Isolation of Arcobacter Species in Water Buffaloes ( Bubalus bubalis)

Abstract

This is the first report of Arcobacter spp. in rectal fecal samples from healthy water buffaloes (Bubalus bubalis) reared on a dairy farm. Arcobacter species were isolated after enrichment, and isolates were identified at species level by multiplex-polymerase chain reaction assay. Thirty samples were examined and Arcobacter spp. were isolated from 96.7% of water buffaloes tested: 38 Arcobacter spp. isolates were obtained, with A. cryaerophilus as the dominant species followed by A. butzleri and A. skirrowii. Nine animals (31%) were colonized by more than one Arcobacter species. The present study indicates that water buffaloes can harbor a variety of Arcobacter spp. and that healthy buffaloes may act as hosts. Water buffalo fecal shedding of Arcobacter spp. may be of significance to human health, considering the potential fecal contamination during harvesting of raw milk and slaughtering.

Introduction

I n recent years concern has grown over the genus Arcobacter because its members have been considered emergent enteropathogens and potential zoonotic agents (Collado and Figueras, 2011). Potential routes of Arcobacter spp. transmission to humans are the consumption of contaminated foods of animal origin and water (Shah et al., 2011). Few surveys have investigated the presence of Arcobacter spp. in bulk tank raw milk, and the reported prevalence rates were 46% in Northern Ireland (Scullion et al., 2006), 5.8% in Malaysia (Shah et al., 2012), and 48% in Italy (Milesi, 2010). The initial source seems to be fecal contamination during the various stages of production processes (Van Driessche and Houf, 2008). Worldwide there is increasing evidence that livestock animals are significant reservoirs of Arcobacter spp. The pathogen has frequently been isolated from the intestinal tracts and fecal samples of different farm animals (pigs, cattle, horses, sheep, and poultry [i.e., chicken, ducks, turkeys, and domestic geese]) (Shah et al., 2011), but no study has hitherto investigated Arcobacter spp. in water buffaloes. The objective of this study was to evaluate the presence of Arcobacter spp. in water buffaloes.

Materials and Methods

The study was carried out on a dairy water buffalo farm located in the Emilia-Romagna region, Italy. The farm included 90 healthy lactating water buffaloes and had farming practices and hygienic conditions comparable to those on a dairy cattle farm. The age of animals ranged from 3 to 5 years. In November 2011, fresh rectal fecal samples (about 50 g) were taken from 30 animals randomly chosen and processed, avoiding cross-contamination. The sampling allows estimation, with a 95% probability, of the prevalence of Arcobacter spp.–positive animals with an expected prevalence of 40% and an accepted error of 15%.

The samples were collected in sterile bags, kept at 5±3°C, and examined within 4 hours after sampling. Isolation was performed using the enrichment procedure described by Houf et al. (2001); briefly, 2 g of feces were put into Arcobacter Broth (Oxoid, Basingstoke, UK) supplemented with 5% lysed horse blood, 0.5% pyruvate, and 0.5% thioglycolic acid as growth supplements and a mix of cefoperazone (16 mg/L), amphotericin B (10 mg/L), 5-fluorulacil (100 mg/L), novobiocin (32 mg/L), and trimethoprim (64 mg/L) as a selective supplement. After 48 h of incubation at 28±1°C, 10 μL of the enrichment broth was streaked onto selective agar plates prepared by suspending 24 g Arcobacter broth (Oxoid, Basingstoke, UK) and 12 g Agar Technical No. 3 (Oxoid), supplemented with selective supplement as described above. The plates were incubated at 28±1°C under microaerobic condition and after 48 h of incubation were checked daily up to 5 days. Different-size colonies of Gram-negative spiral bacteria were subcultured and subjected to presumptive identification using tests that included growth under aerobic conditions, cellular morphology, oxidase, and catalase activity. The DNA of suspected isolates was extracted using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO), subjected to an Arcobacter genus–specific polymerase chain reaction (PCR) using primers ARCO I and ARCO II described by Harmon and Wesley (1996) and, when positive, to species identification by multiplex PCR (Douidah et al., 2010).

Results and Discussion

Arcobacter spp. were isolated from 29 of 30 water buffaloes tested, with an intraherd prevalence of 96.7% (95% confidence interval that with the intraherd prevalence range between 90.0% and 98.9%). A total of 38 isolates of Arcobacter spp. were obtained; of these 26, 7, and 5 were identified as A. cryaerophilus, A. butzleri, and A. skirrowii, respectively, with A. cryaerophilus as the dominant species. Twenty animals (69%) were positive for only one species (17 A. cryaerophilus, 1 A. butzleri, and 2 A. skirrowii), while nine (31%) were positive for two different Arcobacter species (6 for A. butzleri and A. cryaerophilus and 3 for A. cryaerophilus and A. skirrowii). Results are summarized in Table 1.

Table 1.

Number and Percentage of Positive Samples and Arcobacter Species Isolated from 30 Water Buffalo Fecal Samples

	A. butzleri	A. cryaerophilus	A. skirrowii	A. butzleri and A. cryaerophilus	A. skirrowii and A. cryaerophilus	Total positive samples
Number	1	17	2	6	3	29
%	3.3	56.6	6.6	20	10	96.6

This is the first report of the isolation of Arcobacter spp. from water buffalo feces on a dairy farm. We observed that 96.7% of samples were positive for Arcobacter spp., a higher percentage of positive animals than those found in other investigations on dairy cattle ranging from 8% to 41.7% (Wesley et al., 2000; Golla et al., 2002; Van Driessche et al., 2005; Vilar et al., 2010). A comparison of the intraherd prevalence of this study with previously reported data is biased by differences in isolation methods, sample size, test design, and animal host species. Various media and procedures have been used to isolate Arcobacter spp. from different samples (Collado and Figueras, 2011); using a very similar protocol, Van Driessche et al. found an Arcobacter spp. prevalence ranging from 5.9% to 11% in Belgian dairy cows (Van Driessche et al., 2005), whereas the present study disclosed a higher prevalence in water buffalo feces. This difference may be due to different susceptibility to the colonization of the host species or to the different behavior of water buffaloes, which tend to lie in the wet parts of the barn, enhancing the probability of orofecal contact. In addition, individual herd management factors facilitating the circulation of Arcobacter spp. in the herd should not be excluded, and further studies on different farms are required for confirmation.

A. cryaerophilus was the most frequently isolated species, in agreement with the results obtained by Van Driessche et al. (2005). This contrasts with other surveys where A. butzleri was the dominant species isolated from cattle feces (Ongor et al., 2004; Shah et al., 2013), but the different isolation methods used in these surveys may affect the species of Arcobacter detected as most of the isolation methods used are not optimal for all Arcobacter species (Houf et al., 2001). We found more than one Arcobacter spp. in 31% of positive samples. These coinfection results, determined after an isolation method including an enrichment step, may be underestimated as the enrichment step reduces the diversity of Arcobacter species by favoring the faster-growing species (Houf et al., 2002).

Conclusions

Water buffaloes can harbor a variety of Arcobacter spp. In addition, fecal shedding of Arcobacter spp. by water buffaloes may be of significance to human health, considering the potential fecal contamination during harvesting of raw milk and slaughtering. Although pasteurization of milk is recommended, traditional technology involving the use of unpasteurized milk is still employed, and the presence of Arcobacter spp. in water buffalo feces may represent a source of contamination of milk, cheeses, and of meat produced by culling water buffaloes. The results of this study should be interpreted carefully as they were obtained from a single herd investigation, and further studies are required to confirm these preliminary findings.

Disclosure Statement

No competing financial interests exist.

References

Collado

, Figueras

. Taxonomy, epidemiology and clinical relevance of the genus Arcobacter. Clin Microbiol Rev, 2011; 24:174–192.

Douidah

, De Zutter

, Vandamme

et al. Identification of five human and mammal associated Arcobacter species by a novel multiplex-PCR assay. J Microbiol Meth, 2010; 281–286.

Golla

, Murano

, Johnson

et al. Determination of the occurrence of Arcobacter butzleri in beef and dairy cattle from Texas by various isolation methods. J Food Prot, 2002; 65:1849–1853.

Harmon

, Wesley

. Identification of Arcobacter isolates by PCR. Lett Appl Microbiol, 1996; 23:241–244.

Houf

, De Zutter

, Van Hoof

et al. Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods. Appl Environ Microbiol, 2002; 68:2172–2178.

Houf

, Devriese

, De Zutter

et al. Development of a new protocol for the isolation and quantification of Arcobacter species from poultry products. Int J Food Microbiol, 2001; 71:189–196.

Milesi

. Emerging pathogen Arcobacter spp. in food of animal origin [PhD Thesis] Doctoral Program in Animal Nutrition and Food Safety. Milan, Italy: University of Milan, 2010.

Ongor

, Cetinkaya

, Acik

et al. Investigation of Arcobacters in meat and faecal samples of clinically healthy cattle in Turkey. Lett Appl Microbiol, 2004; 38:339–344.

Scullion

, Harrington

, Madden

. Prevalence of Arcobacter spp. in raw milk and retail raw meats in Northern Ireland. J Food Prot, 2006; 69:1986–1990.

10.

Shah

, Saleha

, Murugaiyah

et al. Prevalence and distribution of Arcobacter spp. in raw milk and retail raw beef. J Food Prot, 2012; 75:1474–1478.

11.

Shah

, Saleha

, Zunita

et al.

Arcobacter: An emerging threat to animals and animal origin food products?

Tr Food Sci Technol, 2011; 22:225–236.

12.

Shah

, Saleha

, Zunita

et al. Prevalence, distribution and antibiotic resistance of emergent Arcobacter spp. from clinically healthy cattle and goats. Transbound Emerg Dis, 2013; 60:9–16.

13.

Van Driessche

, Houf

. Survival capacity in water of Arcobacter species under different temperature conditions. J Appl Microbiol, 2008; 105:443–451.

14.

Van Driessche

, Houf

, Vangroenweghe

et al. Prevalence, enumeration and strain variation of Arcobacter species in the faeces of healthy cattle in Belgium. Vet Microbiol, 2005; 105:149–154.

15.

Vilar

, Peña

, Pérez

et al. Presence of Listeria, Arcobacter, and Campylobacter spp. in dairy farms in Spain. Berl Munch Tierarztl Wochenschr, 2010; 123:58–62.

16.

Wesley

, Wells

, Harmon

et al. Fecal shedding of Campylobacter and Arcobacter spp. in dairy cattle. Appl Environ Microbiol, 2000; 66:1994–2000.