Intestinal contents were collected from wild-caught African dwarf crocodiles (Osteolaemus tetraspis)
in 1993 and 1995 which were slaughtered at urban markets in the Congo Republic. The samples were
kept frozen and brought back to Onderstepoort for aerobic culture. Out of 29 specimens, 33 species
of bacteria and 20 species of fungi were isolated. The bacteria included three isolates of Salmonella
and eight isolates of Escherichia coli, most of the latter being rough strains. The flora of individual
specimens contained 1-5 bacterial and 0-5 fungal species. Neither Aeromonas hydrophila nor
Edwardsiella tarda were isolated from any of the samples.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Agnagna, M.

Henton, Marijke M.

Huchzermeyer, Fritz W.

Riley, J.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Intestinal contents were collected from wild-caught African dwarf crocodiles (Osteolaemus tetraspis)
in 1993 and 1995 which were slaughtered at urban markets in the Congo Republic. The samples were
kept frozen and brought back to Onderstepoort for aerobic culture. Out of 29 specimens, 33 species
of bacteria and 20 species of fungi were isolated. The bacteria included three isolates of Salmonella
and eight isolates of Escherichia coli, most of the latter being rough strains. The flora of individual
specimens contained 1-5 bacterial and 0-5 fungal species. Neither Aeromonas hydrophila nor
Edwardsiella tarda were isolated from any of the samples.mn201

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Onderstepoort Journal of Veterinary Research, 67:201-204 (2000) Aerobic intestinal flora of wild-caught African dwarf crocodiles Osteo/aemus tetraspis F.W. HUCHZERMEYER1 *, M.M. HENTON2, J. RILEy3 and M. AGNAGNA4 ABSTRACT HUCHZERMEYER, F.w. , HENTON, M.M. , RILEY, J. & AGNAGNA, M. 2000. Aerobic intestinal flora of wild-caught African dwarf crocodiles Osteolaemus tetraspis. Onderstepoort Journal of Veterinary Research, 67:201-204 Intestinal contents were collected from wild-caught African dwarf crocodiles (Osteolaemus tetraspis) in 1993 and 1995 which were slaughtered at urban markets in the Congo Republic. The samples were kept frozen and brought back to Onderstepoort for aerobic culture. Out of 29 specimens, 33 species of bacteria and 20 species of fungi were isolated. The bacteria included three isolates of Salmonella and eight isolates of Escherichia coli, most of the latter being rough strains . The flora of individual specimens contained 1-5 bacterial and 0-5 fungal species. Neither Aeromonas hydrophila nor Edwardsiella tarda were isolated from any of the samples. Keywords: African dwarf crocodile, Escherichia coli, intestinal flora, Salmonella INTRODUCTION Enteritis, often associated with septicaemia, is a major problem in young farmed crocodiles and sal-monellae are frequently involved (Foggin 1987; 1992a; 1992b; Huchzermeyer 1991; Manolis, Webb, Pinch, Melville & Hollis 1991; Obwolo & Zwart 1993; Van der Walt, Huchzermeyer & Steyn 1997). There is increasing evidence in favour of the protective action of a normal intestinal flora, partially reviewed by Huchzermeyer (1994). Misra, Kumar, Patnaik, Raman & Sinha (1993) reported briefly on the nor-mal gut flora of captive gharials (Gavialis gangeti-GUs). Further knowledge of the normal intestinal flora * Author to whom correspondence is to be directed 1 P.O. Box 12499, Onderstepoort, 0110 South Africa (Formerly of the Pathology Section, Onderstepoort Veterinary Institute) 2 Bacteriology Section, Onderstepoort Veterinary Institute, Pri-vate Bag X04, Onderstepoort, 0110 South Africa 3 Biological Sciences, University of Dundee, Dundee, DD1 4HN, United Kingdom 4 Directeur National de la Faune et Flore, B.P. 2453, Brazzaville, Congo Republic Accepted for publication 13 July 2000-Editor of wild crocodiles could lead to the development of suitable probiotics for use on crocodile farms and would also be useful when assessing the significance of isolates made from diseased animals. Because of the protected status of crocodiles (Ross 1998) as well of the remoteness of most wild croco-dile populations, it is extremely difficult to obtain any biological specimens from them. However, live wild-caught African dwarf crocodiles (Osteolaemus tetra-spis) are regularly brought to urban markets in the Congo Republic to be slaughtered for their meat (Behra 1990). Within the framework of a wider project concerning pathology, parasitology and biology of these reptiles, intestinal content samples were col-lected at the markets with the objective of gaining information on the aerobic intestinal flora of this spe-cies. MATERIALS AND METHODS Samples The internal organs of freshly slaughtered African dwarf crocodiles were purchased at markets in Brazzaville during May 1993 and in Brazzaville and 201 Aerobic intestinal flora of African dwarf crocodiles Osteolaemus tetraspis Impfondo, Congo Republic during April 1995 (Huch-zermeyer & Agnagna 1994). Intestinal contents were collected from these organs, placed into sterile plas-tic tubes and kept frozen in freezing compartments of domestic electric or gas refrigerators (at the time there was no public electricity supply in Impfondo) and in an insulated box with ice packs during trans-port, until they reached the laboratory on the return of the senior author to South Africa. TABLE 1 Aerobic bacteria isolated from intestinal contents of African dwarf crocodiles Isolation and identification procedures Each sample of faeces or intestinal contents was inoculated onto plates containing blood tryptose agar medium (Oxoid, Basingstoke, United Kingdom) in-corporating bovine blood, MacConkey agar no. 1 (Biolab, Box 1998, Halfway House, South Africa), thiosulphate citrate bile salt sucrose (TCBS) agar (Biolab) and potato dextrose agar (Biolab) with and without chloramphenicol (Centaur, Johannesburg, South Africa) and cyclohexamide (BDH, Poole, United Kingdom), as well as into selenite broth (Sigma, St Louis, USA). All the agar plates were incubated aerobically at 25°C and the selenite broth at 42 °C to improve selectivity for Salmonella spp. The plates containing blood tryp-tose agar, MacConkey agar and TCBS agar were incubated for 72 h before discarding, and those con-taining the two types of potato agar for 28 days be-fore discarding. The selenite broth cultures were subcultured onto xylose lactose sodium desoxy-cholate (XLD) agar (Oxoid) after 24 h of culture. The XLD agars were cultured for 24 h at 37°C, and ex-Genus Alcaligenes Bacillus Citrobacter Dermacoccus Enterobacter Enterococcus Escherichia Flavobacterium Klebsiella Kocuria Kurthia Lactobacillus Micrococcus Proteus Salmonella Serratia Staphylococcus Streptococcus Streptomyces Species faecalis alvei cereus circulans coagulans amalonaticus freundii nishinomyaensis agglomerans cloacae gergoviae caecorum durans faaecalis faecium pseudoavium solitarius coli balustinum odoratum oxytoca varians gibsonii sp. luteus mirabilis susp. odorifera chromogenes epidermidis xylosus salivarius sp. amined for colonies resembling Salmonella. Each n = number of isolates bacterial and fungal isolate was identified according to standard methods (Cowan 1974; Campbell & n 1993 n 1995 2 1 11 4 1 1 1 3 1 1 1 7 1 1 1 1 10 1 7 1 8 1 1 2 2 1 3 1 4 6 2 3 1 4 1 2 1 1 Stewart 1980; Krieg & Holt 1984; Sneath, Mair, TABLE 2 Fungal isolates from intestinal contents of African dwarf Sharpe & Holt 1986; Rippon 1988). crocodiles RESULTS The aerobic bacteria and fungi isolated from 21 sam-ples in 1993 and from eight samples in 1995 are presented in Tables 1 and 2, respectively. Bacteria were isolated from all 29 samples, the number of isolates per sample ranging from 1-5 and only one crocodile (specimen 10/93) had a single-species iso-late of Enterococcus faecium. Of the 101 bacterial isolates, 59 were Gram positive and 42 Gram nega-tive, the two most common being Enterococcus spe-cies with 22 isolations and Bacillus spp. with 18 isolations. Most isolates of Escherichia coli were rough, and only one was typable: 051 :K23. The four isolates of Klebsiella oxytoca were each of a differ-ent capsule type: 42, 44, 58 and 60. Details of the three Salmonella isolates have been published in a previous paper (Van der Walt et al. 1997). They were S. wangata, S. yoruba and an unnamed group Ilib Genus Acremonium Arthrinium Aspergillus Beauveria Candida Chrysosporium Cryptococcus Curvularia Fusarium Geotrichum Paecilomyces Penicilllium Phoma Trichoderma Trichosporon Species sp. sp. cia va tus flavus niger sp. guillermondii krusei sp. lipolytica luteolus sp. sp. candidum sp. sp. sp. sp. beige/ii capita tum Salmonella 30:k:enx. n = number of isolates 202 n 1993 n 1995 1 1 5 2 2 3 2 2 1 3 3 3 1 1 1 4 2 7 2 2 1 2 1 Enterobacteriaceae usually occurred as a heavy growth on culture media, whilst Gram negative non-fermenters, such as Alcaligenes spp. and Flavobac-terium spp. were only present in small numbers. Enterococcus spp., Micrococcus spp. and Kurthia spp. were present in heavy growth in only about half the cases; the others occurring in small numbers. Bacillus spp., Lactobacillus spp. and Streptomyces spp. were only present in small numbers in each case where they were isolated. Fungi were isolated from 24 of the 29 samples and the number of isolates per sample ranged from 0-5, with 12 samples yielding a single species each. Specimen 10/93 also had one fungal species, Curvu-laria sp., only. Most fungi were only present in low numbers and Aspergillus spp. and Penicillium spp. were the most frequent isolates. DISCUSSION It is possible that the results were affected by the long enforced preslaughter fast to which the animals had been subjected, with an estimated average time lapse between capture and slaughter of 30 days (Hucl:lzermeyer & Agnagna 1994). After such a long fast the intestinal contents were of minimal quantity. In addition, some market slaughterers routinely dis-carded the very short rectum (rectocolon) during evisceration because of the evil smelling contents of mixed urine and faeces, while the small intestines are sold as edible. Consequently, some of the intestinal content samples had to be taken from the small in-testines and two crocodiles yielded no intestinal con-tent sample whatsoever. It is to be expected that the small intestine would have a less diverse flora than the rectum. Preslaughter cloacal swabbing would have been ideal, but was impracticable as the mar-ket slaughterers were suspicious of our intentions and tended to be uncooperative or to demand extra remuneration for any slight concession given. The bacteria and fungi isolated are similar to the normal intestinal flora of other animal species. Most were present in low numbers, probably as a result of a lack of ingesta. The only genera that were usu-ally present in heavy growth were members of the family Enterobacteriaceae, but Enterobacteriaceae were only isolated from 15 of the 29 cases. Surprisingly few isolates of E. coli (8) and of salmonellae (3) were obtained. Misra et al. (1993) did not find any salmonellae in cloacal swabs from 23 gharials, but had nine isolates of E. coli, while Obwolo & Zwart (1993) obtained eight Salmonella isolates from cloacal swabs of 50 healthy farmed Nile croco-diles in Zimbabwe. In contrast, 49,4 % of all bacte-rial isolates from crocodiles submitted for post mor-tem examination in Zimbabwe over a four-year F.w. HUCHZERMEYER et a/. period were Salmonella spp. (Foggin 1992b). On two farms in Australia 20 % and 81 % of slaughtered Crocodylus porosus and 27,8 % and 55 % of C. johnsoniwere found to harbour salmonellae (Manolis et al. 1991). Eleven Salmonella isolates were reported by Hibberd, Pierce, Hill & Kelly (1996) from 62 dis-eased farmed juvenile C. porosus. The large number and range of Salmonella isolates from farmed croco-diles submitted for necropsy examination in South Africa have been reported by Van der Walt et al. (1997), but our wild-caught animals only yielded three isolates: S. wangata in heavy growth and S. yo-ruba and Salmonella IIlb in small numbers. Edwardsiella tarda which is frequently found asso-ciated with mortality caused by enteritis and septi-caemia in farmed Nile crocodile hatchlings (Foggin 1992a; Huchzermeyer & Henton unpublished data), as well as in farmed American alligators (Gorden, Hazen, Esch & Fliermans 1979), was not isolated from any of our specimens. However, Misra et al. (1993) reported its isolation from three out of 23 gharials. Similarly, Aeromonas hydrophila which has been found associated with bacterial disease in farmed and wild American alligators (Shotts, Gaines, Martin & Prestwood 1972; Gorden et al. 1979; Pe-ters & Cardeilhac 1988) as well as in farmed Nile crocodiles (Foggin 1987), was not present in any of our samples. Hibberd et al. (1996) reported Fusarium spp. isola-tions from 24 of 62 diseased farmed juvenile C. poro-sus, as well as Penicillium spp. from nine and Asper-gillus spp. from eight cases. In this study, Aspergillus spp. and Penicillium spp. were commonly found, al-beit in low numbers, but Fusarium sp. was only iso-lated from one sample. The yeasts, Candida spp., Cryptococcus spp., Trichosporon spp. and Geotri-chum spp. made up 19 of the 52 fungal isolates. They are all considered to be part of the normal flora of mucous membranes. This paper presents the results of the most compre-hensive study of the intestinal flora of any crocodilian species published so far and it is hoped that this is only the beginning. More studies of the gut flora of wild and farmed individuals of the major crocodilian species are required for a better understanding of the function of this flora and the eventual elaboration of crocodile-specific probiotics. ACKNOWLEDGEMENTS This study was supported by the Ostrich and Croco-dile Project of the Onderstepoort Veterinary Institute and by the Brazzaville Office of the Wildlife Conser-vation Society, for which we should like to express our gratitude. Mrs M.M. Engelbrecht is thanked for assisting with the mycological work. 203 Aerobic intestinal flora of African dwarf crocodiles Osteolaemus tetraspis REFERENCES BEHRA, O. 1990. Sex ratio of African dwarf crocodiles (Osteo-laemus tetraspis Cope, 1861) exploited for food in Congo, in: Crocodiles. 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VAN DER WALT, M.L. , HUCHZERMEYER, F.W & STEYN, H.C. 1997. Salmonella isolated from crocodiles and other reptiles during the period 1985-1994 in South Africa. Onderstepoort Journal of Veterinary Research, 64:277-283. 

Aerobic intestinal flora of wild-caught African dwarf crocodiles Osteolaemus tetraspis

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Aerobic intestinal flora of wild-caught African dwarf crocodiles Osteolaemus tetraspis

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