Genetic diversity of the major histocompatibility complex of cotton rats (Sigmodon hispidus) inhabiting an oil refinery complex.

We examined genetic diversity of an immune-response gene within the major histocompatibility complex in cotton rats (Sigmodon hispidus) inhabiting an oil refinery complex. Genetic diversity of MHC-DQA exon-2 was examined within and among contaminated and noncontaminated reference grids. The level of gene diversity within contaminated grids (0.748) was lower than within reference grids (0.818), but the difference was not statistically significant (p > 0.5). Analysis of molecular variance, pairwise FST values, and hierarchical clustering failed to reveal population genetic structure related to contamination. Results of this study indicate either that the level of contaminant-induced selection is insignificant at this major histocompatibility complex (MHC) locus or that gene flow from surrounding areas has obliterated the effects of selection.

Population genetics of the hispid cotton rat (Sigmodon hispidus): patterns of genetic diversity at the major histocompatibility complex.

The hispid cotton rat, Sigmodon hispidus, is a common rodent widely distributed across the southern USA and south into South America. To characterize major histocompatibility complex (MHC) diversity in this species and to elucidate large-scale patterns of genetic partitioning, we examined MHC genetic variability within and among 13 localities, including a disjunct population in Arizona and a population from Costa Rica that may represent an undescribed species. We also tested the hypothesis that populations within the USA are at equilibrium with regard to gene flow and genetic drift, resulting in isolation-by-distance. Using single-strand conformation polymorphism (SSCP) analysis we identified 25 alleles from 246 individuals. Gene diversity within populations ranged from 0.000 to 0.908. Analysis of molecular variance (AMOVA) revealed that 83.7% of observed variation was accounted for by within-population diversity and 16.3% was accounted for by among-population divergence. The disjunct population in Arizona was fixed for a single allele. The Costa Rican population was quite divergent based on allelic composition and was the only population with unique alleles. Within the main portion of the geographical distribution of S. hispidus in the USA there was considerable divergence among some populations; however, there was no significant pattern of isolation-by-distance overall (P = 0.090). Based on the significant divergence of the only sampled population to its east, the Mississippi River appears to represent a substantial barrier to gene flow.

Immunotoxicity risks associated with land-treatment of petrochemical wastes revealed using an in situ rodent model.

Land-treatment of petrochemical wastes is a widely used method to dispose of hazardous and non-hazardous waste by biodegradation. However, no comprehensive assessment of the impact of such disposal techniques on terrestrial ecosystems has been conducted. Despite the presence of suspected immunotoxicants in the soil, wild rodents frequently reside on these waste sites after closure or abandonment. We explored the seasonal sensitivity of the immune system of the hispid cotton rat (Sigmodon hispidus) to in situ exposures on sites land-treated with petrochemical wastes. Animals were monitored on five contaminated land-treatment sites and five ecologically matched-reference sites in Oklahoma, USA, over two seasons (summer and winter). Most hematological parameters were not adversely affected by land-treatment; however, platelet counts were 26% greater in cotton rats from land-treatment sites compared to reference sites in winter. Significant treatment-related differences were observed in total serum protein concentrations, organ mass and organ cellularity, but these differences were not consistent across the five land-treatment units. Lymphoproliferative responses of cotton rat splenocytes stimulated in vitro were elevated for a T-cell mitogen and depressed for a B-cell mitogen in animals from land-treatment compared to reference sites. The ability of splenocytes to proliferate in response to interleukin-2 receptor-binding was not influenced by treatment. Total yields of peritoneal cells, yield of peritoneal macrophages, and yield of peritoneal lymphocytes were influenced to varying degrees by land-treatment. Functionally, in vitro metabolic activity of peritoneal macrophages was 114% greater in cotton rats from land-treatment sites compared to reference sites during summer. These results indicate that petrochemical wastes applied to soils on these five land-treatment sites had variable immunomodulatory effects in resident cotton rats. Immune alterations for some assays were indicative of enhancement on some land-treatment sites while suppressive on other land-treatment sites, which could have been a function of type and concentration of immunotoxicants present on each site and highlights the uniqueness of each land-treatment site.

Fluorosis risks to resident hispid cotton rats on land-treatment facilities for petrochemical wastes.

Land-treatment of petroleum wastes is a widely used industrial practice, yet there has been no comprehensive evaluation of the long-term risks to human or terrestrial ecosystems from such practices. We evaluated cotton rat (Sigmodon hispidus) populations on three sites in Oklahoma (USA) that historically used land-treatment for disposal of various petroleum wastes (July 1995-March 1997). Average concentrations of fluoride in soil from these sites ranged from 878 to 4317 mg/kg. A census of resident cotton rats on land-treatment sites revealed a high incidence (40% overall) of dental lesions compared to reference populations (<1% dental lesions). During winter there was a 34% to 65% increase compared to summer in frequency of dental lesions in cotton rats on two of the three land-treatment sites. Incidence of dental lesions on two land-treatment sites was greater (9-16%) in female cotton rats compared to males. Cotton rats from land-treatment sites had higher concentrations of fluoride in bone and greater severity of dental lesions compared to reference animals. Dental lesions were considered to be most consistent with dental fluorosis because of elevated fluoride in bone. Neither concentration of fluoride in soil nor level of fluoride in bone was a good predictor of severity of dental lesions in cotton rats on land-treatment sites.

Allelic diversity at the Mhc-DQA locus in cotton rats (Sigmodon hispidus) and a comparison of DQA sequences within the family muridae (Mammalia: Rodentia).

The cotton rat (Sigmodon hispidus) is a common murid rodent of the southern United States, Mexico, and Central America. Using single-stranded conformation polymorphism analysis and DNA sequencing techniques, 11 DQA exon 2 alleles were detected among 180 S. hispidus from Caddo County, Oklahoma, USA. The alleles represent a single locus exhibiting a high level of polymorphism. Nucleotide and amino acid distance values among DQA alleles of S. hispidus were higher than those within Mus musculus and species of Rattus. Although the distribution of polymorphic amino acid residues among alleles of S. hispidus was similiar to that of Mus and Rattus, some residues of the alpha-helix region were more variable in S. hispidus. Comparisons of nonsynonymous and synonymous substitutions indicated a trend toward higher numbers of nonsynonymous substitutions; however, this difference was not significant statistically among S. hispidus alleles. To examine evolution of DQA alleles within Muridae, we performed a phylogenetic analysis that included DQA alleles from S. hispidus, Peromyscus leucopus, M. musculus, R. norvegicus, and six Australian species of Rattus. Results depicted monophyly for each genus, and this concordance between species and gene trees represents a lack of evidence for trans-species persistence of alleles among these genera.

Indicators of immunotoxicity in populations of cotton rats (Sigmodon hispidus) inhabiting an abandoned oil refinery.

Wildlife species inhabiting contaminated sites are often exposed to complex mixtures of chemicals, many of which have known effects on physiological and biochemical function. Although sensitivity of the immune system to chemical exposure has been documented in laboratory animal and wildlife species, little work has been conducted on feral wildlife populations inhabiting contaminated sites. Immune function was measured in populations of wild cotton rats (Sigmodon hispidus) inhabiting replicated reference and contaminated study sites at an abandoned oil refinery in Oklahoma four times from 1991 to 1992. Several measures of immunocompetence were examined including immune organ mass and cellularity, hematology, in vivo hypersensitivity, macrophage function, killer cell activity, and lymphoproliferative responsiveness. In vitro proliferation of splenocytes, either spontaneous or induced with concanavalin A (Con A), was the most consistent and reliable indicator of immunotoxicity. Spontaneous proliferation of splenocytes was 48 and 24% higher for cotton rats collected from contaminated than reference sites in September 1991 and September 1992, respectively. Likewise, Con A-induced proliferation of splenocytes ranged form 20 to 53% higher in animals collected from contaminated than reference sites in three of four collection periods. The percentage of splenocytes (mean+/-SE) staining positive for Con A receptors was lower on contaminated sites (73.7+/-1.2%) than reference sites (77.0+/-1.4%) in September 1991. Other measures of immune function including macrophage metabolism, hypersensitivity, blood cellularity, and mass and cellularity of immune organs varied between contaminated and reference sites.

In situ (mesocosm) assessment of immunotoxicity risks to small mammals inhabiting petrochemical waste sites.

Oil refineries inadvertently deposit a variety of complex mixtures of organic hydrocarbons and heavy metals in the soil, many of which are thought to be potent immunotoxicants. Terrestrial ecosystems such as this have not been adequately investigated with respect to wild rodent populations. The primary objective of this study was to use mesocosms to assess the immunotoxicity risks to feral small mammal populations associated with soils contaminated with petroleum refinery wastes. A series of 4-week and 8-week exposure trials using laboratory raised cotton rats (Sigmodon hispidus) were conducted in situ on three contaminated and three reference sites on the Oklahoma Refining Company Superfund Waste Site, Cyril, Oklahoma. Cotton rats exposed to these soils showed significant alterations in selected morphological traits, in vivo humoral immune responses, complement activity, and macrophage activity. However, immune alterations were not great, suggesting that resident small mammals may be a better biomonitoring choice than using mesocosms.

Organochlorine pesticide accumulation and genotoxicity in Mexican free-tailed bats from Oklahoma and New Mexico.

The summer population of Mexican free-tailed bats (Tadarida brasiliensis) at Carlsbad Caverns, New Mexico, declined from an estimated 8.7 million in 1936 to 200,000 in 1974; thereafter, it increased to approximately 700,000 in 1991. This decline has been attributed primarily to organochlorine (OC) pesticide contamination and habitat disturbance. Similar declines have been observed in other populations of this species. This study examined the potential genotoxic effects of OC pesticide contamination on two populations of T. brasiliensis. Pesticide accumulation, frequencies of chromosomal aberrancy, and nuclear DNA content variation in spleen and testicular tissues were examined in specimens collected from Carlsbad Caverns and Vickery Cave, a maternity colony in northwestern Oklahoma, during the summers of 1990 and 1991. Pesticide residues in brain and carcass tissues were identified and quantified by electron capture gas chromatography. Genotoxicity was examined with the standard bone marrow chromosomal aberration assay and flow cytometry. Statistical relationships among pesticide content, observed chromosomal aberrancy, and nuclear DNA content variation were examined. Both populations demonstrated significant levels of DDE contamination; however, the Carlsbad Caverns population showed consistently higher pesticide loads. Males also demonstrated higher levels than females. No statistical differences in chromosomal aberrancy or nuclear DNA content variation were observed among sexes, sites, or collected periods. Positive correlations were detected between brain and carcass DDE concentrations for all bats examined. A significant negative relationship was found between brain DDE concentration and coefficients of variation in spleen DNA content only for males.

Fluorosis in a wild cotton rat (Sigmodon hispidus) population inhabiting a petrochemical waste site.

We have developed an in situ mammalian model for evaluating environmental contamination using wild cotton rats. In a series of experiments, 200 male cotton rats were captured during 4 collection periods (spring 1991 = 35; fall 1991 = 60; spring 1992 = 53; fall 1992 = 52). A total of 103 of these cotton rats were captured from control sites, and the remaining 97 were captured from an abandoned oil refinery. All sites were located in the vicinity of Cyril, Oklahoma. There were alterations in the incisors of cotton rats captured from the refinery site. Normal color of cotton rat incisors is deep yellow-orange, which is imparted by a pigment normally produced by ameloblasts. Grossly, the upper incisors of 37 of 97 rats and lower incisors of 54 of 97 rats were affected. The affected incisors were white, chalky, and thin with striations and erosions of the enamel. Microscopic examination revealed that there were dysplastic and necrotic changes in the ameloblasts. The bone fluoride levels were significantly higher in rats captured from the refinery as compared to the rats captured from the control sites.

Cross-placental transfer of organochlorine pesticides in Mexican free-tailed bats from Oklahoma and New Mexico.

A total of 41 pregnant Mexican free-tailed bats (Tadarida brasiliensis) were collected from Carlsbad Caverns, New Mexico, and Vickery Cave, Oklahoma, in May and June 1990 and May 1991 for organochlorine pesticide analyses. Residues of p,p'-DDE were detected by gas chromatography in 40 embryos at levels highly correlated with brain concentrations (r = 0.496) but were not correlated mother's body tissues (r = 0.060). Embryonic levels also did not correlate with either embryonic fat content (r = 0.018) or maternal fat content (r = 0.300), suggesting that placental membranes offer only marginal protection for developing embryos against exposure to lipophilic organochlorine pesticides.

A new coccidian (Apicomplexa: eimeriidae) in the northern pocket gopher (Thomomys talpoides) and a comparison of oocyst survival in hosts from radon-rich and radon-poor soils.

Forty (93%) of 43 northern pocket gophers (Thomomys talpoides) from the Jemez Mountains, Sandoval County, New Mexico (USA), had coccidian oocysts in their feces when examined. We describe this parasite, Eimeria jemezi, n. sp. Sporulated oocysts were subspheroidal, 13.3 by 12.2 (10 to 17 by 9 to 15) microns, with sporocysts ellipsoidal, 7.1 by 4.4 (5 to 9 by 4 to 5) microns; micropyle and oocyst residuum were absent, but polar bodies, Stieda bodies and sporocyst residua were present. All gophers were collected from two sites of similar habitat 7 km apart. One site (R+) had a high soil radon content (> or = 50 to 70 picocuries (pCi) per liter of air) whereas the other site (R-) had soils that were near average natural levels (1.2 to 1.6 pCi/g uranium nucleotides per gram of soil; < 1.9 pCi/g thorium nucleotides). Twenty-one (88%) of 24 gophers from the R+ site had coccidian oocysts in their intestines when examined, but none of these oocysts ever sporulated, whereas all 19 (100%) gophers from the R- site had coccidian oocysts in their intestines and 16 (84%) of these samples sporulated normally under laboratory conditions. The elevated radon content of the soil may have had an adverse effect on the sporulation of this coccidian while it still was intracellular within its host.

Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat.

Exposure to the mutagen triethylenemelamine on rat bone marrow, blood, and testis was studied using flow cytometry of DAPI-stained nuclei. Increased coefficients of variation (CVs) of the G1 peaks were observed in bone marrow and blood after both 1 d and 5 d exposures. After 5 d exposure and 7 d recovery both tissues had recovered, in some cases to significantly lower CVs. Increased CVs of the 1C peak of testis were observed only after 5 d exposure to the high dose with no subsequently observed recovery. Bone marrow cells also were stained with Hoechst 33258 and Propidium Iodide. No differences among dyes were observed indicating that increased CVs likely are due to DNA damage resulting from interactions with the mutagen rather than differences in how the dyes bind to DNA relative to mutagen binding. This study demonstrates that differences occur among tissues in how quickly they respond and recover from mutagen exposure. Increased CVs, cell cycle alterations, and decreased CVs after recovery are all potentially useful biomarkers of effect for laboratory and field studies in environmental toxicology.

Induction of hepatic CYP1A activity as a biomarker for environmental exposure to Aroclor 1254 in feral rodents.

Specimens of the feral mouse species Reithrodontomys fulvescens trapped from a polychlorinated biphenyl (PCB)-contaminated field location had hepatic ethoxyresorufin (ETR) O-dealkylase activities and immunoreactive CYP1A protein contents which were two- to threefold higher than those measured in animals of the same species and sex collected from non PCB-contaminated reference sites. Specimens with hepatic ETR O-dealkylase activities differing by as little as 50% could readily be assigned as originating from the PCB or reference sites by the use of a specific chemical inhibitor of cytochrome P450IA (CYP1A). The relative levels of ETR O-dealkylase activity in R. fulvescens significantly correlated with hepatic PCB burdens (r = 0.819, P less than 0.01). When the magnitudes of the induced ETR O-dealkylase activities corresponding to given hepatic PCB burdens were compared between the feral animals, F344/NCr rats (Rattus norvegicus) or B6C3F1 mice (Mus musculus) exposed in the laboratory to dietary Aroclor 1254, the order of sensitivity to the inducing effects of PCBs were F344/NCr rat greater than B6C3F1 mouse greater than R. fulvescens.

Flow-cytometric analysis of the effects of triethylenemelamine on somatic and testicular tissues of the rat.

The effects of short-term (24 h) exposure to triethylenemelamine on cellular DNA in five tissues (bone marrow, spleen, kidney, large intestine, and testis) of the rat were studied using flow cytometry. Mean coefficients of variation of the G1 peaks were increased in both the low and high dosage groups relative to controls. Bone marrow exhibited the highest degree of effect, possibly due to the rapid rate of cell division in that tissue, and spleen was next highest. Thus, hematopoietic tissues are highly responsive to short-term, acute exposure to this mutagen. The results of the flow-cytometry assay closely paralleled a simultaneous chromosomal assay conducted on bone marrow of the same rats. These data are interpreted to be consistent with the hypothesis that the observed increase in mean coefficients of variation is due to the clastogenic effects of the mutagen and subsequent unequal distribution of DNA among the daughters of affected cells.