Normal Intestinal Flora of Wild Nile Crocodiles (crocodylus Niloticus) in the Okavango Delta, Botswana

INTRODUCTION Owing to the difficulty in obtaining biological specimens from wild crocodili-ans, very little is known about their normal intestinal flora. The intestinal tract flora isolated from wild-caught African dwarf crocodiles (Osteolaemus tetraspis) has been reported 11. Salmonellae isolated from wild Nile crocodiles from Lake Kariba, and from wild American alligators (Alligator missis-sippiensis) have also been documented 14,21. Other studies have dealt with captive crocodilians, including the normal intestinal flora of captive gharials (Gavialis gangeticus), and the prevalence of salmo-nellae in healthy captive crocodilians


INTRODUCTION
Owing to the difficulty in obtaining biological specimens from wild crocodilians, very little is known about their normal intestinal flora.The intestinal tract flora isolated from wild-caught African dwarf crocodiles (Osteolaemus tetraspis) has been reported 11 .
Salmonellae isolated from wild Nile crocodiles from Lake Kariba, and from wild American alligators (Alligator mississippiensis) have also been documented 14,21 .Other studies have dealt with captive crocodilians, including the normal intestinal flora of captive gharials (Gavialis gangeticus), and the prevalence of salmonellae in healthy captive crocodilians 15,16,18 .
Farmed crocodile hatchlings often fail to develop a normal mixed intestinal flora 10 .In other species, the rapid establishment of bacterial communities of normal flora in the gastrointestinal tract (GIT) is thought to be essential for GIT homeostasis and the prevention of colonization by pathogenic bacteria 4,17 .A deficient intestinal flora is likely to be one of the factors predisposing farmed crocodiles to enteritis.Enteritis, and associated septicaemia, is one of the main causes of mortality in farmed crocodilians 1,6,7 .Deter-mining the normal intestinal tract flora of wild Nile crocodiles is the 1st step towards developing a probiotic for use in farmed Nile crocodiles.

Sampling
Crocodiles were captured in the Panhandle of the Okavango Delta during summer (February 2005).Capture was done at night, using a 4.8 m flat bottomed aluminium boat propelled by a 60 hp engine.Crocodiles were located using a powerful spot-light which, when shone into the crocodile's eyes, reflected back a red glow due to the presence of a retinal tapetum lucidum.Once spotted, the beam of light was kept focused on the crocodile's eyes, making it possible to approach the animal with the boat.Crocodiles estimated to be smaller than 1.2 m total length (TL) were captured by hand.Crocodiles between 1.2 m and 2.3 m were captured using a swivelling noose (Animal Handling Co., USA) which was placed over the snout and pulled tight in the neck region.Crocodiles were then brought onto the boat, jaws were taped shut and the animals were physically restrained.
Twenty-nine animals were randomly selected for cloacal swab collection.Each crocodile was blindfolded and restrained in dorsal recumbency.A cloacal swab was taken by inserting a sterile cotton swab (Transwab, Medical Wire & Equipment Co. Ltd., UK) into the cloaca to a depth of 50-100 mm, rotating the swab, withdrawing it and placing it directly into the sterile transport medium supplied.

Isolation and identification procedures
Cloacal swabs were stored at -10 °C in a domestic gas freezer for up to 1 month.On return from the study site, the swabs were submitted to Golden Vet Lab, Johannesburg.An aerobic bacterial culture and a fungal culture were performed as follows.Each cloacal swab was inoculated onto culture plates containing 5 % bovine blood and MacConkey agar no. 1 (Diagnostic Media Products, South Africa) and also onto thiosulphate citrate bile salt sucrose (TCBS) agar, Mycosel agar, cornmeal agar and Rappaport Vassiliadis (RV) broth (the latter 4 from Selectamedia, South Africa).Anaerobic culture could not be attempted, as many anaerobes would not have survived the storage process.All the agar plates were aerobically cultured at 25 °C, but the RV broth was cultured at 37 °C to improve selectivity for Salmonella isolation.
The bacterial cultures were incubated for 72 hours before discarding, and the fungal cultures for 28 days before discarding.The RV broths were subcultured twice, after 24 hours and after 6 days of incubation, onto xylose lactose sodium desoxycholate (XLD) agar (Selectamedia).The XLD agars were cultured at 37 °C for 24 hours each time, and examined for the presence of colonies resembling Salmonella.Each bacterial and fungal isolate was identified according to standard methods 2,3,12,20,22 .

RESULTS
The bacteria and fungi isolated from each wild specimen are presented in Table 1.The bacteria and fungi are given in the order of frequency in which they were isolated from each cloacal swab, with the 1st-named being the most frequent isolate.
Bacteria were cultured from all 29 specimens.There were a total of 79 isolations, and 16 different species.The number of species cultured per specimen varied from 1 to 4, with only 2 (6.9 %) specimens yielding a single species that could be cultured under the incubation conditions described.Eight crocodiles (27.6 %) had 2 isolates and 15 (51.7 %) yielded 3 isolates.Four isolates were obtained from 4 crocodiles (13.8 %).The mean number of isolations per crocodile was 2.7.
Table 2 shows the number of isolates of each bacterium and the percentage of crocodiles carrying each species.The most commonly isolated species was Microbacterium, found in 21 of the crocodiles (72.4 %), followed by Enterococcus faecalis (14 isolates), Aeromonas hydrophila (10 isolates), and Escherichia coli (9 isolates).No salmonellae were cultured.
Table 3 shows the number of isolates of each fungus, and the percentage of crocodiles carrying each fungus.The most commonly isolated species was Cladosporium, found in 8 crocodiles (27.6 %).

DISCUSSION
The bacterial species most commonly isolated from the wild Nile crocodiles, Microbacterium, is a common soil inhabitant.Neither Microbacterium nor E. faecalis, the 2nd-most frequently isolated species,  11,16 .Enterococcus faecalis was, however, isolated from 1 of the dwarf crocodiles, and other Enterococcus species were isolated from a further 21 of the 29 dwarf crocodiles.
Escherichia coli appears to be a common component in crocodile intestinal tract flora, having also been isolated from 9 of the gharials and 8 of the dwarf crocodiles.Nevertheless, E. coli has been recorded as a cause of septicaemia in crocodilians, including the Nile crocodile.One study found 47 of 409 (11.5 %) bacterial infections to be caused by E. coli 7 .
Aeromonas hydrophila was isolated from 34.5 % of the samples.Aeromonas hydrophila is frequently found associated with mortality caused by enteritis and septicaemia.In Zimbabwe it was the 2nd-most frequent isolate, after Salmonella, from septicaemic Nile crocodiles 7 .It is also an important cause of septicaemia in Crocodylus porosus, Crocodylus johnsoni and Crocodylus novaeguineae 1,13 .Besides E. coli and A. hydrophila, another 5 of the genera isolated (Bacillus, Citrobacter, Proteus, Pseudomonas and Staphylococcus) are known causes of septicaemia in crocodiles 10 .This supports the view that many bacterial septicaemias are caused by normal intestinal tract inhabitants which act as opportunistic pathogens in an immunosupressed host.
No Salmonella were cultured from the wild Nile crocodiles.This is interesting in the light of previous findings.Of 67 wild Nile crocodiles in Lake Kariba, 18 (26.9%) yielded Salmonella 14 .Three of 29 (10.3 %) wild caught African dwarf crocodiles yielded Salmonella, while Salmonella was found in 2 of 71 (2.8 %) wild A. mississippiensis 11,21 .
In farmed crocodilians, Salmonella has frequently been isolated: Obwolo and Zwart found Salmonella in 8 of 50 healthy, 3-year-old, farmed Nile crocodiles, and concluded that Salmonella may represent normal flora in the intestinal tract of crocodiles 18 .By contrast, no Salmonella were found in cloacal swabs from 23 captive gharials 16 .Healthy farmed C. porosus and C. johnstoni were found to carry Salmonella 15 .On 1 farm 20.0 % of C. porosus and 27.8 % of C. johnstoni were carriers, while on another farm the prevalence rate was 81 % and 5 % for the 2 species, respectively.In farmed alligators, Salmonella was isolated from 4 of 29 specimens (14 %) 21 .
In contrast to their frequent occurrence as resident flora, Salmonella was cultured from 202 of 409 farmed Nile crocodiles found to have died from bacterial infections in Zimbabwe 7 .In South Africa, 145 Salmonella isolates, and a wide range of serovars, were reported from farmed Nile crocodiles submitted for necropsy over a 10-year period 24 .
It is clear that while Salmonella can be normal intestinal tract flora in healthy farmed crocodilians, they can also be important pathogens.The role of Salmonella as normal intestinal tract flora in wild crocodilians is unclear.Several factors may account for the apparent absence of Salmonella in this study.The composition of intestinal flora is dependent on ingested food, both the type of food and the amount.Shedding of Salmonella is not necessarily constant.It has been shown that Salmonella could suddenly be excreted from turtles after a period of 6 months with no excretion 5 .Furthermore, cloacal swabbing may underestimate the prevalence of Salmonella compared with faecal swabbing 15 .Logically there will be less efficient horizontal transfer of Salmonella in a natural environment than under intensive conditions.Nevertheless, vertical transmission could occur with equal ease in either environment.Recent findings from C. porosus eggs tend to support the possibility of vertical transmission of Salmonella.Salmonella was cultured in eggs from 12 of 13 clutches on 1 farm.Interestingly, the serotypes isolated were clutch specific 19 .
The fungi isolated from the wild Nile crocodiles are considered environmental.Nevertheless, Cladosporium, Penicillium and Trichoderma have been found in diseased crocodilians 1,8,9,23 .These 3 fungi have also been isolated from the shells of C. porosus eggs, but were not present in the egg yolk 19 .
Fungi were isolated from the intestinal tract of 24 of 29 African Dwarf crocodiles, a far higher occurrence than the present study 11 .
From the limited studies to date, it appears that crocodilian intestinal flora is dynamic and varies according to both crocodilian species and environmental conditions.More studies will be required to improve our understanding of crocodilian intestinal flora, leading to the development of a crocodile-specific probiotic.