PCR-BASED TECHNIQUE FOR IDENTIFICATION AND DETECTION OF TRICHOGRAMMA SPP. (HYMENOPTERA: TRICHOGRAMMATIDAE) WITH SPECIFIC PRIMERS

Abstract The rDNA-ITS2 regions of T. dendrolimi Matsumura and T. ostriniae Pang et Chen (Hymenoptera: Trichogrammatidae) were cloned and sequenced. The homologous sequences available in GenBank were retrieved and analyzed, and then specific primers were designed for molecular identification and detection of T. dendrolimi . Repeated screening showed that PCR amplification by the diagnostic primers enabled the differentiation of not only bulk samples and single adult (male or female), but also eggs and juveniles, which was not possible by conventional methods. The advantage of this system over morphology-based systems is that non-specialists are able to identify individuals or trace specimens efficiently. The derived molecular detection technique was then used to identify 12 specimens collected from different localities on the Chinese mainland; the results showed that this protocol could be applied to molecular monitoring of Trichogramma species in the field. Finally, 1132s of 6 geographical populations of T. dendrolimi (TdCHA, TDJL, TdXZ, TdKH, TdCZ and TdYBL) were cloned and sequenced. The multialignment analysis of intraspecific ITS2 sequences showed that the diagnostic primers have their own theoretical bases.     

基于诊断引物的赤眼蜂鉴定和检测技术

通过对松毛虫赤眼蜂Tichogramma dendrolimi Matsumura和玉米螟赤眼蜂T.ostriniae Pang et Chen（膜翅目：赤眼蜂科）核内可转录第二间隔区（简称：ITS2）的克隆、测序,并获取和分析了GenBank中已登录的同源序列,然后设计了松毛虫赤蜂的特异引物以用于该蜂的分子鉴定和检测,经过反复筛选发现：采用鉴定引物通过PCR扩增不仅可以区分鑫头样品,单头样品（雌蜂或雄峰）,而且可鉴定幼期虫和卵,这用传统方法是无法办到的。该鉴定技术比基于形态学鉴定检测技术用来鉴定了从中国大陆不同地域和寄主上采集到的12个样品,结果表明：该方法可用于赤眼蜂田间分子监测和实验室拟寄生行为研究。     

FLUORESCENCE CONCENTRATION IMMUNOASSAY FOR RAPID DETECTION OF CAMPYLOBACTER SPP. IN CHICKEN RINSE WATER

A total of 215 freshly processed post-chill whole chicken carcasses were assessed for Campylobacter spp. contamination by a fluorescence concentration immunoassay (FCIA) procedure. Whole chicken carcasses were sampled with low volume water rinses from which 5 ml portions were enriched with brucella enrichment broth with or without oxyrase supplement in a test tube enrichment system. After a 24h stationary incubation at 42C, each sample was assayed using a FCIA procedure for the presence or absence of campylobacters. The FCIA procedure indicated Campylobacter spp. contamination in 84% of carcasses using oxyrase supplemented enrichment, while only 47% of the chicken carcasses were positive from nonsupplemented enrichment. The corresponding incidence rates detected by culture method were 92% and 87% for oxyrase supplemented and unsupplemented samples, respectively. The FCIA procedure can be completed in less than 1 h with 48 samples including a positive and a negative control assayed on one plate. In summary, the test tube oxyrase-supplemented stationary enrichment system followed by the use of the FCIA procedure was found to be an effective, rapid method for the detection of Campylobacter spp. in chicken rinse water.     

USE OF POLYMYXIN B IN A CAPTURE ENZYME IMMUNOASSAY FOR DETECTION OF SALMONELLAE SPP. LIPOPOLYSACCHARIDE

A capture enzyme immunoassay for detection of salmonellae sp. lipopolysaccharide was developed. The assay made use of polymyxin B sulfate, passively attached to a polystyrene matrix, to capture lipopolysaccharide. Bound lipopolysaccharride was then detected with a monoclonal antibody, specific for salmonellae spp. followed by goat antimouse antibody conjugated with horseradish peroxidase.
The analytical sensitivity of the assay was approximately 1 ng/ml of lipopolysaccharide. The results are comparable to those obtained with a competitive enzyme immunoassay previously developed. The sensitivity of the polymyxin B assay decreased to 4–5 ng/ml when the salmonellae spp. lipopolysaccharide was mixed with 1–100 μg/ml of Escherichia coli lipopolysaccharide, while this level of heterogeneous lipopolysaccharide, did not decrease the sensitivity of the competitive enzyme immunoassay.
The polymyxin B capture assay was advantageous in that polymyxin B is a standardized reagent that is relatively inexpensive and does not require extensive preparation or containment facilities. The assay is robust; however, because of the light sensitivity of polymyxin B, its stickiness to other reagents and interference by other lipopolysaccharides, this assay requires careful attention to detail and may therefore be an unsuitable assay for field use.     

APPLICABILITY OF RAPID METHODS FOR DETECTION OF SALMONELLA SPP. IN POULTRY FEEDS: A REVIEW

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. More sensitive and rapid detection of contaminated feedstuffs may lead to more selective and therefore less expensive treatment of feeds, reduced rates of transmission to a poultry host and reduced carcass contamination. In order to interrupt the cycle of Salmonella spp. transmission from feed to poultry to the consumer, more rapid detection methods to monitor these sources are needed that provide conclusive results within the time frame of feed mixing or broiler processing. Within the last decade, new variations of selective media have been investigated to increase selectivity without reducing Salmonella spp. recovery. Immunological assay methods may also decrease assay time from 96 h to within 24–30 h. But all commercially available methods still require 16 to 57 h for preenrichment, enrichment, and in some cases, postenrichment to recover sublethally injured cells before the assay can be performed. Among the molecular methods that are currently available, the polymerase chain reaction (PCR) represents a tremendous potential for the detection of low levels of pathogenic bacteria. Once optimized, rapid methods may be used to quickly, reliably and inexpensively screen a variety of feedstuffs and feed components for the presence of Salmonella spp., with the goal of minimizing both the cost of feed treatment and the horizontal transmission of Salmonella spp. from feed to poultry.     

APPLICATION OF GENE AMPLIFICATION IN CONJUNCTION WITH A HYBRIDIZATION SENSOR FOR RAPID DETECTION OF SALMONELLA SPP. AND FECAL CONTAMINATION INDICATORS IN ANIMAL FEEDS

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. To demonstrate the feasibility of rapidly detecting Salmonella spp. and fecal pollution microbial indicators in feed using gene amplification protocols, commercial and mixed feed samples were inoculated with two levels of a marker strain of S. typhimurium. Liquid extracts of the feed samples were used as templates in gene amplification reactions to amplify sequences associated with fecal contamination indicators. The sequence specificity of the amplification products (amplicons) were confirmed using biotin and fluorescein labeled probes in a navel dual probe based hybridization sensor. Using the combination of gene amplification and the hybridization sensor, the presence of sequences associated with fecal contamination were detected in 15 different feed matrices without employing preenrichment steps. Using this detection methodology, fecal pollution can be confirmed in feed at naturally occurring concentrations. The study demonstrates that it is possible to rapidly detect and confirm the presence of pathogenic bacterial genera in feed matrices by combining robust gene amplification reactions with appropriate post amplification detection systems.     

DETECTION OF SALMONELLA SPP. CONTAMINATION IN RAW MATERIALS AND COSMETICS/PHARMACEUTICAL PRODUCTS USING THE BAXTM SYSTEM, A PCR-BASED ASSAY

Abstract A system utilizing the polymerase chain reaction (PCR), the BAX ^TM , was compared and validated against standard selective/enrichment assays to detect the presence of Salmonella spp. in artificially contaminated samples of raw materials and cosmetic/pharmaceutical products. After a 24 h incubation in lactose broth or lactose broth with Tween 20, the inoculated samples were analyzed both by the BAX ^TM system and by standard enrichment/selective methods. Standard enrichment assays required 5–7 days to confirm the presence and identification of Salmonella typhimurium , while the BAX ^TM system reduced the detection time to 30 h. The BAX ^TM system allowed a faster quality control evaluation of those raw materials and cosmetic/pharmaceutical formulations that require Salmonella spp. screening.     

志贺菌流行株药物敏感性及质粒图谱分析

目的：分析志贺菌流行株的质粒图谱及其与细菌药物敏感性的相关性。方法：从菌痢患者粪便标本中分离6株 福氏志力和4株宋内志贺菌,分别对其质粒图谱与药物敏感性进行检测和对其相关性进行分析。结果：不同菌株的质粒图谱具有明显的差异,各菌株的质粒图谱与其对头孢三嗪、头孢唑林、环丙沙星、诺氟沙星、氯霉素的耐药特性无明显相关性。结论：获自患者的福氏志贺菌和宋内志贺菌具有不同的质粒图谱以及抗菌药物敏感性,提示在我市引起菌痢的志贺菌具有不同的来源。     

BIOLOGY AND BEHAVIOUR OF FRIGATEBIRDS FREGATA SPP. ON ALDABRA ATOLL

Both species nested in mixed colonies in mangrove trees. The tops of trees were usually occupied exclusively by minor and the lower parts by ariel, but most nests of both species were in the intermediate parts of the canopy. The main laying season for both species was July to January. A census showed about 27 000 individuals present at the height of the season (1500 breeding pairs of minor, 5350 of ariel). Seasonal variation in numbers could be accounted for almost entirely by the changes in breeding activity of a resident population. Young of both species were fed at or near the nest-site for at least four months after fledging. A recovery near Bombay of a wing-tagged immature ariel shows that this species, at least, undergoes a post-fledging dispersal; it is suggested that young minor either do not disperse, or do so later than ariel. Food samples collected from chicks showed no overall difference between the species, but a seasonal analysis showed that ariel took more squid than minor in the wet season, and in the dry season the two species took different proportions of the two commonest species of flying-fish. Chicks of ariel received smaller meals than minor chicks in the wet season, but similar-sized meals in the dry season; ariel chicks grew more slowly than minor chicks. It is suggested that the timing of the breeding season is related to the need for adults to build up fat reserves to carry them through the courtship, nest-building and laying periods, when they are tied to the colony and so have little opportunity to feed. The evidence for non-annual breeding in frigatebirds is discussed. It is concluded that while successful breeders must breed at intervals of more than 12 months, they could theoretically nest in two successive seasons and that, since breeding success is low, most individuals probably do so. Existing knowledge of the biology of four of the five recognized species of frigatebirds is summarized, and shows that the family is at least as uniform as the tropicbirds and much more so than other Pelicaniformes.     

POLYPHASIC CHARACTERIZATION OF DOLICHOSPERMUM SPP. AND SPHAEROSPERMOPSIS SPP. (NOSTOCALES,CYANOBACTERIA): MORPHOLOGY, 16S rRNA GENE SEQUENCES AND FATTY ACID AND SECONDARY METABOLITE PROFILES1

The genera Dolichospermum (Ralfs ex Bornet et Flahault) Wacklin, L. Hoffm. et Komárek and Sphaerospermopsis Zapomělová, Jezberová, Hrouzek, Hisem, K. ?eháková et Komárk.‐Legn. represent a highly diversified group of planktonic cyanobacteria that have been recently separated from the traditional genus Anabaena Bory ex Bornet et Flahault. In this study, morphological diversity, phylogeny of the 16S rRNA gene, production of fatty acids, and secondary metabolite profiles were evaluated in 33 strains of 14 morphospecies isolated from the Czech Republic. Clustering of the strains based on 16S rRNA gene sequences corresponded to wider groups of species in terms of morphology. The overall secondary metabolite and fatty acid profiles, however, were not correlated to each other and neither were they correlated to the 16S rRNA phylogeny nor the morphology of the strains. Nevertheless, a minor part of the detected secondary metabolites (19% of all compounds) was present only in close relatives and can be thus considered as autapomorphic features.