A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivaxandPlasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

Evaluation of PCR to detect Theileria parva in field-collected tick and bovine samples in Tanzania

The ability of PCR to detect infections of Theileria parva, the cause of East Coast Fever, in field-collected tick and bovine samples from Tanzania was evaluated. PCR-detected infection prevalence was high (15/20, 75%) in unfed adult Rhipicephalus appendiculatus ticks that fed as nymphs on an acutely-infected calf, but low (22/836, 2.6%) in unfed adult R. appendiculatus collected from field sites in Tanzania. Tick infection prevalence was comparable to that in previous studies that used salivary gland staining to detect T parva infection in field-collected host-seeking ticks. Of 282 naturally-exposed zebu calves, seven had PCR-positive buffy coat samples prior to detection of Theileria spp. parasites in stained huffy coat cells or lymph node biopsies. Evidence of Theileria spp. infections was detected in stained smears of lymph node biopsies from 109 calves (38.6%) and huffy coat samples from 81 (28.7%), while huffy coat samples from 66 (23.4%) were PCR-positive for T parva. Implications of these findings for the sensitivity and specificity of the PCR are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Towards science-based sediment quality standards-Effects of field-collected sediments in rainbow trout (Oncorhynchus mykiss).

Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events. Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Field evidence for mechanical transmission of rabbit haemorrhagic disease virus (RHDV) by flies (Diptera : Calliphoridae) among wild rabbits in Australia

Field collected flies were screened for the presence of rabbit haemorrhagic disease virus (RHDV) by applying reverse transcriptase PCR (RT-PCR) in which primers specific to the capsid protein of the virus were used. The virus was detected in flies from locations where rabbit haemorrhagic disease (RHD) was reported and also soon after the release of RHDV in a 'clean' area. Oral and/or anal excretions of flies (flyspots) were found to contain viable virus and oral inoculation of rabbits revealed that a single flyspot was able to cause RHD. We conclude that flyspots are a major potential source of the virus for oral or conjunctival transmission of the virus to rabbits. (C) 1998 Elsevier Science B.V. All rights reserved.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Wolbachia infection and expression of cytoplasmic incompatibility in Armigeres subalbatus (Diptera: Culicidae)

Polymerase chain reaction screening revealed that Armigeres subalbatus (Coquillett), a vector of filariasis, was infected with the intracellular bacteria Wolbachia. Laboratory crosses between infected males and uninfected females resulted in less than half the number of offspring than control crosses between uninfected individuals when young (2- to 3-d-old) males were used in the cross. However, incompatibility was lost when old (14- to 17-d-old) males were used. Field-collected females did not show detectable cytoplasmic incompatibility, and this may be because of the age at which males mate in the field. We used head pigment fluorescence levels to age field males collected from mating swarms, and found that 25-63% of swarming males were older than 13 d. Male age may be one factor influencing the observed low levels of cytoplasmic incompatibility detected in the field.

Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand

We examined the transmission efficiency of 2 strains of Wolbachia bacteria that cause cytoplasmic incompatibility in field populations of Aedes albopictus by polymerase chain reaction assay. We found mainland and island populations throughout Thailand to be superinfected with group A and B bacteria. Of 320 Wolbachia-positive adult mosquitoes, 97.5% were infected with both groups. Single infected individuals of each Wolbachia group were encountered in nearly equal numbers. We screened 550 offspring from 80 field-collected mothers and found the transmission efficiency of group A Wolbachia to be 96.7% and that of group B Wolbachia to be 99.6%. Mothers that did not transmit both Wolbachia infections to all of their offspring were significantly larger in size than those with perfect transmission fidelity. We discuss our findings in relation to the prospects of the use of Wolbachia as a gene-driving mechanism.

Larval diet affects number of femoral 'brush' scales in male Helicoverpa punctigera moths (Lepidoptera : Noctuidae)

Males of Helicoverpa punctigera (Wallengren) show considerable variation in the number of femoral scales on the prothoracic legs. Such intraspecific variation in adult morphology could indicate the presence of undetected sibling species, or it may be related to larval diet. Helicoverpa putactigera is polyphagous, and different host plant species are likely to represent diets of different quality. Femoral lengths and the numbers of femoral scales on the prothoracic legs were therefore determined from: (i) individuals that had been collected as larvae from various host species in the field; and (ii) individuals that had been laboratory-reared, in split-family tests, on different diets, namely cotton, lucerne, sowthistle and artificial diet. Host plant species (and therefore presumably diet quality) influenced femoral length of H. punctigera males and, perhaps in conjunction with this, the number of femoral scales on the fore leg. The rearing experiment indicated, in addition, that the effect of host plant quality varies with larval stage, and that the pattern of this variation across the immature stages is dependent on host plant species. The recorded variation in the morphology of field-collected H. punctigera males is therefore most readily explained as a consequence of different individuals developing (at least for most of their larval life) on different host plant species, with diet quality varying significantly with species. The relevance of these results for insect developmental studies and evolutionary interpretations of host relationships is outlined.

Effect of body size on fecundity of Childers canegrub, Antitrogus parvulus Britton (Coleoptera : Scarabaeidae)

Actual and potential fecundity for Childers canegrub, Antitrogus parvulus Britton, was influenced by the size of females, with the largest females laying the most eggs. Actual or realised fecundity for A. parvulus averaged 18 eggs per female, about half of potential fecundity. Actual fecundity was significantly related to elytron length in a group of laboratory-reared beetles, but not for a group of field-collected beetles. Size was related to potential fecundity for four out of four groups of females collected from emergence traps in the field and for one of two groups reared in the laboratory from field-collected late-instar larvae. As females lay a single batch of eggs, beetle size may be important in the population dynamics of A. parvulus. Populations of A. parvulus with small beetles are potentially less likely to persist and expand than populations with relatively large females.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Overwintering, soil distribution and phenology of Childers canegrub, Antitrogus parvulus (Coleoptera : Scarabaeidae) in Queensland sugarcane

In this study, the question of whether Childers canegrub, Antitrogus parvulus (Britton) overwinters in the subsoil was addressed. Irrigated fields of sugarcane were sampled during a 2-year period near Bundaberg in southern Queensland. Antitrogus parvulus overwintered as second and third instars at each of three sites. During autumn and winter third instars of different allochronic (separated in age by 12 months) populations occurred together and could not be readily separated. Field-collected third instars were reared on ryegrass and separated into two age groups based on the date of pupation. Third instars in the first year of their life cycle (young third instars) remained at shallow depth (100-200 mm) and did not overwinter in the subsoil as once thought. Minimum temperatures during winter were 13-16degreesC and did not prevent young third instars from feeding and gaining weight. Third instars in their second and final year moved downwards from late summer and pupated in the subsoil at 293-425 mm in spring. General phenology was as previously reported with first instar larvae occurring from January until April, second instars from January until November and third instar larvae throughout the year. Prepupae and pupae were found between October and December and adults occurred in soil during November and January. Batches of eggs occurred at a mean depth of 350 mm. First and second instars occurred predominantly at relatively shallow (100-200 mm) depths in the soil profile. All stages tended to be most common under rows of sugarcane rather than in the interrow.

Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10(4.1) and 10(4.3) Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed.

Susceptibility of Aedes aegypti larvae to temephos and Bacillus thuringiensis var israelensis in integrated control

The susceptibility of field collected Aedes aegypti larvae was evaluated in terms of median lethal time (LT50) and final mortality, when treated with temephos, Bacillus thuringiensis var israelensis as well as mixtures of these two agents. Third instar larvae were shown to be more susceptible than early and late fourth instar ones to the entomopathogen. Survival of some individuals when exposed to temephos suggest possible resistance. Temporal synergism in early fourth instar larvae was detected when they were exposed to mixtures of Bti-temephos. The possibility of this integrated treatment is commented on.

Analysis of genetic relatedness between populations of Aedes aegypti from different geographic regions of São Paulo state, Brazil

RAPD markers have been used for the analysis of genetic differentiation of Aedes aegypti, because they allow the study of genetic relationships among populations. The aim of this study was to identify populations in different geographic regions of the São Paulo State in order to understand the infestation pattern of A. aegypti. The dendrogram constructed with the combined data set of the RAPD patterns showed that the mosquitoes were segregated into two major clusters. Mosquitoes from the Western region of the São Paulo State constituted one cluster and the other was composed of mosquitoes from a laboratory strain and from a coastal city, where the largest Latin American port is located. These data are in agreement with the report on the infestation in the São Paulo State. The genetic proximity was greater between mosquitoes whose geographic origin was closer. However, mosquitoes from the coastal city were genetically closer to laboratory-reared mosquitoes than to field-collected mosquitoes from the São Paulo State. The origin of the infestation in this place remains unclear, but certainly it is related to mosquitoes of origins different from those that infested the West and North region of the State in the 80's.