Zebrafish irritant responses to wildland fire-related biomass smoke are influenced by fuel type, combustion phase, and byproduct chemistry.

Human exposure to wildfire-derived particulate matter (PM) is linked to adverse health outcomes; however, little is known regarding the influence of biomass fuel type and burn conditions on toxicity. The aim of this study was to assess the irritant potential of extractable organic material (EOM) of biomass smoke condensates from five fuels (eucalyptus, pine, pine needle, peat, or red oak), representing various fire-prone regions of the USA, burned at two temperatures each [flaming (approximately 640°C) or (smoldering approximately 500°C)] using a locomotor assay in zebrafish (Danio rerio) larvae. It was postulated that locomotor responses, as measures of irritant effects, might be dependent upon fuel type and burn conditions and that these differences relate to combustion byproduct chemistry. To test this, locomotor activity was tracked for 60 min in 6-day-old zebrafish larvae (25-32/group) immediately after exposure to 0.4% dimethyl sulfoxide (DMSO) vehicle or EOM from the biomass smoke condensates (0.3-30 µg EOM/ml; half-log intervals). All EOM samples produced concentration-dependent irritant responses. Linear regression analysis to derive rank-order potency indicated that on a µg PM basis, flaming pine and eucalyptus were the most irritating. In contrast, on an emission-factor basis, which normalizes responses to the amount of PM produced/kg of fuel burned, smoldering smoke condensates induced greater irritant responses (>100-fold) than flaming smoke condensates, with smoldering pine being the most potent. Importantly, irritant responses significantly correlated with polycyclic aromatic hydrocarbon (PAH) content, but not with organic carbon or methoxyphenols. Data indicate that fuel type and burn condition influence the quantity and chemical composition of PM as well as toxicity.

Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish.

Altered dopaminergic signaling causes behavioral changes in mammals. In general, dopaminergic receptor agonists increase locomotor activity, while antagonists decrease locomotor activity. In order to determine if zebrafish (a model organism becoming popular in pharmacology and toxicology) respond similarly, the acute effects of drugs known to target dopaminergic receptors in mammals were assessed in zebrafish larvae. Larvae were maintained in 96-well microtiter plates (1 larva/well). Non-lethal concentrations (0.2-50 µM) of dopaminergic agonists (apomorphine, SKF-38393, and quinpirole) and antagonists (butaclamol, SCH-23390, and haloperidol) were administered at 6 days post-fertilization (dpf). An initial experiment identified the time of peak effect of each drug (20-260 min post-dosing, depending on the drug). Locomotor activity was then assessed for 70 min in alternating light and dark at the time of peak effect for each drug to delineate dose-dependent effects. All drugs altered larval locomotion in a dose-dependent manner. Both the D1- and D2-like selective agonists (SKF-38393 and quinpirole, respectively) increased activity, while the selective antagonists (SCH-23390 and haloperidol, respectively) decreased activity. Both selective antagonists also blunted the response of the larvae to changes in lighting conditions at higher doses. The nonselective drugs had biphasic effects on locomotor activity: apomorphine increased activity at the low dose and at high doses, while butaclamol increased activity at low to intermediate doses, and decreased activity at high doses. This study demonstrates that (1) larval zebrafish locomotion can be altered by dopamine receptor agonists and antagonists, (2) receptor agonists and antagonists generally have opposite effects, and (3) drugs that target dopaminergic receptors in mammals appear, in general, to elicit similar locomotor responses in zebrafish larvae.

Zebrafish developmental screening of the ToxCast™ Phase I chemical library.

Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast™ Phase I chemicals was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concentration range of 1 nM-80 µM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chemicals were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chemical class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.

Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables.

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chemicals using zebrafish behavior at 6 days of age. The behavioral paradigm simultaneously tests individual larval zebrafish under both light and dark conditions in a 96-well plate using a video tracking system. We have found that many variables affect the level or pattern of locomotor activity, including age of the larvae, size of the well, and the presence of malformations. Some other variables, however, do not appear to affect larval behavior including type of rearing solution (10% Hank's vs. 1:3 Danieau vs 60 mg/kg Instant Ocean vs 1× and 1:10× EPA Moderately Hard Water). Zebrafish larval behavior using a microtiter plate format may be an ideal endpoint for screening developmentally neurotoxic chemicals, but it is imperative that many test variables be carefully specified and controlled.

Rearing conditions differentially affect the locomotor behavior of larval zebrafish, but not their response to valproate-induced developmental neurotoxicity.

Zebrafish (Danio rerio) are widely used in developmental research, but still not much is known about the role of the environment in their development. Zebrafish are a highly social organism; thus exposure to, or isolation from, social environments may have profound developmental effects. Details of rearing conditions are often sparse in the zebrafish literature. This study compared (1) the activity of larval zebrafish that were raised individually vs in groups, and (2) the effect of the developmental neurotoxicant valproate. We randomly assigned embryos to single- or group-reared social environments from 0 to 5days post fertilization (dpf), while treating them with or without valproate (final concentration 48µM) from 0 to 2dpf, resulting in a total of four groups (group control, group treated, single control, single treated). At 5dpf all embryos were transferred to singly-housed environments where they remained for locomotor activity testing (alternating periods of light and dark) conducted on day 6. Larvae that had been reared in groups had higher levels of activity in the dark period compared to larvae that had been raised individually. Valproate increased activity in both the singly-reared and group-reared larvae during periods of darkness but not light. Further analyses of dark activity indicated that rearing condition did not differentially affect larval responses to valproate. These results indicate that rearing conditions affected the locomotion of zebrafish larvae, but did not alter the effect of the developmental neurotoxicant valproate.

Acute neuroactive drug exposures alter locomotor activity in larval zebrafish.

As part of the development of a rapid in vivo screen for prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae by assessing the acute effects of prototypic drugs that act on the central nervous system. Initially, we chose ethanol, d-amphetamine, and cocaine, which are known, in mammals, to increase locomotion at low doses and decrease locomotion at higher doses. Wild-type larvae were individually maintained in 96-well microtiter plates at 26 degrees C, under a 14:10 h light:dark cycle, with lights on at 0830 h. At 6 days post-fertilization, ethanol (1-4% v/v), d-amphetamine sulfate (0.1-20.0 microM) or cocaine hydrochloride (0.2-50.0 microM) were administered to the larvae by immersion. Beginning 20 min into the exposure, locomotion was assessed for each animal for 70 min using 10-minute, alternating light (visible light) and dark (infrared light) periods. Low concentrations of ethanol and d-amphetamine increased activity, while higher concentrations of all three drugs decreased activity. Because ethanol effects occurred predominately during the light periods, whereas the d-amphetamine and cocaine effects occurred during the dark periods, alternating lighting conditions proved to be advantageous. These results indicate that zebrafish larvae are sensitive to neuroactive drugs, and their locomotor response is similar to that of mammals.

Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol.

The increasing use of zebrafish (Danio rerio) in developmental research highlights the need for a detailed understanding of their behavior. We studied the locomotion of individual zebrafish larva (6 days post-fertilization) in 96-well microtiter plates. Movement was recorded using a video-tracking system. Time of day results indicated locomotion, tested in darkness (infrared), decreased gradually from early morning to a stable level between 13:00 and 15:30 h. All further studies were conducted in early-to-late afternoon and lasted approximately 1 h. Each study also began with a period of darkness to minimize any unintended stimulation caused by transferring the plates to the recording platform. Locomotion in darkness increased initially to a maximum at 4 min, then decreased steadily to a low level by 20 min. Locomotion during light was initially low and then gradually increased to a stable level after 20 min. When 10-min periods of light and dark were alternated, activity was low in light and high in dark; curiously, activity during alternating dark periods was markedly higher than originally obtained during either extended dark or light. Further experiments explored the variables influencing this alternating pattern of activity. Varying the duration of the initial dark period (10-20 min) did not affect subsequent activity in either light or dark. The activity increase on return to dark was, however, greater following 15 min than 5 min of light. Acute ethanol increased activity at 1 and 2% and severely decreased activity at 4%. One-percent ethanol retarded the transition in activity from dark to light, and the habituation of activity in dark, while 2% ethanol increased activity regardless of lighting condition. Collectively, these results show that locomotion in larval zebrafish can be reliably measured in a 96-well microtiter plate format, and is sensitive to time of day, lighting conditions, and ethanol.

The ESRC research ethics framework and research ethics review at UK universities: rebuilding the Tower of Babel REC by REC.

The history of the National Health Service research ethics system in the UK and some of the key drivers for its change into the present system are described. It is suggested that the key drivers were the unnecessary delay of research, the complexity of the array of processes and contradictions between research ethics committee (REC) decisions. It is then argued that the primary drivers for this change are and will be replicated by the systems of research ethics review being put in place at UK universities in response to the Economic and Social Research Council research ethics framework. It is argued that this is particularly problematic for multi-centre review and for researchers who switch institutions. Finally, some potential solutions to this problem and their feasibility are discussed.

Time course of cholinesterase inhibition in adult rats treated acutely with carbaryl, carbofuran, formetanate, methomyl, methiocarb, oxamyl or propoxur.

To compare the toxicity of seven N-methyl carbamates, time course profiles for brain and red blood cell (RBC) cholinesterase (ChE) inhibition were established for each. Adult, male, Long Evans rats (n=4-5 dose group) were dosed orally with either carbaryl (30 mg/kg in corn oil); carbofuran (0.5 mg/kg in corn oil); formetanate HCl (10 mg/kg in water); methomyl (3 mg/kg in water); methiocarb (25 mg/kg in corn oil); oxamyl (1 mg/kg in water); or propoxur (20 mg/kg in corn oil). This level of dosing produced at least 40% brain ChE inhibition. Brain and blood were taken from 0.5 to 24 h after dosing for analysis of ChE activity using two different methods: (1) a radiometric method which limits the amount of reactivation of ChE activity, and (2) a spectrophotometric method (Ellman method using traditional, unmodified conditions) which may encourage reactivation. The time of peak ChE inhibition was similar for all seven N-methyl carbamate pesticides: 0.5-1.0 h after dosing. By 24 h, brain and RBC ChE activity in all animals returned to normal. The spectrophotometric method underestimated ChE inhibition. Moreover, there was a strong, direct correlation between brain and RBC ChE activity (radiometric assay) for all seven compounds combined (r(2)=0.73, slope 1.1), while the spectrophotometric analysis of the same samples showed a poor correlation (r(2)=0.09). For formetanate, propoxur, methomyl, and methiocarb, brain and RBC ChE inhibitions were not different over time, but for carbaryl, carbofuran and oxamyl, the RBC ChE was slightly more inhibited than brain ChE. These data indicate (1) the radiometric method is superior for analyses of ChE activity in tissues from carbamate-treated animals (2) that animals treated with these N-methyl carbamate pesticides are affected rapidly, and recover rapidly, and (3) generally, assessment of RBC ChE is an accurate predictor of brain ChE inhibition for these seven pesticides.

Neurobehavioral effects of chronic dietary and repeated high-level spike exposure to chlorpyrifos in rats.

This study aimed to model long-term subtoxic human exposure to an organophosphorus pesticide, chlorpyrifos, and to examine the influence of that exposure on the response to intermittent high-dose acute challenges. Adult Long-Evans male rats were maintained at 350 g body weight by limited access to a chlorpyrifos-containing diet to produce an intake of 0, 1, or 5 mg/kg/day chlorpyrifos. During the year-long exposure, half of the rats in each dose group received bi-monthly challenges (spikes) of chlorpyrifos, and the other half received vehicle. Rats were periodically tested using a neurological battery of evaluations and motor activity to evaluate the magnitude of the acute response (spike days) as well as recovery and ongoing chronic effects (non-spike days). Effects of the spikes differed as a function of dietary level for several endpoints (e.g., tremor, lacrimation), and in general, the high-dose feed groups showed greater effects of the spike doses. Animals receiving the spikes also showed some neurobehavioral differences among treatment groups (e.g., hypothermia, sensory and neuromotor differences) in the intervening months. During the eleventh month, rats were tested in a Morris water maze. There were some cognitive deficits observed, demonstrated by slightly longer latency during spatial training, and decreased preference for the correct quadrant on probe trials. A consistent finding in the water maze was one of altered swim patterning, or search strategy. The high-dose feed groups showed more tendency to swim in the outer annulus or to swim very close to the walls of the tank (thigmotaxic behavior). Overall, dietary exposure to chlorpyrifos produced long-lasting neurobehavioral changes and also altered the response to acute challenges.

Wellness perception in persons with traumatic brain injury and its relation to functional independence.

OBJECTIVES: To test the reliability and validity of a perceptual wellness measure in persons after traumatic brain injury (TBI) and to determine whether a relation exists between functional independence and wellness perceptions in the same population. DESIGN: Survey research. SETTING: A private, residential brain injury program. PARTICIPANTS: A convenience sample of 49 patients (43 men, 6 women) with TBI whose mean age was 32.1 years (range, 18-61yr) and mean time since injury was 10.47 years (range, 1-21yr). MAIN OUTCOME MEASURES: The Perceived Wellness Survey (PWS) assessed wellness. The PWS has 6 subscales measuring physical, psychologic, emotional, intellectual, spiritual, and social wellness. The FIM instrument was used to measure functional status. RESULTS: The mean PWS score (15.99) for the sample was comparable to published samples of adults (mean, 15.31-16.51); however, the reliability of the composite score (alpha = .58) and the subscales (alpha = .32-.64) was less than that obtained in previous samples (composite alpha = 0.91; subscale alpha = .64-.81). The correlations between the PWS and the FIM scores were not significant. CONCLUSIONS: The PWS in its composite form is a reliable measure for use with persons with TBI. The finding that perceived wellness and functional independence were not related suggests that these constructs are unique and thus should both be measured. The measurement of perceptions will enable the provider to consider a client holistically and to develop programs that address quality of life issues. Further, because perceptions influence behaviors, understanding a person's perceptions in multiple dimensions may provide a useful and necessary framework for developing intervention programs that address behavioral and cognitive issues that are important to that person.

Evaluation of ewe vaccination as a tool for increasing bighorn lamb survival following pasteurellosis epizootics.

We conducted field and laboratory experiments to evaluate whether treating pregnant bighorn ewes with a combination of an experimental Pasteurella trehalosi and Mannheimia haemolytica (formerly P. haemolytica) vaccine and a commercially-available bovine P. multocida and M. haemolytica vaccine would increase lamb survival following a pneumonia epidemic. Three free-ranging bighorn herds affected by pasteurellosis outbreaks between November 1995 and June 1996 were included in the field experiment. Post-epidemic lamb survival was low in all three herds in 1996, with November lamb:ewe ratios of < or = 8:100. In March 1997, thirty-six ewes (12/herd) were captured and radiocollared. Half of the ewes captured in each herd were randomly selected to receive both vaccines; the other half were injected with 0.9% saline solution as controls. Lambs born to radiocollared ewes were observed two or more times per week and were considered to have survived if they were alive in October 1997, about 6 mo after birth. Lamb survival differed among herds (range 22% to 100%), and survival of lambs born to vaccinated ewes was lower (P = 0.08) than survival of lambs born to unvaccinated ewes. Bronchopneumonia (pasteurellosis) was the dominant cause of mortality among lambs examined. We concurrently evaluated vaccine effects on survival of lambs born to seven captive ewes removed from the wild during the 1995-96 epidemic. Antibody titers were high in captive ewes prior to vaccination, and vaccines failed to enhance antibody titers in treated captive ewes. None of the captive-born lambs survived. These data suggest that, using existing technology, vaccinating bighorn ewes following pneumonia epidemics has little chance of increasing neonatal survival and population recovery.

Use of a polymerase chain reaction method to detect the leukotoxin gene lktA in biogroup and biovariant isolates of Pasteurella haemolytica and P trehalosi.

OBJECTIVE: To determine whether Pasteurella haemolytica and P trehalosi isolates possess the structural gene for Pasteurella leukotoxin lktA and whether beta-hemolytic activity of these isolates correlated with detection of the lktA gene. SAMPLE POPULATION: 147 P haemolytica isolates from 21 biovariant groups and 101 P trehalosi isolates from 7 biovariant groups. In addition, P multocida and organisms from 7 other genera were tested to establish specificity of the procedure. PROCEDURE: Isolates were observed for beta-hemolysis. A polymerase chain reaction (PCR) procedure was used to amplify the RTX domain of the Pasteurella lktA gene. RESULTS: The lktA gene was detected in 108 (44%) isolates, including 15 associated with respiratory tract disease. All but 2 (98%) of the isolates that had the lktA gene were beta-hemolytic when grown on sheep blood agar. The remaining 140 isolates were negative for the lktA gene and hemolytic activity. CONCLUSIONS AND CLINICAL RELEVANCE: Hemolytic activity of P haemolytica and P trehalosi isolates correlated with detection of the lktA gene for all but 2 isolates. However, 56% of isolates tested were negative for the lktA gene and beta-hemolytic activity. Leukotoxin production and secretion is a major virulence factor when other conditions are favorable for disease development. Therefore, identification of strains that possess the lktA gene may aid in the evaluation of the pathogenic potential of Pasteurella strains carried by wild and domestic animals.

Gestational exposure to chlorpyrifos: comparative distribution of trichloropyridinol in the fetus and dam.

Chlorpyrifos (O,O'-diethyl O-[3,5,6-trichloro-2-pyridyl] phosphorothionate) is a commonly used anticholinesterase insecticide, and therefore the potential for human exposure is high. The present time course and dose response studies were conducted to delineate the toxicokinetics of chlorpyrifos and its metabolites in the pregnant rat and fetus. Time-pregnant, Long-Evans rats were treated orally with chlorpyrifos during late gestation (Gestational Days 14-18). Following euthanasia the level of chlorpyrifos and its metabolites, chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol (TCP), were measured in both fetal and maternal brain and liver (limits of quantitation: 59.2, 28.8, and 14.0 ng/g tissue, respectively). In addition, cholinesterase inhibition was also measured in the same tissues for comparison. TCP was the only component detected. The highest level of TCP and the lowest level of cholinesterase activity showed the same time of peak effect: 5 h after the last dose. The concentration of TCP in the maternal liver was approximately fivefold higher than the TCP concentration in fetal liver, but, paradoxically, the concentration of TCP in the fetal brain was two- to fourfold higher than the TCP concentration in the maternal brain. The half-life of the TCP was identical in all tissues examined (12-15 h). These toxicokinetic results suggest that the fetal nervous system may be exposed to a higher concentration of chlorpyrifos than the maternal nervous system when the dam is orally exposed to chlorpyrifos during late gestation.

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine.

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.

Sample preparation and high-performance liquid chromatographic analysis of deoxyribonucleoside triphosphates in individual rat embryos.

A rapid, robust, and sensitive method has been developed to measure concentrations of deoxyribonucleoside triphosphates in individual, day 14 rat embryos by modifying and optimizing existing methods for cellular extracts. Significant changes include: (i) oxidative degradation of ribonucleoside triphosphates using methylamine at lower pH (decreased from 6.5 to 4.0) to improve poor HPLC peak shape of early eluting nucleotides; (ii) glass fiber disc solid-phase extraction of the reaction mixture, which dramatically reduces impurities that interfere with nucleotide measurement, eliminates the necessity of column regeneration, and allows mobile phase recycling; and (iii) lower ionic strength (reduced from 0.4 to 0.26 or 0.12 M ammonium phosphate) and higher pH (increased from 3.25 to 5.55 or 6.98, respectively) mobile phase, conditions which are less destructive to the column's bonded phase and silica support, thereby contributing to longer column life. Enhancements include: (i) filtration of the sample prior to HPLC injection and addition of an in-line filter, guard column, and saturating precolumn of silica in the mobile phase flow, which aids substantially in extending column life and improves chromatographic stability, and (ii) inclusion of an internal standard to correct for mechanical losses. Limits of determination at a signal to noise ratio of 6:1 range from 5.5 to 12 pmol on-column or 0.41 to 0.87 pmol/mg of embryonic tissue depending on the specific nucleotide. Recoveries are quantitative for all nucleotides, and interassay variabilities are between 5 and 7% when quantified by peak height. The method has also been applied successfully to analysis of murine erythroleukemic cell cultures and this, when coupled with the embryo results, suggests its general utility.

Biovariants of isolates of Pasteurella from domestic and wild ruminants.

A total of 608 bacterial isolates previously identified as Pasteurella haemolytica biotypes A and 3, P. trehalosi, and P. multocida, were separated into 73 distinct biovariants using 21 phenotypic characteristics. The largest group (54%) of wildlife isolates was identified as biogroup 2 and biogroup 2 variants. Biogroup 2 and biogroup 2 variants accounted for only 17% of isolates from domestic ruminants and were all from sheep. In contrast, 43% of isolates from domestic ruminants were identified as biogroup 1 and biogroup 1 variants, whereas only 6% of isolates from wildlife were identified in these groups. The majority of biogroup 1 isolates from wild ruminants were from 1 group of bighorn sheep in Arizona that were geographically separated from other wildlife sampled. Similarly, 1 biogroup 2 variant, 2E, was cultured only from free-ranging Dall sheep in Alaska. Twelve percent of domestic isolates and 6% of wildlife isolates were indole positive. The remaining isolates from wildlife (33%) and domestic animals (30%) were distributed among 53 distinct biovariants. None of these individual biovariants represented >4% of the total isolates. Phenotypic characterization was valuable for distinguishing between isolates from different hosts and from different geographic areas and can be used to assist in epidemiologic studies.

Pasteurella spp. in sympatric bighorn and domestic sheep.

Domestic sheep were sighted at different times from 1991 to 1993 on four Nevada (USA) ranges occupied by bighorn sheep. Nasal and pharyngeal swab samples were collected from both sheep species and cultured to determine if any strains of Pasteurella spp. were shared on range conditions after contact of the two species. Pasteurella spp. were isolated from all 38 bighorn sheep and 16 of 17 domestic sheep included in this study. The isolates were characterized on the bases of species, biotype, serotype, biogroup, and restriction enzyme analyses (REA) as well as ribotyping of bacterial DNA. A P. haemolytica biotype 3, biogroup 11 isolate from a domestic sheep had biochemical, REA, and ribotype profiles which were identical to those of isolates from three bighorn sheep on the same range. None of the other isolates were found to be common to the two sheep species. Disease was not detected in any of the bighorn populations. However, bighorn sheep populations were extirpated on two ranges while increasing on the other two, including the range on which P. haemolytica biotype 3, biogroup 11 strain was isolated. Declining sheep numbers were not correlated with the presence of any one strain of Pasteurella spp from the sheep.