Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated <Emphasis Type="Italic">Neorickettsia</Emphasis> sp. in Europe

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.     

Exploration of natural foci of tularemia and plague in Armenia using the serological examination of bird droppings and excrements of predatory mammals]

Forty strains of tularemia and 619 of plague microbes were isolated in 1974 in bacteriological examination of natural plague and tularemia foci from a great number of small mammals and their ectoparasites. At the same time in serological examination (in the antibody neutralization test) of bird pellets, 52 mummified cadavers, and 34 excretion samples of mammalian beasts of prey collected in Armenia (its central and North-Western part) in 1973 the antigen of tularemia microbe was revealed in 73, 8, and 3, and of plagye--in 42, 5, and 1 cases, respectively. In one of the sites examined the number of positive findings failed to exceed 10--17%, this indicating a low intensity of the epizootic in the majority of the places. Judging by the mean titres of the serological test, which varied from 1:12 to 1:1428 in examination for tularemia and from 1:12 to 1428 in examination for plague, it was possible to detect both epizootics coursing during the examination, and those which occurred several monts ago. Tularemia and plague foci were not infrequently present at the same territories, and these diseases could involve the same individual animals of Microtus arvalis (Pall.). The data obtained pointed to the greater effectiveness of examination of bird's pellets and excretions of mammalian beasts of prey for the reconnaisance investigation of the natural foci of plague and tularemia in comparison with the bacteriological methods applied usually.     

Rapid species identification of pygmy rabbits (Brachylagus idahoensis) from faecal pellet DNA

The pygmy rabbit (Brachylagus idahoensis) is a small lagomorph of the western United States that specializes in sagebrush (Artemisia spp.) habitat. Intensive habitat loss and modification have increased the vulnerability of pygmy rabbit populations, but the current geographic distribution and population status remain unclear. To aid in detection and population monitoring, we developed a species identification test that uses mitochondrial DNA species-specific primers to distinguish among six sympatric lagomorph species using DNA isolated from faecal pellets. Applying this test, we successfully identified the species of origin for all pellet samples that produced a positive PCR result (77% of 283 pellets collected). Pellets collected during the winter (December-February) had higher PCR success rate (93%) than pellets collected at other times of the year (72%). This test, using non-invasive genetic sampling of faecal pellets, provides an efficient method for assessing site occupancy and distribution of pygmy rabbits and other lagomorphs across large geographic areas.     

Indirect hemagglutination test of Rickettsia orientalis: its specificity for reference strains, Karp, Gilliam and Kato.

Amberlite XE-64 and bovine serum albumin-treated rickettsia suspension, which was prepared from the chicken yolk sacs infected with each of 3 reference strains of Rickettsia orientalis, Karp, Gilliam and Kato, was sonicated at 10 KC for 15 min at 4 degrees C and centrifuged at 10,000 rpm for 40 min at 0 degrees C. The supernatant was used to sensitize the formalinized and tanned sheep red blood cells (SFTSRC). This antigen (2.5%) could be preserved for at least 1 wk at 4-8 degrees C and at least a month, if merthiolate (1:10,000) was added. Each SFTSRCP of 3 reference strains was found to be specific in indirect hemagglutination (IHA) reaction with each homologous immune serum and there was little cross reaction with the heterologous one. This minor cross reaction might be due to the presence of a small amount of soluble antigen in SFTSRC. No cross IHA reaction with the typhus fever immune serum was observed. The IHA test was considered to be useful for the diagnosis of scrub typhus.     

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Rook Spring Seed Dispersal in the Agricultural Landscape – Frugivory, Granivory or Accidental Transport?

Seed dispersal seems to be extremely important in agrocoenoses where suitable habitats (patches) are surrounded by an unfavourable environment (matrix). The role of the rook Corvus frugilegus, an omnivorous bird, in seed dispersal was studied in the agricultural landscape of Eastern Poland. We analyzed 739 pellets produced by regurgitation, which were collected under breeding colonies in April, May and June. Our goal was to i) assess the structure of the seed pool in pellets; ii) evaluate the temporal variation in the pellet seed pool on two different time scales; iii) compare the species composition of seeds in pellets and vegetation under the rook nests. Seeds were present in 18 % of pellets; 571 seeds were found, half of them belonging to dry-fruited species, without any obvious adaptations to endozoochory. These seeds could be an additional source of food, or they could have been accidentally swallowed during foraging for other food items. Taking into consideration the abundance of the rook population, we assessed the mean number of seeds transferred by one bird to be from 4 seeds per month in April and up to 160 seeds in June. The most important factor responsible for qualitative and quantitative structure of seed pool in pellets is the time when pellets were regurgitated. The type and availability of food determines the number and species structure of dispersed seeds. The comparison of the species structure of the seed pool in pellets and of the herb layer under the breeding colonies showed that the rook could effectively disperse seeds of weeds, meadow and ruderal species, that could germinate under the dense canopy of trees at the studied sites.     

Isolation of a spotted fever group Rickettsia, Rickettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Evidence of High Tolerance to Ecologically Relevant Lead Shot Pellet Exposures by an Upland Bird

Upland birds that display grit ingestion behavior are potentially at risk of detrimental effects and death from lead poisoning at trap and skeet ranges and other areas where vast quantities of spent lead shot pellets abound. Because commonly cited force-feeding pellet exposure studies deviate from true field conditions, their results may not reflect true risks faced by upland birds. In particular, studies that use new shot pellets and administer more pellets than would be reasonably ingested, critically interfere with the understanding of actualized pellet exposures. In this study, northern bobwhites (Colinus virginianus), a frequent test species in shot pellet research, were dosed in an ecologically-relevant manner (i.e., with spent shot and with no more than three pellets). Notably, the 56-day post-dosing observation period, during which a battery of physiological measures were recorded, exceeded that of related studies. Despite a sustained suppression of a lead poisoning indicator, the data suggest upland birds can withstand spent shot pellet exposures. Data detected a survivorship ≥95%, absence of illness, demonstrated tolerance for extremely high blood lead concentrations, and unaffected blood parameters. In conjunction with ecological considerations (e.g., spatial scale and animal behavior), concern about bird population losses from the incidental ingestion of spent shot pellets is potentially overstated.     

Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets

The impact on antigen uptake and antibody response by the addition of absorption enhancers to Vibrio anguillarum O2 antigen was studied in oral vaccination trials of African catfish (Clarias gariepinus). Oral vaccination was achieved by feeding lag time coated pellets. The lag time coat prevents premature release of the encapsulated vaccine in the tank, before ingestion of the pellets by the fish. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Feeding of bacterin-layered pellets without absorption enhancers resulted in a rather low antigen uptake and antibody levels. The addition of absorption enhancers such as sodium salicylate, sodium caprate and vitamin E TPGS increased the serum antigen levels and specific antibody levels in the systemic circulation. Skin mucus antibody levels were higher after oral vaccination compared to the IP and control group. The addition of absorption enhancers in the oral groups further increased the antibody levels obtained in the skin mucus.     

Isolation of spotted fever group rickettsia from Apodemus speciosus in an endemic area in Japan.

A spotted fever group rickettsia was isolated from Apodemus mice captured near the site where a person had been infected in Miyazaki Prefecture. Antigenic characteristics of the isolate designated as strain TO-1 were compared with those of Rickettsia japonica (strain YH) and Rickettsia montana (ATCC VR611) by the indirect fluorescent antibody test with 14 serum specimens obtained from Apodemus speciosus, immune rat antisera against the strains TO-1 and YH, and three patients' sera. The titers of these sera measured with strain TO-1 were identical to those with strain YH, suggesting the antigenic similarity between these two organisms. It was also suggested that Apodemus speciosus is likely to be an important vertebrate host for a spotted fever group rickettsia in Japan.     

Large-scale features affect spatial variation in seed dispersal by birds in juniper woodlands

Fruit–frugivore interactions have received great attention from evolutionary, ecological and applied perspectives. However, despite the fact that large-scale approaches may offer new insights on fruit–frugivore interactions, little work has been devoted to exploring the factors shaping large-scale variation of seed dispersal. This paper studies the spatial variation in seeds regurgitated within pellets by wintering thrushes (genusTurdus) in a set of 26 Spanish juniper (Juniperus thurifera) woodlands scattered along a latitudinal gradient during two winters of contrasting cone and bird abundance. It explicitly explores whether pellet rain variation among woodlands was related to changes in vegetation cover, temperature, day length duration and bird abundance. Top regression models in the Akaike information criterion framework explained 34 % and 70 % of variance in pellet numbers in low and high crop and bird abundance years, respectively. In both winters, the spatial variation in pellets correlated to thrush abundance and day length duration. Pellet abundance linked to cone distribution only in the year of high crop, in which many migratory, extra-Iberian thrushes arrived to exploit the resource. Strong dependence of seed dispersion on large-scale features affecting bird numbers (e.g. the arrival of extra-Iberian migratory birds) and feeding behaviour (e.g. latitudinal variation in day length duration) suggests a primary role for variables acting at large scale on this mutualistic interaction.     

IgE antibody against hepatitis B surface antigen in mice

Hepatitis B surface antigen (HBs antigen) was examined for elicitation of IgE production by injection into mice. The Prausnitz-Küstner (PK)-type skin test in the rat was employed for detection of IgE antibody to HBs antigen, because no sufficient purified HBs antigen was available as the challenging antigen for the passive cutaneous anaphylaxis (PCA) test in rats. The positive PK test was considered to be due to IgE antibody, since the active principle was inactivated by heating the sera at 56 C for 30 min, did not bind to protein A and was eliminated by anti-mouse IgE antisera. These data indicate that the PK-type test in rats can be used for detection of mouse IgE antibody when the amount of a test sample is not sufficient for the PCA test in rats.     

Relative Sensitivity of Gel Diffusion, Complement Fixation, and Immunoelectroosmophoresis Tests for Detection of Hepatitis-Associated Antigen and Antibody

The immunoelectroosmophoresis (IEOP) test was compared with gel diffusion and complement fixation (CF) tests for sensitivity in detecting hepatitis-associated antigen (HAA) in the sera of hepatitis patients, for titration of HAA, and for detection of antibody to HAA. The IEOP test was found to be slightly more sensitive than either gel diffusion or CF tests for detection of antigen in the patients' sera. Titers of HAA demonstrated by IEOP were higher than those seen in gel diffusion tests but lower than CF titers. The gel diffusion test with an "enhancement" pattern was found to be more reliable than the other two procedures for detection of low levels of anti-HAA, due to the greater inhibitory effect of an antigen excess in the IEOP system and the possible masking of low levels of antibody by anticomplementary activity in the CF test system. Staining of immunoprecipitates in the IEOP test contributed little to the sensitivity of the test for detection of HAA.     

Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Analysis of pellets of the Grey heron, Ardea cinerea, from colonies in Belgium

Analyses of the contents of pellets of Grey herons from different breeding colonies and breeding seasons indicated that the mammal and invertebrate species included in the heron diet varied between colonies and between years. The large differences in mammal species found in neighbouring heronries suggested the existence of important small-scale variability in the exploitation of mammals. Almost no fish and bird remains were found in the pellets. We believe that most invertebrates, even the smallest ones, were actively taken by the herons. Identification of mammal species composition by means of skulls and hair remains gave different results. The shape of a pellet seemed to be dependent upon the (moisture?) conditions during its formation.     

Colorful Drying

Drying is one of the standard unit operations in the pharmaceutical industry and it is important to become aware of the circumstances that dominate during the process. The purpose of this study was to test microcapsulated thermochromic pigments as heat indicators in a fluid bed drying process. The indicator powders were manually granulated with α-lactose monohydrate resulting in three particle-size groups. Also, pellets were coated with the indicator powders. The granules and pellets were fluidized in fluid bed dryer to observe the progress of the heat flow in the material and to study the heat indicator properties of the indicator materials. A tristimulus colorimeter was used to measure CIELAB color values. Color indicator for heat detection can be utilized to test if the heat-sensitive API would go through physical changes during the pharmaceutical drying process. Both the prepared granules and pellets can be used as heat indicator in fluid bed drying process. The colored heat indicators give an opportunity to learn new aspects of the process at real time and could be exploded, for example, for scaling-up studies.     

Acute toxicity of lead, steel, and an iron-tungsten-nickel shot to mallard ducks (Anas platyrhynchos)

Twenty mallards (Anas platyrhynchos) of both sexes were dosed by oral gavage with Heavi-Shot (H-S; Environ-Metal, Inc., Sweet Home, Oregon, USA) pellets, 20 with steel shot, and 10 with lead (Pb) pellets, all of equal size. All pellets were fired from a shotgun into an absorbent material, retrieved, and weighed prior to introduction into the ducks. Birds were fed whole kernel corn and grit and observed for signs of toxicity for 30 days following dosing. Hevi-Shot pellets lost an average of 6.2% of their mass and steel shot pellets lost 57% of their mass in the birds' gizzards. Almost all (90%) of the Pb shot dosed birds died before the end of the study, while no mortality was observed in the steel or H-S dosed groups. Even though total food consumption differed between the H-S and steel shot groups, mean bird weight change was not different. There were no significant morphologic or histopathologic abnormalities of the liver and kidney in the H-S and steel shot groups. Results indicated that mallards dosed orally with eight No. 4 H-S pellets were not adversely affected over a 30-day period, and that H-S provides another environmentally safe nontoxic shot for use in waterfowl hunting.     

Highly sensitive electroimmunodiffusion method of detecting antigens on cellulose acetate film]

A highly sensitive electro-immunodiffusion test suggested by the authors for antigen detection on cellulose-acetate films consists of three stages: antigen concentration in a discontinuous buffer system on cellulose-acetate films; antigen detection on the same films by immunodiffusion using standard test system; to detect the precipitation bands the washed films are stained with protein dyes in case the reaction takes place in the zone of vision, or subject to further treatment by means of "plating" the precipitates with antiglobulin antibody or by radioautography. The method permits one to reveal the nanogram levels of alfa-fetoprotein and it may be applied for detection of antigens with different molecular weights and electrophoretic mobility.     

流行性出血热冻干致敏人O型血球的研制及其应用

本文应用致敏的人O型血球研究反向间接血凝(RPHA)和反向间接血凝抑制(RPHI)方法用以检测流行性出血热抗原抗体,并试验成功用pH9.0硼酸盐水制备灭活鼠脑病毒液作为抗原。为抗原制备提供了一种简便的方法。以上RPHA法用于检测组织培养内病毒与用荧光法检测细胞内病毒抗原法结果一致,用RPHI检测病人血清抗体效价,特异性高,敏感性与IFA相同。该致敏血球和抗原是冻干制品,稳定性好、使用方便,是一种代替荧光检测病毒抗原和抗体的良好制品。