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Trop Anim Health Prod (2017) 49:777–782 DOI 10.1007/s11250-017-1262-3

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Arcobacter spp. in fecal samples from Brazilian farmed caimans (Caiman yacare, Daudin 1802) Maria Gabriela Xavier Oliveira 1 & Leandro Nogueira Pressinotti 2 & Giovane Spínola Carvalho 2 & Mirela Caroline Vilela Oliveira 1 & Luisa Zanolli Moreno 1 & Carlos Emilio Cabrera Matajira 1 & Alessandro Spínola Bergamo 2 & Victor Manuel Aleixo 3 & Alexandre Caixeta Veiga 2 & Elvis de Souza Corsino 3 & Ana Paula Guarnieri Christ 4 & Maria Inês Zanolli Sato 4 & Andrea Micke Moreno 1 & Terezinha Knöbl 1

Received: 21 June 2016 / Accepted: 6 March 2017 / Published online: 21 March 2017 # Springer Science+Business Media Dordrecht 2017

Abstract The aim of this study was to perform the identification and molecular characterization of Arcobacter cryaerophilus and Arcobacter butzleri isolated from caiman (Caiman yacare), kept at a production farm, in Brazil. Forty fecal samples were analyzed. After isolation and identification, 21/40 strains of A. butzleri and 19/40 strains of A. cryaerophilus were subjected to PCR for potential virulence gene detection. The results of the PCR showed 38/40 strains positive for the cadF, cj1349, ciaB, and tlyA genes, 39/ 40 strains positive for the pldA gene, and 40/40 strains positive for the mviN gene. None of the strains presented the irgA gene. Hemagglutinin (hecA gene) and hemolysin (hecB) genes were detected in 21/40 and 16/40 strains, respectively. The SE-AFLP showed a great genetic diversity, but some clonally groups were disseminated in various tanks. These data reveal that the strains presented the same virulence traits described from Arcobacter isolated from food-borne disease in humans.

* Terezinha Knöbl [email protected] 1

Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87-Cidade Universitária, São Paulo, SP, Brazil

2

Universidade do Estado de Mato Grosso, Av. São João s/n-Cavalhada, Cáceres, MT, Spain

3

Instituto Federal de Educação Ciência e Tecnologia de Mato Grosso Campus Cáceres, Av. dos Ramires s/n–Distrito Industrial, Cáceres, MT, Spain

4

Companhia Ambiental do Estado de São Paulo (CETESB), Av. Prof. Frederico Hermann Jr, 345, Pinheiros, São Paulo/SP 05459-900, Brazil

Keywords Reptile . Pathogens . Alligators . Campylobacteriaceae

Introduction Caiman yacare is a Crocodylia member of the Alligatoridae family which lives in Argentina, Bolivia, Paraguay, and Brazil. The conservation status of C. yacare is considered lower risk/least concern by the IUCN Red List (IUCN 2015). The current estimated population of caiman in the Pantanal region is over 3 million. Males grow until the asymptotic snout-vent length of 129.2 ± 3.2, while females grow until 87.0 ± 3.2 cm. They are opportunistic predators that usually eat aquatic vertebrates and invertebrates (Kuhnert et al. 2012). Until a few decades ago, the illegal harvesting of C. yacare was focused on obtaining the skins for the leather market, but now it is more desirable to obtain the tail meat encouraged by tourists and local population searching for wild meat (Campos et al. 2010). Since the 1990s, Brazil adopted a policy for legal ranching management of C. yacare in order to prevent illegal actions. The ranching strategy is focused on the legal production of C. yacare for commercialization of meat and leather skins, thus, avoiding predation and contributing to the in situ conservation. Following the trend of this wild meat market, specialized farms commercialize in nature caiman meat and other manufactured products such as mortadella and sausage, which adds value to the species by-products (Fernandes et al. 2014). The commercial farms promote economic development in the poorest villages of Pantanal (Brazil), and the regulation and inspection of slaughter also has an impact on human health by

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avoiding the consumption of wild meat without sanitary inspection. From a nutritional standpoint, caiman meat, as well as the meat of many other wild animals, has two major advantages: high protein level and low intramuscular fat content (Vicente-Neto et al. 2010). However, microbiological risks must be considered, mainly because opportunistic bacteria reside in the digestive tract of the animals and are likely to contaminate the meat products during the processing chain. Reptiles are a reservoir for many bacterial species, including Enterobacteriaceae, Erysipelothrix rhusiopathiae, Pseudomonas spp., Bacillus spp., Clostridium spp., Pasteurellaceae, Staphylococcus spp., Streptococcus spp., Acinetobacter spp., Coxiella burnetii, Mycoplasma spp., Chlamydiae, and Mycobacterium spp. However, most published data involve reptiles kept as pets (Campos et al. 2010). Information concerning bacterial pathogens in wild and captive caimans is still scarce. While public health relevance of Salmonella spp. in reptile meat consumption has been previously documented (Uhart et al. 2011), risks associated with the contamination of C. yacare meat by members of Campylobacteraceae are unknown. Arcobacter spp. belong to the Campylobacteraceae family and are an ubiquitous organism present in water, food products, and feces from healthy or sick humans and domestic and wildlife animals (Hsu and Lee 2015; Talay et al. 2016). Some species of the Arcobacter genus are commensals; however, Arcobacter butzleri and Arcobacter cryaerophilus are recognized as opportunistic human pathogens and were found more regularly in diarrheic stools than in nondiarrheic stools (Vandenberg et al. 2004; Lehner et al. 2005; Ho et al. 2006; Prouzet-Mauléon et al. 2006). Although their pathogenicity remains to be fully elucidated, these pathogenic species have been considered as potential zoonotic food-borne agents (Ho et al. 2006), but the relevance for public health is currently unclear (Hänel et al. 2016). Food-borne Arcobacter diseases have been associated with consumption or manipulation of poultry and pork; however, this pathogen has been isolated from seafood, dairy products, and some vegetables (Fernandez et al. 2015). Wildlife animals carry Arcobacter spp. and potential pathogenic species were described in cloacal and fecal samples from lizards, snakes, and chelonians but never in crocodilian members (Hsu and Lee 2015). This study analyzed the feces of caimans intended for human consumption for the presence of potential pathogenic species from the Arcobacter genus.

Materials and methods Samples and bacterial strains isolation Forty fecal swabs were collected from C. yacare from a farm in Brazil. The swabs were placed in Stuart transport medium (Copan Diagnostics Inc., Corona, CA, USA) and refrigerated

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until analysis. The animals were allocated in four tanks with ten animals per tank (n = 10) and kept in a closed and covered shed measuring 4.0 m2, with uneven floor and water available in one third of this area. The swabs were homogenized in 2 mL of peptone water, and 1 mL of this broth was inoculated in 9 mL of Johnson and Murano (JM) broth (Johnson and Murano 1999). The tubes were incubated in aerobic conditions for 48 h at 30 °C. After incubation, a 10 μL aliquot of the broth was deposited on a sterile membrane (0.45 μm), which was subsequently placed on the surface of JM selective agar. After 1 h, the filter was removed, and the plate was streaked and incubated in aerobic conditions for 48–72 h at 30 °C. Characteristics colonies were stored at −80 °C in skim milk tryptone glucose glycerol broth for further analysis. MALDI-TOF MS identification Suspected colonies were submitted to matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification. For sample preparation, bacterial proteins were extracted using an ethanol/formic acid protocol (Kuhnert et al. 2012). The protein suspension (1 μL) was transferred to a polished steel MALDI target plate (Bruker Daltonik) and allowed to dry at room temperature. The sample was overlaid with 1 μL of matrix (10 mg α-cyano-4-hydroxycinnamic acid per mL in 50% acetonitrile/2.5% trifluoroacetic acid), and mass spectra in the 2–20 kDa range were acquired using a Microflex™ mass spectrometer (Bruker Daltonik). For MALDI-TOF MS analysis, the spectra were loaded into a MALDI BioTyper™ 3.0 and compared with the manufacturer’s library, which resulted in the log (score) value. Standard Bruker interpretative criteria were applied: scores ≥2.0 and scores ≥1.7 were accepted for the species assignment but scores ≤2.0 were accepted for genus identification. Identification and determination of virulence genes by PCR A. cryaerophilus and A. butzleri identification was confirmed by polymerase chain reaction (PCR) as previously described by Douidah et al. (2010). One Arcobacter strain from each positive animal was submitted to the PCR method to determine the presence of the putative virulence genes that codify for the following: adhesion (cadF and cj1349), invasion (ciaB), phospholipase (pldA), hemolysin (tlyA), regulation of an outer membrane protein associated with iron uptake (irgA), and hemagglutinin (hecA), as well as genes associated with hemolysin activation (hecB) and a virulence marker (mviN) (Douidah et al. 2012). The amplification mixture consisted of 10 mM Tris-HCl buffer (pH 8.3), MgCl 2, 200 mM deoxynucleotide triphosphates, DNA primers,


0.5 U Taq DNA polymerase, and ultrapure water at a final volume of 25 μL. The amplified products were separated by electrophoresis in 1.5% agarose gel stained with BlueGreen® (LGC Biotecnologia, São Paulo, Brazil) and identified through 100 bp DNA ladder (New England BioLabs Inc., Ipswich, MA, USA). Single-enzyme amplified fragment length polymorphism (SE-AFLP) One Arcobacter strain from each positive animal was genotyped using SE-AFLP. Restriction endonuclease digestion and ligation were performed as described by McLauchlin et al. (2000). To 10 μL of extracted DNA, 24 U of Hind III (New England BioLabs Inc., Ipswich, MA, USA) and ultrapure water were added, yielding a final volume of 20 μL. This reaction was incubated overnight at 37 °C. Then, 5 μL of digested DNA was added to 0.2 μg of adapter ADH1 and ADH2 oligonucleotides, 1 U of T4 DNA ligase (New England BioLabs Inc., Ipswich, MA, USA), and ultrapure water, yielding a final volume of 20 μL. The mixture was incubated for 3 h at room temperature. Ligated DNA was heated to 80 °C for 10 min, and 5 μL was used for each PCR reaction. PCR was performed using a final volume of 50 μL, which contained 5 μL of ligated DNA, 2.5 mM of MgCl2, 30 pmol of primer (HI-G), and 1 U of Taq polymerase, in 1× PCR buffer. This mixture was denatured at 94 °C for 4 min with subsequent steps of 35 cycles of 1 min at 94 °C, 1 min at 60 °C, and 2.5 min at 72 °C. Electrophoresis was performed using a 2.0% agarose gel at 24 V for 26 h. The amplified products were stained with GelRedTM (New England BioLabs Inc., Ipswich, MA, USA) and compared to a 100 bp DNA ladder (New England BioLabs Inc., Ipswich, MA, USA). Statistical analysis SE-AFLP band patterns were analyzed using Dice coefficient by means of Bionumerics 7.5 software (Applied Maths NV, Saint-Martens-Latem, Belgium) to generate a dendrogram. Similarity value of 90% cutoff was used to analyze SEAFLP clusters (Van Belkum et al. 2007).

Results and discussion All 40 fecal samples were positive to Arcobacter spp. isolation. A total of 52.5% (21/40) of tested animals were positive to isolation of A. butzleri and 47.5% (19/40) were positive for A. cryaerophilus isolation (Fig. 1). Considering the identification of 40 isolated strains according the specie A. butzleri and A. cryaerophilus, there was 100% agreement between the PCR and MALDI-TOF identification methods.

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This paper reports, for the first time, the isolation of Arcobacter species from captive caiman, with 100% of frequency. Gilbert et al. (2014) screened captive reptiles by PCR for the Arcobacter genus and detected a prevalence of 19.9%, including Lacertilia (lizards—14.1%), Serpentes (snakes— 13%), and Testudines (chelonia—30.5%) suborders. The highest frequency of occurrence in captive caiman may be influenced by handling practices. Enclosure features, the semi-aquatic habitats of the animals, and feeding them with bovine viscera may favor bacterial infection. Fernandez et al. (2015) investigated the frequency of A. butzleri in animals and food samples in southern Chile. While 10.7% of fecal samples were positive, the frequency of chicken meat contamination was 72%. The persistence of Arcobacter spp. in food-processing environments and crosscontamination in the slaughterhouse were discussed by Hsu and Lee (2015). Thus, the caiman production chain requires care management and hygiene practices to reduce this high contamination of carcasses in order to avoid Arcobacter transmission by wild meat consumption. Further studies are required to determine the association of the presence of Arcobacter spp. in the stool of reptiles and eventual contamination of meat once industry technologies are available that can prevent survival of the agent in the meat and there are methods for reducing the associated risks. Sfaciotte et al. (2015) analyzed samples of vacuum-packed and frozen meats in a commercial store in Paraná, Brazil and concluded that all caiman meats are appropriate for consumption based on the requirements of health legislation standards (for the counts of mesophilic aerobic bacteria, aerobic psychrophilic bacteria, coliforms, Escherichia coli, and Staphylococcus spp.). The risk assessment and the use of technology to make food safe are fundamental in villages that depend on this activity for economic development. Pathogenicity of Arcobacter spp. can be dependent on many factors, mainly host resistance and virulence of strains. Only limited information is available about the pathogenesis of infection by zoonotic species of Arcobacter. The in vitro interactions of Arcobacter isolates on human colon cell line HT-29/B6 were demonstrated by Karadas et al. (2016). Açik et al. (2016) showed that A. butzleri induced enteritis, congestion, and necrosis in the liver, kidney, and spleen in zebrafish. Gölz et al. (2016) showed that infected gnotobiotic mice had an increased secretion of pro-inflammatory cytokines in small and large intestine and serum samples. The authors pointed out that A. butzleri is more than a commensal in vertebrate hosts. Furthermore, data from genome sequencing highlighted several virulence markers, and nine putative genes (cadF, cj1349, ciaB, pldA, tlyA, irgA, hecA, hecB, and mviN) have been employed for pathogenic strains identification by PCR (Ferreira et al. 2016). The results of our study showed that all isolates (100%) were positive for the virulence gene associated with peptidoglycan

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Fig. 1 Dendogram based on AFLP of 40 strains of Arcobacter spp. isolated from fecal samples of C. yacare


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biosynthesis protein (mviN) (Table 1). The frequency of genes related to adhesion, invasion, hemolysin, and phospholipase was 95–100%. The hemagglutinin gene and hemolysin activator were detected in 52.5 and 40% of the isolates, respectively. All isolates were negative for the iron regulator-encoding gene. The virulence profile suggests that these strains isolated from the fecal samples of caiman presented similar genetic virulence traits, compared to the Arcobacter spp. isolated from humans with gastroenteric disease (Tabatabaei et al. 2014). The dendrogram obtained by SE-AFLP (Fig. 1) showed a great heterogeneity, with 40 strains grouped in 30 different genotypic profiles. However, there was a positive correlation between the SE-AFLP profile and the Arcobacter species. Plainly, the upper portion of the dendogram grouped 19/19 strains of A. cryaerophilus. The similarity of these strains ranged from 40 to 100%, with a clonal group (90–100% of similarity) com posed by seven (7/19) strains of A. cryaerophilus. In the lower portion of the dendrogram, 20/21 strains of A. butzleri were grouped, with a similarity ranging from 55 to 100%. Considering a cut of 90% of similarity, we identified three clonal groups: two strains with two groups each and one group with five strains. The SE-AFLP data showed that, in addition to the great diversity of genotypes, some clones were spread between different tanks. The clone comprising the species A. cryareophilus was detected in tanks 1, 2, and 4; while the clones of five strains of A. butzleri were disseminated in tanks 1, 3, and 4. These findings highlight potential horizontal transmission of the agent during captive breeding.

Conclusion The presence of potential virulent strains of A. butzleri and A. cryaerophilus in the fecal samples of C. yacare highlights eventual risks associated with meat contamination. Actions to prevent contamination in farms and slaughterhouses should be

Table 1

Virulence genes in Arcobacter spp.

Virulence factor

Gene

Positive strains/n total

Adhesion

cadF cj1349 ciaB pldA tlyA irgA hecA hecB mviN

38/40 38/40 38/40 39/40 38/40 0/40 21/40 16/40 40/40

Invasion Phospholipase Hemolysin Iron regulator Hemagglutinin Hemolysin activating Peptidoglycan biosynthesis protein

adopted because these bacteria are considered emerging foodborne pathogens. Acknowledgements This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo–FAPESP (grant 2014/07837-6). CAPES and CNPq research grants are gratefully acknowledged. M.G.X.O. and C.E.C.M. and L.Z.M. are recipients of FAPESP fellowships (2014/06584-7, 2015/26159-1 and 2013/17136-2, respectively). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Statement of the welfare of animals All procedures performed were in accordance with the ethical standards of the School of Veterinary Medicine and Animal Science of University of São Paulo and were approved by the Ethics Committee on Use of Animal for Research.

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