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Quantitative real-time reverse transcription-PCR analysis of deformed wing virus infection in the honeybee (Apis mellifera L.).

Deformed wing virus (DWV) can cause wing deformity and premature death in adult honeybees, although like many other bee viruses, DWV generally persists as a latent infection with no apparent symptoms. Using reverse transcription (RT)-PCR and Southern hybridization, we detected DWV in all life stages of honeybees, including adults with and without deformed wings. We also found DWV in the parasitic mite Varroa destructor, suggesting that this mite may be involved in the transmission of DWV. However, the detection of the virus in life stages not normally associated with mite parasitism (i.e., eggs and larvae) suggests that there are other modes of transmission. The levels of DWV in different life stages of bees were investigated by using TaqMan real-time quantitative RT-PCR. The amounts of virus varied significantly in these different stages, and the highest levels occurred in pupae and in adult worker bees with deformed wings. The variability in virus titer may reflect the different abilities of bees to resist DWV infection and replication. The epidemiology of DWV is discussed, and factors such as mite infestation, malnutrition, and climate are also considered.

Animals↗

Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread.

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.

Apyrase↗

Identification of coagulase-negative staphylococci with the API staph system.

A kit for the identification of staphylococci based on the biochemical criteria proposed by Kloos and Schleifer (W.E. Kloos and K.H. Schleifer, J. Clin. Microbiol., 1:82-88, 1975) is now available commercially. The system was used to identify 100 strains of coagulase-negative staphylococci isolated from various body sites as the primary etiological agent of clinical infection. The increasing importance of staphylococci and their resistance to antibiotics provided the rationale for such an investigation. Over 90% of the Staphylococcus isolates were easily identified as to their species on the basis of their reaction profile to 19 biochemical tests included in the kit. The remainder, which showed minor variations, could also be assigned to the various species. Identification of the isolates was as follows: S. epidermidis, 54; S. haemolyticus, 5; S. simulans, 2; S. hominis, 1; S. capitis, 4; S. cohnii, 2; S. warneri, 2; S. xylosus, 8; and S. saprophyticus, 22. Antibiotic sensitivity patterns were determined for each of the isolates. Novobiocin resistance was detected in strains of S. saprophyticus and S. xylosus, a property hitherto recognized in Micrococcus sp. type 3 causing bacteriuria in young women. Resistance to penicillin was widespread among strains of several species, whereas resistance to tetracycline was mainly confined to strains of S. epidermidis. General resistance to sulfamethoxazole and nalidixic acid was found among all strains, with almost uniform sensitivity to the other drugs tested.

Anti-Bacterial Agents↗

Evaluation of the API latex conjugate reagent for serological typing of Salmonella and Shigella species.

The serological types of Salmonella and Shigella isolates were determined by using the SerImm Sure latex conjugates (Analytab Products, Plainview, N.Y.) and the standard slide agglutination test. The Salmonella latex conjugates correctly typed 114 of the 115 Salmonella isolates found in serogroups A through F; 50% these isolates were from serogroups B. The Shigella latex conjugates correctly typed 87 of the 92 Shigella isolates; 87% of these isolates were from serogroups B and D.

Agglutination Tests↗

API QuadFERM+ with rapid DNase for identification of Neisseria spp. and Branhamella catarrhalis.

The QuadFERM+ system (Analytab Products, Plainview, N.Y.), a 2-h carbohydrate degradation method for the identification of Neisseria spp., was evaluated along with a rapid DNase test for confirmation of Branhamella catarrhalis. QuadFERM+ identified 100% of 82 N. gonorrhoeae and 96% of 54 N. meningitidis strains. The two misidentified meningococcal strains were biochemically atypical and were also misidentified by the conventional method. Of 26 N. lactamica strains, 25 (96%) were correctly identified. Of 21 Neisseria spp., 14 (67%) produced carbohydrate reactions in agreement with the conventional procedure, and 7 strains produced detectable acid in the QuadFERM+ from maltose and sucrose but not glucose. All 9 N. cinerea and 30 B. catarrhalis strains were asaccharolytic by QuadFERM+. The rapid DNase test was positive for all B. catarrhalis strains and negative for all other organisms. Two beta-lactamase-positive N. gonorrhoeae strains and 25 (93%) of 27 beta-lactamase-positive B. catarrhalis strains were detected by the 2-h acidometric beta-lactamase test on the strip. QuadFERM+ with rapid DNase is a simple and easily interpretable method for identification of these organisms in the clinical laboratory.

Bacteriological Techniques↗

Evaluation of API An-IDENT and RapID ANA II systems for identification of Actinomyces species from clinical specimens.

We compared the accuracy of the An-IDENT system (bioMerieux Vitek, Inc., Hazelwood, Mo.) and the RapID ANA II system (Innovative Diagnostic Systems, Norcross, Ga.) with that of conventional biochemical tests for the identification of 85 strains of Actinomyces species. In our hands, the overall accuracy of the An-IDENT was 59% and that of the RapID ANA II was 24%. The error rate for the An-IDENT was 18% and that for the RapID ANA II was 38%. The results of this study suggest that although the An-IDENT was more accurate than the RapID ANA II (P < 0.005), neither system, in our hands, was able to identify Actinomyces species with an acceptable degree of accuracy. It is recommended that suspected Actinomyces isolates be identified by conventional testing.

Actinomyces↗

Prevalence and phylogeny of Kakugo virus, a novel insect picorna-like virus that infects the honeybee (Apis mellifera L.), under various colony conditions.

We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), from the brains of aggressive worker honeybees that had counterattacked a giant hornet. To survey the prevalence of KV in worker populations engaged in various labors, we quantified KV genomic RNA. KV was detected specifically from aggressive workers in some colonies, while it was also detected from other worker populations in other colonies where the amount of KV detected in the workers was relatively high, suggesting that KV can infect various worker populations in the honeybee colonies. To investigate whether the KV strains detected were identical, phylogenetic analysis was performed. There was less than a 2% difference in the RNA-dependent RNA polymerase (RdRp) sequences between KV strains from aggressive workers and those from other worker populations, suggesting that all of the viruses detected were virtually the same KV. We also found that some of the KV-infected colonies were parasitized by Varroa mites, and the sequences of the KV strains detected from the mites were the same as those detected from the workers of the same colonies, suggesting that the mites mediate KV prevalence in the honeybee colonies. KV strains had approximately 6% and 15% sequence differences in the RdRp region from deformed wing virus and Varroa destructor virus 1, respectively, suggesting that KV represents a viral strain closely related to, but distinct from, these two viruses.

Animals↗