Occurrence and diversity of Campylobacter species in captive chelonians

Journal Pre-proof Occurrence and diversity of Campylobacter species in captive chelonians Carlotta De Luca, Gregorio Iraola, Ilias Apostolakos, Elena Boetto, Alessandra Piccirillo

PII:

S0378-1135(19)30435-3

DOI:

https://doi.org/10.1016/j.vetmic.2019.108567

Reference:

VETMIC 108567

To appear in:

Veterinary Microbiology

Received Date:

26 April 2019

Revised Date:

19 December 2019

Accepted Date:

26 December 2019

Please cite this article as: De Luca C, Iraola G, Apostolakos I, Boetto E, Piccirillo A, Occurrence and diversity of Campylobacter species in captive chelonians, Veterinary Microbiology (2019), doi: https://doi.org/10.1016/j.vetmic.2019.108567

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Occurrence and diversity of Campylobacter species in captive chelonians

Carlotta De Lucaa,1, Gregorio Iraolab,c, Ilias Apostolakosa, Elena Boettoa, Alessandra Piccirilloa,*

Department of Comparative Biomedicine and Food Science, University of Padua,

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a

Legnaro (PD), Italy.

Center for Integrative Biology, Universidad Mayor, Santiago de Chile, Chile.

present address: Christian Doppler Laboratory for Innovative Poultry Vaccines (IPOV),

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c

Unidad de Bioinformática, Institut Pasteur de Montevideo, Montevideo, Uruguay.

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b

*Corresponding

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University of Veterinary Medicine, Vienna, Austria.

author: Alessandra Piccirillo. Department of Comparative Biomedicine

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and Food Science, University of Padua, viale dell’Università, 16 - 35020 Legnaro (PD), Italy. Tel. +39 049 8272968 - Fax. +39 049 8272973 - E-mail:

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[email protected].

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Highlights Presence of campylobacters potentially pathogenic for humans in captive reptiles.

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Three taxa of Campylobacter recently described in chelonians were all isolated.

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Data suggest reptilian-associated Campylobacter are not pathogenic for chelonians.

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It is crucial to further investigate the epidemiology of Campylobacter in chelonians.

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ABSTRACT

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The aim of this study was to investigate the occurrence and diversity of

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Campylobacter species in chelonians. From July 2016 to September 2017, a total of 452 individuals from a large variety of tortoises (n = 366) and turtles/terrapins (n = 86) kept

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in private collections and breeding centres, wildlife rescue centres, zoos, pet shops, and veterinary clinics from Northern Italy were sampled and subjected to microbiological

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examination. Campylobacter genus and species confirmation was performed by single and multiplex PCRs. Out of 452 samples, five (1.1%) tested positive: three for C. iguaniorum (two Testudo graeca and one Testudo hermanni), one for C. fetus subsp. testudinum (Stigmochelys pardalis) and one for C. geochelonis (Testudo hermanni). This

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study suggests that Campylobacter spp. are not common in chelonians, but a variety of species can be detected in these hosts, including those potentially pathogenic for humans. Further studies are needed to understand the epidemiology and the pathogenic potential for both animals and humans of reptile-associated Campylobacter spp.

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Keywords: Campylobacter fetus subsp. testudinum, Campylobacter iguaniorum, Campylobacter geochelonis, chelonian, tortoise, turtle, zoonosis.

INTRODUCTION

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The genus Campylobacter is composed by Gram negative, microaerophilic

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bacteria that inhabit the intestinal tract of various animals, as either commensals or

pathogens (Rukambile et al., 2019). Human campylobacteriosis is the most frequently

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reported foodborne zoonosis in the European Union (EFSA & ECDC, 2018). Sources of

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human infections are consumption of raw or undercooked meat, unpasteurized milk and untreated water, or direct contact with colonized animals that carry Campylobacter

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asymptomatically (EFSA & ECDC, 2018).

The growing number of reptiles kept as pets and their potential role as reservoir of

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zoonotic microorganisms prompted studies that aimed to evaluate the occurrence of Campylobacter spp., resulting in the description of several novel species and subspecies in the last few years (Fitzgerald et al., 2014; Gilbert et al., 2015; Piccirillo et al., 2016). Until 2013, C. fetus was considered the most common species inhabiting the intestinal

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tract of chelonians with pathogenic potential for humans (Harvey and Greenwood, 1985; Tu et al., 2004). In 2014, Fitzgerald et al. performed phenotypical analyses on a cluster of C. fetus strains isolated from both reptiles and diseased humans demonstrating that they represented a novel subspecies designated as C. fetus subsp. testudinum. This subspecies has been isolated from chelonians like Terrapene carolina and squamata like Tiliqua

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nigrolutea or Heterodon nasicus (Dingle et al., 2010; Fitzgerald et al., 2014). Following this discovery, C. fetus isolated from human infections has been increasingly identified as subsp. testudinum (Patrick et al., 2013; Choi et al., 2016). In the following years, two new other Campylobacter species were identified in reptiles. First, C. iguaniorum was isolated from lizards such as Iguana iguana and Pogona vitticeps (Gilbert et al., 2014), and tortoises like Stigmochelys pardalis (Benejat et al., 2014). Second, during another

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screening study to determine the occurrence of Campylobacter spp. in reptiles in 2016,

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three strains isolated from different tortoises and initially characterized as C. fetus subsp. fetus (Giacomelli and Piccirillo, 2014), were subsequently characterized by phenotypic

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and genetic analyses as a novel species named C. geochelonis (Piccirillo et al., 2016).

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So far, epidemiological information about Campylobacter species in chelonians is limited to data from a small number of studies carried out in restricted geographic regions

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(Wang et al., 2013; Giacomelli and Piccirillo, 2014; Gilbert et al., 2014). Expanding the knowledge on the presence of Campylobacter spp. in chelonians is important for both

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veterinary and public health due to the zoonotic potential and unknown pathogenicity of most of these species in these animals. Therefore, the aim of this study was to determine the occurrence and diversity of Campylobacter species in Northern Italy.

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MATERIALS AND METHODS Sample collection

From July 2016 to September 2017, a total of 452 cloacal swabs were collected

from 32 different chelonian species (Supplementary Table 1). Three hundred sixty-six tortoises and 86 turtles/terrapins were sampled individually. The animals were kept in

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private collections and breeding centres (n = 19; 264 individuals sampled), a wildlife rescue centre (n = 1; 39), zoos (n = 3; 108), pet shops (n = 2; 22), or submitted to private veterinary clinics (n = 2; 16); in addition, three dead animals were sampled upon arrival at the Veterinary Hospital of the University of Padua. All facilities were located in Veneto and Lombardia regions (Northern Italy). All sampled individuals were apparently healthy, except for most of those submitted to the veterinary clinics. At sampling, data

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regarding the gender, the age, the origin, and the health status, as well as information

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regarding the management of animals were collected. Campylobacter spp. isolation and identification

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Swabs were transported to the laboratory in Amies Transport Medium

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supplemented with charcoal (Copan, Italy), refrigerated and processed within 24 hrs. Campylobacter isolation and identification for microbiological screening was done

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according to the protocol previously described by Giacomelli and Piccirillo (2014). DNA was extracted from colonies suspected to be Campylobacter spp. by boiling

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single colonies in 100 μl of sterile RNase/DNase free water (Sigma-Aldrich, Italy) for 20 min. Extracted DNA was quantified using Nanodrop 2000 and the DNA concentration of each sample was adjusted to 20 ng/µl. DNAs were initially screened using a multiplex PCR to identify Campylobacter genus and the following species: C. coli, C. fetus, C.

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jejuni, C. hyointestinalis, C. lari and C. upsaliensis (Yamazaki-Matsune et al., 2007). Isolates positive for Campylobacter genus only were further analysed by single PCR using primers specifically designed in this study: C. iguaniorum (Cig-F: 5’GGCACGACACACACAGTAGA-3’; Cig-R: 5’-GCATTGCTAGTCCCACCAGT-3’), C. fetus subsp. testudinum (Cft-F: 5’-ACTTCTTCGCTGCTGAGCTA-3’; Cft-R: 5’-

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GATTTGCAGCAATCCACGCA-3’) and C. geochelonis (GEO-F: 5’AAGCGGCTCTATCAAGGCTC-3’; GEO-R: 5’-AGTTGCCCTGCTAACCTACG-3’). Multiplex PCRs were performed using the Qiagen Multiplex PCR Kit (Qiagen, Italy); single PCRs were performed using the DreamTaq Green PCR Master Mix (ThermoFisher Scientific, Italy) in a final volume of 25 μl containing 1 μl of bacterial DNA. C. fetus subsp. testudinum (in-house control), C. iguaniorum (kindly provided by Dr. Gilbert,

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Utrecht University, The Netherlands) and C. geochelonis (DSM 102159 and LMG 29375

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reference strains) were used as positive controls. All PCR reactions were carried out in a 2720 Thermal Cycler (Applied Biosystems, Italy). Following PCR, amplicons were

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separated by electrophoresis in a 3% (w/v) agarose gel (Sigma-Aldrich, Italy) and

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visualized by Gel Doc® XR (Bio-Rad, Italy), after SYBR® Safe DNA Gel Stain

RESULTS

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(Invitrogen, Italy) staining.

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Out of 452 chelonians screened for Campylobacter spp., five (1.1% of sampled individuals) tested positive: three (0.66%) for C. iguaniorum (from two Testudo graeca and one Testudo hermanni), one (0.22%) for C. fetus subsp. testudinum (from Stigmochelys pardalis) and one (0.22%) for C. geochelonis (from Testudo hermanni). At

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the chelonian species level, 8.3% (1/12) of Stigmochelis pardalis were positive for C. fetus subsp. testudinum, 4.8% (2/42) of Testudo graeca for C. iguaniorum, and 1.2% (2/168) of Testudo hermanni for C. geochelonis (0.6%, 1/168) and C. iguaniorum (0.6%, 1/168), respectively.

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The three tortoises positive for C. iguaniorum were healthy females, aged 6 to 30 years, living in a wildlife rescue centre and free to roam among several other Testudo. C. fetus subsp. testudinum was isolated from a healthy female breeder of Stigmochelys pardalis kept in a private breeding centre together with other tortoises, belonging to five different species (Astrochelys radiata, Geochelone elegans, Centrochelys sulcata, T. hermanni and T. marginata). C. geochelonis was isolated from a female of Testudo

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hermanni admitted to a veterinary clinic because affected by Septicemic Cutaneous

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Ulcerative Disease (SCUD), a cutaneous bacterial infection involving the carapace and

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the skin, associated with an altered immune status of the animal (Jacobson, 2007).

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DISCUSSION

The study aimed to investigate the occurrence and diversity of Campylobacter

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spp. in chelonians. Individuals from various chelonian species were sampled; only 1.1% (5/452) tested positive for the bacteria. Campylobacter was only recovered from

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terrestrial species of Chelonii. In the limited number of similar studies, Campylobacter spp. occurrence ranged from 9.7% (10/103) in Taiwan (Wang et al., 2013) and 10.2% (5/49) in Italy (Giacomelli and Piccirillo, 2014) to 25.3% (39/154) in The Netherlands (Gilbert et al., 2014). By contrast, Campylobacter was not detected in 200 free-living

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turtles belonging to the aquatic species Emys orbicularis and Trachemis scripta in Spain (Marin et al., 2013), accordingly to our findings. It is worth mentioning that different isolation and detection procedures were employed in these studies, including direct PCR, which proved a more sensitive method than culturing (Gilbert et al., 2014). However, direct PCR has limitations as well, since positivity implies merely the presence of

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bacterial DNA and not of the viable strain thus hampering further analysis. The low prevalence reported in our study may also be explained by intermittent shedding of intestinal microorganisms described in reptiles (DuPonte et al., 1978; Burnham et al., 1998). Three novel species and subspecies of Campylobacter (C. iguaniorum, C. fetus subsp. testudinum and C. geochelonis) have been recently reported to colonize the

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intestinal tract of chelonians (Giacomelli and Piccirillo, 2014; Gilbert et al., 2014), with

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all of them detected in the present study. Here, we report for the first time the presence of C. iguaniorum in Testudo hermanni. This species was found in healthy female tortoises

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living in close contact with each other and other Testudo in a wildlife rescue centre.

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Despite no other tortoises living in the same facility tested positive, a common source of infection or a horizontal transmission (i.e. faecal-oral) route may be hypothesized.

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Furthermore, C. fetus subsp. testudinum was isolated from an animal belonging to the species Stigmochelys pardalis representing a new host for this subspecies. In addition to

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the abovementioned studies, another research group reported only C. fetus in chelonians from Taiwan (Wang et al., 2013). Taken together, present and previous data suggest that Chelonii, besides being infrequently colonized by Campylobacter spp., are mainly carriers of reptilian-associated campylobacters, rather than clinically relevant,

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thermophilic species. Therefore, we speculate that some of these species, like C. geochelonis, may be markedly host-restricted to chelonians. However, C. fetus subsp. testudinum seems to possess a broader host range (reptiles and humans), while C. iguaniorum has also been isolated from an alpaca (Vicugna pacos) (Miller et al., 2016). The different body temperatures among mammals, birds and reptiles could explain the

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heterogeneous distribution of Campylobacter species among hosts. In fact, the optimal growth temperature for most campylobacters ranges between 30 and 45 ºC (Vandamme et al., 2015), but these new strains isolated from reptiles show optimal growth at 25 ºC (Fitzgerald et al., 2014; Gilbert et al., 2014; Piccirillo et al., 2016), which could be attributed to an evolutionary adaptation to these animals (Gilbert et al., 2015; Gilbert et al., 2016). To support this hypothesis, the genetic divergence among C. fetus isolated

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from reptilian and mammalian hosts and even among strains isolated from humans and

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cattle (Iraola et al., 2017) is well established (Tu et al., 2001; Tu et al., 2005; Gilbert et al., 2016).

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It is interesting to notice that all the tortoises testing positive for Campylobacter

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were females, although further studies are necessary to assess whether the presence of the microorganism is related to the gender, since the low occurrence hinders any statistical

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analysis for risk factor of Chelonian colonization. All the Campylobacter-positive tortoises were healthy animals, with the only exception of T. hermanni positive for C.

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geochelonis, whose SCUD lesions were unrelated to the presence of the bacterium in the intestine. To our knowledge, no studies reporting Campylobacter-associated disease in chelonians are currently available and very rarely these bacteria have been isolated from animals showing clinical signs of disease (Benejat et al., 2014). Therefore, evidence so

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far shows that Campylobacter are not pathogenic for Chelonii.

CONCLUSION Chelonians seem to be susceptible to reptilian-associated campylobacters, since no other species was detected in the present study. Given the overall low number of

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studies on the topic and the high variability of results, the role of chelonians as reservoir of Campylobacter spp. is not yet elucidated and may be affected by multiple factors, such as gender, environment and contact with other animals. However, the presence of Campylobacter in the intestinal tract of Chelonii appears to be predominant in terrestrial species compared to aquatic animals. For these reasons, owners of Chelonii should always pay attention to a correct hygiene while handling their animals, as reported by the

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Centre for Disease Control and Prevention (CDC, 2019)

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(https://www.cdc.gov/campylobacter/pets.html). In addition, it has recently been

described how dietary probiotics affect the intestinal microbiota of turtles, including

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Campylobacter, suggesting that acting on the diet of pet reptiles may represent an option

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to avoid colonization (Rawski et al., 2016). Further studies should be also undertaken to clarify the pathogenic potential of reptilian-associated Campylobacter spp. for both

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FUNDING

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animals and humans that is still largely unknown.

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This work was supported by the University of Padua (DOR1707179).

ACKNOWLEDGEMENTS The Authors wish to thank Dr. Marco Bedin, Dr. Diego Cattarossi, Dr. Gino

Conzo, Dr. Davide Guadagnini, Dr. Marco Martini, Dr. Camillo Sandri and Dr. Ferdinando Zanin for the help in sample collection. We are also grateful to Parco Natura

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Viva, Parco Faunistico Le Cornelle and Tropicarium Park of Jesolo for giving access to chelonian samples kept in the zoological gardens.

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