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Escherichia coli and fecal-coliform bacteria as indicators of recreational water quality

In 1986, the U.S. Environmental Protection Agency (USEPA) recommended that Escherichia coli (E. coli) be used in place of fecal-coliform bacteria in State recreational water-quality standards as an indicator of fecal contamination. This announcement followed an epidemiological study in which E. coli concentration was shown to be a better predictor of swimming-associated gastrointestinal illness than fecal-coliform concentration. Water-resource managers from Ohio have decided to collect information specific to their waters and decide whether to use E. coli or fecal-coliform bacteria as the basis for State recreational water-quality standards. If one indicator is a better predictor of recreational water quality than the other and if the relation between the two indicators is variable, then the indicator providing the most accurate measure of recreational water quality should be used in water-quality standards. Water-quality studies of the variability of concentrations of E. coli to fecal-coliform bacteria have shown that (1) concentrations of the two indicators are positively correlated, (2) E. coli to fecal-coliform ratios differ considerably from site to site, and (3) the E. coli criteria recommended by USEPA may be more difficult to meet than current (1992) fecal-coliform standards. In this study, a statistical analysis was done on concentrations of E. coli and fecal-coliform bacteria in water samples collected by two government agencies in Ohio-- the U.S. Geological Survey (USGS) and the Ohio River Valley Water Sanitation Commission (ORSANCO). Data were organized initially into five data sets for statistical analysis: (1) Cuyahoga River, (2) Olentangy River, (3) Scioto River, (4) Ohio River at Anderson Ferry, and (5) Ohio River at Cincinnati Water Works and Tanners Creek. The USGS collected the data in sets 1, 2, and 3, whereas ORSANCO collected the data in sets 4 and 5. The relation of E. coli to fecal-coliform concentration was investigated by use of linear-regression analysis and analysis of covariance. Log-transformed E. coli and fecal-coliform concentrations were highly correlated in all data sets (r-values ranged from 0.929 to 0.984). Linear regression analysis on USGS and ORSANCO data sets showed that concentration of E. coli could be predicted from fecal-coliform concentration (coefficients of determination (R2) ranged from 0.863 to 0.970). Results of analysis of covariance (ANCOVA) indicated that the predictive equations among the three USGS data sets and two ORSANCO data sets were not significantly different and that the data could be pooled into two large data sets, one for USGS data and one for ORSANCO data. However, results of ANCOVA indicated that USGS and ORSANCO data could not be pooled into one large data set. Predictions of E. coli concentrations calculated for USGS And ORSANCO regression relations, based on fecal-coliform concentrations set to equal Ohio water-quality standards, further showed the differences in E. coli to fecal-coliform relations among data sets. For USGS data, a predicted geometric mean of 176 col/100 mL (number of colonies per 100 milliliters) was greater than the current geometric-mean E. coli standard for bathing water of 126 col/100mL. In contrast, for ORSANCO data, the predicted geometric mean of 101 col/100 mL was less than the current E. coli standard. The risk of illness associated with predicted E. coli concentrations for USGS and ORSANCO data was evaluated by use of the USEPA regression equation that predicts swimming-related gastroenteritis rates from E. coli concentrations.1 The predicted geometric-mean E. coli concentrations for bathing water of 176 col/100 mL for USGS data and 101 col/100 mL for ORSANCO data would allow 9.4 and 7.1 gastrointestinal illnesses per 1,000 swimmers, respectively. This prediction compares well with the illness rate of 8 individuals per 1,000 swimmers estimated by the USEPA for an E. coli concentration of 126 col/100 mL. Therefore, the

Water-Resources Investigations Report↗

Victims and vectors: highly pathogenic avian influenza H5N1 and the ecology of wild birds

The emergence of highly pathogenic avian influenza (HPAI) viruses has raised concerns about the role of wild birds in the spread and persistence of the disease. In 2005, an outbreak of the highly pathogenic subtype H5N1 killed more than 6,000 wild waterbirds at Qinghai Lake, China. Outbreaks have continued to periodically occur in wild birds at Qinghai Lake and elsewhere in Central China and Mongolia. This region has few poultry but is a major migration and breeding area for waterbirds in the Central Asian Flyway, although relatively little is known about migratory movements of different species and connectivity of their wetland habitats. The scientific debate has focused on the role of waterbirds in the epidemiology, maintenance and spread of HPAI H5N1: to what extent are they victims affected by the disease, or vectors that have a role in disease transmission? In this review, we summarise the current knowledge of wild bird involvement in the ecology of HPAI H5N1. Specifically, we present details on: (1) origin of HPAI H5N1; (2) waterbirds as LPAI reservoirs and evolution into HPAI; (3) the role of waterbirds in virus spread and persistence; (4) key biogeographic regions of outbreak; and (5) applying an ecological research perspective to studying AIVs in wild waterbirds and their ecosystems.

Avian Biology Research↗

Spiral swimming behavior due to cranial and vertebral lesions associated with Cytophaga psychrophila infections in salmonid fishes

C. psychrophila infections of the cranium and anterior vertebrae in salmonid fishes were associated with ataxia, spiral swimming along the axis of the fish, and death. The syndrome was observed in 2-10% of underyearling coho salmon Oncorhynchus kisutch, rainbow trout Salmo gairdneri, and steelhead trout S. gairdneri at several private, state, and federal hatcheries in Washington and Oregon, USA, between 1963 and 1987. Affected fish did not recover and ultimately died. Histological examination consistently revealed subacute to chronic periostitis, osteitis, meningitis, and ganglioneuritis. Inflammation and periosteal proliferation of the anterior vertebrae at the junction of the vertebral column with the cranium with extension into the cranial case was a consistent feature. The adjacent nervous tissue, particularly the medulla, was often compressed by the proliferative lesion, and this may have caused the ataxia. Though bacteria were seldom observed in these lesions. C. psychrophila was isolated in culture from the cranial cavity of all affected fish that were tested. Epidemiological observations suggested that this bacterium is the causative agent because the spiral swimming behaviour and lesions were observed only in populations that had recovered from acute C. psychrophila infections.

Diseases of Aquatic Organisms↗

Investigating seagrass in Toxoplasma gondii transmission in Florida (Trichechus manatus latirostris) and Antillean (T. m. manatus) manatees

Toxoplasma gondii is a feline protozoan reported to cause morbidity and mortality in manatees and other marine mammals. Given the herbivorous nature of manatees, ingestion of oocysts from contaminated water or seagrass is presumed to be their primary mode of infection. The objectives of this study were to investigate oocyst contamination of seagrass beds in Puerto Rico and determine the seroprevalence of T. gondii in Antillean ( Trichechus manatus manatus ) and Florida ( T. m. latirostris ) manatees. Sera or plasma from Antillean (n = 5) and Florida (n = 351) manatees were tested for T. gondii antibodies using the modified agglutination test. No T. gondii DNA was detected via PCR in seagrass samples (n = 33) collected from Puerto Rico. Seroprevalence was 0%, suggesting a lower prevalence of T. gondii in these manatee populations than previously reported. This was the first study to investigate the potential oocyst contamination of the manatee diet, and similar studies are important for understanding the epidemiology of T. gondii in herbivorous marine mammals.

Diseases of Aquatic Organisms↗

Two distinct phylogenetic clades of infectious hematopoietic necrosis virus overlap within the Columbia River basin

Infectious hematopoietic necrosis virus (IHNV), an aquatic rhabdovirus, causes a highly lethal disease of salmonid fish in North America. To evaluate the genetic diversity of IHNV from throughout the Columbia River basin, excluding the Hagerman Valley, Idaho, the sequences of a 303 nt region of the glycoprotein gene (mid-G) of 120 virus isolates were determined. Sequence comparisons revealed 30 different sequence types, with a maximum nucleotide diversity of 7.3% (22 mismatches) and an intrapopulational nucleotide diversity of 0.018. This indicates that the genetic diversity of IHNV within the Columbia River basin is 3-fold higher than in Alaska, but 2-fold lower than in the Hagerman Valley, Idaho. Phylogenetic analyses separated the Columbia River basin IHNV isolates into 2 major clades, designated U and M. The 2 clades geographically overlapped within the lower Columbia River basin and in the lower Snake River and tributaries, while the upper Columbia River basin had only U clade and the upper Snake River basin had only M clade virus types. These results suggest that there are co-circulating lineages of IHNV present within specific areas of the Columbia River basin. The epidemiological significance of these findings provided insight into viral traffic patterns exhibited by IHNV in the Columbia River basin, with specific relevance to how the Columbia River basin IHNV types were related to those in the Hagerman Valley. These analyses indicate that there have likely been 2 historical events in which Hagerman Valley IHNV types were introduced and became established in the lower Columbia River basin. However, the data also clearly indicates that the Hagerman Valley is not a continuous source of waterborne virus infecting salmonid stocks downstream.

Diseases of Aquatic Organisms↗

Ticks without borders: Microbiome of immature neotropical tick species parasitizing migratory songbirds along northern Gulf of Mexico

Introduction: The long-distance, seasonal migrations of birds make them an effective ecological bridge for the movement of ticks. The introduction of exotic tick species to new geographical regions can cause the emergence of novel tick-borne pathogens. This study examined the prevalence of exotic tick species parasitizing migratory songbirds at stopover sites along the northern Gulf of Mexico using the mitochondrial 12S rRNA gene. Methods: Overall, 421 individual ticks in the genera Amblyomma , Haemaphysalis , and Ixodes were recorded from 28 songbird species, of which Amblyomma and Amblyomma longirostre were the most abundant tick genera and species, respectively. A high throughput 16S ribosomal RNA sequencing approach characterized the microbial communities and identified pathogenic microbes in all tick samples. Results and discussion: Microbial profiles showed that Proteobacteria was the most abundant phylum. The most abundant pathogens were Rickettsia and endosymbiont Francisella , Candidatus Midichloria , and Spiroplasma . Permutation multivariate analysis of variance revealed that the relative abundance of Francisella and Rickettsia drives microbial patterns across the tick genera. We also noted a higher percentage of positive correlations in microbe-microbe interactions among members of the microbial communities. Network analysis suggested a negative correlation between a) Francisella and Rickettsia and, b) Francisella and Cutibacterium . Lastly, mapping the distributions of bird species parasitized during spring migrations highlighted geographic hotspots where migratory songbirds could disperse ticks and their pathogens at stopover sites or upon arrival to their breeding grounds, the latter showing mean dispersal distances from 421–5003 kilometers. These findings spotlight the potential role of migratory birds in the epidemiology of tick-borne pathogens.

Alabama, Louisiana↗

Host vs. pathogen evolutionary arms race: Effects of exposure history on individual response to a genetically diverse pathogen

Introduction: Throughout their range, bighorn sheep ( Ovis canadensis ) populations have seen significant disease-associated declines. Unfortunately, understanding of the underlying epidemiological processes driving the disease dynamics in this species has hindered conservation efforts aimed at improving the health and long-term viability of these populations. Individual response to pathogen exposure emerges from dynamic interactions between competing evolutionary processes within the host and pathogen. The host’s adaptive immune system recognizes pathogens and mounts a defensive response. Pathogens have evolved strategies to overcome adaptive immune defenses including maintaining high genetic diversity through rapid evolution. The outcomes of this evolutionary warfare determine the success of pathogen invasion of the host and ultimately the success of conservation efforts. Methods: During an epizootic dominated by a single strain, we explore these host-pathogen dynamics by examining the variation in effects of pathogen invasion on captive bighorn sheep with differing histories of exposure to genetically diverse strains of Mycoplasma ovipneumoniae (Movi). We monitored clinical signs of disease and sampled animals and their environment to detect spread of Movi among 37 bighorn sheep separated into nine pens based on known exposure Results: We documented Movi transmission within and across pens and we detected Movi DNA in air, water, and invertebrate samples. Higher levels of antibody to Movi prior to the epizootic were associated with a lower likelihood of presenting clinical signs of pneumonia. Nonetheless, higher antibody levels in symptomatic individuals were associated with more severe progressive disease, increased probability and speed of pneumonia-induced mortality, and reduced likelihood of returning to a healthy state. Bighorn sheep with previous exposure to a strain other than the predominant epizootic strain were more likely to recover. Discussion: Our results indicate that Movi-strain variability was sufficient to overwhelm the adaptive host immunological defenses. This outcome indicates, in free-ranging herds, past exposure is likely insufficient to protect bighorn sheep from infection by new Movi strains, although it influences the progression of disease and recovery within the herd. Therefore, given Movi-strain variability and the lack of immunological protection from past exposure, focusing management efforts on minimizing the introduction of Movi into bighorn herds, through separation of domestic and bighorn sheep and avoidance of management activities that create commingling of bighorn sheep carrying differing Movi strains, will likely be the most effective approach for reducing the effects of disease and achieving bighorn sheep conservation goals.

Idaho, Oregon, South Dakota, Washington↗

A novel gonadotropic microsporidian parasite (Microsporidium clinchi n. sp.) infecting a declining population of pheasantshell mussels (Actinonaias pectorosa) (Unioinidae) from the Clinch River, USA

Freshwater mussels of the order Unionida are among the most endangered animal groups globally, but the causes of their population decline are often enigmatic, with little known about the role of disease. In 2018, we collected wild adult pheasantshell ( Actinonaias pectorosa ) and mucket ( Actinonaias ligamentina ) during an epidemiologic survey investigating an ongoing mussel mass mortality event in the Clinch River, Virginia and Tennessee, USA. Histopathology and transmission electron microscopy showed a novel microsporidian parasite primarily infecting the ovary of pheasantshell. Sequencing of the small subunit rRNA gene produced a 1333 bp sequence with the greatest similarity to Pseudonosema cristatellae (AF484694.1; 86.36%; e-value = 0), a microsporidium infecting the freshwater bryozoan ( Cristatella mucedo ). Microsporidia were observed in 65% (17/26) of the examined female pheasantshell ( A. pectorosa ) and in no (0/2) female muckets ( A. ligamentina ) and occurred at mortality and non-mortality sites. Our findings indicate that a novel parasite, Microsporidium clinchi n. sp., is present in pheasantshell in the Clinch River, and while likely not a cause of mass mortality, could reduce fecundity and recruitment in this declining population and threaten the success of reintroductions. Surveillance of M. clinchi n. sp. and evaluation of broodstock and their progeny for microsporidia would therefore be prudent.

Tennessee, Virginia↗

Hirsutonosema embarrassi n. gen. n. sp. (Phylum Microsporidia) in the Ovary of Mucket (Actinonaias ligamentina), Plain Pocketbook (Lampsilis cardium), and Fatmucket (Lampsilis siliquoidea) (Unionidae) from the Embarrass River, Wisconsin, USA

During an epidemiological survey following a mortality event of freshwater mussels in 2018 in the Embarrass River, Wisconsin, USA, we identified a novel microsporidian parasite in the ovaries of mucket ( Actinonaias ligamentina ), plain pocketbook ( Lampsilis cardium ), and fatmucket ( Lampsilis siliquoidea ) (Unionidae). Histopathology showed round-to-oval microsporidian spores in the cytoplasm of oocytes in 60% (3/5) of female mucket, 100% (4/4) of female plain pocketbook, and 50% (1/2) of female fatmucket. Using transmission electron microscopy, we found that mature spores were round-to-oval and measured 4.13 ± 0.64 µm (3.14–5.31) long by 2.88 ± 0.37 µm (2.36–3.68) wide. The spores had a thin electron-dense exospore with a spiky “hairy” coat, a thick electron lucent endospore, diplokaryotic nuclei, a polar vacuole, and 27–28 polar filaments arranged in 1–3 rows. Sequencing of the small subunit rRNA produced a 1356 bp sequence most similar to that of Pseudonosema cristatellae (92%), and phylogenetic analysis grouped it within the freshwater Neopereziida. Genetic, morphological, and ultrastructural characteristics did not closely match those of other Pseudonosema spp., and a new genus and species, Hirsutonosema embarrassi n. gen. n. sp., were designated. Additional studies could evaluate host susceptibility, distribution, seasonality, transmission, and lethal or sub-lethal effects of this parasite on freshwater mussels.

Wisconsin↗

Monkeypox virus in animals: Current knowledge of viral transmission and pathogenesis in wild animal reservoirs and captive animal models

Mpox, formerly called monkeypox, is now the most serious orthopoxvirus (OPXV) infection in humans. This zoonotic disease has been gradually re-emerging in humans with an increasing frequency of cases found in endemic areas, as well as an escalating frequency and size of epidemics outside of endemic areas in Africa. Currently, the largest known mpox epidemic is spreading throughout the world, with over 85,650 cases to date, mostly in Europe and North America. These increased endemic cases and epidemics are likely driven primarily by decreasing global immunity to OPXVs, along with other possible causes. The current unprecedented global outbreak of mpox has demonstrated higher numbers of human cases and greater human-to-human transmission than previously documented, necessitating an urgent need to better understand this disease in humans and animals. Monkeypox virus (MPXV) infections in animals, both naturally occurring and experimental, have provided critical information about the routes of transmission; the viral pathogenicity factors; the methods of control, such as vaccination and antivirals; the disease ecology in reservoir host species; and the conservation impacts on wildlife species. This review briefly described the epidemiology and transmission of MPXV between animals and humans and summarizes past studies on the ecology of MPXV in wild animals and experimental studies in captive animal models, with a focus on how animal infections have informed knowledge concerning various aspects of this pathogen. Knowledge gaps were highlighted in areas where future research, both in captive and free-ranging animals, could inform efforts to understand and control this disease in both humans and animals.

Viruses↗

Detection and genetic characterization of red-spotted grouper nervous necrosis virus and a novel genotype of nervous necrosis virus in black sea bass from the U.S. Atlantic coast

Nervous necrosis virus (NNV) causes a neurologic disease in a wide range of marine fish and poses serious disease risks to marine aquaculture worldwide. Little is known about the presence of NNV along the Atlantic coast of the United States, aside from the presence of barfin flounder nervous necrosis virus (BFNNV) in coldwater species in the northern part of this range. Herein we conducted surveillance for NNV from 2020 to 2022 in the mid-Atlantic region of the United States in black sea bass Centropristis striata , a serranid fish that is found throughout the eastern U.S. coast. Molecular detection methods have identified and characterized red-spotted grouper nervous necrosis virus (RGNNV) sequences at low prevalence throughout the years. Further, in 2022, a higher prevalence of a novel NNV genotype, tentatively named black sea bass nervous necrosis virus (BSBNNV), was characterized for the first time. Though virus isolation was unsuccessful, this study was the first to genetically identify NNV in this region and in this species. These findings highlight the need for further research on NNV to understand epidemiology and virulence in the context of marine fisheries and an emerging marine aquaculture industry in the United States.

New Jersey↗

Assessing monkeypox virus prevalence in small mammals at the human-animal interface in the Democratic Republic of the Congo

During 2012, 2013 and 2015, we collected small mammals within 25 km of the town of Boende in Tshuapa Province, the Democratic Republic of the Congo. The prevalence of monkeypox virus (MPXV) in this area is unknown; however, cases of human infection were previously confirmed near these collection sites. Samples were collected from 353 mammals (rodents, shrews, pangolins, elephant shrews, a potamogale, and a hyrax). Some rodents and shrews were captured from houses where human monkeypox cases have recently been identified, but most were trapped in forests and agricultural areas near villages. Real-time PCR and ELISA were used to assess evidence of MPXV infection and other Orthopoxvirus (OPXV) infections in these small mammals. Seven (2.0%) of these animal samples were found to be anti-orthopoxvirus immunoglobulin G (IgG) antibody positive (six rodents: two Funisciurus spp.; one Graphiurus lorraineus ; one Cricetomys emini ; one Heliosciurus sp.; one Oenomys hypoxanthus , and one elephant shrew Petrodromus tetradactylus ); no individuals were found positive in PCR-based assays. These results suggest that a variety of animals can be infected with OPXVs, and that epidemiology studies and educational campaigns should focus on animals that people are regularly contacting, including larger rodents used as protein sources.

Viruses↗

White-nose syndrome surveillance and bat monitoring activities in North Coast and Cascades Network parks 2016–2024

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome (WNS) in bats, has caused serious declines in bat populations across North America. We conducted WNS surveillance in five different park units in the North Coast and Cascades Network (NCCN) from 2016 to 2024, following the initial detection of Pd and WNS in Washington State in 2016. We captured and swabbed bats, swabbed roost materials, and collected guano and tested these samples for Pd DNA using qPCR. We confirmed WNS through histopathology of tissue samples and carcasses. We detected Pd at five locations in Mount Rainier National Park, starting in 2017. We confirmed WNS at four of these locations, with the first clinical signs detected in 2022. We detected Pd for the first time in Olympic and North Cascades National Parks in 2024. From these efforts, we generated information that can be compared to other datasets, helping us advance our knowledge of WNS/Pd epidemiology. We also conducted three field and laboratory-based experiments to inform early detection/rapid response (EDRR) planning. The first was a field experiment using non-infective Pd DNA to evaluate the rate of DNA degradation and the probability of detecting Pd DNA in the field. Experimental degradation rates for Pd DNA ranged from 1.6% to 8.2% and were lower in protected sites. The second was a laboratory-based experiment to understand Pd growth on four different substrates. We detected increasing levels of Pd in autoclaved guano and in plywood, suggesting these substrates may be environmental reservoirs. Pd remained stable in fresh guano but in soil it decreased, suggesting microbial interactions that may influence Pd growth in these substrates. We also collected wood shavings from a Pd positive bat box in June and August to evaluate viable Pd persistence in wood in a summer roost. Despite the characterization that Pd required cold conditions to persist, viable Pd was present in wood shavings collected during the summer season. Finally, we evaluated the National White-Nose Syndrome Decontamination Protocol through experiments. We found that ethanol was not effective as a sporicidal agent in any of the concentrations we tested and that a 1:10 dilution of bleach did not kill Pd spores, though higher concentrations did. These findings resulted in changes to the national protocol.

Oregon, Washington↗

Studies on avian malaria in vectors and hosts of encephalitis in Kern County, California. I. Infections in avian hosts

An epizoological study of Plasmodium infections in wild birds of Kern County, California, in the years 1946 through 1951 greatly extended knowledge of the occurrence of these parasites and their behavior in nature. Examination of 10,459 blood smears from 8,674 birds representing 73 species resulted in the observation of Plasmodium spp. in 1,094 smears representing 888 individual birds of 27 species. Seven species of Plasmodium were found: relictum, elongatum, hexamerium, nucleophilum, polare, rouxi and vaughani. Plasmodium relictum was by far the most frequently observed species, occurring in at least 79 per cent of the infected birds. Twelve new host species are recorded for this parasite. Sufficient morphological variation was observed to indicate that two strains of this species probably exist in nature. Numerous new host records were made of plasmodia with elongate gametocytes. The finding of parasites believed to be P. rouxi in two new host species represents the first record of the occurrence of this Plasmodium outside of Algeria. Multiple smears were obtained from a number of individual birds over varying time periods. Evidence of prolonged parasitemia was unusual, but some individuals had parasitemia on consecutive months and even for three successive years. In most individuals, parasitemias were of short duration. The inoculation of blood from wild birds into canaries led to the demonstration of many infections not observed on blood smear examination of donors. Use of these two complementary techniques led to more complete host records and a truer picture of the prevalence of infection. Three age classes of birds were studied--nestling, immature (less than 1 year of age) and adult. Parasites were observed in all three groups but infections in the younger individuals were most susceptible to interpretation. As to time of onset, numerous records were obtained of infection in nestling birds. Prevalence rates in immature birds after a single season's exposure ranged from 64 to 100 per cent in the house finch and 17 to 68 per cent in the English sparrow in different areas and years. Marked differences were found in the prevalence rates in different summer months, years and areas. It is believed these differences reflect variation in a number of environmental factors. This study indicates the extensive distribution of Plasmodium infection in a wide range of wild avian hosts. The observations are of possible importance in epidemiological studies of other arthropod-borne diseases such as the viral encephalitides for which these birds serve as hosts.

California↗

Guidelines for the field evaluation of desert tortoise health and disease

Field evaluation of free-ranging wildlife requires the systematic documentation of a variety of environmental conditions and individual parameters of health and disease, particularly in the case of rare or endangered species. In addition, defined criteria are needed for the humane salvage of ill or dying animals. The purpose of this paper is to describe, in detail, the preparation, procedures, and protocols we developed and tested for the field evaluation of wild desert tortoises ( Gopherus agassizii ). These guidelines describe: preparations for the field, including developing familiarity with tortoise behavior and ecology, and preparation of standardized data sheets; journal notes to document background data on weather conditions, temperature, rainfall, locality, and historic and recent human activities; procedures to prevent the spread of disease and parasites; data sheets for live tortoises to record tortoise identification, location, sex, body measurements and activity; health profile forms for documenting and grading physical abnormalities of tortoise posture and movements, general condition (e.g., lethargy, cachexia), external parasites, and clinical abnormalities associated with shell and upper respiratory diseases; permanent photographic records for the retrospective analysis of progression and regression of upper respiratory and eye diseases, analysis of shell lesions and evaluation of growth and age; and indications and methods for salvaging ill or dying tortoises for necropsy evaluation. These guidelines, tested on 5,000 to 20,000 tortoises over a 10 to 27 yr period, were designed to maximize acquisition of data for demographic, ecological, health and disease research projects; to reduce handling and stress of individual animals; to avoid spread of infectious disease; to promote high quality and consistent data sets; and to reduce the duration and number of field trips. The field methods are adapted for desert tortoise life cycle, behavior, anatomy, physiology, and pertinent disease; however the model is applicable to other species of reptiles. Comprehensive databases of clinical signs of disease and health are crucial to research endeavors and essential to decisions on captive release, epidemiology of disease, translocation of wild tortoises, breeding programs, and euthanasia.

Journal of Wildlife Diseases↗

Using amplified fragment length polymorphism analysis to differentiate isolates of Pasteurella multocida serotype 1

Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida , kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1 isolates cultured during a laboratory challenge study of Mallards ( Anas platyrhynchos ) and collected from wild birds and environmental samples during avian cholera outbreaks were characterized using amplified fragment length polymorphism (AFLP) analysis, a whole-genome DNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratory challenge demonstrated that P. multocida underwent genetic changes during a 3-mo period. Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmental samples revealed that isolates were distinguishable from one another based on regional and temporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocida isolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool to advance the study of avian cholera epidemiology. Further application of AFLP technology to the examination of wild bird avian cholera outbreaks may facilitate more effective management of this disease by providing the potential to investigate correlations between virulence and P. multocida genotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission.

California, Iowa, Missouri, Nebraska, Utah, Wiscon↗

Prevalence of antibodies to type A influenza virus in wild avian species using two serologic assays

Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

Alaska, California, Delaware, Georgia, Michigan, M↗

Avian influenza shedding patterns in waterfowl: implications for surveillance, environmental transmission, and disease spread

Despite the recognized importance of fecal/oral transmission of low pathogenic avian influenza (LPAI) via contaminated wetlands, little is known about the length, quantity, or route of AI virus shed by wild waterfowl. We used published laboratory challenge studies to evaluate the length and quantity of low pathogenic (LP) and highly pathogenic (HP) virus shed via oral and cloacal routes by AI-infected ducks and geese, and how these factors might influence AI epidemiology and virus detection. We used survival analysis to estimate the duration of infection (from virus inoculation to the last day virus was shed) and nonlinear models to evaluate temporal patterns in virus shedding. We found higher mean virus titer and longer median infectious period for LPAI-infected ducks (10–11.5 days in oral and cloacal swabs) than HPAI-infected ducks (5 days) and geese (7.5 days). Based on the median bird infectious dose, we found that environmental contamination is two times higher for LPAI- than HPAI-infectious ducks, which implies that susceptible birds may have a higher probability of infection during LPAI than HPAI outbreaks. Less environmental contamination during the course of infection and previously documented shorter environmental persistence for HPAI than LPAI suggest that the environment is a less favorable reservoir for HPAI. The longer infectious period, higher virus titers, and subclinical infections with LPAI viruses favor the spread of these viruses by migratory birds in comparison to HPAI. Given the lack of detection of HPAI viruses through worldwide surveillance, we suggest monitoring for AI should aim at improving our understanding of AI dynamics (in particular, the role of the environment and immunity) using long-term comprehensive live bird, serologic, and environmental sampling at targeted areas. Our findings on LPAI and HPAI shedding patterns over time provide essential information to parameterize environmental transmission and virus spread in predictive epizootiologic models of disease risks.

Journal of Wildlife Diseases↗