A rapid dot immunoassay for the detection of serum antibodies to eastern equine encephalomyelitis and St. Louis encephalitis viruses in sentinel chickens

A dot enzyme-linked immunosorbent assay utilizing a novel membrane, polyvinylidene difluoride, is described. This assay was developed for the rapid detection of serum antibodies to eastern equine encephalomyelitis virus and St. Louis encephalitis virus in sentinel chickens. Antigens were spot-filtered through the membrane. Membranes were dipped into small vials of sera. Antigen-antibody complexes were detected with enzyme-conjugated antiglobulin which, when exposed to substrate, produced a colored insoluble product. The antibody detection protocol was completed within 50 min and was compared with a standard plate enzyme immunoassay. Chickens were experimentally infected with eastern equine encephalomyelitis and St. Louis encephalitis and bled on a daily basis. The dot immunoassay correctly identified 99% (123/124) of the eastern equine encephalomyelitis virus and 100% (67/67) of the St. Louis encephalitis virus antisera. Sera from sentinel chicken flocks in Maryland were also assayed. These data indicate that the dot immunoassay should be considered as an alternative to current assays for the screening of sera for antibodies to virus antigens. This assay could easily be performed in the field and allows for the screening of antibodies to several different viruses in one test.     

Evaluation of domestic pigeons as sentinels for detecting arbovirus activity in southern California.

Flocks of sentinel domestic pigeons (Columbia livia) detected increases in St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) virus activity in southern California concurrently with flocks of sentinel chickens. However, occasional low-titered, transient seroconversions to both WEE and SLE viruses also occurred in pigeons during periods when virus activity was not detected by seroconversions in sentinel chickens, by virus isolation from Culex mosquitoes, or by human disease. Moreover, SLE virus seroconversions detected in pigeons by a hemagglutination inhibition (HI) test frequently could not be confirmed either by a plaque-reduction neutralization test (PRNT) on the same sera, or by an HI test on the next monthly serum sample from the same bird. Experimental infection studies, in which pigeons were inoculated subcutaneously with SLE (SOUE 16-84) virus, confirmed that pigeons developed low-titered and transient HI antibodies that were detectable infrequently by PRNT. In contrast, experimental infection with WEE (BFS 1703) virus produced elevated antibody responses that were detectable by HI for 8-12 weeks and by PRNT for at least 25 weeks. Pigeons infected with SLE virus rarely developed detectable viremias, whereas most birds infected with WEE virus developed viremias on postinfection day 1 that persisted for two or three days. Host-preference studies indicated that pigeons were less attractive as bait in lard can traps to host-seeking Culex mosquitoes than were chickens, and that blood-engorged Culex females collected near sentinel locations fed more frequently upon galliform than columbiform birds. Collectively, these results indicated that sentinel pigeons would not provide an adequate replacement for sentinel chickens to monitor WEE or SLE viruses, and would be a dead-end host for SLE virus.     

Arbovirus surveillance in six states during 1972.

A virus surveillance project was established and maintained during 1972 along 10 major river drainages in six states. Mosquitoes, biting flies, and blood specimens from sentinel equines were collected during 83 field trip visits to 141 arthropod collecting sites and 22 sentinel locations from April into December 1972. There were 173,074 mosquitoes tested and 303 arboviruses isolated from 11 of 41 species. From 13,388 biting flies tested, 8 arbovirus isolations were obtained in 1 of 5 species. There was no isolation of Venezuelan equine encephalitis (VEE) virus. Western equine encephalitis (WEE) virus isolates were the most numerous and were followed by Turlock, St. Louis encephalitis, Hart Park, California encephalitis, and Bunyamwera (BUN) group viruses. The first isolation of WEE from the mosquito Cullex (Mel). erraticus is reported, as is the extension of the ranges for Buttonwillow virus from California to New Mexico and Texas. Also a single isolation of the BUN group from Culicoides variipennis extends the range of this virus-vector relationship from California to Texas. New distribution records for mosquito species previously unreported for Arizona, Louisana, New Mexico, and Oklahoma are reported. The sentinel burros detected WEE serologic conversions at two sites in New Mexico and at one in Texas. The surveillance project provided state and federal officials with current information on the status of arbovirus activity, including the absence of VEE activity during 1972, and it demonstrated the existence of the potential for WEE epizootics and epidemics throughout a wide geographic area of the Western United States.     

Investigation of the southern limits of Murray Valley encephalitis activity in Western Australia during the 2000 wet season

Western Australia experienced its worst-ever outbreak of the mosquito-borne Murray Valley encephalitis (MVE) virus during the 2000 wet season. Highest-on-record rainfall throughout much of the state during the 2000 wet season gave rise to extensive mosquito breeding and increased MVE virus transmission, resulting in nine cases of encephalitis. Activity of MVE virus in Western Australia is monitored by detecting MVE virus-specific antibodies in serum from sentinel chickens, located at towns and communities throughout the north of the state. However, during 2000, all 28 flocks of chickens seroconverted to MVE virus, including a flock located >600 km further south than MVE virus activity had ever previously been recorded. Furthermore, the majority of the nine cases of encephalitis occurred outside the enzootic Kimberley region. We therefore undertook a major serosurvey of domestic chicken flocks both south and east of the previously defined regions of virus activity. The results suggest that MVE virus activity extended as far south as the Midwest and northern Goldfields during 2000. A new southern limit of activity of MVE virus is therefore proposed. The results have implications for managing outbreaks of MVE virus in Western Australia and have enabled us to locate additional sentinel flocks as part of the MVE surveillance program for future years.     

Simulation studies of St. Louis encephalitis virus in south Florida

Two simulation models were used to investigate the epidemiology of St. Louis encephalitis virus (SLEv) in south Florida, one including sentinel hosts (chickens) and amplification hosts (wild birds), while the other one included age structure in the amplification host population. The overall population size of the vector, Culex nigripalpus, was a major factor in the likelihood of epizootics for both models, but the seasonal dynamics of the vector alone did not explain variation in transmission. Interactions between seasonal factors in the mosquito and reproduction in the wild amplification avian hosts appeared to be important in the likelihood of epizootics. Biased feeding between sentinel and amplification hosts affected the time course of virus prevalence and may have implications for the interpretation of sentinel data. The time of virus introduction strongly affected the timing of outbreaks but did not affect the likelihood of epizootics. In most cases, the outbreak occurred immediately after virus introduction; however, in some cases the outbreak was delayed until the mosquito population increased. This has implications for the timing of control strategies directed against mosquito populations.     

Persistence of mosquito-borne viruses in Kern County, California, 1983-1988

The persistence of arboviruses was studied from 1983 to 1988 in mixed agriculture, marsh, riparian, and foothill habitats in Kern County, CA. Western equine encephalomyelitis (WEE) virus was isolated frequently during 1983 from Culex tarsalis and Aedes melanimon and was detected by the seroconversion of sentinel chickens. WEE virus then disappeared, even though vector competence studies during 1984-1986 showed that Cx. tarsalis was able to transmit WEE virus. St. Louis encephalitis (SLE) virus was detected sporadically in 3 of the 6 years of the study by isolation from Cx. tarsalis and/or by sentinel chicken seroconversion. When mosquito pools were screened for virus in suckling mice, Turlock (TUR) and Hart Park (HP) viruses were isolated from Cx. tarsalis during each summer. Vertical transmission of HP was indicated by the isolation of virus from a pool of male Cx. tarsalis. California encephalitis (CE) virus was isolated repeatedly from host-seeking Ae. melanimon females, males, and adults reared from field-collected immatures, verifying vertical transmission in nature. Horizontal transmission of CE virus among both jackrabbits (Lepus californicus) and desert cottontails (Sylvilagus auduboni) appeared to amplify Ae. melanimon infection rates during the summer of 1985, but elevated herd immunity depressed infection rates during 1986. Thus, CE, HP, and TUR viruses persisted in Kern County, while WEE virus appeared to become extinct and required reintroduction. The sporadic occurrence of SLE virus activity remains unexplained, but its persistence may require both vertical transmission and reintroduction.     

Previous infection with West Nile or St. Louis encephalitis viruses provides cross protection during reinfection in house finches

House finches are competent hosts for both West Nile and St. Louis encephalitis viruses and frequently become infected during outbreaks. In the current study, House finches were infected initially with either West Nile or St. Louis encephalitis viruses and then challenged 6 weeks post infection with either homologous or heterologous viruses. Although mortality rates were high during initial infection with West Nile virus, prior infection with either virus prevented mortality upon challenge with West Nile virus. Prior infection with West Nile virus provided sterilizing immunity against both viruses, whereas prior infection with St. Louis encephalitis virus prevented viremia from St. Louis encephalitis virus, but only reduced West Nile virus viremia titers. Immunologic responses were measured by enzyme immunoassay and plaque reduction neutralization tests. Heterologous challenge with West Nile virus in birds previously infected with St. Louis encephalitis virus produced the greatest immunologic response, markedly boosting antibody levels against St. Louis encephalitis virus. Our data have broad implications for free-ranging avian serological diagnostics and possibly for the recent disappearance of St. Louis encephalitis virus from California.     

The spatial-temporal distribution of drought, wetting, and human cases of St. Louis encephalitis in southcentral Florida

Using a dynamic hydrology model, we simulated land surface wetness conditions at 42 sites in 28 counties in southcentral Florida from 1990 to 1998 and compared these simulations with the incidence of human cases of St. Louis encephalitis (SLE) within these counties. Within counties, drought four months prior and wetting one-half month prior were significantly associated with human cases of SLE. Simulated land surface wetness conditions resolved transmission loci in both space and time, and May drought was significantly associated with the subsequent occurrence of human SLE cases. These findings are consistent with previous results associating simulated land surface wetness conditions with the transmission of SLE virus as measured in sentinel chickens, and support our working hypothesis that springtime drought facilitates SLE virus amplification in mosquito and wild bird populations.     

Temporal Connections between Culex tarsalis Abundance and Transmission of Western Equine Encephalomyelitis Virus in California

Definition of targets for vector control requires an understanding of the relationship between vector abundance and the intensity of arbovirus transmission. Using an extensive surveillance dataset with observations from sentinel chicken flocks and mosquito traps paired in time and space, hierarchical autoregressive logistic regression models were developed to predict the probability of seroconversion in chickens for western equine encephalomyelitis virus (WEEV) based on the relative abundance of the principal vector, Culex tarsalis. After adjustments for confounders, the abundance of Cx. tarsalis 29–42 d before the date of chicken sampling was credibly associated with the risk of WEEV transmission in both the Central and Coachella Valleys, and a doubling of relative Cx. tarsalis abundance was associated with a 58% increase in the odds of seroconversion. The critical time windows identified in our study highlight the need for surveillance of vector populations and forecasting models to guide proactive vector control measures before the detection of transmission to sentinel chickens.     

Detection of Venezuelan equine encephalitis virus in rural communities of Southern Florida by exposure of sentinel hamsters.

Venezuelan equine encephalitis (VEE) virus strains were recovered from sentinel hamsters exposed in close proximity to homes in rural South Florida. Sentinel hamster surveillance methods over extended periods offer one effective way of uncovering VEE virus activity in relation to human habitation.     

California state Mosquito-Borne Virus Surveillance and Response Plan: a retrospective evaluation using conditional simulations

The California Mosquito-Borne Virus Surveillance and Response Plan recently was developed to provide a semi-quantitative means for assessing risk for western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses and to provide intervention guidelines for mosquito control and public health agencies during periods of heightened risk for human infection. West Nile virus recently has arrived in California, and the response plan also will provide a baseline for assessing the risk for human and equine infection with this virus. In the response plan, overall risk is calculated by averaging risk due to 1) environmental conditions, 2) adult mosquito vector abundance, 3) vector infection rates, 4) sentinel chicken seroconversion rates, 5) equine cases (for WEE), 6) human cases, and 7) the proximity of virus activity to populated areas. Overall risk is categorized into three levels: normal season, emergency planning, or epidemic conditions. We evaluated this response plan using historical data from years with no, enzootic, and epidemic activity of WEE and SLE in several areas of California to determine whether calculated risk levels approximated actual conditions. Multiple methods of risk calculation were considered for both viruses. Assessed risk based on cumulative temperature, rainfall, and runoff levels over the entire season provided more or equally accurate assessments than biweekly assessments based solely on the previous half-month. For WEE, during years with enzootic activity or early-season periods of years with WEE epidemic activity, combining horse and human cases as a single risk factor improved the model's ability to forecast pending WEE activity, but separating the two factors allowed a better indication of WEE activity during epidemics and periods with no activity. For SLE, assignment of higher risk to drier conditions as measured by rainfall and runoff yielded the most accurate representation of actual virus activity during all recent study periods.     

Correlation of Culex tarsalis population indices with the incidence of St. Louis encephalitis and western equine encephalomyelitis in California.

Mosquito population indices from California for the period 1953-1973 were analyzed to determine their association with activity of St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) viruses. Culex tarsalis female populations, as measured by New Jersey light trap indices (LTI), correlated positively with the incidence rates of encephalitis in humans, and were a reliable means of forecasting the years of highest incidence. The critical level of C. tarsalis in urban areas below which no human cases of SLE and WEE were detected was an LTI of 0.1. Critical urban levels of C. tarsalis associated with significant human SLE or WEE incidence ranged between LTIs of 6.4 (for rural mosquito abatement districts [MADs] with large resident human populations). Peaks in annual incidence of SLE and WEE in humans occurred during years when seasonal average C. tarsalis female populations in urban areas reached a LTI of 21. Peaks in weekly incidence of SLE and WEE were associated, respectively, with weekly LTIs of 21 and 81 in urban traps. Isolation rates of SLE virus from mosquito pools and transmission of the virus to enzootic hosts were highest when urban LTIs were between 10 and 19.9 and between 5 and 9.9, respectively. The WEE viral isolations and enzootic transmission rates were highest when LTIs in urban areas were 1-4.9.     

Arboviral infections in the United States.

Arboviruses are important considerations in the differential diagnosis of encephalitis and other acute infections. Alterations in the environment and in human behaviors contribute to changing patterns of arboviral transmission. These trends, the periodic epidemic resurgence of arboviral diseases such as St. Louis encephalitis, and the discovery of new arboviruses present a continued challenge to infectious disease clinicians.     

The house sparrow (Passer domesticus) as a sentinel for St. Louis encephalitis virus

Birds are the primary hosts for St. Louis encephalitis (SLE) virus in most of North America. Because the increased prevalence of antibody in House Sparrows (Passer domesticus) has been related to human cases, this species has been frequently used as a sentinel of SLE virus activity in urban areas. This study investigated the susceptibility of House Sparrows to two strains of SLE virus, measured antibody profiles, and evaluated the use of House Sparrows in an urban surveillance system. House Sparrows were susceptible to both strains of SLE virus inoculated, although not equally, and produced viremias sufficient to infect vector mosquitoes. Both hemagglutination-inhibiting (HI) and neutralizing (N) antibody developed rapidly and to high titers within 2 weeks after inoculation. Detectable humoral antibody began to disappear by 3 months, but persisted for 2 years in 27% for HI and 36% for N antibody of the surviving birds. However, all of the surviving birds were resistant to reinfection with SLE virus at 2 years after inoculation. The titer of HI antibody appeared to be useful in determining recent exposure to SLE virus. The experimental data on HI antibody development and persistence was related to field serologic data from House Sparrows. The monthly prevalences of SLE antibody for independent samples of sera from House Sparrows collected in Memphis, Tennessee, in 1980 were similar. SLE amplification in the House Sparrow population was delayed until September. The Memphis arbovirus surveillance system detected the amplification quickly, and responded with increased adult mosquito control in the focal areas. Urban surveillance of SLE utilizing House Sparrows as sentinels is discussed.     

Australian encephalitis in the Northern Territory: clinical and epidemiological features, 1987–1996

Background: The last epidemic of Australian encephalitis occurred in 1974. Since then, cases have been reported from the Kimberley of Western Australia (WA).
Aims: To describe the epidemiology and clinical features of Australian encephalitis in the Northern Territory (NT) of Australia.
Methods: Review of cases of Australian encephalitis presenting to Royal Darwin Hospital from 1987–1996 and review of sentinel chicken surveillance for Australian encephalitis viruses.
Results: Sixteen patients were identified; ten from the NT and six from WA. Cases occurred in the years 1987, 1988, 1991 and 1993. Infection was acquired throughout northern NT below latitude 20°S in the months March to July. All infections were due to Murray Valley encephalitis (MVE) virus. Eleven of the patients were children. Distinguishing features were spinal cord and brainstem involvement and the absence of seizures in adults. CT scanning was normal and EEG showed no focal activity. Five died (31%) and four (25%) have residual neurological disability. Sentinel chicken surveillance since 1992 shows yearly seroconversion to MVE virus throughout northern NT; human cases occurred simultaneously with chicken seroconversion in 1993.
Conclusions: Australian encephalitis is endemic in the NT; the areas at risk are norm of Tennant Creek. Outbreaks are seasonal and occur every few years. Young children are most at risk. Mortality and morbidity are high. Prevention of disease is by avoidance of mosquito exposure and vector control measures.     

Short report: detection probability of arbovirus infection in mosquito populations

An important component of arbovirus surveillance is monitoring the vector for presence of the pathogen. Intervention and preventive programs need early detection of arboviral activity in mosquito populations. In this report, we examine the factors affecting the probability of detection of mosquito infections. Since arbovirus infection rates in mosquito populations are very low, observations of zero-infected mosquito samples are common. Using statistical models, we describe methods to estimate the probability of detection and upper bounds of confidence intervals of mosquito infection rates as measures of confidence for observations of zero infection. Our results show that detection of low levels of mosquito infections requires large samples (greater than 1,600 individuals) for a high probability (0.8) of detection. Due to focal transmission of arboviruses, grouping samples over different sampling sites and times is often inappropriate for detection of mosquito infection. We emphasize sample size as a key determinant in detection of mosquito infections and recommend intensified entomologic surveys at sentinel sites to detect arboviral activity.     

Isolation of St. Louis encephalitis virus in South Brazil.

Eight strains of a flavivirus identified as St. Louis encephalitis (SLE) virus were isolated from wild rodents, birds, and sentinel mice in three locations in the State of S?o Paulo, Brazil from 1967--1969. No illness attributable to SLE virus infection was detected among the local inhabitants, although about 5% of the local population had neutralizing antibodies to this virus.     

Elevated Japanese encephalitis virus activity monitored by domestic sentinel piglets in Thailand

Twenty-nine domestic piglets from pig farms located in three provinces of Thailand between 2003 and 2004 were used as sentinel animals for Japanese encephalitis virus (JEV) circulation. Piglets were used as sentinel to underline, on one hand, the role of domestic pigs as JEV amplifying host and, on another hand, to point out the interest of using sentinel animals for Japanese encephalitis surveillance. JEV activity was demonstrated through i/ antibody detection using a specific ELISA test for the identification of Immunoglobulins of class M and G, ii/ virus isolation on cell culture, after experimental mosquito inoculation for virus amplification. Almost 100% and 83% of the piglets, respectively, had specific IgG and IgM JEV antibodies and 35% yielded a virus isolate. Piglets of the growing farm industry act as virus amplifier increasing the risk of transmission for the human community. Conclusively, since piglets JEV infection appears early in life and is generally clinically unnoticed, it represents an exceptional sentinel model for human health threats, which has to be considered by health authorities.