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.     

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.     

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.     

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.     

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.     

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%).     

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.     

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.     

Characterization of Pasteurella multocida isolates from wetland ecosystems during 1996 to 1999

We cultured 126 Pasteurella multocida isolates, 92 from water and 34 from sediment samples collected from wetlands in the Pacific and Central flyways of the United States between 1996 and 1999. Most (121) of the isolates were P. multocida serotype 1, but serotypes 3, 3/4, 10, and 11 were also found. Many (82) of the isolates were further characterized by DNA fingerprinting procedures and tested in Pekin ducks for virulence. Almost all the serotype 1 isolates we tested caused mortality in Pekin ducks. Serotype 1 isolates varied in virulence, but the most consistent pattern was higher mortality in male ducks than in females. We found no evidence that isolates found in sediment vs. water, between Pacific and Central flyways, or during El Ni?o years had consistently different virulence. We also found a number of non-serotype 1 isolates that were avirulent in Pekin ducks. Isolates had DNA fingerprint profiles similar to those found in birds that died during avian cholera outbreaks.     

Multi-species patterns of avian cholera mortality in Nebraska's Rainwater Basin

Nebraska's Rainwater Basin (RWB) is a key spring migration area for millions of waterfowl and other avian species. Avian cholera has been endemic in the RWB since the 1970s and in some years tens of thousands of waterfowl have died from the disease. We evaluated patterns of avian cholera mortality in waterfowl species using the RWB during the last quarter of the 20th century. Mortality patterns changed between the years before (1976-1988) and coincident with (1989-1999) the dramatic increases in lesser snow goose abundance and mortality. Lesser snow geese (Chen caerulescens caerulescens) have commonly been associated with mortality events in the RWB and are known to carry virulent strains of Pasteurella multocida, the agent causing avian cholera. Lesser snow geese appeared to be the species most affected by avian cholera during 1989-1999; however, mortality in several other waterfowl species was positively correlated with lesser snow goose mortality. Coincident with increased lesser snow goose mortality, spring avian cholera outbreaks were detected earlier and ended earlier compared to 1976-1988. Dense concentrations of lesser snow geese may facilitate intraspecific disease transmission through bird-to-bird contact and wetland contamination. Rates of interspecific avian cholera transmission within the waterfowl community, however, are difficult to determine.     

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.     

Recovery and identification of Pasteurella multocida from mammals and fleas collected during plague investigations

During the 12-yr period, 1973-1984, 243 isolates of Pasteurella multocida were recovered or identified from specimens submitted for plague tests. Of the isolates, 79% were from rodents, 10% from lagomorphs, and 7% from carnivores; eight isolates were recovered from pools of rodent or carnivore fleas, and two were recovered from cat-bite wounds of human patients. No correlations of host or geographic sources, season, or biotypic or serotypic characteristics were found. Of the rodent strains serotyped, most were found to be serotypes 1A or 3A, which suggests a possible epizootiologic role for rodents in outbreaks of avian cholera that commonly involve these serotypes.     

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.     

Outer membrane protein H for protective immunity against Pasteurella multocida

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.     

Avian cholera in a southern giant petrel (Macronectes giganteus) from Antarctica

A southern giant petrel (Macronectes giganteus) was found dead at Potter Peninsula, King George Island, South Shetland, Antarctica. The adult male was discovered approximately 48 hr after death. Macroscopic and microscopic lesions were compatible with avian cholera and the bacterium Pasteurella multocida subsp. gallicida, serotype A1 was isolated from lung, heart, liver, pericardial sac, and air sacs. In addition, Escherichia coli was isolated from pericardial sac and air sacs. This is the first known report of avian cholera in a southern giant petrel in Antarctica.     

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.     

Serological types of Pasteurella multocida isolated from turkeys and chickens in Canada

Outbreaks of fowl cholera continue to plague the Canadian poultry industry despite widespread immunization against the causative agent, Pasteurella multocida. Fowl cholera bacterins currently employed by domestic poultry growers contain three serological types, namely, serotypes 1, 3, and 4. In this study a total of 84 strains of P. multocida were isolated in Canada from outbreaks of fowl cholera in turkeys and chickens. Serotyping was accomplished using the gel diffusion precipitin test. Based on the gel diffusion precipitation patterns, 27 serotypes containing one to six antigenic determinants were recognized. The most prevalent serotype both in turkeys and chickens appeared to be type 3. Significantly, greater than 20% of P. multocida isolates failed to react with antisera raised against serotypes 1, 3, and 4.     

Feather corticosterone reveals effect of moulting conditions in the autumn on subsequent reproductive output and survival in an Arctic migratory bird

For birds, unpredictable environments during the energetically stressful times of moulting and breeding are expected to have negative fitness effects. Detecting those effects however, might be difficult if individuals modulate their physiology and/or behaviours in ways to minimize short-term fitness costs. Corticosterone in feathers (CORTf) is thought to provide information on total baseline and stress-induced CORT levels at moulting and is an integrated measure of hypothalamic–pituitary–adrenal activity during the time feathers are grown. We predicted that CORTf levels in northern common eider females would relate to subsequent body condition, reproductive success and survival, in a population of eiders nesting in the eastern Canadian Arctic during a capricious period marked by annual avian cholera outbreaks. We collected CORTf data from feathers grown during previous moult in autumn and data on phenology of subsequent reproduction and survival for 242 eider females over 5 years. Using path analyses, we detected a direct relationship between CORTf and arrival date and body condition the following year. CORTf also had negative indirect relationships with both eider reproductive success and survival of eiders during an avian cholera outbreak. This indirect effect was dramatic with a reduction of approximately 30% in subsequent survival of eiders during an avian cholera outbreak when mean CORTf increased by 1 standard deviation. This study highlights the importance of events or processes occurring during moult on subsequent expression of life-history traits and relation to individual fitness, and shows that information from non-destructive sampling of individuals can track carry-over effects across seasons.     

Epizootiological features of avian cholera on the north coast of California

An avian cholera (Pasteurella multocida) epizootic was observed among wildfowl at the Centerville Gun Club, Humboldt County, California (USA) in January 1978. Compared to their live populations and use of the area, coots (Fulica americana) died in proportionately greater numbers than any other species. Coots collected by gunshot were evaluated for sex and age composition, and morphometry from November 1977 through mid-January 1978 at this site. There was no substantial difference in the sex, age or morphometry between birds dying of avian cholera and from those dying from gunshot. Assuming coots dying of gunshot are representative of the general population, it appears there was little selection among coots by P. multocida. There was evidence for a sequential mortality similar to that reported previously at this site: coots were the first birds to die, followed by American wigeon (Anas americana) and northern pintails (A. acuta acuta); northern shovelers (A. clypeata) and mallards (A. platyrhynchos) died late in the epizootic.     

Marine ecosystems and cholera

Historically, most of the major epidemics or outbreaks of cholera around the world have originated in coastal regions. The most dramatic of recent outbreaks of cholera occurred in India and Bangladesh in 1991, followed by an outbreak of cholera after almost a century without cholera in South America in 1991. Both of these recent epidemics were reported first in the coastal regions of India and Peru, respectively. Cholera epidemics are seasonal, occurring during the spring and fall months. Outbreaks of cholera in noncholera epidemic areas have been ascribed to travel and shipping activities, but there is compelling evidence that V. cholerae always is present in the aquatic environment and proliferates under nonepidemic conditions while attached to, or associated with, eucaryotic organisms. It is hypothesized that climate directly influences the incidence and geographic distribution of the cholera bacterium.