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Journal of Wildlife Diseases, 40(3), 2004, pp. 377-382
© Wildlife Disease Association  2004
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ARE WETLANDS THE RESERVOIR FOR AVIAN CHOLERA?

Michael D. Samuel1,2,3, Daniel J. Shadduck1 and Diana R. Goldberg1

1 US Geological Survey, Biological Resources Division, National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin 53711, USA
3 Corresponding author (email: mdsamuel{at}wisc.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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.

  Key words:  Avian cholera, disease reservoir, Pasteurella multocida, wetlands.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Avian cholera kills thousands of waterfowl annually in North American wetlands; however, the reservoir for the bacteria that causes the disease (Pasteurella multocida) remains uncertain (Botzler, 1991). Two important reservoirs—a reservoir is defined as a ‘‘place where the infective agent can survive on a year-round basis’’ (Botzler, 1991)—have been suggested as a source of avian cholera in waterfowl populations: carrier birds and epizootic wetland sites. Although neither of these hypotheses has been thoroughly or consistently investigated, several observations have contributed to the idea that soil or water at specific wetland areas may serve as the reservoir for this disease. First, although the disease has occurred in many areas, there has been a consistent pattern of recurrence of winter and spring avian cholera outbreaks in northern California (USA), the Rain-water Basin in Nebraska (USA), in Texas (USA) (Fig. 1Go), and in the prairies of Canada (Wobeser et al., 1979; Wobeser, 1992). In addition, a number of researchers have isolated P. multocida from wetlands where avian cholera outbreaks were occurring (Rosen and Bischoff, 1950; Rosen, 1969; Korschgen et al., 1978; Price and Brand, 1984; Backstrand and Botzler, 1986; Samuel et al., 2003), indicating that wetlands can be contaminated with bacteria during outbreaks. Second, several researchers have observed that P. multocida can survive for considerable time periods (e.g., weeks to >1 yr) in the laboratory (Bendheim and Evan-Shoshan, 1975; Awad et al., 1976; Bredy and Botzler, 1989; Price et al., 1992), providing the theoretical potential for long-term survival under favorable environmental conditions. And third, researchers have also found that survival of P. multocida in the laboratory can depend on the water or sediment characteristics (Rosen and Bischoff, 1950; Bredy and Botzler, 1989; Price et al., 1992) and that wetland water chemistry may be associated with outbreak areas (Windingstad et al., 1988), providing the possibility that differential survival of bacteria may occur among wetlands.



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  		FIGURE 1. Distribution of avian cholera epizootics reported in wild waterfowl (1944–97) in the United States and shown in shaded circles, which represent the relative magnitude of mortality events (National Wildlife Health Center, unpubl. data). Fall wetland sample locations 1995–98 shown with stars. All sampled wetlands had avian cholera outbreaks during the previous winter–spring. Each star may represent multiple wetlands and multiple years of sampling (see Table 1Go for details).

 
We investigated the hypothesis that wet-lands are the most likely reservoir for P. multocida and thus play an important role in the epizootiology of avian cholera. If wetlands serve as reservoirs for the bacterium that causes avian cholera, then P. multocida should be present in these wet-lands before migratory birds arrive. In addition, the patterns of consistent disease mortality in specific locations and isolation of P. multocida from wetlands with outbreaks imply that wetlands with recent mortality are a potential reservoir for the disease agent. Each fall, from 1995 to 1998, we sampled wetlands throughout the United States where avian cholera outbreaks were reported during the previous winter or spring. We collected sediment and water samples and attempted isolation of P. multocida from each wetland. Although we attempted to isolate all P. multocida serotypes, we were primarily concerned with serotype 1, which typically causes avian cholera mortality in the Pacific, Central, and Atlantic flyways (Brogden and Rhoades, 1983; Windingstad et al., 1983; Hirsh et al., 1990; Wilson et al., 1995). If wetlands are an important reservoir for avian cholera, we expected to isolate P. multocida during the fall, prior to use by migratory waterfowl.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
During 1995–98, wetlands where avian cholera mortality, confirmed by diagnostic pathology and culture, occurred were selected for sampling the next fall to determine whether P. multocida could be isolated from water or sediment. We selected wetlands where estimated mortality was at least 100 waterbirds because we believed that these losses increased the likelihood that P. multocida was present in the wet-land during the outbreak. We sampled wet-lands during September to November, depending on geographic location, prior to major in-fluxes of migratory waterfowl, which could also be a reservoir for the disease (Botzler, 1991; Samuel et al., 1999a, b).

We collected water and sediment samples for isolation of P. multocida using the methods described in Samuel et al. (2003). We used the cryopreservation method and quality assurance procedures described by Samuel et al. (2003) to preserve water and sediment samples prior to attempting isolation of P. multocida. In addition to water and sediment samples, we collected water samples for chemical, turbidity, and dissolved protein analyses and we measured other water quality characteristics (temperature, pH, redox potential, conductivity, and dissolved oxygen) using a Yellow Springs Instruments 610 DM water quality meter and 600 XL probe (Yellow Springs, Ohio, USA).

Following field collection, samples were transported to the US Geological Survey National Wildlife Health Center (NWHC), Madison, Wisconsin (USA), in liquid nitrogen vapor shippers. Cryovials containing the water and sediment samples were transferred to liquid nitrogen tanks for storage until they could be conveniently processed in the laboratory. Attempted isolation of P. multocida followed the methods described in Moore et al. (1998) and Samuel et al. (2003). Suspect bacterial colonies were identified by methods described in Samuel et al. (1997).

We calculated the proportion of water and sediment samples and the proportion of sampled wetlands that had detectable P. multocida. We also calculated the exact 95% confidence intervals on these prevalence data (Zar, 1984).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
During September–November of 1995–98, we sampled 44 wetlands in six different states in the United States (Table 1Go). Many of the sampled wetlands were distributed throughout enzootic avian cholera areas in the Klamath Basin, Central Valley, and San Joaquin Valley of California and the Rain-water Basin in Nebraska (Fig. 1Go). We also sampled wetlands in areas with less frequent occurrences of avian cholera, including Swan Lake National Wildlife Refuge (NWR) in Missouri, Lac Qui Parle Wildlife Management Area (WMA) in Minnesota, the eastern San Francisco Bay in California, Stillwater NWR in Nevada, and the panhandle of Texas (all sites in the United States). Reported avian cholera mortalities from our sampled wetlands varied from approximately 90 to >8,000 during the previous winter–spring period (Table 1Go).


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  		TABLE 1. Time period of sample collection, geographic location of wetland, and number of waterbirds reported dead during previous avian cholera outbreaks at wetlands sampled during the subsequent fall.
 
We were unable to isolate P. multocida from any of the water or sediment samples collected during our fall sampling. Thus, estimated prevalence was 0.0% for water, sediment, and wetlands. Assuming that each of the water or sediment samples we collected had independent probabilities of containing P. multocida, then the 95% confidence interval on the proportion of water or sediment samples with detectable concentrations of P. multocida was between 0.0% and 0.63%. Alternatively, the 95% confidence interval on the proportion of wetlands with detectable P. multocida was 0.0% to 6.2%.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Whether wetlands or waterbirds serve as a reservoir for avian cholera has been a controversial issue with important implications for understanding the epizootiology of this disease. In part, debate about the reservoir for the disease has persisted because of lack of consistent research investigating the proposed hypotheses. We were unable to recover P. multocida from any of the 440 water and 440 sediment samples we collected from the wetlands we sampled during the fall, prior to the return of migratory waterfowl populations.

We believe our research provides the strongest evidence to date that wetlands are not the primary reservoir for the P. multocida serotypes that cause avian cholera. However, we acknowledge some limitations of our sampling methods and the difficulty of proving the hypothesis that wetlands never serve as a reservoir for the disease. Nevertheless, we believe several lines of evidence support our conclusion. In previous laboratory and field studies (Moore et al., 1998) we found the preservation and culturing methods used in our investigations to be highly sensitive for detecting P. multocida at concentrations of two to 18 organisms per milliliter in wet-land water samples. This method was superior to the standard mouse inoculation method used for previous work (Moore et al., 1998). Concurrent to this study, we attempted to isolate P. multocida from wet-lands where avian cholera outbreaks occurred, using the same sampling and isolation procedures described in this study. During the winters of 1996–99, we recovered 51 P. multocida isolates (49 serotype 1) from 20 (46%) of the 44 wetlands with outbreaks. Pasteurella multocida was isolated from 31 (7%) of 440 water samples and from 20 (4.5%) of 440 sediment samples collected during or shortly following epizootic events. Representative wetland isolates were tested for virulence in Pekin ducks (four ducks per isolate), with most of the isolates being pathogenic (Samuel et al., 2003). In addition, 17 of the wet-lands we sampled during the fall were also sampled the previous spring during avian cholera outbreaks. During spring we recovered P. multocida serotype 1 from 41% (seven) of these wetlands, and from 10% (17) of the water and 2.4% (four) of the sediment samples, but none of the samples collected in the fall. For these samples collected in the fall, 95% confidence intervals indicated that <1% of the water and sediment samples and <7% of the epizootic wetlands we sample contained detectable bacteria. Thus, although we cannot prove that wetlands we sampled were completely free of P. multocida, our data do not support the hypothesis that wetlands are an important reservoir for avian cholera because the bacteria were not present in sufficient amounts or in a sufficient number of wetlands to ensure annual infection of migrating waterbirds. Based on serologic studies conducted on lesser snow geese (Chen caerulescens caerulescens), we have also found that many geese were infected with the P. multocida serotype 1 bacteria, but survived the infection (Samuel et al., 1999a, b). Further research has also confirmed that birds, especially snow geese, are carriers of pathogenic strains of P. multocida and that enzootic transmission occurs year round in these waterfowl (Samuel et al., unpubl. data).

A persistent issue in the epizootiology of avian cholera has been the identification of a reservoir for the disease agent. Ambiguity about whether birds or wetlands are the primary source of P. multocida has inhibited our understanding about factors such as transmission, carrier birds, and persistence of disease in waterfowl populations. Our study provides evidence that wetlands are not a likely reservoir for the bacteria, although wetlands likely play an important role in disease transmission. Alternatively, other studies give support to the hypothesis that carrier birds are an important reservoir for the disease. Future bird research should focus on their role in maintaining disease throughout the year and the role of environmental conditions and other stressors on the initiation of disease outbreaks. Research on wetlands should evaluate the role of wetlands in disease transmission and determine which factors influence the growth of P. multocida. Strategies for prevention and control of avian cholera outbreaks should consider that carrier birds are the most likely source of disease outbreaks and disease spread. Management actions that decrease potential disease transmission by separating carrier species from other species, reducing stress factors that may precipitate epizootic events, and reducing densities of waterfowl offer potential strategies to minimize the impact of avian cholera on waterfowl and other bird populations.


   ACKNOWLEDGMENTS
 
We are grateful to M. McCollum and S. Smith for assistance with fieldwork. Many state and federal wildlife biologists provided considerable logistic support and assistance to our study; in particular we thank J. Beckstrand, G. Mensik, G. Mack, G. Gerstenberg, and K. Sandie for their help. D. Johnson and M. Moore provided laboratory assistance. R. G. Botzler, G. Wobeser, C. J. Brand, and R. G. McLean provided many helpful suggestions for improvement of the manuscript.


   FOOTNOTES
 
2 Current address: US Geological Survey, Wisconsin Cooperative Wildlife Research Unit, Department of Wildlife Ecology, 204 Russell Lab, 1630 Linden Drive, University of Wisconsin, Madison, Wisconsin 53706, USA Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
AWAD, F. I., A. A. SALEM, AND A. A. FAYED. 1976. Studies on the viability of Pasteurella multocida type 1 under simulated environmental conditions in Egypt. Egyptian Journal of Veterinary Sciences 13: 57–69.

BACKSTRAND, J. M., AND R. G. BOTZLER. 1986. Survival of Pasteurella multocida in soil and water in an area where avian cholera is enzootic. Journal of Wildlife Diseases 22: 257–259.[Medline]

BENDHEIM, U., AND A. E. EVAN-SHOSHAN. 1975. Survival of Pasteurella multocida and Pasteurella anatipestifer in various natural media. Refuah Veterinarith 32: 40–46.

BOTZLER, R. G. 1991. Epizootiology of avian cholera in wildfowl. Journal of Wildlife Diseases 27: 367–395.[Abstract]

BREDY, J., AND R. G. BOTZLER. 1989. The effects of six environmental variables on the survival of Pasteurella multocida in water. Journal of Wildlife Diseases 25: 232–239.[Abstract]

BROGDEN, K. A., AND K. R. RHOADES. 1983. Prevalence of serologic types of Pasteurella multocida from 57 species of birds and mammals in the United States. Journal of Wildlife Diseases 19: 315–320.[Abstract]

HIRSH, D. C., D. A. JESSUP, K. P. SNIPES, T. E. CARPENTER, D. W. HIRD, AND R. H. MCCAPES. 1990. Characteristics of Pasteurella multocida isolated from waterfowl and associated avian species in California. Journal of Wildlife Diseases 26: 204–209.[Abstract]

KORSCHGEN, C. E., H. C. GIBBS, AND H. L. MENDALL. 1978. Avian cholera in eider ducks in Maine. Journal of Wildlife Diseases 14: 254–258.[Abstract]

MOORE, M. K., D. J. SHADDUCK, D. R. GOLDBERG, AND M. D. SAMUEL. 1998. A cryopreservation method for Pasteurella multocida from wetland samples. Journal of Wildlife Diseases 34: 182–185.[Abstract]

PRICE, J. I., AND C. J. BRAND. 1984. Persistence of Pasteurella multocida in Nebraska wetlands under epizootic conditions. Journal of Wildlife Diseases 20: 90–94.[Abstract]

———, B. S. YANDELL, AND W. P. PORTER. 1992. Chemical ions affect survival of avian cholera organisms in pondwater. Journal of Wildlife Management 56: 274–278.

ROSEN, M. N. 1969. Species susceptibility to avian cholera. Bulletin of the Wildlife Disease Association 5: 195–200.

———, AND A. I. BISCHOFF. 1950. The epidemiology of fowl cholera as it occurs in the wild. Transactions of the North American Wildlife Conference 15: 147–154.

SAMUEL, M. D., D. R. GOLDBERG, D. J. SHADDUCK, J. I. PRICE, AND E. G. COOCH. 1997. Pasteurella multocida serotype 1 isolated from a lesser snow goose: Evidence of a carrier state. Journal of Wildlife Diseases 33: 332–335.[Abstract]

———, D. J. SHADDUCK, D. R. GOLDBERG, V. BARANYUK, L. SILEO, AND J. I. PRICE. 1999a. Antibodies against Pasteurella multocida in snow geese in the western Arctic. Journal of Wildlife Diseases 35: 440–449.[Abstract]

———, J. Y. TAKEKAWA, G. SAMELIUS, AND D. R. GOLDBERG. 1999b. Avian cholera mortality in lesser snow geese nesting on Banks Island, Northwest Territories. Wildlife Society Bulletin 27: 780–787.

———, D. J. SHADDUCK, D. R. GOLDBERG, M. A. WILSON, D. O. JOLY, AND M. A. LEHR. 2003. Characterization of Pasteurella multocida isolates from wetland ecosystems during 1996 to 1999. Journal of Wildlife Diseases 39: 798–807.[Abstract]

WILSON, M. A., R. M. DUNCAN, G. E. NORDHOLM, AND B. M. BERLOWSKI. 1995. Pasteurella multocida isolated from wild birds of North America: A serotype and DNA fingerprint study of isolates from 1978 to 1993. Avian Diseases 39: 587–593.[Medline]

WINDINGSTAD, R. M., R. M. DUNCAN, AND D. THORNBURG. 1983. Outbreak of avian cholera on the wintering grounds of the Mississippi Valley Canada goose flock. Journal of Wildlife Diseases 19: 95–97.[Abstract]

———, S. M. KERR, R. M. DUNCAN, AND C. J. BRAND. 1988. Characterization of an avian cholera epizootic in wild birds in western Nebraska. Avian Diseases 32: 124–131.[Medline]

WOBESER, G., B. HUNTER, B. WRIGHT, D. J. NIEMAN, AND R. ISBISTER. 1979. Avian cholera in Saskatchewan, spring 1997. Journal of Wildlife Diseases 15: 19–24.[Abstract]

———. 1992. Avian cholera and waterfowl biology. Journal of Wildlife Diseases 28: 674–682.[Medline]

ZAR, J. H. 1984. Biostatistical analysis, 2nd Edition. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, pp. 378–379.

Received for publication 8 August 2003.



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