Effects of sertraline on behavioral indices of crayfish Orconectes virilis.

Sertraline, a selective serotonin re-uptake inhibitor, is a widely prescribed antidepressant in North America. Though sertraline is continuously released from wastewater treatment plant discharge to surface water, effects of aqueous exposure of sertraline on behavioral responses of aquatic animals are largely unknown. Our study explored the effects of aqueous exposures of sertraline on antagonistic bouts and predator response behavior of virile crayfish (Orconectes virilis). Crayfish were either exposed or not exposed to waterborne sertraline and then size-matched for paired antagonistic bouts to determine if sertraline affects the aggression of each crayfish. We investigated the effect of sertraline on responses to visual predator cues and determined whether sertraline acts as an olfactory cue. Our results demonstrate that crayfish exposed to sertraline are more aggressive when paired with control crayfish but, when sertraline crayfish are paired, there is no change in aggression. Attraction response to sertraline in behavioral mazes was also observed, which may represent a maladaptive behavior, and in an ecological context may result in crayfish moving to areas with elevated levels of sertraline. However, aqueous exposure to sertraline had no effect on predator responses of crayfish. Future research is warranted to determine whether such medicine released in wastewater treatment plant effluents produces long-term ecologically important consequences for aquatic animals residing in urbanized aquatic ecosystems.

Inactivation of infectious bursal disease and Newcastle disease viruses at temperatures below 0 C using chemical disinfectants.

This study evaluated the effectiveness of bleach, Surface Decontamination Foam (SDF), and Virkon in inactivating infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) at temperatures below 0 C. To simulate the influence of organic load on the effectiveness of disinfectants, as would be encountered in disinfecting farm vehicles and equipment, the viruses were suspended in preparations containing light or heavy levels of organic matter. A small volume of the viral suspension was applied to the upper surface of stainless steel carrier disks and these were then air dried. The dried virus inoculum was covered with disinfectant to which propylene glycol had been added to prevent freezing. The disks were incubated at various temperatures for periods up to 24 hr. With NDV, at -10 C all three disinfectants in both organic preparations achieved a 5 log 10 reduction within 5 min. Results with SDF were similar at -25 and -10 C. To achieve comparable reduction with Virkon and bleach at -25 C, contact periods up to 2 or 24 hr, respectively, were required. With IBDV, to achieve a 5 log 10 to reduction by treatment with Virkon or SDF at -20 C, contact periods of 2 or 24 hr, respectively, were required in both organic preparations. It was concluded that at temperatures as low as -20 to -25 C, SDF was the most effective disinfectant for killing NDV and Virkon was most effective for killing IBDV. The finding that a contact period of up to 2 hr was required to kill IBDV, whereas NDV was killed within 5 min, attests to the greater stability of the former virus.

Suggesting a testing strategy for possible endocrine effects of drug metabolites.

Most pharmaceuticals are extensively metabolized by organisms, which results in internal exposure to mixtures of parent compounds and various metabolites. Many of these metabolites are considered non-toxic, but some metabolites retain toxic properties of the parent compound or elicit other undesirable outcomes. Unfortunately, the effects of metabolites are often not considered when endocrine activities of chemicals are evaluated in vitro. In this study two approaches, an "effect-based" and a "compound-by-compound" testing design, were used to determine the effects of metabolites of the antidepressant sertraline on aromatase enzyme activity. In the "effect-based" approach, a mixture of sertraline metabolites, produced by liver microsomes, inhibited aromatase, but was less potent than sertraline. In the "compound-by-compound" testing design, three specific metabolites were evaluated individually and in mixtures. Though two N-desmethylated metabolites were more potent aromatase inhibitors than sertraline, hydroxyl ketone sertraline did not inhibit the enzyme and mixtures of these metabolites and sertraline were less potent than predicted from a concentration addition model. Our findings highlight the importance of considering aromatase inhibition, and potentially other biological activities, of pharmaceutical metabolites produced by liver microsome preparations and then comparing such observations to studies of specific metabolites available for testing in pure form. Subsequently, a five step integrated strategy for screening of the potential endocrine effects of drugs and their metabolites are proposed.

Infectious bursal disease virus as a surrogate for studies on survival of various poultry viruses in compost.

Infectious bursal disease virus (IBDV) is resistant to many environmental stresses and often persists on farms for months. This study investigated survival of a vaccine strain of IBDV in the bursa of Fabricius and splenic tissue from experimentally infected chickens and in splenic tissue and manure that had been inoculated with the virus. The specimens buried in compost were contained within nylon mesh bags, and the tissues were enclosed within the abdominal cavity of chicken carcasses. Extracts of composted specimens were inoculated into Vero cell cultures, and real-time reverse transcriptase PCR was used to quantify the virus in the cultures. By day 7 in compost, the temperature had been slightly above 55 C for 2.6 days and IBDV had been inactivated in specimens that had been inoculated with virus but had survived in tissues that had been taken from infected chickens. By day 14, the temperature had been above 55 C for 8.8 days and the virus was inactivated in all specimens. The results suggest that composting of poultry carcasses and manure would help to break the cycle of infection with IBDV and that the virus could be valuable as a surrogate for predicting the inactivation of less resistant viruses during composting.

Ultrafast laser diode thermal desorption method for analysis of representative pharmaceuticals in soil leachate samples.

We developed and evaluated a novel analytical method combining ambient ionization technique - laser diode thermal desorption with chemical ionization (LDTD-APCI) and tandem mass spectrometry detection. The LDTD/APCI-MS/MS method was developed for determination of representative pharmaceuticals from different classes (carbamazepine, sulfamethoxazole, irbesartan, fexofenadine) in leachate samples from soil sorption experimentation. We then optimized laser pattern, laser energy and spiked sample volume, which are crucial parameters for this LDTD/APCI-MS/MS method. We further identified utility of a chelating agent (Na2-EDTA) to obtain the highest achievable and reproducible signal of target analytes. Achieved method performance parameters (LODs, LOQs, trueness and precision) were comparable with those obtained from LC-MS/MS. However, application of this novel LDTD/APCI-MS/MS method reduced analysis time by two orders of magnitude (to 12 s), compared to more conventional LC-MS/MS approaches, without use of organic solvents. We expect this novel method will reduce costs and increase throughput for future analyses of pharmaceuticals in the environment while advancing a timely principle of green chemistry.

Survival of avian influenza and Newcastle disease viruses in compost and at ambient temperatures based on virus isolation and real-time reverse transcriptase PCR.

In four composting experiments, survival of avian influenza (AI) and Newcastle disease (ND) viruses was assessed by virus isolation in embryonated chicken eggs (ECEs) and by real-time reverse transcriptase-polymerase chain reaction. Specimens contained in nylon mesh bags consisted of 20-g samples of chicken manure, used litter, or feed that had been inoculated with allantoic fluid containing an AI virus (H6N2, Expt. 1) or an ND vaccine virus (Expt. 2). Other specimens consisted of 20-g samples of infected ECEs that had been homogenized and mixed with corn silage. As a control, allantoic fluid diluted in phosphate-buffered saline was contained in sealed vials. Except for the feed, in which the AI virus was inactivated soon after the specimen was inoculated, on day 0 the specimens buried in compost or placed outside at ambient temperatures contained at least 5.0 log10 of virus and 7.7 log10 of viral RNA. By day 7, temperatures in compost ranged from 50 C to 65 C, and viruses had been killed in all specimens in bags. In comparison, viruses in sealed vials remained viable to day 10. Viral RNA in mesh-bag specimens had been degraded to nondetectable levels by day 10, but it was still detected in sealed vials on day 21. In specimens that were held at ambient temperatures (13 C-28 C), the viruses in mesh-bag specimens were inactivated by day 21, but their RNA was still detected. In comparison, the viruses in sealed vials survived to day 21. In Expts. 3 and 4, viruses were inactivated in carcass specimens and in whole ECEs during composting. In an in vitro experiment, the time required for a 1-log10 reduction of viruses was significantly shorter (P < 0.05) in water extracts from compost than in phosphate buffers at temperatures of 25 C to 45 C. This study provided evidence that microbial activity during composting contributed to the rapid killing of AI and ND viruses and to the degradation of their viral RNA.

Structural characterization and serological specificities of lipopolysaccharides from Salmonella enterica serovar Gallinarum biovar Pullorum standard, intermediate and variant antigenic type strains.

The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti-S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.

Development of methods for detection and quantification of avian influenza and Newcastle disease viruses in compost by real-time reverse transcription polymerase chain reaction and virus isolation.

Composting has been used for disposal of poultry carcasses and manure following outbreaks caused by avian influenza virus (AIV) and Newcastle disease virus (NDV), but methods are needed to test for survival of the viruses in compost to ensure biosecurity. Methods developed in the present study include extracting viruses from compost and purifying viral RNA. The extracted viruses were detected by virus isolation using embryonated chicken eggs, and the purified RNA was detected by real-time reverse transcription PCR (RRT-PCR). The virus isolation and the RRT-PCR methods were evaluated with 3 compost preparations that were produced from chicken manure mixed with corn silage, wood shavings, or wheat straw. The detection limits of both methods were 1,700 and 1,000 embryo lethal doses of AIV and NDV per gram of compost, respectively. The copy number of viral RNA quantified by RRT-PCR was highly correlated with the amount of virus in compost. The results suggested that the RRT-PCR method may be used as an alternative to the virus isolation method for rapid detection and accurate quantification of AIV and NDV in compost.

Select antibiotics in leachate from closed and active landfills exceed thresholds for antibiotic resistance development.

Though antibiotic resistance (ABR) represents a major global health threat, contributions of landfill leachate to the life cycle of antibiotics and ABR development are poorly understood in rapidly urbanizing regions of developing countries. We selected one of the largest active landfills in Asia and two landfills that have been closed for 20 years to examine antibiotic occurrences in leachates and associated hazards during wet and dry season sampling events. We focused on some of the most commonly used human antibiotics in Hong Kong, one of the most populous Asian cities and the fourth most densely populated cities in the world. Seven antibiotics (cephalexin [CLX], chloramphenicol [CAP], ciprofloxacin [CIP], erythromycin [ERY], roxithromycin [ROX], trimethoprim [TMP], sulfamethoxazole [SMX]) were quantitated using HPLC-MS/MS generally following previously reported methods. Whereas CLX, CAP, ROX and SMX in leachates did not exceed ABR predicted no effect concentrations (PNECs), exceedances were observed for CIP, ERY and TMP in some study locations and on some dates. In fact, an ABR PNEC for CIP was exceeded in leachates during both sampling periods from all study locations, including leachates that are directly discharged to coastal systems. These findings highlight the importance of developing an advanced understanding of pharmaceutical access, usage and disposal practices, effectiveness of intervention strategies (e.g., leachate treatment technologies, drug take-back schemes), and contributions of landfill leachates to the life cycle of antibiotics and ABR development, particularly in rapidly urbanizing coastal regions with less advanced waste management systems than Hong Kong.

In vitro study of Salmonella enteritidis and Salmonella typhimurium definitive type 104: survival in egg albumen and penetration through the vitelline membrane.

Salmonella enteritidis and Salmonella typhimurium definitive type 104 (DT104) have been detected in the chicken oviduct, and their survival in egg albumen at the chicken body temperature of 42 degrees C may be important in oviductal and transovarian contamination of intact shell eggs. Eight S. enteritidis and 24 S. typhimurium DT104 strains were tested for their in vitro survival in egg albumen. The concentration of the organisms declined more rapidly when incubated at 42 degrees C than at 37 degrees C and dropped to nondetectable levels within 96 h at the higher, but not at the lower, temperature. In another experiment, 3 S. enteritidis and 3 S. typhimurium DT104 strains were randomly selected, and dosages of 20 and 200 cells of each strain were inoculated onto the vitelline membranes of egg yolks, which were then submerged in the original albumen and incubated for 24 h at 42 degrees C. Under these conditions, the organisms survived in albumen but did not penetrate the vitelline membrane. However, in a similar experiment, penetration did occur when the specimens were incubated at 30 degrees C for 72 h. The results suggest that low numbers of S. enteritidis and S. typhimurium DT104 can be expected to survive in egg albumen during the 24-h period of egg formation in the oviduct but would be unlikely to invade the yolk.before oviposition. However, depending on storage conditions following oviposition, S. enteritidis, as well as S. typhimurium DT104, could survive longer and may eventually invade the egg yolks.

Simple extraction of Campylobacter lipopolysaccharide and protein antigens and production of their antibodies in egg yolk.

Antigens were heat extracted from Campylobacter jejuni (LI04) and C. coli (LI020) in the presence of ethylenediaminetetraacetate (EDTA) and were recovered in the supernatant of a low-speed centrifugation. The method is simpler, safer and more efficient in extracting lipopolysaccharide (LPS) antigens than the hot phenol method. The extracted antigens (LPS plus several proteins) elicited production of antigen-specific antibodies in the egg yolk of immunized hens. Antibodies purified by polyethyleneglycol fractionation were used to detect antigens fractionated on SDS polyacrylamide gel electrophoresis.

Salmonella detection by the polymyxin-cloth enzyme immunoassay using polyclonal and monoclonal detector antibodies.

Several commercially available O-antigen polyclonal antisera and a monoclonal antibody to the core region of lipopolysaccharide (LPS) were examined as sources of detector antibodies in a polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) for Salmonella. In this assay, polymyxin-coated polyester cloth captured the LPS antigens from Salmonella broth cultures, followed by immunoenzymatic detection of the captured LPS using specific antibodies. Pools of polyvalent antisera reacted with all of the Salmonella strains tested, but also gave cross-reactions with some non-Salmonella bacteria. On the other hand, the monoclonal antibody gave positive reactions with all of the Salmonella tested except serogroup O-strains, but did not react with any of the non-Salmonella bacteria. The monoclonal antibody supplemented with a single factor serogroup O:35 rabbit antiserum was able to detect the serogroup O-strains without causing any cross-reactions with the non-Salmonella bacteria. As an example of the applicability of this assay system, low levels of Salmonella cells spiked into various food samples were successfully detected after an overnight enrichment in broth.

Rapid detection and enumeration of Salmonella in chicken carcass rinses using filtration, enrichment and colony blot immunoassay.

A strategy was developed for 24-h detection and enumeration of Salmonella spp. on processed chicken carcasses. Carcasses were rinsed with saline and the rinses spiked with known numbers of serogroup B, C, D or E Salmonella. The total rinse volume was passed through two filter units of decreasing pore size. These removed most of the extraneous material while permitting rapid passage of more than 77% of the Salmonella. At least 100 ml of the filtrate was passed through a third filter unit containing a nitrocellulose capture membrane. Captured bacteria were selectively enriched by incubating the nitrocellulose membrane on filter pads soaked in Rappaport-Vassiliadis broth and then on pads soaked in brilliant green broth containing sulfadiazine and novobiocin. A colony blot immunoassay using two anti-Salmonella monoclonal antibodies was used to identify and enumerate the captured Salmonella. As few as five Salmonella colony forming units per carcass rinse could be detected. An evaluation of this system with 24 field samples indicated that the specificity was comparable to and the sensitivity higher than that of standard culture procedures.

Simple and economical culture of Campylobacter jejuni and Campylobacter coli in CO2 in moist air.

Strains of Campylobacter jejuni and C. coli representing the 18 serogroups (Lior) most commonly isolated from humans in Canada were grown on solid media in an atmosphere of 10% CO2 in moist air, 99% relative humidity. When the growth of all 18 serogroups on Mueller Hinton agar in a microaerobic atmosphere (5% O2, 10% CO2 and 85% N2) was compared with the growth of all 18 serogroups on the same media in 10% CO2 in moist air, colony sizes were significantly larger (p less than 0.05) for strains grown in 10% CO2 in moist air. No significant difference in colony numbers was seen between the two atmospheres. The addition of blood to the media significantly enhanced the growth of the campylobacters in both types of atmospheres (p less than 0.05). This simple CO2 atmosphere permitted the use of a common CO2 incubator thereby reducing the cost and difficulty of culturing these organisms.

Assay of Salmonella in enrichment cultures of foods, feeds and environmental samples by the polymyxin-cloth enzyme immunoassay.

A variety of foods, animal feeds and environmental samples were analyzed for the presence of Salmonella using the polymyxin-cloth enzyme immunoassay (p-CEIA) system. Salmonella lipopolysaccharide (LPS) antigens were captured from test samples on polymyxin-coated polyester cloth, followed by immunoenzymatic detection of bound antigens using a monoclonal antibody recognizing an LPS common core oligosaccharide. Dairy and egg products, animal feeds and environmental samples from food processing plants were pre-enriched for 24 h, followed by selective enrichment for a further 24 h in either tetrathionate brilliant green (TBG), selenite cystine (SC) or brain-heart infusion broth containing 0.5% yeast extract, 0.5% cholate and 0.3% selenite (BYCS). The samples were assayed by the p-CEIA after each stage of enrichment. After selective enrichment, the p-CEIA gave results which were in complete agreement with the standard culture technique in the analysis of all foods examined. On the other hand, a combination of selective enrichment and the p-CEIA out-performed the Modified Semi-Solid Rappaport Vassiliadis (MSRV) method in screening pre-enrichment cultures of feeds and environmental samples. Use of the new selective medium BYCS prior to performing the p-CEIA gave the highest recovery of Salmonella from feeds and environmental samples.

Characterization of monoclonal antibodies for the rapid detection of foodborne campylobacters.

The specificity of 97 monoclonal antibodies (MAbs) to the Campylobacter jejuni Lior serogroup 6 reference strain was assessed using an indirect enzyme linked immunosorbent assay (ELISA). Four MAbs, M316, M337, M357 and M637, reacted with whole cells of the C. jejuni, C. coli and C. lari reference strains of the 20 most common Lior serogroups and 25 recent C. jejuni and C. coli isolates, and did not react with most of the 42 other Campylobacter and non-Campylobacter spp. tested. Immunoblot analysis revealed that MAbs M337 and M357 reacted with a protein component with molecular mass of approximately 62 kiloDaltons (kDa) while M316 and M637 reacted with protein components of approximately 92 and 31 kDa, respectively. The detection limit of M357 in an indirect ELISA was 10(5) colony forming units. These four highly specific MAbs may be useful reagents of an immunoassay for the rapid detection of thermophilic campylobacters in foods and clinical samples.

Evaluation of culture enrichment procedures for use with Salmonella detection immunoassay.

To design efficient culture strategies for use with immunoassays to detect Salmonella in food, the growth of these organisms was investigated according to the Bacteriological Analytical Manual (BAM) and enrichment-immunoassay (EI) culture procedures. The cultures were further evaluated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The BAM procedure includes pre-enrichment in nutrient broth (NB) for 16 h followed by selective enrichment in either Rappaport-Vassiliadis (RV) or tetrathionate brilliant green (TBG) broth for 16 h. The EI procedure includes pre-enrichment in NB for 4 h, selective enrichment in RV for 16 h and post-enrichment in NB for 4 h. The effects of different incubation times for pre- and post-enrichment, and different culture media for selective enrichment (TBG and RV) and post-enrichment in NB and Brain Heart Infusion broth (BHI) on the growth of the bacteria and ELISA titers in the EI procedure were also investigated. Salmonella enteritidis and S. typhimurium inoculated at different initial concentrations between 0.1 and 35 CFU/ml grew to similar concentrations of 10(7) to 10(8) colony forming unit (CFU)/ml in pure culture and generally 2 to 4 fold lower concentrations (P<0.05) in mixed culture using spiked chicken rinse. In the BAM procedure, the concentration of Salmonella cultured in RV was higher (P<0.01) than that in TBG. The cultures in TBG showed positive results for ELISA, but those in RV were generally negative. In the EI procedure, the ELISA titers from cultures post-enriched in NB or BHI were higher (P<0.01) when TBG, as compared to RV, was used for selective enrichment. Post-enrichment in BHI yielded higher numbers of Salmonella and higher ELISA titers than those in NB (P<0.05) for post-enrichment. This study demonstrated that in both culture procedures small numbers of Salmonella could be increased to at least 10(7) CFU/ml which is detectable by most ELISAs, and that the type of the culture media used may have a significant impact on ELISA results.

Molecular discrimination of Campylobacter coli serogroup 20 biotype I (Lior) strains.

Plasmid, protein and restriction endonuclease analysis (REA) profiles and multilocus enzyme electrophoresis were used to effect a molecular discrimination of twenty-seven Campylobacter coli serogroup 20, biotype 1 (Lior) strains. These strains were not outbreak-associated but were isolated from a number of different countries and different animal and environmental sources. Each of the techniques was able to discriminate, to various degrees, between the serogroup 20, biotype 1 strains. The choice of a particular technique depends to a large extent on the level of discrimination desired, the previous experiences of the investigator and on the laboratory facilities at hand. REA profiles demonstrated the greatest degree of discrimination between these strains. Plasmid and protein profiles could discriminate reasonably well. Multilocus enzyme electrophoresis (allozyme typing) and protein profiles may prove effective in subgrouping serogroup 20, biotype 1 strains.

Electrophoretic and immunoblot analysis of Campylobacter fetus lipopolysaccharides.

Proteinase K-digested cell lysates from 25 Campylobacter fetus subspecies fetus and C. fetus subsp. venerealis strains were examined by SDS-PAGE and immunoblotting. Three SDS-PAGE lipopolysaccharide (LPS) profiles were observed. Two profiles were consistent with those previously reported for serogroup A and serogroup B and AB isolates and were distinguished by the relative mobility of bands in the O-chain region and by a strong reaction on immunoblots with homologous antisera. The third profile was similar but had faster migrating O-chain bands. Immunoblot reactions using homologous and heterologous adsorbed antisera showed that the O-antigen of the C. fetus subsp. fetus reference strain and other profile 2-type LPS strains was distinct from the O-antigens of strains with profile 1- or profile 3-type LPS. O-antigens of strains with profile 1- and profile 3-type LPS had shared epitopes. One strain had core components but no detectable O-antigens. Common core LPS antigens appear to be present in all strains and antibodies to common core LPS epitopes may be useful reagents for rapid detection of C. fetus.