Frequency of Animal Leptospirosis in the Southern United States and the Implications for Human Health.

Leptospirosis is a zoonotic disease with symptoms in humans and animals, ranging from subclinical to serious and fatal. The disease occurs worldwide, but there is limited recognition of the public and animal health risks it poses in the southern United States. A systematic review of the frequency of animal leptospirosis in 17 states and jurisdictions covering the southern continental United States was performed to advance our understanding of the pathogen's distribution and identify transmission patterns that could be targeted for prevention efforts. Fifty-two articles, spanning >100 years, met the analysis criteria. A wide range of techniques were used to measure seroprevalence and isolate the bacteria. The assessment identified exposure to Leptospira spp and Leptospira spp infection among a diverse range of species, spanning 22 animal families within 14 states, suggesting that the pathogen is distributed throughout the southern region. Disease frequency trends were assessed among animals in various habitats (all habitats, nonwild habitats, and wild habitats). The frequency of Leptospira spp detection in animals in wild habitats increased slightly over time (<0.2%/year). We identified reports of 11 human leptospirosis illness clusters and outbreaks in the southern United States. Exposure to potentially contaminated surface waters were documented for at least seven of the events, and interactions with infected or likely infected animals were documented for at least six of the events. This analysis highlights the need for stronger partnerships across the public and animal health fields to enhance diagnostics, surveillance, and reporting. The early identification of leptospirosis in animals may serve as an indicator of environmental contamination and trigger prevention measures, such as vaccinating companion animals and livestock, use of potable water, and the wearing of waterproof protective clothing near water that may be contaminated.

Lack of influence by endosymbiont Wolbachia on virus titer in the common bed bug, Cimex lectularius.

BACKGROUND: The common bed bug, Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius. METHODS: We used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia. RESULTS: There was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia-positive and Wolbachia-cured groups. CONCLUSIONS: These findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of Wolbachia, and our conclusions underscore the need for future research.

A Comparison of the chemical sensitivities between in vitro and in vivo propagated juvenile freshwater mussels: Implications for standard toxicity testing.

Unionid mussels are ecologically important and are globally imperiled. Toxicants contribute to mussel declines, and toxicity tests using juvenile mussels-a sensitive life stage-are valuable in determining thresholds used to set water quality criteria. In vitro culture methods provide an efficient way to propagate juveniles for toxicity testing, but their relative chemical sensitivity compared with in vivo propagated juveniles is unknown. Current testing guidelines caution against using in vitro cultured juveniles until this sensitivity is described. Our objective was to evaluate the relative sensitivity of juvenile mussels produced from both in vitro and in vivo propagation methods to selected chemicals. We conducted 96-h acute toxicity tests according to ASTM International guidelines with 3 mussel species and 6 toxicants: chloride, nickel, ammonia, and 3 copper-based compounds. Statistically significant differences between in vitro and in vivo juvenile 96-h median effect concentrations were observed in 8 of 17 tests, and in vitro juveniles were more sensitive in 6 of the 8 significant differences. At 96 h, 4 of the 8 statistically different tests for a given chemical were within a factor of 2, which is the intralaboratory variation demonstrated in a recent evaluation of mussel toxicity tests. We found that although differences in chemical sensitivity exist between in vitro and in vivo propagated juvenile mussels, they are within normal toxicity test variation. Therefore, in vitro propagated juvenile mussels may be appropriate for use in ASTM International-based toxicity testing. Environ Toxicol Chem 2018;37:3077-3085. © 2018 SETAC.

Key metabolites in tissue extracts of Elliptio complanata identified using (1)H nuclear magnetic resonance spectroscopy.

We used (1)H nuclear magnetic resonance spectroscopy to describe key metabolites of the polar metabolome of the freshwater mussel, Elliptio complanata. Principal components analysis documented variability across tissue types and river of origin in mussels collected from two rivers in North Carolina (USA). Muscle, digestive gland, mantle and gill tissues yielded identifiable but overlapping metabolic profiles. Variation in digestive gland metabolic profiles between the two mussel collection sites was characterized by differences in mono- and disaccharides. Variation in mantle tissue metabolomes appeared to be associated with sex. Nuclear magnetic resonance spectroscopy is a sensitive means to detect metabolites in the tissues of E. complanata and holds promise as a tool for the investigation of freshwater mussel health and physiology.

Integration of environmental stewardship and local economic development to enhance community health.

Environmental groups working to preserve natural ecosystems and groups working to enhance local economic development often find themselves on philosophically opposite sides of the negotiation table. Case histories of cooperative engagement are provided that serve as examples of how environmental stewardship is compatible with local economic development and community health.

Effects of opioids and anesthetic drugs on body temperature in cats.

OBJECTIVE: To determine which class of opioid alone or in conjunction with other anesthetic drugs causes post-anesthetic hyperthermia in cats. STUDY DESIGN: Prospective, randomized, crossover study. ANIMALS: Eight adult, healthy, cats (four spayed females and four castrated males weighing 3.8 +/- 0.6 kg). METHODS: Each cat was instrumented with a wireless thermistor in the abdominal cavity. Temperature in all phases was recorded every 5 minutes for 5 hours. Population body temperature (PBT) was recorded for approximately 8 days. Baseline body temperature is the final 24 hours of the PBT. All injectable drugs were given intramuscularly. The cats were administered drugs in four phases: 1) hydromorphone (H) 0.05, 0.1, or 0.2 mg kg(-1); 2) morphine (M) (0.5 mg kg(-1)), buprenorphine (BUP) (0.02 mg kg(-1)), or butorphanol (BUT) (0.2 mg kg(-1)); 3) ketamine (K) (5 mg kg(-1)) or ketamine (5 mg kg(-1)) plus hydromorphone (0.1 mg kg(-1)) (KH); 4) isoflurane in oxygen for 1 hour. Fifteen minutes prior to inhalant anesthetic, cats received either no premed (I), hydromorphone (0.1 mg kg(-1)) (IH), or hydromorphone (0.1 mg kg(-1)) plus ketamine (5 mg kg(-1)) (IHK). RESULTS: Mean PBT for all unmedicated cats was 38.9 +/- 0.6 degrees C (102.0 +/- 1 degrees F). The temperature of cats administered all doses of hydromorphone increased from baseline (p < 0.03) All four opioids (H, M, BUP and BUT) studied increased body temperature compared with baseline (p < 0.005). A significant difference was observed between baseline temperature values and those in treatment KH (p < 0.03). Following recovery from anesthesia, temperature in treatments IH and IHK was different from baseline (p < 0.002). CONCLUSIONS AND CLINICAL RELEVANCE: All of the opioids tested, alone or in combination with ketamine or isoflurane, caused an increase in body temperature. The increase seen was mild to moderate (<40.1 degrees C (104.2 degrees F) and self limiting.

Correlation of mannitol fermentation with virulence-associated genotypic characteristics in Vibrio vulnificus isolates from oysters and water samples in the Gulf of Mexico.

Vibrio vulnificus strains (n = 469) isolated from the Gulf of Mexico oysters and waters over a period of 2 years were subjected to phenotypic and genotypic characterizations. Of the strains that could be definitively genotyped (n = 465), 58% were classified as genotype A, 29% as genotype B, and 13% as genotype A/B by 16S rRNA genotyping. When the same strain bank was characterized by virulence-correlated gene (vcg) typing, 65% were genotype E while 35% were genotype C. Further analysis focusing on strains falling into typical genotype categories (i.e., 16S rRNA types A or B, excluding type A/B strains) showed a high degree of concordance (93%) when comparing the two genotyping methods. d-Mannitol fermentation was also predictive of genotype, with an 86% agreement between 16S rRNA genotype and mannitol fermentation patterns, and an 85% agreement between vcg genotype and mannitol fermentation patterns. d-Mannitol fermentation should be considered as a simple and less expensive alternative to screen V. vulnificus isolates for virulence potential, particularly when analyzing large strain banks.

Domestic wastewater influent profiling using mitochondrial real-time PCR for source tracking animal contamination.

Real-time PCR amplifying mammalian and avian mitochondrial DNA (mtDNA) was developed to characterize domestic and light industrial wastewater influent from two municipal wastewater treatment facilities (WWTF) over a 24-week period. Influent samples were assayed with species-specific primers and dual-labeled probes for human, bovine, swine, dog, cat, Canada goose and white-tailed deer to detect and quantify eukaryotic mtDNA contributors to local municipal wastewaters. Human (mean=9.6 x 10(4) copies/ml) and dog (mean=5.3 x 10(2) copies/ml) mtDNA were detected in all 24 samples (12 samples/site). Bovine and swine mtDNA were detected sporadically and at lower levels than human mtDNA, means=3.0 x 10(4) and 9.5 x 10(2) copies/ml, respectively. Domestic cat, Canada goose and white-tailed deer were detected only once in 24 samples. Mitochondrial DNA concentrations were compared to other bacterial, chemical and spectrophotometric parameters. Human mtDNA was positively correlated with ammonia concentration (P=0.01) and initial OD(600) reading (P=0.02) at one WWTF. Bovine mtDNA was positively correlated with biological oxygen demand (BOD) (P=0.02), final DNA concentration (P=0.03), initial and final humic acid concentrations (P=0.01, P=0.01), and final OD(600) (P=0.03) at one WWTF and total suspended solids (TSS) (P=0.04, P=0.09) at both facilities. Fecal coliforms were not positively or negatively correlated with mtDNA concentrations of any species assayed. For source tracking purposes, a combination of human (10(5) copies/ml) and dog mtDNA signal (10(2) copies/ml) could be indicative of municipal domestic wastewater contamination of environmental waters.

Assessment of the effect of varying soil organic matter content on the bioavailability of malathion to the common nightcrawler, Lumbricus terrestris L.

This study investigated the effect of soil organic matter content on the bioavailability of malathion to the common nightcrawler, Lumbricus terrestris. Earthworms were exposed for 72 h to malathion on two soil types, 8% organic matter and 55% organic matter. Two different measures of bioavailability, malathion body burdens and tissue cholinesterase activities, were then measured in the malathion exposed animals. There were no significant differences in body burden or cholinesterase levels in L. terrestris exposed to malathion on soils with differing organic matter content. This suggests that absorption into organic matter is not a limiting factor of malathion bioavailability to earthworm species.

Mitochondrial multiplex real-time PCR as a source tracking method in fecal-contaminated effluents.

Multiplex real-time PCR amplifying fecal mitochondrial DNA (mtDNA) combined with rapid, crude DNA preparations are promising additions to surface water source tracking methods. Amplification of eukaryotic mitochondrial DNA identifies the fecal source directly and can be used in conjunction with other intestinal microbial methods to characterize effluents. Species-specific primers and dual-labeled probes for human, swine, and bovine NADH dehydrogenase subunit 5 (ND5) genes were created for multiplex real-time PCR in feces and effluent slurries. The linear range of the multiplex assay was 10(2)-10(7) mtDNA copies for human, bovine, and swine effluent in combination (equal volumes). PCR amplification efficiencies for bovine, human, and swine mtDNA when assayed in combination were 93, 107, and 92% respectively. Linear regression correlation coefficients (r2) were 0.99 for all standard curves except for human mtDNA in combination (r2 = 0.95). Multiplex amplification of bovine, human, and swine mtDNA (ND5) exhibited no cross-reactions between the effluents from three species of interest. Also, no cross-reactions were observed with effluents of other vertebrates: sheep, goat, horse, dog, cat, Canada goose, broiler, layer, turkey, and tilapia. Performed as a blind test, the PCR operator was able to correctly identify all but two effluent challenge samples (10/12 or 83% correct) with no false positives (22/22 or 100% correct). The multiplex assay had a tendency to detect the species of highest mtDNA concentration only. Better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly. Real-time PCR detection limit was calculated as 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent. Some carry-over mtDNA PCR signal from consumed beef, but not pork, was found in feces of human volunteers.

Polychlorinated biphenyl exposure causes gonadal atrophy and oxidative stress in Corbicula fluminea clams.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants that have been linked to oxidative and other toxic effects in both humans and wildlife. Due to recent environmental health concerns at a PCB contaminated Superfund site near Raleigh, NC, we used a common clam species (Corbicula fluminea) as surrogates to isolate the effects of PCBs on threatened bivalves native to the region. Under controlled laboratory conditions, clams were exposed to 0, 1, 10, or 100 ppb Aroclor 1260 in the ambient water for 21 days. Measured biomarkers spanned a range of effective levels of biological organization including low molecular weight antioxidants, lipid-soluble antioxidants, and whole tissue radical absorption capacity. These data were augmented by use of histological evaluation of whole samples. Aroclor 1260 significantly increased reduced glutathione (GSH) and total protein concentrations at all treatments levels. Significant decreases were measured in all treatments in gamma -tocopherol and total oxidant scavenging capacity (TOSC) and alpha -tocopherol values in the 100 ppb exposure. Histologically, Aroclor 1260 caused significant gonadal atrophy, effacement of gonad architecture with accumulations of Brown cells, and inflammation and necrosis in digestive glands and foot processes. Our results indicate that oxidative mechanisms play a significant role in the decreased health of these clams due to exposure to Aroclor 1260. The changes in the gonads of exposed clams suggest that a serious threat to bivalve reproduction exists due to PCB exposure.

Seminal and endocrine characteristics of male Pallas' Cats (Otocolobus manul) maintained under artificial lighting with simulated natural photoperiods.

Pallas' cats (Otocolobus manul) have a pronounced reproductive seasonality controlled by photoperiod. Previous studies of reproduction in captive Pallas' cats exposed to natural light showed a breeding season of December-April. This study evaluated the impact of artificial lighting timed to simulate natural photoperiods on male reproductive seasonality of four Pallas' cats housed indoors. Semen evaluation, blood collection, and body weight measurements were conducted every 1-2 months from November 2000-June 2001. Fecal samples were collected from each male twice weekly to assess testosterone and corticoid concentrations. Mean values for reproductive traits (sperm attributes, testicular volume) were highest from February-April, the defined breeding season. Fecal testosterone concentrations were highest from mid-January to mid-March. Male Pallas' cats managed indoors under simulated photoperiods experienced a delayed onset of the breeding season by 1-2 months and a decreased length of the breeding season. Over the course of the study, fecal corticoid concentrations did not seem to differ among seasons. Although mating attempts during this study were unsuccessful, subsequent pairings of male and female Pallas' cats in the same research colony during the 2002 and 2003 breeding seasons produced viable offspring. These results suggest that male Pallas' cats, housed indoors under simulated photoperiods, exhibit distinct reproductive cyclic patterns, characterized by a delayed and truncated breeding season. Adrenocortical activity varied among individuals, but did not adversely affect reproductive parameters. Housing Pallas' cats indoors under simulated photoperiods may represent a viable strategy for maintaining breeding success while limiting disease exposure. Zoo Biol 0:1-13, 2007. (c) 2007 Wiley-Liss, Inc.

Temporal and spatial variability in stable isotope compositions of a freshwater mussel: implications for biomonitoring and ecological studies.

Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue delta(15)N and delta(13)C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve delta(13)C values correlated with site ambient particulate organic matter (POM) delta(13)C values, and site mean bivalve delta(15)N values correlated with site ambient water dissolved delta(15)N-NO(3) values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in delta(15)N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph delta(15)N compositions responded to a shift in ambient dissolved delta(15)N-NO(3), although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies.

Renal biopsy: a retrospective study of methods and complications in 283 dogs and 65 cats.

Renal biopsy often is required to establish a definitive diagnosis in dogs and cats with renal disease. In this retrospective study, we determined the complications of renal biopsy as well as factors that may be associated with development of complications and procurement of adequate renal biopsy specimens in 283 dogs and 65 cats. Data extracted from medical records at 4 institutions were evaluated using logistic regression. Proteinuria was the most common indication for renal biopsy in dogs. Complications were reported in 13.4 and 18.5% of dogs and cats, respectively. The most common complication was severe hemorrhage; hydronephrosis and death were uncommon. Dogs that developed complications after renal biopsy were more likely to have been 4 to < 7 years of age and > 9 years, to weigh < or = 5 kg, and to have serum creatinine concentrations > 5 mg/dL. The majority of biopsies from both dogs (87.6%) and cats (86.2%) were considered to be of satisfactory quality. Biopsies from dogs were more likely to be of high quality if they were obtained when the patient was under general anesthesia and more likely to contain only renal cortex if they were obtained by surgery. We concluded that renal biopsy is a relatively safe procedure, with a low frequency of severe complications. Hospital practices and patient variables have the potential to impact both the quality of the specimen obtained and the rate of complications.

Comparison of individual and pooled sampling methods for detecting bacterial pathogens of fish.

Examination of finfish populations for viral and bacterial pathogens is an important component of fish disease control programs worldwide. Two methods are commonly used for collecting tissue samples for bacteriological culture, the currently accepted standards for detection of bacterial fish pathogens. The method specified in the Office International des Epizooties Manual of Diagnostic Tests for Aquatic Animals permits combining renal and splenic tissues from as many as 5 fish into pooled samples. The American Fisheries Society (AFS) Blue Book/US Fish and Wildlife Service (USFWS) Inspection Manual specifies the use of a bacteriological loop for collecting samples from the kidney of individual fish. An alternative would be to more fully utilize the pooled samples taken for virology. If implemented, this approach would provide substantial savings in labor and materials. To compare the relative performance of the AFS/USFWS method and this alternative approach, cultures of Yersinia ruckeri were used to establish low-level infections in groups of rainbow trout (Oncorhynchus mykiss) that were sampled by both methods. Yersinia ruckeri was cultured from 22 of 37 groups by at least 1 method. The loop method yielded 18 positive groups, with 1 group positive in the loop samples but negative in the pooled samples. The pooled samples produced 21 positive groups, with 4 groups positive in the pooled samples but negative in the loop samples. There was statistically significant agreement (Spearman coefficient 0.80, P < 0.001) in the relative ability of the 2 sampling methods to permit detection of low-level bacterial infections of rainbow trout.

Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae).

Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.

Reference ranges for hemolymph chemistries from Elliptio complanata of North Carolina.

Hemolymph chemistries may be useful nonlethal measures of bivalve health. The prognostic value of hemolymph, however, depends on a comparison of chemistry results to reference ranges from healthy individuals. Currently, knowledge of expected hemolymph values in healthy and unhealthy freshwater mussels is extremely limited. The purpose of this study was to develop a set of reference ranges for clinical evaluation of hemolymph from a freshwater mussel species common to southeastern USA. We collected hemolymph from 380 Elliptio complanata from 19 apparently healthy populations from northwest of Raleigh, North Carolina, during May through July 2001. We present reference ranges for hemolymph parameters ammonia, glucose, calcium, magnesium, phosphorus, aspartate aminotransferase (AST), bicarbonate, protein and cell count, and for tissue glycogen. We compare the subpopulations of mussels from regions with an agricultural riparian buffer to those surrounded predominantly by forested lands. We further present correlations noted between hemolymph chemistries and physical or physiologic parameters. The only statistically significant differences between populations contiguous to agricultural and forested lands were in hemolymph calcium and glucose concentrations. Other statistically significant correlations identified were between gravidity and hemolymph protein concentration and tissue glycogen content, as well as between gravidity and parasite burden, and between shell length and hemolymph glucose, AST, calcium and bicarbonate concentrations. The results of this study will aid the interpretation of health measures from populations of E. complanata of similar geographic and seasonal origin.