Correlations of daily activity with avian cholera mortality among wildfowl.

We tested the hypothesis that wildfowl activities can influence the risk of avian cholera (Pasteurella multocida infection) for susceptible birds at Centerville, Humboldt County, California (USA). Avian cholera mortality characteristics from past epizootics were correlated with variations in flock size, habitat use and 11 feeding and nonfeeding behaviors among six empirically defined groups of wildfowl: American coots (Fulica americana), tundra swans (Cygnus columbianus), American wigeon (Anas americana), northern pintails (A. acuta), northern shovelers (A. clypeata)/mallards (A. platyrhynchos), and teal (A. discors, A. crecca, A. cyanoptera). The position of these wildfowl groups in past mortality sequences was directly correlated with mean flock size, time spent on land, and time spent grazing on land or in shallow water. We propose that variations in bird density, habitat use and frequency of grazing may serve as predisposing factors to avian cholera among wildfowl.     

Antibodies against Pasteurella multocida in snow geese in the western Arctic.

To determine if lesser snow geese (Chen caerulescens caerulescens) are a potential reservoir for the Pasteurella multocida bacterium that causes avian cholera, serum samples and/or pharyngeal swabs were collected from > 3,400 adult geese breeding on Wrangel Island (Russia) and Banks Island (Canada) during 1993-1996. Pharyngeal swab sampling rarely (> 0.1%) detected birds that were exposed to P. multocida in these populations. Geese with serum antibody levels indicating recent infection with P. multocida were found at both breeding colonies. Prevalence of seropositive birds was 3.5% at Wrangel Island, an area that has no recorded history of avian cholera epizootics. Prevalence of seropositive birds was 2.8% at Banks Island in 1994, but increased to 8.2% during 1995 and 1996 when an estimated 40,000-60,000 snow geese were infected. Approximately 50% of the infected birds died during the epizootic and a portion of the surviving birds may have become carriers of the disease. This pattern of prevalence indicated that enzootic levels of infection with P. multocida occurred at both breeding colonies. When no avian cholera epizootics occurred (Wrangel Island, Banks Island in 1994), female snow geese (4.7%) had higher antibody prevalence than males (2.0%).     

Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.     

Epizootiology of avian cholera in wildfowl

Pasteurella multocida, the cause of avian cholera, has naturally infected over 100 species of free-living birds. Among wild birds, avian cholera has its greatest impact on North American wildfowl. Epizootics usually are explosive in onset and may involve thousands of birds. The disease has been reported in every month of the year among wildfowl. Disproportionate mortality, with some species suffering proportionately greater mortality than others, has been a common feature of this disease. Presence of animal organic matter plays a significant role in the survival of P. multocida. There are conflicting reports or a lack of information on the role of host sex, age, body size, other physical features, genetic variation or behavioral differences, as predisposing factors to infection by P. multocida. There also are ambiguities on the relationship between season, precipitation, temperature, nutritional stress, water quality, other microorganisms, and environmental contaminants, and the occurrence of avian cholera in wildfowl. Two competing hypotheses for the year-round reservoir of wildfowl strains of P. multocida are ambient soil or water of enzootic sites, and carrier animals; most current evidence favors the role of carrier animals. Transmission most likely occurs by ingestion of contaminated water, inhalation of bacteria-rich aerosols, or both. While many techniques have been proposed to prevent or control avian cholera, none have been rigorously tested to determine their effectiveness.     

Are wetlands the reservoir for avian cholera?

Wetlands have long been suspected to be an important reservoir for Pasteurella multocida and therefore the likely source of avian cholera outbreaks. During the fall of 1995-98 we collected sediment and water samples from 44 wetlands where avian cholera epizootics occurred the previous winter or spring. We attempted to isolate P. multocida in sediment and surface water samples from 10 locations distributed throughout each wetland. We were not able to isolate P. multocida from any of the 440 water and 440 sediment samples collected from these wetlands. In contrast, during other investigations of avian cholera we isolated P. multocida from 20 of 44 wetlands, including 7% of the water and 4.5% of the sediment samples collected during or shortly following epizootic events. Our results indicate that wetlands are an unlikely reservoir for the bacteria that causes avian cholera.     

Avian cholera in waterfowl: the role of lesser snow and ross's geese as disease carriers in the Playa Lakes Region

We collected samples from apparently healthy geese in the Playa Lakes Region (USA) during the winters of 2000-01 and 2001-02 to determine whether carriers of Pasteurella multocida, the bacterium that causes avian cholera, were present in wild populations. With the use of methods developed in laboratory challenge trials (Samuel et al., 2003a) and a serotype-specific polymerase chain reaction method for identification of P. multocida serotype 1, we found that a small proportion of 322 wild birds (<5%) were carriers of pathogenic P. multocida. On the basis of serology, an additional group of these birds (<10%) were survivors of recent avian cholera infection. Our results confirm the hypothesis that wild waterfowl are carriers of avian cholera and add support for the hypothesis that wild birds are a reservoir for this disease. In concert with other research, this work indicates that enzootic infection with avian cholera occurs in lesser snow goose (Chen caerulescens caerulescens) populations throughout their annual cycle. Although fewer Ross's geese (Chen rossii) were sampled, we also found these birds were carriers of P. multocida. Even in the absence of disease outbreaks, serologic evidence indicates that chronic disease transmission and recent infection are apparently occurring year-round in these highly gregarious birds and that a small portion of these populations are potential carriers with active infection.     

Associations between water quality, Pasteurella multocida, and avian cholera at Sacramento National Wildlife Refuge

We studied patterns in avian cholera mortality, the presence of Pasteurella multocida in the water or sediment, and water chemistry characteristics in 10 wetlands at the Sacramento National Wildlife Refuge Complex (California, USA), an area of recurrent avian cholera epizootics, during the winters of 1997 and 1998. Avian cholera outbreaks (>or=50 dead birds) occurred on two wetlands during the winter of 1997, but no P. multocida were recovered from 390 water and 390 sediment samples from any of the 10 wetlands. No mortality events were observed on study wetlands during the winter of 1998; however, P. multocida was recovered from water and sediment samples in six of the 10 study wetlands. The pH levels were higher for wetlands experiencing outbreaks during the winter of 1997 than for nonoutbreak wetlands, and aluminum concentrations were higher in wetlands from which P. multocida were recovered during the winter of 1998. Water chemistry parameters (calcium, magnesium, sodium, and dissolved protein) previously linked with P. multocida and avian cholera mortality were not associated with the occurrence of avian cholera outbreaks or the presence of P. multocida in our study wetlands. Overall, we found no evidence to support the hypothesis that wetland characteristics facilitate the presence of P. multocida and, thereby, allow some wetlands to serve as long-term sources (reservoirs) for P. multocida.     

Outbreaks of avian cholera in Hope Bay, Antarctica

During austral summers 1999-2000 and 2000-01, two outbreaks of avian cholera occurred in the Hope Bay area (63 degrees 24'S, 56 degrees 59'W), located on the tip of the Antarctic Peninsula. Eighty-six dead birds were found: five kelp gulls (Larus dominicanus), 36 skuas (Stercorarius sp.), and 45 Adelie penguins (Pygoscelis adeliae). The carcasses were studied using clinical, pathological, and microbiological criteria. Water samples from ponds where birds were settled and samples from 90 healthy birds also were analyzed during the second outbreak. Pasteurella multocida isolates were identified by biochemical tests, capsular type, somatic serotype, and susceptibility to nine antibiotics. Molecular subtyping was performed by ApaI and SmaI pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC-PCR). In February 2000, mortality in skuas was 16% and 2% in kelp gulls. In the 2000-01 breeding season, mortality in south polar skuas was 47%, 24% in brown skuas, 1.4% in kelp gulls, and 0.01% in Adelie penguins. All birds had lesions of avian cholera. In kelp gulls the presentation was chronic, whereas skuas and penguins suffered subacute and acute disease, respectively. Fifty-five isolates recovered from dead birds and one from water were identified as P. multocida gallicida, type A:1. The strains presented a unique molecular pattern by PFGE and ERIC-PCR. A possible hypothesis to explain the origin of the outbreaks was that nonbreeder kelp gulls carried P. multocida gallicida to Hope Bay, and avian cholera was transmitted through water to skuas and penguins. This study reports avian cholera in new bird species, their potential role in the transmission of the disease, and the different responses of these species to the disease.     

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.     

Attempts to identify the source of avian vacuolar myelinopathy for waterbirds

Attempts were made to reproduce avian vacuolar myelinopathy (AVM) in a number of test animals in order to determine the source of the causative agent for birds and to find a suitable animal model for future studies. Submerged vegetation, plankton, invertebrates, forage fish, and sediments were collected from three lakes with ongoing outbreaks of AVM and fed to American coots (Fulica americana), mallard ducks and ducklings (Anas platyrhynchos), quail (Coturnix japonica), and laboratory mice either via gavage or ad libitum. Tissues from AVM-affected coots with brain lesions were fed to ducklings, kestrels (Falco sparverius), and American crows (Corvus brachyrhynchos). Two mallards that ingested one sample of Hydrilla verticillata along with any biotic or abiotic material associated with its external surface developed brain lesions consistent with AVM, although neither of the ducks had clinical signs of disease. Ingestion of numerous other samples of Hydrilla from the AVM affected lakes and a lake with no prior history of AVM, other materials (sediments, algae, fish, invertebrates, and water from affected lakes), or tissues from AVM-affected birds did not produce either clinical signs or brain lesions in any of the other test animals in our studies. These results suggest that waterbirds are most likely exposed to the causative agent of AVM while feeding on aquatic vegetation, but we do not believe the vegetation itself is the agent. We hypothesize that the causative agent of AVM might either be accumulated by aquatic vegetation, such as Hydrilla, or associated with biotic or abiotic material on its external surfaces. In support of that hypothesis, two coots that ingested Hydrilla sampled from a lake with an ongoing AVM outbreak in wild birds developed neurologic signs within 9 days (ataxia, limb weakness, and incoordination), and one of two coots that ingested Hydrilla collected from the same site 13 days later became sick and died within 38 days. None of these three sick coots had definitive brain lesions consistent with AVM by light microscopy, but they had no gross or histologic lesions in other tissues. It is unclear if these birds died of AVM. Perhaps they did not ingest a dose sufficient to produce brain lesions or the lesions were ultrastructural. Alternatively, it is possible that a separate neurotoxic agent is responsible for the morbidity and mortality observed in these coots.     

Avian botulism during late autumn and early spring in Saskatchewan

Two outbreaks of botulism in central Saskatchewan in which mortality of waterfowl continued into late autumn and then recurred in the same marshes the following spring are described. Small numbers of birds were involved in each instance. Dabbling ducks (predominantly mallards, Anas platyrhynchos and pintails, Anas acuta) and American coots, Fulica americana were affected most commonly in autumn, whereas only diving ducks (predominantly lesser scaup, Aythya affinis) were found to be involved in spring. Live maggots present in carcasses despite sub-freezing temperatures were the probable source of intoxication in the autumn; the source of toxin in the spring was not determined.     

Avian cholera exposure and carriers in greater white-fronted geese breeding in Alaska, USA

We conducted a 3-yr study (2001-03) on greater white-fronted geese (Anser albifrons frontalis) breeding in Alaska, USA, to determine the exposure of this population to Pasteurella multocida and the potential role of these birds as disease carriers. We tested sera from nearly 600 adult geese for antibodies to P. multocida serotype 1. We found a low prevalence (<5%) of positive antibodies in adult geese, and based on the short duration of detectable antibodies, these findings indicate recent infection with P. multocida. Prevalence was similar to serologic results from both breeding and wintering lesser snow geese. We also collected oral (n=1,035), nasal (n=102), and cloacal (n=90) swab samples to determine the presence of avian cholera carriers in this population. We were unable to isolate P. multocida serotype 1 from any of the birds sampled. Based on comparison with other waterfowl species, we concluded that these geese may be exposed to avian cholera during the winter or spring migration but are unlikely to play a significant role as carriers of the bacterium causing avian cholera.     

Avian disease at the Salton Sea

A review of existing records and the scientific literature was conducted for occurrences of avian diseases affecting free-ranging avifauna within the Salton Sea ecosystem. The period for evaluation was 1907 through 1999. Records of the U.S. Department of Agriculture, Bureau of Biological Survey and the scientific literature were the data sources for the period of 1907–1939. The narrative reports of the U.S. Fish and Wildlife Service's Sonny Bono National Wildlife Refuge Complex and the epizootic database of the U.S. Geological Survey's National Wildlife Health Center were the primary data sources for the remainder of the evaluation. The pattern of avian disease at the Salton Sea has changed greatly over time. Relative to past decades, there was a greater frequency of major outbreaks of avian disease at the Salton Sea during the 1990s than in previous decades, a greater variety of disease agents causing epizootics, and apparent chronic increases in the attrition of birds from disease. Avian mortality was high for about a decade beginning during the mid-1920s, diminished substantially by the 1940s and was at low to moderate levels until the 1990s when it reached the highest levels reported. Avian botulism (Clostridium botulinum type C) was the only major cause of avian disease until 1979 when the first major epizootic of avian cholera (Pasteurella multocidia) was documented. Waterfowl and shorebirds were the primary species affected by avian botulism. A broader spectrum of species have been killed by avian cholera but waterfowl have suffered the greatest losses. Avian cholera reappeared in 1983 and has joined avian botulism as a recurring cause of avian mortality. In 1989, avian salmonellosis (Salmonella typhimurium) was first diagnosed as a major cause of avian disease within the Salton Sea ecosystem and has since reappeared several times, primarily among cattle egrets (Bubulcus ibis). The largest loss from a single epizootic occurred in 1992, when an estimated 155000 birds, primarily eared grebes (Podiceps nigricollis), died from an undiagnosed cause. Reoccurrences of that unknown malady have continued to kill substantial numbers of eared grebes throughout the 1990s. The first major epizootic of type C avian botulism in fish-eating birds occurred in 1996 and killed large numbers of pelicans (Pelecanus occidentalis & P. erythrorhynchos). Avian botulism has remained as a major annual cause of disease in pelicans. In contrast, the chronic on-Sea occurrence of avian botulism in waterfowl and shorebirds of previous decades was seldom seen during the 1990s. Newcastle disease became the first viral disease to cause major bird losses at the Salton Sea when it appeared in the Mullet Island cormorant (Phalacrocorax auritus) breeding colony during 1997 and again during 1998.     

Improving the reliability of molecular sexing of birds using a W-specific marker

Molecular techniques for identifying sex of birds utilize length differences between CHD-Z and CHD-W introns, but in some cases these methods can lead to sexing errors. Here we show that an additional W-specific primer can be used in conjunction with a pre-existing sexing primer pair to dramatically improve the reliability of molecular sexing methods. We illustrate the approach with American coots (Fulica americana), a species with CHD-Z polymorphism that could not be accurately sexed using traditional methods. We developed a reverse primer GWR2 designed to sit within the intron of the W chromosome and amplify a distinctively small DNA fragment that serves as a W-specific marker. Analysis of known-sex individuals indicates that this W-specific primer provides an efficient and reliable protocol to identify the sex of F. americana. The development of such sex-specific primers will likely increase the reliability of molecular sexing methods in other birds as well. Comparisons between CHD-Z alleles of coots and common moorhens (Gallinula chloropus) revealed that CHD-Z polymorphism evolved separately in these two closely related species. We discuss the implications of repeated evolution of CHD-Z polymorphisms among birds.     

Experimental vacuolar myelinopathy in red-tailed hawks

Avian vacuolar myelinopathy (AVM) was recognized in 1994 as a cause of wild bird mortality when 29 bald eagles (Haliaeetus leucocephalus) succumbed to the disease at DeGray Lake, Arkansas (USA). The cause of AVM and its source remain undetermined despite extensive diagnostic and research investigations. Two years later, when AVM killed 26 eagles in the same area in Arkansas, it became apparent that American coots (Fulica americana) had identical neurologic signs and lesions, and it was hypothesized that eagles acquired AVM via ingestion of affected coots. In order to test this hypothesis, we fed coot tissues (brain, liver, kidney, muscle, fat, and intestinal tract) to rehabilitated, non-releasable red-tailed hawks (Buteo jamaicensis). Five hawks received tissues from coots with AVM lesions, and one hawk received tissues from coots without brain lesions that had been collected at a site where AVM never has been documented. All hawks received 12-70 g/day (mean = 38 g) of coot tissues for 28 days. All six hawks remained clinically normal during the study. The birds were euthanatized on day 29 and microscopic lesions of AVM were found in all hawks that received tissues from affected coots, but not in the hawk that received tissues from unaffected coots. This marks the first time that AVM has been produced in birds under laboratory conditions and proves that birds of prey can acquire AVM via ingestion of tissues from affected coots.     

Pasteurella multocida from outbreaks of avian cholera in wild and captive birds in Denmark

An outbreak of avian cholera was observed among wild birds in a few localities in Denmark in 2001. The highest mortalities were among breeding eiders (Somateria mollissima) and gulls (Larus spp.). Pulsed-field gel electrophoresis (PFGE) was conducted using ApaI and SmaI as restriction enzymes and restriction enzyme analysis (REA) using HpaII. The Pasteurella multocida subsp. multocida strain isolated from birds in this outbreak was indistinguishable from a strain that caused outbreaks in 1996 and 2003. Most isolates from domestic poultry had other PFGE patterns but some were indistinguishable from the outbreak strain. Among 68 isolates from wild birds, only one PFGE and one REA pattern were demonstrated, whereas among 23 isolates from domestic poultry, 14 different SmaI, 12 different ApaI, and 10 different HpaII patterns were found. The results suggest that a P. multocida strain has survived during several years among wild birds in Denmark.     

Epizootiologic studies of avian vacuolar myelinopathy in waterbirds

Epizootic avian vacuolar myelinopathy (AVM) was first recognized as a neurologic disease in bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana) in Arkansas, USA in 1994 and 1996, respectively, but attempts to identify the etiology of the disease have been unsuccessful to date. Between 1998 and 2001, wing clipped sentinel birds (wild American coots and game farm mallards [Anas platyrhynchos]) were released at Lake Surf, North Carolina, a lake with recurrent outbreaks of AVM, in order to gain a better understanding of the epizootiology of the disease. As early as 5-7 days post-release, sentinel coots and mallards showed neurologic signs of disease and were confirmed with AVM upon histologic examination of their brains. Serial releases of sentinel mallards during the summer, fall, and winter of 2000-01 demonstrated that exposure to the causative agent at a threshold sufficient to manifest disease was seasonal and occurred over about a 2 mo period, during November and December. Our findings that disease onset can be very rapid (5-7 days) and that exposure to the causative agent of AVM is site-specific, seasonal (late fall to early winter), and occurs over a relatively short duration (several months) supports the hypothesis that the disease is caused by a chemical substance, most likely of natural origin.     

Avian cholera in eider ducks in Maine

Outbreaks of avian cholera (Pasteurella multocida) occur frequently in common eiders (Somateria mollissima dresseri) in Maine during early summer. Studies over a seven year period show that over 90% of the loss occurred in incubating females and might be associated with their weakened condition because females do not feed during the incubation period. High nesting densities also may contribute to the losses. The exact source of P. multocida is unknown although carrier birds were found.     

Description of the oocysts of Eimeria paludosa (Apicomplexa: Eimeriidae) from Fulica americana (Aves: Gruiformes), with comments on synonyms of eimerian species from related birds

Between November and December 1988, fecal and intestinal contents were collected from 25 northern American coots, Fulica americana americana, in Arkansas and Texas, and examined for coccidial parasites. Seventeen (68%) of the coots were infected with Eimeria paludosa, herein described; for the first time, photomicrographs of the species are presented. Sporulated oocysts are ovoid, 16.5 x 12.6 (15-23 x 11-14) microns, with a lightly to heavily pitted single-layered wall; an oocyst residuum is absent, but a prominent micropyle is present. A large, or several smaller, polar granule(s) is present, usually located beneath the micropyle. Sporocysts are elongate-ovoid, 10.8 x 6.2 (10-12 x 5-7) microns, with Stieda and substieda bodies. A sporocyst residuum is present, normally composed of very fine faint granules scattered among the sporozoites or, rarely, as a spherical mass. Sporozoites are elongate, 8.7 x 2.7 (7-11 x 2-3) microns, in situ. Each sporozoite contains a spherical-ellipsoid posterior refractile body and occasionally a spherical anterior refractile body. A nucleus is located immediately anterior to the posterior refractile body. The occurrence of E. paludosa in F. a. americana is a new host and geographic record for the parasite. In addition, several of the previously described eimerian species from gruiform birds are proposed to be synonyms of E. paludosa.     

Comparison of methods to detect Pasteurella multocida in carrier waterfowl

We conducted laboratory challenge trials using mallard ducks (Anas platyrhynchos) to compare methods for detecting carriers of Pasteurella multocida, the bacterium that causes avian cholera, in wild birds. Birds that survived the initial infection were euthanized at 2-4 wk intervals up to 14 wk post challenge. Isolates of P. multocida were obtained at necropsy from 23% of the birds that survived initial infection. We found that swab samples (oral, cloacal, nasal, eye, and leg joint) were most effective for detecting carrier birds up to 14 wk post infection. No detectable differences in isolation were observed for samples stored in either 10% dimethysulfoxide or brain heart infusion broth. The frequency of detecting carriers in our challenge trials appeared to be related to mortality rates observed during the trial, but was not related to a number of other factors including time after challenge, time delays in collecting tissues postmortem, and route of infection. In our trials, there was little association between antibody levels and carrier status. We concluded that swabs samples collected from recently dead birds, stored in liquid nitrogen, and processed using selective broth provide a feasible field method for detecting P. multocida carriers in wild waterfowl.