Comparison of gross visual and microscopic assessment of four anatomic sites to monitor Besnoitia tarandi in barren-ground caribou (Rangifer tarandus)

The objective of this study was to establish a standardized protocol to monitor Besnoitia tarandi prevalence and intensity in barren-ground caribou (Rangifer tarandus) herds by: 1) calculating the relative sensitivity and specificity of the gross visual assessment of four anatomical sites compared with microscopic evaluation, and 2) determining which of four anatomical sampling sites was the most sensitive for detecting B. tarandi cysts by microscopy. Sampled tissues consisted of the conjunctiva of the left eye and skin sections from the rostrum, metatarsus, and thigh from 312 harvested caribou. Diagnosis of infection with B. tarandi was based on observation of at least one cyst by microscopic examination. For each tissue, the maximal density of cysts (number of B. tarandi cysts/mm(2) in the section examined) was calculated for a measured area consisting of the dermis extending from the epidermis of the skin to the base of the hair follicles and adnexal structures. For the conjunctiva, the entire submucosa was evaluated. Gross visual evaluation markedly underestimated B. tarandi prevalence in caribou with a relative sensitivity ranging from 0.29 in the conjunctiva to 0.13 in the skin section from the thigh, whereas relative specificities ranged from 0.98 to 1.00. The metatarsus and rostrum skin sections had the highest probabilities of cyst detection of all four anatomical sampling sites. The metatarsus harbored significantly higher densities of B. tarandi cysts than the rostrum, thigh, or conjunctiva. In conclusion, microscopic evaluation of a skin section from the anterior aspect of the mid-third portion of the metatarsal region could be used as a standardized comparative indicator of density of B. tarandi infection in Rangifer.     

The biological aspects of botfly invasions in reindeer]

Field and laboratory studies established the development time of all the phases of Oedemagena tarandi and Cephenemyia trompe in different climatic zones of their habitat. The phenological and ecological features of O. tarandi and C. trompe in the aforesaid zones were identified which are the basis for scientifically grounded prophylactic measures. Data on potential and actual fecundity make it possible to calculate their reproduction potential and to forecast the invasion intensity of the reindeer in the next season.     

Trapping oestrid parasites of reindeer: the response of Cephenemyia trompeand Hypoderma tarandi tobaited traps

Abstract. At 340–360 km North of the Arctic Circle in Norway, Hypoderma tarandi (L.) and Cephenemyia trompe (Modeer) females were caught in baited traps from 10 July to 21 August. During three summers, adverse climatic conditions inhibited flight activity of these oestrids on 56–68% of the days. Flies were not caught prior to or after these dates, nor at winds above 8 m/s, temperatures below 10C, light intensities below 20,000 lux, or during periods of rain or snow. C0₂-baited insect flight traps caught significantly more H. tarandi females than non-baited traps. However, neither a white reindeer hide or reindeer interdigital pheromone glands enhanced the attraction of C0₂to H. tarandi or C. trompe. Hypoderma tarandi females also were attracted to mobile people, but not to stationary individuals. There were no significant differences in the numbers of C.trompe or H.tarandi caught in C0₂-baited traps in a birch/willow woods, on the treeless vidda (=tundra-like biome), or at woodsrvidda ecotone sites. Flies were caught in traps on days when the nearest reindeer herds were 25–100 km away. Significantly more H.tarandi and C.trompe were caught from 09.30 to 14.30 hours than from 14.30 to 19.30 hours; no flies were caught from 20.00 to 07.00 hours (Norwegian Standard Time = NST). Because CO_z-baited traps caught from hundreds to thousands of mosquitoes, blackflies and Culicoides midges, when climatic conditions inhibited oestrid activity, reindeer aggregations and movements attributed to insect attacks during warm sunny days may be largely in response to attacks by H.tarandi and C.trompe.     

Preliminary evaluation of four immunological tests for the early diagnosis of Hypoderma tarandi causing hypodermosis in reindeer

294 serum samples from five Norwegian reindeer herds were examined for antibodies against Hypoderma tarandi L. The first and second larval instars of H. tarandi were tested as antigens in immunodiffusion tests, passive haemagglutination and an Enzyme-Linked Immunosorbent Assay (ELISA). The latter technique, using first instar antigens, produced the best results. A significant difference (P less than 0.1%) was observed between the antibody value of naive reindeer bred in France and those from infected Norwegian herds. No correlation was observed between the antibody titre and the number of warbles recovered at necropsy from the 294 Norwegian reindeer.     

Factors affecting size, longevity and fecundity in the reindeer oestrid flies Hypoderma tarandi (L.) and Cephenemyia trompe (Modeer)

1. Laboratory reared reindeer oestrid flies Hypoderma tarandi and Cephenemyia trompe (Diptera: Oestridae) were weighed to determine progressive weight loss and death weights at treatments with various temperature and humidity conditions.
2. Four individual measurements of size were taken: larval weight, wet weight of newly eclosed flies, wing length, and weight of flies after dehydration and fat extraction. In H. tarandi, males were bigger than females (except for wing length), whereas the reverse was true for C. trompe .
3. Size variation was not significantly related to conditions (temperature, humidity, duration) during the pupal stage, but individual reindeer produced flies (both species) of different mean sizes. These size differences were not correlated with larval burden (= number of larvae per individual host), but are hypothesized to be connected to unknown host quality factors.
4. Longevity of flies kept in vials and subjected to various temperature and humidity conditions revealed that C. trompe lived significantly longer than H. tarandi (range: 4–44 and 1.2–27 days, respectively) at 5–33 °C. Male H. tarandi survived longer than females; female C. trompe survived longer than males. Longevity was not significantly correlated to any of the size measures.
5. Most flies had a large portion of their fat reserves left at death.
6. In H. tarandi , mean number of eggs was 609 ± SD 73 (range 354–772, n = 119). Egg number was slightly dependent on larval size, but not on wet weight of newly eclosed flies or wing length. In C. trompe , mean number of eggs was 960 ± SD 208 (range 493–1349, n = 31).
7. The possible adaptive value of large size in oestrids is questioned. Benefits of flexibility in size in oestrids are hypothesized.     

Abiotic factors influencing embryonic development, egg hatching, and larval orientation in the reindeer warble fly, Hypoderma tarandi

Wild-caught, tethered females of the reindeer warble fly, Hypoderma tarandi (L.) (= Oedemagena tarandi (L.)), (Diptera, Oestridae) were stimulated to oviposit on hairs of a reindeer hide. Newly laid eggs incubated at constant temperatures and relative humidities hatched within 3 days to 2 weeks, depending on the experimental conditions. Over a range of 7-40 degrees C, hatching only occurred between 20 and 37 degrees C. Eggs held at 100% relative humidity had lower hatchability and longer time to hatch relative to eggs held at 77% relative humidity. The average number of degree-days for hatching was 50.35. Between 20 and 33 degrees C there was a temperature-dependent linear trend in developmental rate, and the proportion of eggs hatching was highest, and least variable, at the mid-temperature ranges. The temperature range found in the natural host micro-habitat where H. tarandi commonly affix their eggs (close to the skin at the base of a host hair) was consistent with the experimental temperature treatments that produced the highest hatching rate. Newly emerged larvae displayed positive thermotaxis, while showing no phototaxic or geotaxic behaviour. Results indicate that constraints of the host environment, coupled with temperature-dependent hatching success, may impose a selective pressure on oviposition behaviour.     

The standardization of the reverse indirect haemagglutination test for the assay of the viral antigen of Japanese encephalitis vaccine

The reverse indirect haemagglutination (RIHA) test has been standardized for the assay of the viral antigen of purified Japanese encephalitis (JE) vaccine. Glutaraldehyde fixed sheep erythrocytes were sensitized with ammonium sulphate purified antibodies to JE vaccine raised in mice. The sensitivity of the erythrocytes fell to about one hundredth of the initial sensitivity in the first two days after preparation. After initial loss in sensitivity the stability of the cells became stabilized and the cells retained their titre for one year at 4-8 degrees C. The initial loss in sensitivity was not reduced by storing the cells at -70 degrees C, but after freeze drying the sensitized cells with a stabilizer one day after their preparation the cells retained their sensitivity. The RIHA test has been found to be a highly reproducible and sensitive method for detecting viral antigen in 5-10 ng of protein nitrogen. The sensitivity of the test was affected by the origins of the erythrocytes, i.e. from the different sheep from which they were drawn. To obtain results more rapidly, goose erythrocytes were used in place of sheep erythrocytes and the sensitized goose erythrocytes gave RIHA results in only 40 min.     

Age-related changes of the mineral component content in the hemolymph of 2d- and 3d-stage larvae of the reindeer warble fly (Hypodermatidae)

A mineral components concentration in the haemolymph of the IInd- and IIIrd-stage larvae of Oedemagena tarandi depends on the physiological state of parasites. Metabolism of mineral components is most intensive in a young actively growing organism and during moulting. The moulting is characterized by the decrease in the concentration of inorganic phosphorus and potassium and by the increase of calcium, magnesium and natrium.     

Oral myiasis in a child by the reindeer warble fly larva Hypoderma tarandi

We describe a case of human oral myiasis by a first-stage larva of the reindeer warble fly, Hypoderma (Oedemagena) tarandi (L.) (Diptera: Oestridae), in a 12-year-old girl. In September the patient complained of erythema, swelling and conjunctivitis of the right eyelid. Symptoms subsided spontaneously but re-occurred in October. In December she presented with acute swelling of the right corner of the mouth. Later that day a living larva protruded from this swelling. The patient had been on vacation in Norway during the previous summer months.     

Redescription of Besnoitia tarandi (Protozoa: Apicomplexa) from the reindeer (Rangifer tarandus)

Besnoitia tarandi tissue cysts were found in naturally-infected reindeer (Rangifer tarandus) from Finland. Infectivity of its tissue cysts, bradyzoites, and tachyzoites to animals and cell culture was studied. The bradyzoites and tissue cysts were not infectious to out-bred mice, rabbits or gerbils. When fed tissue cysts, neither cats nor dogs excreted oocysts. However, the parasite was lethal to interferon-gamma gene knock out mice irrespective of the route of inoculation. The parasite was grown successfully in African Green Monkey cells from tissues of two reindeer for the first time. Non-dividing, uninucleate tachyzoites from smears from cell cultures were 5.6 x 1.4 microm (4.5-7.4 x 1.0-1.9, n=50) in size. Longitudinally-cut bradyzoites in tissue sections measured 7.4 x 1.3 microm (6.5-7.8 x 1.0-1.6, n=30). Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and equids (Besnoitia bennetti) in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. tarandi was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collection.     

Trapping oestrid parasites of reindeer: the relative age, fat body content and gonotrophic conditions of Cephenemyia trompeand Hypoderma tarandifemales caught in baited traps

Abstract. Dissection of flies caught in northern Norway revealed that only mated, gravid females of H.tarandi (L.) and larviparous C.trompe (Modeer) were caught in host-mimicking C0₂-baited traps. Trapped females had the same gonotrophic and fat body (FB) conditions as females caught on and around reindeer. Most trapped females of both species were of middle to old age, having only one-half to no FB reserves left and only a few to moderate numbers of eggs or larvae remaining. Most young females trapped also had previously oviposited or larviposited at least once, and some newly eclosed, mated females were present throughout the fly season. Based on the known location of reindeer herds, it was evident that trapped flies that had recently oviposited or larviposited had dispersed into the trapping area from 25–100 km away. Declining FB reserves sustained wild-caught C.trompe females (and in utero larvae) in the laboratory for 14 days and H.tarandi females for 18 days. Reserve FB also was depleted during long flights. Females of both species that flew for the longest times (5.1–11.7 h) on a laboratory flight mill had low FB reserves, but nearly maximum numbers of eggs or larvae. Conversely, most females that flew for less than 5 h on the flight mill had little or no FB remaining, and few eggs or larvae. The large FB reserves accumulated as larvae feed in the vertebrate host enable the non-feeding adults to survive and infect their hosts even after prolonged periods of flight-inhibiting climatic conditions.     

Molecular epidemiological investigation of enterohaemorrhagic Escherichia coli isolates in Japan

We have established several measures for control and prevention of EHEC infection including designation of the disease as notifiable since there was the sudden increase in the incidence of infection with EHEC O157:H7 in Japan in 1996, involving multiple outbreaks. Improvements in methodologies for isolation of these organisms and enhanced laboratory screening have revealed a variety of sources in food and animals. Although there seems to be a bovine reservoir for O157 EHEC in Japan as well as North America and UK, different foods have been linked to EHEC infection including salads, radish sprouts and salmon roe. There is clear evidence that divergent clones of EHEC O157:H7 are prevalent throughout Japan based on laboratory surveillance, however, we still need to better define the role of EHEC serogroups other than Escherichia coli O157 as important causes of human infection.     

The serodiagnosis of infections caused by Verocytotoxin-producing Escherichia coli

Patients with haemolytic uraemic syndrome (HUS) and haemorrhagic colitis (HC) produce serum antibodies to the lipopolysaccharides (LPS) of Escherichia coli O157 and certain other E. coli serogroups. Patients may also make salivary antibodies to the LPS of E. coli O157. Serological tests based on these antibodies can be used to provide evidence of infection in the absence of culturable VTEC or the toxins they produce. Serum antibodies to LPS persist for several months following onset of disease, enabling both current and retrospective serological testing. The LPS of E. coli O157 shares epitopes with strains of Brucella abortus, Yersinia enterocolitica O9, Vibrio cholerae O1 Inaba, group N Salmonella and certain strains of Citrobacter freundii and E. hermanni. Serological tests for serum antibodies to E. coli O157 should be evaluated in the light of these cross-reactions. Serological tests to supply evidence of infection with E. coli O157 have been shown to provide a valuable adjunct to bacteriological procedures for detecting culturable VTEC and VT. The use of well characterized LPS antigens in association with the techniques of ELISA and immunoblotting provide valuable procedures for detecting evidence of infection with E. coli O157 and possibly other VTEC.     

Distribution Patterns of Wolbachia Endosymbionts in the Closely Related Flower Bugs of the Genus Orius: Implications for Coevolution and Horizontal Transfer

Vertical transmission is the primary route of the endosymbiont Wolbachia for its own spread among invertebrate hosts, but horizontal transmission between different hosts is believed to have occurred multiple times. However, it is not well known how Wolbachia commonly spread among closely related hosts. We focused on the closely related species of the minute pirate bugs belonging to the genus Orius, which are important biological control agents in agricultural crops because they are the most useful natural enemy of various tiny pests, such as thrips. Here, we examined five Orius species (Orius sauteri, Orius nagaii, Orius minutus, Orius strigicollis, and Orius tantillus) from eight geographic localities in Japan for Wolbachia infection. Two distinct strains, wOus1 and wOus2, were detected based on Wolbachia surface protein (wsp) gene sequencing. Furthermore, multilocus sequence typing revealed that each of the strains comprised two variants that differed in a single nucleotide. The overall distribution patterns of the two Wolbachia strains were found to differ among host species: prevalent double infection with wOus1 and wOus2 in O. strigicollis; fixation of single infection with wOus2 in O. nagaii; occurrence of single infection with wOus1 in O. sauteri; prevalence of single infection with wOus1 in O. minutus with an exception in a single population; and lack of Wolbachia infection in O. tantillus. Such differences in the distribution patterns of Wolbachia may reflect the evolutionary history of Wolbachia infection among Orius species and/or ecological and physiological differences among the Orius species that determine the invasiveness and maintenance of the two Wolbachia strains.     

PCR assay for the specific amplification of Oesophagostomum bifurcum DNA from human faeces

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodule worm) is of major human health significance in northern Togo and Ghana where the human hookworm, Necator americanus, also exists at high prevalence. Accurate diagnosis of O. bifurcum infection in humans is central to studying the epidemiology and controlling the parasite. To overcome limitations of current copro-diagnostic methods, we have developed an alternative, molecular approach. Utilising genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal DNA, we have established a two-step, semi-nested PCR method for the specific amplification of minute amounts (fg) of O. bifurcum DNA from human faecal samples. Using a panel of 155 well-defined faecal and DNA samples, the assay achieved a sensitivity of 94.6% and a specificity of 100%. This PCR assay will be useful for the diagnosis of O. bifurcum infection and as a molecular tool for elucidating the epidemiology of human oesophagostomiasis.     

施钾量与AM真菌接种效应的关系

以非灭菌土壤为生长基础,通过烤烟盆栽试验研究了施钾量与菌根侵染及接种生物效应之间的关系,结果表明,施用钾肥能显著提高AM真菌对烤烟的侵染率,不施钾肥菌根侵染率最低,AM真菌对烤烟生长影响较小,低中等施钾量（0.375-1.125kK2O/kg土）显著促进了AM真菌的侵染,改善了宿主钾营养,增加植株生长量,过高施钾量（1．5gK2O/kg土）抑制AM的侵染,使烟株干物重及其含钾量有所下降。     

肠出血性大肠杆菌O157感染防治研究进展

肠出血性大肠杆菌 (EHEC)感染是一种重要的新发传染病 ,O15 7是EHEC的一个主要菌型 ,感染该菌可使人患腹泻、出血性结肠炎 (HC)、溶血性尿毒综合征 (HUS)等 ,死亡率较高。EHECO15 7感染在许多国家包括我国都有暴发流行。EHECO15 7产生的粘附因子Intimin可引起粘附擦拭 (A/E)损伤 ,并可产生致死性的毒素Stx。抗生素治疗可使患者并发HUS危险性增加 ,临床上无特效的治疗药物 ,疫苗研究将对EHECO15 7的控制起重要作用。     

Aggregation of entomopathogenic nematodes, heterorhabditis spp. and steinernema spp., among host insects at 9 and 20 degrees C and effects on efficacy

The negative binomial distribution (NBD) with density-dependent k was used to test and describe the distribution of nematodes in Otiorhynchus sulcatus, Galleria mellonella, and Spodoptera exigua at 9 and 20 degrees C. Aggregation was greater in O. sulcatus than in the other two hosts. In case of G. mellonella exposed to the heterorhabditid HF85, the distribution of nematodes in the insects was random. There was only a small effect of low temperature on aggregation; this effect was stronger for O. sulcatus than for the other two hosts. Aggregation of nematodes among insects in a population increases the infection chance of already infected insects and decreases the infection chance of uninfected insects. Consequently, more nematodes will have to penetrate O. sulcatus than G. mellonella or S. exigua to cause the same proportion of infection in a population. The relevance of aggregation of nematodes for control O. sulcatus is discussed. Copyright 1999 Academic Press.     

Species identification of Hypoderma affecting domestic and wild ruminants by morphological and molecular characterization

Cuticular structures and the sequence of the cytochrome oxidase I gene were compared for Hypoderma bovis (Linnaeus), Hypoderma lineatum (De Villers), Hypoderma actaeon Brauer, Hypoderma diana Brauer and Hypoderma tarandi (Linnaeus) (Diptera, Oestridae). Third-stage larvae of each species were examined by scanning electron microscopy revealing differences among species in the pattern and morphology of spines on the cephalic and thoracic segments, by spine patterns on the tenth abdominal segment, and by morphology of the spiracular plates. The morphological approach was supported by the molecular characterization of the most variable region of the cytochrome oxidase I (COI) gene of these species, which was amplified by polymerase chain reaction and analysed. Amplicons were digested with the unique restriction enzyme, BfaI, providing diagnostic profiles able to simultaneously differentiate all Hypoderma species examined. These findings confirm the utility of morphological characters for differentiating the most common Hypoderma larvae and reconfirm the power of the COI gene for studying insect identification and systematics.