Diagnostic applications of molecular and serological assays for bluetongue and African horse sickness.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

West Nile Virus Activity in a Winter Roost of American Crows (Corvus brachyrhynchos): Is Bird-To-Bird Transmission Important in Persistence and Amplification?

Since its emergence in North America, West Nile virus (WNV) has had a large impact on equines, humans, and wild bird communities, yet gaps remain in our understanding of how the virus persists at temperate latitudes when winter temperatures preclude virus replication and host-seeking activity by mosquito vectors. Bird-to-bird transmission at large communal American Crow roosts could provide one mechanism for WNV persistence. Herein, we describe seasonal patterns of crow and Culex mosquito abundance, WNV infection rates, and the prevalence of WNV-positive fecal samples at a winter crow roost to test the hypothesis that bird-to-bird transmission allows WNV to persist at winter crow roosts. Samples were collected from large winter crow roosts in the Sacramento Valley of California from January 2013 until August 2014, encompassing two overwintering roost periods. West Nile virus RNA was detected in local crow carcasses in both summer [13/18 (72% WNV positive)] and winter [18/44 (41% WNV positive)] 2013-2014. Winter infections were unlikely to have arisen by recent bites from infected mosquitoes because Culex host-seeking activity was very low in winter and all Culex mosquitoes collected during winter months tested negative for WNV. Opportunities existed for fecal-oral transfer at the overwintering roost: most carcasses that tested positive for WNV had detectable viral RNA in both kidney and cloacal swabs, suggesting that infected crows were shedding virus in their feces, and >50% of crows at the roost were stained with feces by mid-winter. Moreover, 2.3% of fecal samples collected in late summer, when mosquitoes were active, tested positive for WNV RNA. Nevertheless, none of the 1,119 feces collected from three roosts over two winters contained detectable WNV RNA. This study provided evidence of WNV infection in overwintering American crows without mosquito vector activity, but did not elucidate a mechanism of WNV transmission during winter.

Lack of Evidence for Laboratory and Natural Vertical Transmission of Bluetongue Virus in Culicoides sonorensis (Diptera: Ceratopogonidae).

Culicoides sonorensis (Wirth & Jones) is the principal North American vector of bluetongue virus (BTV). BTV infection of livestock is distinctly seasonal (late summer and fall) in temperate regions of the world such as California, which has led to speculation regarding vertical transmission of the virus within the midge vector as a potential mechanism for interseasonal maintenance ("overwintering") of the virus. To evaluate potential vertical transmission of BTV in its midge vector, we fed adult midges BTV-spiked blood and used a BTV-specific quantitative reverse transcriptase polymerase chain reaction assay to evaluate parent, egg, and progeny stages of laboratory-reared C. sonorensis for the presence of viral nucleic acid. Whereas BTV nucleic acid was weakly detected in egg batches of virus-fed female midges, virus was never detected in subsequent progeny stages (larvae, pupae, and F1 generation adults). Similarly, BTV was not detected in pools of larvae collected from the waste-water lagoon of a BTV-endemic dairy farm in northern California during the seasonal period of virus transmission. Collectively, these results indicate that BTV is not readily transmitted vertically in C. sonorensis, and that persistence of the virus in long-lived parous female midges is a more likely mechanism for overwintering of BTV in temperate regions.

Spatial variation in host feeding patterns of Culex tarsalis and the Culex pipiens complex (Diptera: Culicidae) in California.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) is now endemic in California across a variety of ecological regions that support a wide diversity of potential avian and mammalian host species. Because different avian hosts have varying competence for WNV, determining the blood-feeding patterns of Culex (Diptera: Culicidae) vectors is a key component in understanding the maintenance and amplification of the virus as well as tangential transmission to humans and horses. We investigated the blood-feeding patterns of Culex tarsalis Coquillett and members of the Culex pipiens L. complex from southern to northern California. Nearly 100 different host species were identified from 1,487 bloodmeals, by using the mitochondrial gene cytochrome c oxidase I (COI). Cx. tarsalis fed on a higher diversity of hosts and more frequently on nonhuman mammals than did the Cx. pipiens complex. Several WNV-competent host species, including house finch and house sparrow, were common bloodmeal sources for both vector species across several biomes and could account for WNV maintenance and amplification in these areas. Highly competent American crow, western scrub-jay and yellow-billed magpie also were fed upon often when available and are likely important as amplifying hosts for WNV in some areas. Neither species fed frequently on humans (Cx. pipiens complex [0.4%], Cx. tarsalis [0.2%]), but with high abundance, both species could serve as both enzootic and bridge vectors for WNV.

Ability of transstadially infected Ixodes pacificus (Acari: Ixodidae) to transmit West Nile virus to song sparrows or western fence lizards.

The hypothesis that Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) may serve as a reservoir and vector of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in California was tested by determining the ability of this tick species to become infected with the NY99 strain of WNV while feeding on viremic song sparrows, to maintain the infection transstadially, and then to transmit WNV to recipient naive song sparrows and western fence lizards during the nymphal stage. The percentage of ticks testing positive by reverse transcription-polymerase chain reaction (RT-PCR) decreased from 77% of 35 larvae at day 6 after ticks were transferred to donor song sparrows (day of detachment) to 23% of 35 nymphs at 59 d postinfestation (approximately 19 d after molting to the nymphal stage). However, the percentage of ticks positive by RT-PCR from which infectious virus was recovered by Vero cell assay decreased from 59% on day 6 to 12% on day 59, even though there was no statistically significant decrease in the quantity of RNA within positive ticks. Attempts to improve the sensitivity of plaque assays by blind passage through C6/36 cell cultures were unsuccessful. These data indicated that ticks maintained viral RNA but not necessarily infectious virus over time. Nymphs from larvae that fed on song sparrows with peak viremias ranging from 7.2 to 8.5 log10 plaque-forming units (PFU) per ml were used in transmission attempts. From one to seven RNA-positive nymphal ticks engorged and detached from each of four recipient song sparrows or western fence lizards. Blood samples from sparrows and lizards remained negative, indicating that transmission did not occur. An additional four lizards inoculated with 1,500 PFU of WNV developed moderate viremias, ranging from 4.2 to 5.6 log10 PFU/ml. Our data and data from previous studies collectively indicated that ixodid ticks were not able to experimentally transmit WNV and therefore most likely would not be important vectors in WNV transmission cycles.

Deposition of pyrethrins and piperonyl butoxide following aerial ultra-low volume applications in the Coachella Valley, California.

Data on adulticide deposition were collected during studies optimizing aerial ultra-low volume applications and droplet size in the desert environment of the Coachella Valley, Riverside County, California. Pyrenone 25-5 and BVA Spray 13 oil were applied by a single-engine, fixed wing aircraft equipped with 2 Micronair AU5000 atomizers. Data recorded by a portable weather station documented that weather conditions were suitable for application. Adulticide residue was collected using 24-cm-diameter filter papers positioned along 2-3 transects, with 3 positive controls held outside of the treated zone. The trace amounts of 2 major insecticidal components (pyrethrin I and II) and the synergist piperonyl butoxide (PBO) were detected from samples near the center of the spray zone by high-performance liquid chromatography (HPLC); pyrethrin deposition was highest at the center, 156 microg/m2, and it was not detectable 60 m beyond the center of the transect, whereas PBO deposition was 5,000 microg/m2 at the center but was not detectable beyond 150 m. Droplet diameters on spinning Teflon slides were larger than expected for the rated output of the atomizers. For these single swath trials, the lack of swath overlap due to drift resulted in low mortality in sentinel mosquitoes. Detection of residues was limited to the centroid of droplet densities on spinning glass slides and with mortality among sentinel mosquitoes, indicating HPLC may be useful in detecting postspray residues.

Nesting Ardeid colonies are not a focus of elevated West Nile virus activity in southern California.

A large nesting colony of Ardeid birds at the Finney-Ramer Wildlife Refuge in Imperial County, California, did not appear to be a focus of West Nile virus (WNV) amplification during the summer of 2004. Blood samples taken during June and July from 155 nestlings of four species of Ardeid birds (cattle egrets, black-crowned night herons, great egrets, and snowy egrets) and five nestling double-crested cormorants yielded a single WNV isolation from a 3-week-old cattle egret. Antibody was detected by enzyme immunoassay from 20 nestlings (13%), 14 (70%) of which were confirmed as positive by plaque reduction neutralization test (PRNT). However, titration end points against WNV and St. Louis encephalitis virus (SLEV) were similar precluding viral identification. The grouping of positives within few nests, highest PRNT titers in youngest birds (<1 weeks of age), the decline of titer with nestling age, and the lack of antibody specificity indicated that antibody may have been acquired maternally and did not represent new infections. Infection rates in Culex tarsalis mosquitoes collected near the Ardeid colony at Ramer Lake (3.1 per 1,000) were statistically similar to rates estimated at the nearby Wister Unit wetlands (5.3 per 1,000) that lacked an Ardeid nesting colony. Black-crowned night heron nestlings experimentally infected with the NY99 strain of WNV produced viremias >5 log10 plaque forming units (PFU)/mL and were considered moderately competent hosts, whereas cattle egret nestlings had viremias that remained <5 log10 PFU/mL and were incompetent hosts.

Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission.

The ability of the invading NY99 strain of West Nile virus (WNV) to elicit an elevated viremia response in California passerine birds was critical for the effective infection of Culex mosquitoes. Of the bird species tested, Western scrub jays, Aphelocoma coerulescens, produced the highest viremia response, followed by house finches, Carpodacus mexicanus, and house sparrows, Passer domesticus. Most likely, few mourning, Zenaidura macroura, or common ground, Columbina passerine, doves and no California quail, Callipepla californica, or chickens would infect blood-feeding Culex mosquitoes. All Western scrub jays and most house finches succumbed to infection. All avian hosts produced a lower viremia response and survived after infection with an endemic strain of St. Louis encephalitis virus. Culex species varied in their susceptibility to infection with both viruses, with Culex stigmatosoma Dyar generally most susceptible, followed by Culex tarsalis Coquillett, and then Culex p. quinquefasciatus Say. Populations within Culex species varied markedly in their susceptibility, perhaps contributing to the focality of WNV amplification. Transmitting female Cx. tarsalis expectorated from six to 3,777 plaque-forming units (PFU) of WNV during transmission trials, thereby exposing avian hosts to a wide range of infectious doses. Highly susceptible house finches and moderately susceptible mourning doves were infected by subcutaneous inoculation with decreasing concentrations of WNV ranging from 15,800 to <0.3 PFU. All birds became infected and produced comparable peak viremias on days 2-3 postinoculation; however, the rise in viremia titer and onset of the acute phase of infection occurred earliest in birds inoculated with the highest doses. WNV virulence in birds seemed critical in establishing elevated viremias necessary to efficiently infect blood feeding Culex mosquitoes.

Prevalence of antibodies to western equine encephalomyelitis and St. Louis encephalitis viruses in residents of California exposed to sporadic and consistent enzootic transmission.

Sera from outpatients attending county health department clinics in areas of California with consistent (Imperial Valley) and sporadic (Sacramento Valley) enzootic transmission of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses exhibited neutralizing antibody prevalence rates of 1.3% (n = 690) and 0.5% (n = 1,066) for WEE and 11.0% and 0.8% for SLE, respectively. Seroprevalence for SLE virus in Imperial County increased as a function of both age and years of residence, indicating that this virus was endemic with a low rate of annual infection. Of 26 sera that tested positive for SLE virus antibody by an enzyme immunoassay, but were negative by plaque reduction neutralization test, 14 (53%) had neutralizing antibody that reacted with > or = one type of dengue (DEN) virus. The DEN virus infections presumably were acquired elsewhere because neither the vectors nor DEN virus transmission occurs in California. The low prevalence of neutralizing antibody for WEE and SLE in the California human population indicated that despite recent increases in enzootic transmission, contact between humans and infectious mosquitoes have remained low.

Host selection patterns of some Pakistan mosquitoes.

The host selection patterns of 18 species of Pakistan mosquitoes are described, emphasizing the presumed malaria vectors, Anopheles culicifacies, An. fluviatilis, and An. stephensi, and the probable vectors of West Nile virus, Culex pipiens fatigans and Cx. tritaeniorhynchus. All species tested, with the exception of Cx, p. fatigans, were considered to be essentially zoophilic. Few human feeds were recorded for An. annularis (0.7%), An. culicifacies (0.5%), An. fluviatilis (1.1%), An. nigerrimus (14.3%), and Cx. bitaeniorhynchus (2.8%), while Cx. p. fatigans (37.6%) commonly fed on man. No human positive feeds were recorded for An. stephensi. Temporal or spatial changes in host selection patterns were not discerned with the exception of Cx. p. fatigans, whose feeding patterns varied opportunistically with host availability. Cx. p. fatigans females collected from houses fed more commonly on man than those resting in cattle sheds or in agricultural fields. Cx. p. fatigans resting in cattle sheds during winter fed mostly on birds and bovids, changing to man and bovids during the spring and then to man and birds during summer. The relationships between these results and vector-borne disease transmission in Pakistan are discussed.

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.

Prevalence of antibodies to mosquito-borne encephalitis viruses in residents of the Coachella Valley, California.

Sera from 19 (2.6%) and 118 (16.4%) of 719 outpatients attending clinics in the southeastern Coachella Valley, California during 1993 and 1994 exhibited IgG antibodies to western equine encephalomyelitis and St. Louis encephalitis (SLE) viruses, respectively, using enzyme immunoassays. However, only seven (1.0%) and 36 (5.0%) outpatients were positive by plaque-reduction neutralization tests (PRNTs), and seven (1.0%) and 84 (11.7%) outpatients were positive by sera hemagglutination inhibition assays, respectively. None were positive for IgM antibodies indicative of recent infection or were diagnosed clinically with central nervous system disease. Prevalence of PRNT antibody to SLE increased as a function of patient age, but did not vary significantly in relation to years of residence, sex, race, postal zip code, occupation, or month of collection.

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.

Anopheles culicifacies Giles: a release-recapture experiment with cohorts of known age with implications for malaria epidemiology and genetical control in Pakistan.

A mark-release-recapture experiment with the progeny of wild collected Anopheles culicifacies was performed during the monsoon season of 1978 at the village of Khano-Harni, a malarious area situated in Lahore District, Punjab Province, Pakistan. The recapture rates of males and females released at breeding and resting sites were statistically comparable and totalled 8.0% for females and 5.9% for males collected resting in houses and cattle sheds. Mating occurred mostly on the night following the evening of release when the females were about two days old. Insemination rates for females released at resting and breeding sites were statistically comparable. The initial gonotrophic cycle required four days; subsequent cycles occurred at two-day intervals with re-feeding on the same night as oviposition. Constant daily survivorship estimated from the decline in recaptures per day was 0.568 for females and 0.676 for males. Female survivorship estimated by vertical age grading methods ranged from 0.472 to 0.820. Horizontal life tables were calculated from the decline in recaptures and from vertical age-grading techniques and indicated that female or male life expectancy at emergence was less than 2.5 days. Population size was calculated using the Lincoln Index, Jackson's positive and negative methods and Itô's modification of Jackson's positive method and averaged from 1045.5 to 1049.6 females and from 449.1 to 1323.7 males. The relationship of these results to malaria transmission and genetical control is discussed.

West Nile virus in Pakistan. III. Comparative vector capability of Culex tritaeniorhynchus and eight other species of mosquitoes.

Eight species of mosquitoes from Pakistan were compared with Culex tritaeniorhynchus as experimental vectors of West Nile (WN) virus. When fed by the membrane or cotton-pledget methods on a dose of WN virus 100% infective for Cx tritaeniorhynchus, 95% and 73% of the females of Cx fuscocephala and Cx pseudovishnui became infected, respectively. Cx quinquefasciatus, Cx univittatus, Aedes albopictus, Ae. caspius, Ae. indicus and Ae. lineatopennis were all significantly less susceptible than Cx tritaeniorhynchus. In agreement with the single dose comparisons, the median per os infective dose of WN virus for Cx fuscocephala, Cx pseudovishnui and Ae. caspius was substantially greater than for Cx tritaeniorhynchus. The median parenteral infective dose for all six species tested was less than 1 SMICLD50. Both Cx tritaeniorhynchus and Cx quinquefasciatus were more susceptible to infection with WN virus when fed on viraemic chickens than when fed on defibrinated blood using cotton pledgets or membranes. After infection by intrathoracic inoculation, only Ae. indicus and Ae. lineatopennis showed a reduced ability to transmit WN virus when compared to Cx tritaeniorhynchus.

West Nile virus in Pakistan. 1. Sero-epidemiological studies in Punjab Province.

Serum samples collected during 1978-79 from residents of the Chiniot and Changa Manga National Forest (CMF) areas of Punjab Province, Pakistan, had over-all neutralizing (N) antibody positive rates for West Nile (WN) virus of 32.8% (n = 192) and 38.5% (n = 239), respectively. Comparison of the age-specific antibody rates indicated that the pattern of exposure to infection was different in the two areas. Samples from a 1968 serosurvey of residents of the CMF area had an age-specific N antibody profile similar to the 1978 CMF sample, but both the over-all N and haemagglutination-inhibition (HI) antibody positive rates were much higher in the 1968 sample. When tested against antigen prepared from the Pakistan I-746 strain of WN virus, the percentage of sera HI antibody positive and the geometric mean titre of the sera were significantly higher than when tested against the Egypt-101 antigen. One of 124 and 11 of 50 sera from the 1978 and 1968 samples from CMF exhibited detectable HI antibody against dengue-3 virus, respectively, indicating cross-reacting flavivirus antibody was present. None of the positive sera had a higher titre against dengue-3 than against WN virus, but four of the 1968 sera reacted to equal titre against both antigens. During the 1978-79 CMF survey, serum samples from domestic and wild animals were tested for WN virus antibody. Of the 317 wild birds captured, 85 were N-antibody positive. The only frequently bled mammal was the Indian cow, from which 21 of 58 samples were positive for WN antibody.

Effect of temperature on Culex tarsalis (Diptera: Culicidae) from the Coachella and San Joaquin Valleys of California.

The F1 progeny of Culex tarsalis Coquillett females from the Coachella (CV) and San Joaquin (SJV) valleys collected during April 1991, and April, July, and October 1993 were reared and maintained as adults at 5 constant temperatures ranging from 14 to 38 degrees C. CV F1 progeny exhibited smaller body size, enhanced survival during spring, and higher autogeny rates than SJV F1 progeny; however, upper and lower thermal tolerance limits, immature developmental rates and survivorship, and adult life table parameters were relatively similar for both strains. Mosquitoes from both sites exhibited midsummer changes in immature developmental rates and survivorship, adult wing length, life expectancy at emergence, and generation time. These data indicated that temperature may select for both spatial and temporal changes in mosquito biology.

Landscape affects the host-seeking patterns of Culex tarsalis (Diptera: Culicidae) in the Coachella Valley of California.

Effective arbovirus transmission requires that the principal vertebrate hosts and vectors have frequent contact. Vegetation and other landscape features used by roosting or nesting birds at night dictate their exposure to nocturnally active host-seeking Culex tarsalis Coquillett and therefore to western equine encephalomyelitis and St. Louis encephalitis viruses. Precipitin tests on 645 Cx. tarsalis that were collected resting and host-seeking near the Salton Sea in Coachella Valley, CA, indicated that passeriform birds (64%) and rabbits (25%) were the most frequent bloodmeal hosts and that the percentage of females feeding on birds varied temporally as an inverse fuction of mosquito abundance. Blood meals were not taken from communally roosting water birds. The spatial distribution of host-seeking females then was investigated by deploying dry ice baited traps within seven sites representative of habitats found along the Salton Sea. Mosquito catch was greatest at traps within elevated vegetation such as Tamarisk, mesquite, cattails, and orchards and lowest at traps positioned at snags over water, sand bars, open fields, and within housing in a small rural community. These data indicate that host-seeking Cx. tarsalis females congregated at specific landscape features that were not necessarily associated with large concentrations of potential bloodmeal hosts.

Population ecology and dispersal of Culex tarsalis (Diptera: Culicidae) in the Coachella Valley of California.

During 5 bimonthly experiments in 1993, 55,548 host-seeking and 22,563 newly emerged Culex tarsalis Coquillett females were marked with fluorescent dust and released at a marsh along the Salton Sea. Overall, 3,758 (6.7%) host-seeking and 37 (0.2%) newly emerged females were recaptured in dry ice-baited traps and walk-in red boxes operated for 7-12 consecutive days after release. The recapture of newly emerged females was unexpectedly low and insufficient for further analysis. The recapture rates and dispersiveness of females collected host-seeking within the study area were not different from females collected host-seeking at a site 16 km SE of the release site, indicating that Cx. tarsalis may not rely on memorized flight paths. Loss rates of host-seeking females were high, because many females readily dispersed from the marsh study area during host-seeking flights. Estimates of population size ranged from 914,000 in February to 4,000 in July and were well correlated with catch size in CO2 traps. The wing length and fructose positivity rate of released females did not vary as a function of age or dispersal distance at recapture. Parity of released females increased over time, but nullipars were collected during all recapture periods, perhaps indicating difficulty in blood meal acquisition. Cohort dispersal progressed at the rate of about 0.2 km/d and was sufficient to disseminate arboviruses in the southern Coachella Valley.