Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA.

Rabbit serum samples from eleven different research facilities were evaluated for the presence of immunoglobulin G against Pasteurella multocida by using an enzyme-linked immunosorbent assay (ELISA). Each facility which submitted serum samples also provided a brief history of each rabbit colony tested. Rabbits from colonies reported to have endemic P. multocida or of undetermined status had 83 (58.9%) of 141 rabbits that were positive. Colonies reported to be free from P. multocida had 110 (92.4%) of 119 rabbits that were negative by ELISA. The ELISA test described here showed a high degree of agreement (92-94%) with two other P. multocida ELISAs at different diagnostic facilities. This study confirms that an ELISA testing for serum antibodies against the P. multocida is a reliable diagnostic tool to screen colonies for P. multocida.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

Successful rederivation of contaminated immunocompetent mice using neonatal transfer with iodine immersion

There is an ongoing need to eradicate intercurrent disease from research mouse colonies. Commonly used surgical methods, however, are expensive and time-consuming. The purpose of this study was to determine the percentage of litters that could be rederived from infected mouse colonies by neonatal transfer. We immersed neonatal mice in a dilute iodine solution and transferred them to disease-free foster mothers within 48 h of birth. Donor and foster mothers were evaluated for pathogens by serology and fecal polymerase chain reaction (PCR) assay. Of 55 donor mothers, 100% were positive serologically and 59% were positive by fecal PCR for one or more tested organisms, including mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and Helicobacter hepaticus. At 4 to 6 weeks after neonatal transfer, 95% of foster mothers (which served as sentinels for the transferred pups) tested free of pathogens, the exceptions being one case of mouse parvovirus 1 and two of Helicobacter spp. We suggest that cross-fostering is a viable low-cost method for rederivation of mouse colonies contaminated with pathogens such as mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and H. hepaticus.     

Mutagenicity of blue rayon extracts of human bile in the Ames test

The mutagenicity of human bile was examined in the Ames Salmonella/microsome assay. Bile samples were obtained from the gallbladders resected from patients with cholelithiasis, choledocholithiasis, gallbladder cancer, extrahepatic bile duct cancer and other disease. For extraction of mutagenic components, the bile samples were treated with blue rayon and the adsorbed materials were assayed with Salmonella typhimurium TA98 in the presence of S9 mix. Twenty-four bile samples were tested and positive mutagenic activity was found in 14 samples. A 200-μl bile equivalent material gave 6.3 times as many revertant colonies as the solvent control. With several samples that had undergone two cycles of blue rayon extraction, clear dose-response relationships in mutagenicity were demonstrated.     

Spontaneous, mutagen-induced and adenovirus-induced anchorage independent tumorigenic variants of mouse cells

Normal C57 Black mouse embryo cells did not form colonies in agarose, but rare variant (ar+) cells able to grow in agarose were detected. Fluctuation analysis showed that ar+ variants arose by spontaneous mutation in the cultured cells. The frequency of ar+ variants was increased by treating cells with N-methyl-N'nitro-N-nitrosoguanidine or ethyl methane sulphonate, or by abortive infection by human adenovirus type 5. Induced ar+ cells were fibroblastic; most grew slowly and had slightly reduced saturation density and increased serum requirement, but formed colonies in agarose. Fourteen of twenty ar+ clones induced by Ad5 were T antigen negative and two of these were also negative when tested for viral DNA. Six clones contained a few cells that were T antigen positive when first tested, but were negative when retested later. The ar+ variants were tumorigenic in athymic and in normal syngeneic mice. The results suggest that the ar+ phenotype can arise by spontaneous or chemically-induced mutation, and can be induced by adenovirus by a process different from classical transformation.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Development of Panel of Monoclonal Antibodies Specific to Urochordate Cell Surface Antigens

Monoclonal antibodies are an important tool in the study of botryllid ascidians’ immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.     

The advantage of enzyme-linked immunosorbent assay (ELISA) as a method of microbiological monitoring for rat virus (RV).

An enzyme-linked immunosorbent assay (ELISA) was tested to detect antibodies against rat virus (RV). The purified ELISA antigens were prepared from rat embryonic cells infected with RV-13 (prototype strain) and UT-2 (Japanese isolate), respectively. Western blotting analysis confirmed that both of these antigens had three structural polypeptides (81 K, 61 K, and 59 K). Eleven laboratory and wild rat colonies in Japan were tested for rat virus contamination, serologically. No significant differences in the sero-positive ratio and the distributions of ELISA titers were demonstrated in the ELISA, using antigens from RV-13 and UT-2. ELISA was more sensitive and specific for detecting antibodies against RV from rat serum rather than hemagglutination inhibition (HI) test. This study also confirmed that the RV contaminated widely in colonies of laboratory and wild rats in Japan, and suggested that RV would have to be checked during the microbiological monitoring of laboratory rats.     

The possibility of assaying Wistar rat bone marrow CFUs in a xenogeneic (rat-to-mouse) system

The possibility of replacing the space-consuming rat-to-rat colony-forming unit (CFUs) assay by rat-to-mouse assay systems was examined using Wistar rat bone marrow. After considering the published results on the responsiveness of mouse strains to hemopoietic xenografts and on the ways to abrogate "xenogeneic resistance', we tested C57B1/6J and C3H/He mice conditioned by cyclophosphamide (CY) and/or whole-body irradiation in the following combinations: 850 rad C57Bl; 850 rad + CY C57Bl; 800 rad + CY C3H. A linear relationship between the number of cells injected and the macroscopical spleen colony count could be demonstrated with all three combinations. However, we observed a high number of endogenous colonies in the 850 rad C57B1 system. The results were confirmed by karyotype analysis. Colony yield and seeding efficiency with 800 rad + CY C3H were comparable to the rat-to-rat assay, but were considerably lower in the case of 850 rad + CY C57B1. In the latter system, the colonies were primarily erythroid.     

Prevalence of Yersinia pestis in rodents and fleas associated with black-tailed prairie dogs (Cynomys ludovicianus) at Thunder Basin National Grassland, Wyoming

Rodents (and their fleas) that are associated with prairie dogs are considered important for the maintenance and transmission of the bacterium (Yersinia pestis) that causes plague. Our goal was to identify rodent and flea species that were potentially involved in a plague epizootic in black-tailed prairie dogs at Thunder Basin National Grassland. We collected blood samples and ectoparasites from rodents trapped at off- and on-colony grids at Thunder Basin National Grassland between 2002 and 2004. Blood samples were tested for antibodies to Y. pestis F-1 antigen by a passive hemagglutination assay, and fleas were tested by a multiplex polymerase chain reaction, for the presence of the plague bacterium. Only one of 1,421 fleas, an Oropsylla hirsuta collected in 2002 from a deer mouse, Peromyscus maniculatus, tested positive for Y. pestis. Blood samples collected in summer 2004 from two northern grasshopper mice, Onychomys leucogaster, tested positive for Y. pestis antibodies. All three positive samples were collected from on-colony grids shortly after a plague epizootic occurred. This study confirms that plague is difficult to detect in rodents and fleas associated with prairie dog colonies, unless samples are collected immediately after a prairie dog die-off.     

Cryptosporidium sp. in a free ranging house gecko (Hemidactylus turcicus) in Israel

A house gecko, Hemidactylus turcicus, from an Israeli locality was found infected by Cryptosporidium sp. Infection was recovered in the anterior gut, upon examination by a transmission electron microscope. It comprised from 2.9 x 3.3 microns undivided and 2.6-3.3 x 3.8-3.9 microns dividing meronts, 1.9-2.3 x 2.0-2.6 microns young macrogamonts, and a 3.3 x 3.9 microns sporulated oocyst containing in cross section 4 sporozoites and a residuum. Oocyst dimensions were smaller and apparently not conspecific with the Cryptosporidium sp. reported from the introduced H. turcicus in Texas.     

Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria

Microbial transglutaminase (MTGase) is a commercial enzyme that has been applied to many protein containing foods to improve their textural property. The screening of MTGase-producing microorganisms from various sources might lead to the discovery of a new MTGase with different characteristics. This report demonstrates the use of a direct detection method for MTGase-producing bacteria grown on an agar plate by filter paper disc (FPD) assay. The principle of the assay is the formation of a red burgundy color by the hydroxamate-ferric complex. The color developed intensity was linearly correlated by the concentration of hydroxamic acid in the range of 0.1-0.8 μM and was visually scored at 4 levels: 0, 1, 2 and 3. Streptoverticillium mobaraense DSM 40847, a positive MTGase-producer, was chosen for the verification and improving of the proposed method. The colonies grown on the nutrient agar plate at 37°C for 24 h were covered with FPDs and 30 μl of substrates (CBZ-Gln-Gly and hydroxylamine). After incubation, 10 μl of the ferric-TCA-HCl solution was placed on the FPD. The optimal time taken to catalyze the formation of CBZ-Gln-Gly-hydroxamic acid by the MTGase and the time taken for the hydroxamate-ferric complex to form color were 180 and 60 min, respectively. Using this assay, 30 of 189 colonies isolated from wastewater and floating-floc samples showed MTGase-positive colonies which were well correlated to the quantitative screening of MTGase activity (R(2) = 0.9758). The results revealed that the FPD assay could be used for the qualitative screening of MTGase-producing bacteria.     

Problems associated with the identification of bordetella bronchiseptica.

Bordetella bronchiseptica, isolated from rodent nasopharygeal swabs, failed to produce characteristic colonies after 24 hours incubation at 37 degrees C. 4-7 days incubation at 37 degrees C was required to achieve positive motility test results, when isolates later identified as B. bronchiseptica were tested by Craigie tube and soft agar stab methods. The biochemical tests used to identify suspected B. bronchiseptica are specified.     

Nutritional ecology of the Formosan subterranean termite (Isoptera: Rhinotermitidae): growth and survival of incipient colonies feeding on preferred wood species

The wood of 11 plant species was evaluated as a food source significantly impacting the growth and survival of incipient colonies of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Colonies of C. formosanus feeding on pecan, Carya illinoensis (Wangenh.), and red gum, Liquidambar styraciflua L., produced significantly more progeny than colonies feeding on other wood species tested. Progeny of colonies feeding on pecan and American ash, Fraxinus americana L., had significantly greater survival than progeny of colonies feeding on other wood species. Colonies feeding on a nutritionally supplemented cellulose based matrix showed similar fitness characteristics as colonies feeding on the best wood treatments. These results indicate that differences observed in colony fitness can be partially explained by nutritional value of the food treatment, raising the possibility that wood from different tree species have different nutritional values to the Formosan subterranean termites. Colonies feeding on loblolly pine, Pinus taeda L., and ponderosa pine, Pinus ponderosa Laws., had significantly lower survival and produced significantly fewer workers and soldiers than colonies feeding on other wood species. Colony survival from 90 to 180 d of age and from 90 to 360 d of age was significantly correlated with the number of workers present at 90 d of colony age, indicating that colony survival depends on the presence of workers. Wood consumption in a multiple-choice study was significantly correlated with colony fitness value. This suggests that feeding preference of C. formosanus is at least partially influenced by the nutritional value of the food source.     

Detection of gentoxicity of benzidine and its derivatives with the Escherichia coli DJ 702 lacZ reversion mutagenicity assay

AIMS: The feasibility of Escherichia coli DJ 702 lacZ mutagenicity assay to detect genotoxicity of benzidine and its derivatives was evaluated. METHODS AND RESULTS: DJ 702 strain was grown overnight at 30 degrees C in Luria-Bertani (LB) medium containing some components, such as chloramphenicol, ampicillin, delta-aminolevulinic acid, isopropyl-beta-d-thiogalactoside, and trace element mix. The mixtures of a bacterial culture and tested chemical at indicated doses were incubated at 30 degrees C for 30 min. Subsequently, 2 ml of molten top agar was added and the resulting mixtures were immediately poured onto a minimal lactose (ML) plate. Plates were incubated at 30 degrees C for 48 h. The number of colonies was determined by visual scoring. In this study, results showed that all the tested chemicals were mutagenic to DJ 702 strain. CONCLUSIONS: E. coli lac mutagenicity assay using DJ 702 strain can detect the mutagenicity of benzidine and its derivatives. SIGNIFICANCE AND IMPACT OF THE STUDY: We detected the mutagenicity of benzidine and its derivatives in E. coli lac mutagenicity assay using DJ 702, indicating that this assay may be used to detect benzidine and its derivatives in a powerful, sensitive, and convenient mutagenesis assay.     

Heat curing Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae) of a fitness-reducing microsporidium

Laboratory colonies of the predatory mite Metaseiulus occidentalis in Gainesville, FL were found to be infected with an undescribed microsporidium. Experiments were performed to quantify the effect of infection on the fitness of M. occidentalis and to determine if heat treatment can cure mites of the microsporidium. The colonies tested were derived from an isofemale line so that differences in performance could be attributed to the presence of microsporidia. A subcolony of an uninfected isofemale line was infected with the microsporidium by feeding females infected eggs from another colony of M. occidentalis. Infected mites had a shorter mean (+/-SD) female life span (7.4 +/- 2.9 vs. 10.0 +/- 2.8 days), lower mean oviposition (1.6 +/- 0.7 vs. 2.2 +/- 0.4 eggs/day), and a male-biased sex ratio (43 +/- 16% vs. 57 +/- 15% female progeny). The infection was reduced temporarily in colonies initiated from mites that were reared in a growth chamber at 33 degrees C from egg to adult, but healthy colonies only were established from the progeny of the heat-treated adults. These colonies remained free of infection for 10 weeks.     

The role of humoral factors in the regression of leukemia in chickens as measured by in vitro colony formation

Leukemic myeloblasts induced by avian myeloblastosis virus in the chicken formed small compact (type II) colonies in semi-solid agar medium. Normal yolk sac cells from 12-day old embryos formed large diffuse (type I) colonies under the same conditions. Type I colony formation (but not type II) was strictly dependent upon the presence in the medium of a colony stimulating factor (CSF) present in fresh chicken serum or conditioned medium. Serum CSF levels were determined for normal, leukemic, and birds which had spontaneously regressed from myeloblastic leukemia. When type I colony formation was used as the assay, serum CSF levels of leukemic birds were found to be significantly lower than levels in either normal or regressed birds. When the same sera were tested for their ability to induce type II colonies, leukemic birds demonstrated a significantly higher CSF level than either normal or regressed sera. Regressed chickens had serum CSF levels similar to normal birds.