate preparation from infected BHK cells which was subsequently probed in an indirect ELISA format with polyclonal antibodies representative of each serogroup. Bound immunoglobulin was detected by the use of a recombinant streptococcal protein G-HRPO conjugate and subsequent reaction with the chromogenic substrate. All reference orbiviruses tested in the SG-ELISA were identified and were in agreement with the serogroups originally designated. Minimal inter-serogroup cross-reactions were observed. One-way cross-reactions were observed between Warrego and Mitchell River viruses.

The use of an indirect ELISA with protein G-peroxidase conjugate and a blocking ELISA to demonstrate recent bluetongue infection in sheep.

The humoral immune response of sheep infected with bluetongue virus serotypes 3, 9 and 16 was monitored by plaque inhibition (PI), blocking ELISA (BELISA) and indirect ELISA over a period of 63 days post-infection. Results indicated that testing of a single plasma or serum sample by both a BELISA and an indirect ELISA using a recombinant streptococcal protein G (PrG) peroxidase conjugate enabled an assessment of the proximity of a recent infection based on the failure of PrG to bind ovine IgM class antibodies. When BELISA and indirect ELISA results were expressed as a ratio, values indicative of recent infection (> or = 5) were observed for an average duration of 16.5 days (range 8 to 23 days) following the initial detection of antibody by BELISA. This approach has potential to improve diagnosis of a wide range of virus infections by providing an indicator for the relationship of serological status with a recent infection. However, where reinfection may occur, as with bluetongue virus, alternative methods may be required for definitive diagnosis.

A monoclonal antibody blocking ELISA detects antibodies specific for epizootic haemorrhagic disease virus.

The isolation of a monoclonal antibody (1G9/C9) with specificity for the epizootic haemorrhagic disease (EHD) serogroup has enabled the development of a highly sensitive and specific blocking ELISA (B-ELISA) for the detection of serum antibodies to EHD viruses. The assay was sensitive to blocking antibodies present in hyperimmune reference antisera to all six EHD serotypes tested but was unaffected by reference antisera to 19 South African and eight Australian serotypes of the related orbivirus bluetongue virus (BTV). The sensitivity of the EHD B-ELISA exceeded that of an indirect ELISA (I-ELISA) for EHD-specific antibody detection. Serum antibody titres to BTV and EHD in experimental and field sera, including a sentinel herd from which virus isolations were made, were examined in both the BTV and EHD B-ELISA tests. These results showed the B-ELISA was only sensitive to antibodies specific for the homologous serogroup in each case, even where sequential and mixed infections with each virus type occurred.

Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep.

An antigen-capture ELISA (Ag-ELISA) was developed to detect bluetongue virus (BTV) antigen directly from blood samples. Four blood preparations [whole blood, buffy coat, washed red blood cells (RBC) and plasma] taken pre-inoculation and on days 6 to 9 post-inoculation (PI) were used in the ELISA to study antigenaemia in forty sheep, each experimentally infected with one of 20 South African BTV serotypes. Seventeen of the 20 serotypes were detected and 27 of the 40 sheep were at some stage Ag-ELISA positive. Over the period of sampling, Ag-ELISA positive results were most frequently returned from whole blood taken on days 6 and 7 PI. However in some instances the quantity and/or duration of BTV antigenaemia was greater in buffy coat and washed RBC preparations. In a selection of samples examined, positive Ag-ELISA results were generally obtained when samples had an infectious virus titre in eggs of > 10(3.2) egg lethal doses (ELD50/ml). The appearance and duration of detectable antigenaemia was compared with the development of clinical signs and antibody responses of infected sheep. On days 6 and 7 PI the presence of fever (> 40 degrees C) was indicative to the likelihood of detectable antigenaemia. After day 5 PI antigenaemia declined and clinical signs of swollen face and inflamed feet appeared together with the first detectable antibody response. The Ag-ELISA, when used in conjunction with clinical observations and serologic data, should be useful as a rapid diagnostic procedure for bluetongue disease.

Implementation of internal laboratory quality control procedures for the monitoring of ELISA performance at a regional veterinary laboratory.

Quality control (QC) procedures for antigen detection enzyme-linked immunosorbent assays (ELISAs) for hog cholera (HC) virus, foot and mouth disease (FMD) virus, and an antibody detection ELISA for FMD virus were established at a regional veterinary laboratory in northern Thailand. A recently developed computer software package, QCEL, was used to facilitate management and analysis of QC data. The program was used to assess test performance by producing Shewhart-CUSUM control charts which monitored control data for unacceptable fluctuations or trends. QCEL-generated control charts and analyses are presented and discussed. The use of a simple integrated computerised system for storage and analysis of QC control data provided the laboratory with the opportunity to achieve increased confidence in the results of tests performed.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.

Use of combined Shewhart-CUSUM control charts in internal quality control of enzyme-linked immunosorbent assays for the typing of foot and mouth disease virus antigen.

An enzyme-linked immunosorbent assay (ELISA) for the typing of foot and mouth disease virus (FMDV) antigen was employed for the routine laboratory diagnosis of FMD at a regional veterinary laboratory in northern Thailand. An objective procedure was developed to monitor the test performance of the ELISA, using absolute test control limits in a Shewhart-CUSUM (cumulative sum) control chart method. The procedure detected significant data trends and 'beyond control limit' situations for each antigen typing system (types O, A and Asia 1), using an assay variable (gamma i). Retrospective analysis using Shewhart-CUSUM control charts of data from 42 ELISAs demonstrated that control limits were exceeded in two assays for FMDV type A. The Shewhart-CUSUM control chart is a simple and reliable internal quality control method for the detection of significant random and systematic variation in assays.

A comparison of type O foot and mouth disease virus field isolates from northern Thailand.

A survey of type O foot and mouth disease (FMD) virus isolates from northern Thailand was undertaken to determine the relationship between field viruses and the vaccine in use, and to gauge the range of antigenic variation among field viruses. Isolates were collected from the two most recent epizootics, 1986-1987 and 1989-1990, and assessed using a two-dimensional neutralisation test to determine their relationship to FMD type O1 Bangkok 1960 (O BKK/60) reference (vaccine challenge) virus. The critical r value for the survey was 0.259 and all isolates tested were found to have an r value considerably greater than this (range 0.66 to 0.80). The results showed close antigenic relationships between the isolates and the reference virus, and indicated a relatively small range of antigenic variation between the isolates.

The dynamics and impact of foot and mouth disease in smallholder farming systems in South-East Asia: a case study in Laos.

There is a general lack of data on the different patterns of dynamics and impact of foot and mouth disease (FMD) in South-East Asia and the impact the disease has on different sectors, in particular the smallholder sector in which livestock play such an important role. A pilot study was conducted of a recent outbreak of FMD that swept across the southern part of Laos during the second half of 1999. The main objectives of the study were to investigate the possible routes of transmission of the disease and the impact of FMD on the predominantly smallholder rice/livestock production system of Savannakhet Province. The study was performed by group interviews of farmers in ten villages, located in five districts across the width of the Province, and of district and provincial veterinary officials. Results suggested that the infection had probably been introduced from the eastern border and had spread rapidly west, along a principal trading route of pigs, cattle and buffalo. In the process, many villages adjacent to this trading route became infected and the disease spread rapidly within infected villages. The disease had a significant impact on the agricultural system, but the impact would have been much greater had the epidemic occurred during the season of paddy field preparation. Mortality was observed in young buffalo, cattle and pigs, and long periods of morbidity were observed in buffalo, often requiring extended treatment. The sale of livestock for cash was severely restricted, creating additional repercussions on that sector. It was concluded that the most appropriate approach to FMD control would be to prevent infected animals from entering the principal trading routes for pigs, cattle and buffalo. This will require the involvement of all the stakeholders of the livestock industry, including traders and veterinary authorities. A further tactic to be considered would be to protect livestock systems adjacent to these trading routes by vaccination. An economic study of the market incentives of both traders and smallholders is recommended and this approach is advocated in other parts of South-East Asia where livestock trading routes present the major risk of FMD outbreaks.

Establishment of a typing enzyme-linked immunosorbent assay for foot and mouth disease antigen, using reagents against viruses endemic in Thailand.

Antisera were produced at a central laboratory in Thailand against the endemic serotypes (O, A and Asia 1) of foot and mouth disease (FMD) virus. At a regional veterinary laboratory, these antisera were used in an indirect sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and serotyping of FMD virus (FMDV) antigen. ELISA readings of < 0.10 optical density (OD) units were considered negative. This was verified using fifty tissue samples which were known to be negative for FMDV. The highest mean sample value for three different dilutions was 0.02 OD units. Of a total of 93 samples submitted for antigen typing, 80 (86%) tested positive by ELISA and 13 (14%) were negative. No FMDV was detected in ELISA-negative samples following attempted tissue-culture virus isolation.

Epidemic of blindness in kangaroos--evidence of a viral aetiology.

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.

A study of risk factors for infection with HPAI H5N1 in small poultry farms in Thailand using a questionnaire survey.

A questionnaire was used to collect data on small poultry farm management and wild bird observed in poultry keeping areas to identify putative risk factors for infection with HPAI H5N1. The study was conducted in 2008 in four subdistricts of central Thailand that had experienced outbreaks of HPAI H5N1 in poultry. Descriptive and inferential analyses including univariable analyses and multivariable logistic regression were used to identify putative risk factors. Risk factors included purchasing native chickens/fighting cocks from commercial hatcheries, replacing or restocking birds individually, and observing lesser whistling ducks (Dendrocygna javanica) on the farm daily. Selecting healthy animals when purchasing animals to ensure that they were disease free was a protective factor. To fully understand the epidemiology of infection of small poultry farms with HPAI H5N1, control of movement of domestic poultry and serological and virological testing of the poultry population should be applied.