Oral and Cloacal Helicobacter Detection in Wild and Captive Orinoco Crocodiles ( Crocodylus intermedius ) in Venezuela

Introduction

The Orinoco crocodile (Crocodylus intermedius), one of the most threatened crocodile species, has been reduced to a few wild populations in the Venezuelan and Colombian Llanos (Seijas and Chávez 2000). In Venezuela, the Orinoco crocodile was given protected status in the 1970s and release program had been created in 1990, but their populations have not recovered (Boede and Sogbe 2000, Espinosa-Blanco et al. 2017).

Bacterial infections are common complications of animal bites, and bacteria recovered from infected bite wounds reflect the oral microbiota of the biting animal (Abrahamian and Goldstein 2011). In the United States and Australia, the encounters of humans with crocodilians have increased and bacterial infections of human wounds inflicted by these reptiles have been reported (Abrahamian and Goldstein 2011, Smith et al. 2017). Some authors have reported the presence of bacteria with medical importance to humans in oral and cloacal samples of wild crocodiles in Botswana and Mexico (Lovely and Leslie 2008, Charruau et al. 2012). American alligator microbiota shows differences between the farm-raised and wild populations in correlation to diet (Keenan et al. 2013).

In Venezuela, human settlements are growing close to remaining populations, where Crocodylus intermedius still lives, finding zones of human–crocodile sympatry, increasing the probabilities of encounters and accidents between humans and crocodiles and possibility risk of pathogens transmission. In addition, studies realized in Venezuelan captive breeding centers (CBCs) report the presence of bacteria with medical importance to humans in Orinoco crocodile and American crocodile (Crocodylus acutus) (Boede and Velasco 1993, Boede and Sogbe 2000). These researches showed evidence that human contact could be altering the bacterial community in crocodiles. However, we did not find reports about the presence of Helicobacter species in Orinoco crocodile comparing wild and captive populations. Helicobacter spp. are gram-negative, microaerophilic, spiral-shaped bacteria associated with gastrointestinal diseases in humans and animals. Helicobacter pylori, a human pathogen, is the most studied species. Wild and domestic animals had been reported as reservoirs of Helicobacter species, but crocodiles were not included (Harbour and Sutton 2008, Schrenzel et al. 2010). Our aim was to detect by PCR Helicobacter genus and H. pylori species from Orinoco crocodile in Venezuelan wild and captive populations.

Materials and Methods

Fieldwork was conducted from February to July 2016 at two wild localities (Cojedes River System [09°21′54′′N; 68°42′32.4′′W] and Capanaparo River [06°59′34.8′′N; 67°17′38.4′′W]) and two CBCs (Masaguaral ranch [08°34′19′′N; 67°34′57′′W] and University of the Llanos UNELLEZ [08°36′45′′N; 69°26′45.08′′W]).

Captures, total length (TL), and sex of animals were determined following the methodology described by Espinosa-Blanco et al. (2017). Crocodilians size categories were estimated by Seijas and Chávez (2000).

All animals were captured and released under Venezuelan Ministry of Environment permits (0314 and DGDB 00057). The oral and cloacal sterile cotton swab samples of five individuals in each locality (n = 20) were collected. DNA was extracted using PowerSoil DNA Isolation kit (MO BIO, Inc., Carlsbad, CA). To confirm the presence of bacterial DNA, we used 16S rRNA gene primers by PCR (8F and 1525R; Contreras et al. 2007). Positive samples for bacterial DNA were tested for Helicobacter spp. DNA. Helicobacter DNA was amplified using genus-specific primers for 16S rRNA gene (HeliF and HeliR) (Contreras et al. 2007). Positive samples for Helicobacter genus DNA were then tested for H. pylori DNA, amplifying glmM gene (glmMF and glmMR) (Contreras et al. 2007).

Amplifications were performed using a Ready-To-Go PureTaq PCR kit (Amersham Biosciences, NJ) in a GeneAMP 9700 (Applied Biosystems, CA) thermal cycler according to the manufacturer's recommendations for each primer set. The positive control was H. pylori DNA from a clinical strain, and the negative control was sterile water.

A PCR inhibition assay was used for our samples with negative bacterial PCR results adding DNA from positive control to PCR.

Helicobacter genus-specific fragments (∼260 base pairs [bp]) from two cloacal samples were purified and sequenced at Macrogen, Inc. (Seoul, Korea) and sequences were deposited in GenBank (MH037130 and MH037131).

Results and Discussion

We caught 8 males, 11 females, and 1 non-sexed Orinoco crocodiles. Males ranged from 76 to 398 cm TL and females ranged from 58 to 350 cm TL. Size structure categories were II–IV from wild crocodiles and IV–V from captive crocodiles. Bacterial DNA was confirmed in 20 oral and 19 cloacal samples. A PCR inhibition assay was performed on the cloacal sample (animal #46), which was negative bacterial PCR and no inhibition was detected, suggesting that DNA concentration was low.

Helicobacter DNA was detected in cloacal swabs from Masaguaral Ranch CBC and in oral and cloacal swabs from UNELLEZ CBC (Table 1). These results indicated that this bacterial genus is only present in farm-raised crocodiles. In addition, H. pylori was detected in two cloacal swabs from UNELLEZ (animals #49 and 53; Table 1). Detection of H. pylori in two Orinoco crocodiles from CBCs suggests that human manipulation could be altering bacterial community of Orinoco crocodiles.

Non-pylori Helicobacter amplicons from two cloacal samples were sequenced. One amplicon (animal #42) had 97% similarity with Helicobacter himalayensis (NR_135861). The second amplicon (animal #52) had 96% similarity with an uncultured Helicobacter species (GU462202). These results suggest that more of one Helicobacter species could be present in cloacal samples. Therefore, we need to use other molecular tools to determine bacterial diversity in these samples.

Helicobacter genus has been associated with gastrointestinal diseases in birds and mammals (Harbour and Sutton 2008); however, in reptiles, it has not been identified as causal agent of illnesses (Gilbert et al. 2017). According to Huchzermeyer (2003), the captivity can act as stressor in crocodiles, which may increase their susceptibility to bacterial infections. Since in this study, Helicobacter was detected only in apparently healthy captive crocodiles, more studies are needed to determine whether Helicobacter spp. can cause gastrointestinal or systemic disorders in this reptile. This is the first report of high prevalence of Helicobacter species, including H. pylori in C. intermedius from CBCs. The Orinoco crocodiles infected are adult animals often used for reproduction, but not to be released. However, it is necessary to study animals that will be released, because H. pylori is a waterborne pathogen (Azevedo et al. 2008), and infected crocodiles could be introducing this bacterium to waters by fecal contamination, representing a possible risk to humans that use rivers. Furthermore, crocodile attacks or incidental bites on humans could be an additional infection source. The inclusion of microbiological analysis in crocodile conservation program is necessary to reduce the risk of microbial contamination.