Introduction

Farming of the American alligator (Alligator mississippiensis) is a major component of the aquaculture industry in Florida, Louisiana and other southeastern states (4). Captive breeding has gained economic significance for alligator farmers due to the expense and often limited number of hatchlings available from the wild. In order for the farmer to successfully and competitively breed alligators, attention to proper husbandry practices including diet, temperature, stocking densities and disease control is required. In this report we describe the isolation of four species of fungi from the egg membranes and chorioallantois of eggs from two Florida alligator farms and from free-ranging Louisiana alligators. The predominant organism was identified as a Fusarium S.

Materials and Methods

A total of 368 eggs was collected and incubated in our laboratory at 31 C under conditions of high humidity (near saturation). Three hundred six (306) eggs originated from freeranging Louisiana alligators, and two clutches of 24 and 38 eggs, respectively, were received from two commercial Florida farms. Prior to incubation each egg was marked and disinfected with 1% Nolvasan solution. Brown, circular lesions up to 0.5 cm in diameter were observed on the majority of eggs from one farm and on 6 eggs from wild alligators. These lesions were usually located on or near the chorioallantois (8). The number and size of lesions varied considerably depending on the origin of the specimen. Samples from the egg membranes and the chorioallantois from 27 eggs were submitted for microbiological and histological examination. Scrapings from both membranes were inoculated on differential media and incubated at 37 C. Sabouraud's agar plates were kept at room temperature. Samples of soil and water from a farm with a high incidence of affected eggs were submitted for microbiological examination. Tissue samples of the egg membrane and the chorioallantois were taken from affected and unaffected eggs, fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of 6 um were stained by the Brown and Hopps modification of the Gram stain and by the Giemsa method. Fungal elements were identified in the egg membranes and chorioallantois. Periodic acid Schiff and methenamine silver stains were used to confirm their presence. Fertile eggs showing lesions were incubated and monitored daily. Unaffected infertile eggs were inoculated with pure suspensions of the isolated fungi in order to determine their ability to produce characteristic lesions on the egg membrane.

Results

Bacteriological studies usually resulted in the isolation of gram negative organisms belonging to the family Enterobacteriaceae. Enterobacter cloacae, Citrobacter s., Proteus s., Pseudomonas aerucrinosa, and an a-hemolytic streptococcus were isolated. Tryptic soy agar with 5% sheep blood and Sabouraud's dextrose agar both typically showed rapid heavy growth of white cottony colonies. Sabouraud's agar containing cycloheximide was inhibitory; whereas, Sabouraud's agar with chloramphenicol allowed growth. Lactophenol cotton blue-stained tape mounts of these colonies resulted in the demonstration of branching septate hyphae and multicelled macroconidia as well as elongated microconidia. These characteristics are compatible with Fusarium species and speciation resulted in the identification of Fusarium oxvsporum as the predominant organism isolated from eggs. The ubiquity of this fungus was demonstrated by the ease with which it was isolated from soil and water samples. Fusarium oxvsporum was isolated from all 27 eggs. Additional fungal species were isolated from only 2 of these eggs, both of which yielded Penicillium fellucanum, Paecili aviotti and Aspergillus niger, in addition to the Fusarium s. Histological examination showed thickened egg membranes and hypertrophic chorioallantoic membranes containing numerous septate branching filamentous structures compatible with Fusarium sipecies. Inoculation of disinfected, nonaffected infertile eggs with a pure suspension of Fusarium oxvsporum or the other isolated fungal species, resulted in only Fusarium oxvsporum demonstrating the ability to create lesions on the egg membrane. Embryos from grossly affected eggs showed retarded embryonic development and embryonic death. Surviving embryos were smaller at hatch compared to hatchlings from unaffected eggs.

Discussion

Fusarium s. are distributed throughout the world and considered to be common soil organisms (3). They are recognized as opportunistic pathogens and have been described as the cause of wound infections and keratomycosis in humans (4). Infections due to Fusarium s. have also been reported in crocodiles, sea turtles, fish and other species of animals (1,2,5,7). The large number of infected eggs from captive alligators is probably caused by the ubiquity of Fusarium s., high stocking densities of captive breeders and the lack of regular water and soil decontamination in breeding pens. The route of egg invasion by fungal elements is uncertain. Fusarium s. have been shown to be a cause of vulvovaginitis in swine. A study to detect Fusarium s. in the reproductive tracts of female alligators could provide evidence for infection of the eggs while in the oviduct. Another plausible source of infection is highly colonized soil. Predisposing factor for this route of infection may be stress or nutritional deficiencies of the female alligator resulting in improper egg shell composition and/or immunodeficiency. These factors may allow fungal elements enter and infect the egg membrane. Further investigation is needed to fully characterize the pathogenesis of this disease: Froducers may also improve sanitation and hygiene in order to minimize exposure of both female alligators and their eggs to this organism.

Acknowledgements

We thank Dr. H. Jean Shadomy, Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia for her expert assistance and for the identification of Fusarium oxysporum.

References

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2.  Austwick,P.K.C., 1984. Fusarium infections in human and animals. In Moss, M. and J.E. Smith (eds.). The Applied Mycology of Fusarium. New York, Cambridge University Press, pp. 129-140.

3.  Braude Abraham I., Infectious Diseases and Medical Microbiology, W.B. Saunders Company 1986, p. 605-607.

4.  Cardeilhac P.T., Florida Department of Agriculture and Consumer Services: Final Reports for the Aquaculture Market Development Aid Program 1987-1988; Husbandry and Preventative Medicine Practices that Increase Reproductive Efficiency of Breeding Colonies of Alligators, p.90-114.

5.  Kuttin E.S.; J. Mullar, et al 1978. Mykosen bei Krokodilen. Mykosen, 21:39-48.

6.  Lennete Edwin H.; Manual of Clinical Microbiology, 4th Edition, American Society for Microbiology, 1985, p. 592-593.

7.  Rippon John W.; Medical Mycology 3rd Edition, W.B. Saunders Company 1988, p. 732-735.

8.  Webb Grahame J.W. ; Manolis Charlie S. and Whitehead Peter J.: Wildlife Management: Crocodiles and Alligators, Surrey Beatty & Sons PTY Limited 1987, p. 417-444.