Isolation and Seroprevalence of Aeromonas spp. Among Common Food Animals Slaughtered in Nagpur,Central India

Abstract

Aeromonads are ubiquitous foodborne pathogens with a global distribution. Animal-origin foods and contaminated animals are the main sources of Aeromonas infection to humans. So far little is known about the occurrence of Aeromonas spp. in food-producing animals in India. The present study was conducted to determine the prevalence and seroprevalence of Aeromonas species from 50 each of meat, blood, and sera samples collected from cattle, buffaloes, goats, and pigs slaughtered in and around Nagpur, Central India. Alkaline peptone water and ampicillin dextrin agar were used to isolate Aeromonas spp. An indirect enzyme-linked immunosorbent assay (ELISA) was standardized by use of whole-cell antigen (WC) and outer membrane protein (OMP) of Aeromonas hydrophila (MTCC 646). Aeromonads were isolated from 44 (22%) of the meat samples, and 1 (0.5%) from the blood samples. Seroprevalence by indirect ELISA-based WC antigen was estimated as 68% in cattle, 44% in buffaloes, 60% in goats, and 30% in pigs. OMP-based ELISA yielded a seroprevalence of 56%, 48%, 52%, and 22% in cattle, buffaloes, goats, and pigs, respectively. The results revealed that OMP-based ELISA and WC-based ELISA were in agreement with one another. Isolation along with high seropositivity demonstrates the presence of foodborne Aeromonas spp. in the Nagpur city of Central India.

Introduction

The genus Aeromonas is an important foodborne pathogen showing a worldwide distribution (Merino et al., 1995). Aeromonas spp. are Gram-negative, nonsporulating, facultative anaerobic and rod-shaped bacteria. The important pathogenic species are A. hydrophila, A. caviae, and A. veronii. Aeromonas spp. mainly cause gastroenteritis in immunocompromised patients (Janda and Abbott, 1998). These organisms may also cause peritonitis, cholangitis, meningitis, septic arthritis, osteomyelitis, myositis, ocular infections, urinary tract infections, pneumonia, and hemolytic uremic syndrome (Hochedez et al., 2010).

Aeromonas spp. are present in the normal microbiota of fish (Vivekanandhan et al., 2005) and may be present in other food animals such as shellfish, poultry, cattle, and pigs (Ibrahim and MacRae, 1991; Pin et al., 1994; Neyts et al., 2000; Yadav and Kumar, 2000). As a result, foods of animal origin and contaminated animals may play a significant role in the transmission of the aeromonads to humans (Igbinosa et al., 2012). Higher prevalence of Aeromonas spp. may be possible in the Indian slaughterhouses or food markets due to poor sanitation, and to hygienic and temperature menace (Yadav and Kumar, 2000; Vivekanandhan et al., 2005; Subashkumar et al., 2006). Although Aeromonas spp. have been reported from a number of sources in India (Vivekanandhan et al., 2005; Nagar et al., 2011), relatively few studies have assessed the prevalence of Aeromonas spp. in food animals and none have reported the seroprevalence of Aeromonas spp. To address this gap, the present study was undertaken to evaluate the prevalence and seroprevalence of Aeromonas spp. in cattle, buffaloes, goats, and pigs slaughtered in and around Nagpur city, Maharashtra, Central India.

Diagnoses by conventional cultural methods are time consuming and labor intensive. Immunological methods have an advantage over the conventional cultural methods. In the past, serodiagnosis has been done by use of various antigens (i.e., lipopolysaccharide [Thuvander et al., 1993], whole cell [WC], and outer membrane protein [OMP] [Sachan and Agarwal, 2002; Shome et al., 2005]). However, WC- and OMP-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Aeromonas spp. in food animals are still poorly studied (Arora et al., 2006). Hence, systematic work has to be done to understand the risk of Aeromonas transmission through food animals.

Materials and Methods

Sample collection

From December 2011 to July 2012, 50 meat and blood samples were collected from slaughtered cattle, buffaloes, goats, and pigs in Nagpur district, in the Vidarbha region of Maharashtra state, Central India.

Each meat sample (10 g) was randomly collected from the carcass (neck, loin, brisket, and flank) before fabrication in sterilized polyethylene sachets, and blood (10 mL) was collected from the same animal during exsanguination in sterilized test tubes. Meat and blood samples were processed immediately for isolation of Aeromonas spp. Blood samples were centrifuged (1,841 g, 5 min) and sera were collected in 1.5-mL centrifuge tubes and stored at −20°C in 0.01% thimerosal (Merthiolate™; Sigma Aldrich, Steinheim, Germany) until further analysis.

Isolation and identification of Aeromonas spp.

Meat samples (10 g) were aseptically homogenized with 90 mL of sterile normal saline solution, using a stomacher sterile blender (Lab Med, UK). One milliliter of homogenate was inoculated in 9 mL of alkaline peptone water (APW) broth for enrichment. Similarly, the blood samples were added with APW and incubated at 37°C for 18–24 h (Agarwal et al., 2003).

A loopful of enriched inoculum from APW was directly streaked on ampicillin dextrin agar plates. The plates were incubated for 24 h at 37°C. After incubation, the typical yellow small mucoid honey-drop–like colonies were observed. These colonies were presumptively identified as Aeromonas spp. The presumptive Aeromonas colonies were identified up to species level based on the selected biochemical tests by Abbott et al. (2003).

Serodiagnosis of Aeromonas infection

Preparation of WC antigen

WC antigen from the standard strain of A. hydrophila (Microbial Type Culture Collection Strain No. 646, MTCC 646) was prepared by the heat-killed method (Sachan and Agarwal, 2002). One hundred milliliters of brain-heart infusion (BHI) broth was inoculated with the standard strain of A. hydrophila and incubated in a shaker for 12 h at 37°C. Then the culture was heated at 100°C for 2 h, followed by centrifugation at 10,000 rpm for 10 min at 4°C. After centrifugation the cell pellet was collected and washed twice with sterile normal saline. Finally, packed cells were resuspended in 2 mL of normal saline and stored at −20°C until further analysis.

Extraction of OMP

OMP was extracted by sodium lauroyl sarcosinate (sarkosyl) treatment (Arora et al., 2006). The standard strain of A. hydrophila (MTCC 646) was inoculated in BHI broth and incubated in a shaker for 12 h at 37°C, followed by centrifugation at 9,727 g for 30 min at 4°C. After centrifugation, the cell pellet was collected and treated with 10 mM tris(hydroxymethyl) aminomethane buffer containing 0.3% (wt/vol) NaCl (pH 8.0). Then the cell pellet was sonicated 3 times at 10 Am (amplitude) for 45 s. The sonicated material was centrifuged at 10,000 rpm for 2 min. The supernatant was transferred to new tubes and centrifuged for 1 h at 67,973 g at 4°C. Resulting pellets of cell envelop suspensions were incubated overnight at 4°C with 3% (wt/vol) sodium lauroyl sarcosinate (Sigma-Aldrich, St. Louis, MO) in 10 mM Tris buffer. Finally, OMP was obtained by centrifugation at 17,000 rpm for 1 h. The cell pellet was washed twice with distilled water and stored at −20°C. The protein content of OMP was estimated by a modified Lowry's method (Schacterle and Pollack, 1973).

The WC and OMP were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). Thirty micrograms of WC and 20 μg of OMP were used for SDS-PAGE. Coomassie blue and silver stainings were used for characterization.

Raising of antisera against WC antigen

Hyperimmune serum was raised against WC antigen in an adult healthy rabbit (Sachan and Agarwal, 2002). The density of cells was adjusted with McFarland standard nephelometric tubes to 10⁹ cells/mL. Adult healthy rabbits weighing 2–2.5 kg were inoculated intravenously with 0.25, 0.5, 1, and 2 mL at 4-day intervals. The rabbits were test bled 14 days after inoculation.

Indirect plate ELISA

The indirect plate ELISA was performed to evaluate the samples (Engvall and Perlman, 1971). The ELISA test was standardized by a checkerboard analysis. In brief, polyvinyl microtiter plates (Nunc, Denmark) were coated with various cell concentrations of 10⁶–10⁷ cells/mL of WC antigen and 1–2 μg/mL of OMP and then incubated for 12 h at 4°C. Then 1:400 to 1:3200 dilutions of hyperimmune and healthy sera were added and incubated for 2 h at 37°C. Then 1:2000–1:8000 (for pig 1:20,000) anti-rabbit HRPO conjugate (Merck, India) was added and incubated for 1 h at 37°C. One milligram per milliliter solution of O-phenylene-diamine dihydrochloride (OPD) (Sigma-Aldrich) was used as a substrate. 2N H₂SO₄ was used to stop the reaction. The plates were read at 492 nm by an ELISA reader (Multiskan Go; Thermofisher, Finland).

Cross-reactions were carried out by coating the plates with 10⁷ cells/mL of Listeria monocytogenes (MTCC 1143), Vibrio parahaemolyticus (MTCC 451), V. cholera (MTCC 3906), and Staphylococcus aureus (MTCC 1144). The serum samples of cattle, buffaloes, goats, and pigs were screened according to the standardized conditions of WC- and OMP-based indirect ELISA.

Statistical analysis

Results were expressed as prevalence and corresponding 95% exact CI. The association between the results of the WC- and OMP-based indirect ELISA was assessed using Spearman rank order correlation coefficient.

Results

Isolation from food animals

Aeromonas spp. were identified in 44 (22%) of the 200 raw meat samples examined (Table 1). Out of 200 blood samples processed for isolation, only 1 blood sample was positive for A. hydrophila. This positive blood sample originated from a pig.

Table 1.

Prevalence of Aeromonas Species in Raw Meat Samples in Nagpur, India

		Aeromonas spp.		Aeromonas hydrophila		Aeromonas sobria		Aeromonas caviae
Source	Total samples processed	Positive	Percentage (95% CI)	Positive	Percentage (95% CI)	Positive	Percentage (95% CI)	Positive	Percentage (95% CI)
Beef	50	14	28 (16–42)	7	14 (6–27)	6	12 (5–24)	1	2 (0–11)
Carabeef	50	10	20 (10–34)	3	6 (1–17)	5	10 (3–22)	2	4 (0–14)
Chevon	50	8	16 (7–29)	1	2 (0–11)	5	10 (3–22)	2	4 (0–14)
Pork	50	12	24 (13–38)	4	8 (2–19)	1	2 (0–11)	7	14 (6–27)
Total	200	44	22	15	8	17	9	12	6

Serodiagnosis of Aeromonas infection

The checkerboard analysis of WC antigen showed that 10⁶ cells/mL showed an optimal reaction at 1:3200 dilution of anti-WC serum, in all species. Anti-species HRPO conjugate in cattle, buffaloes, and goats showed an optimal reaction at 1:8000. In pigs the optimal dilution was at 1:20,000. The OMP-based indirect ELISA conditions were standardized at 1 μg/mL. For all species, serum dilution was at 1:1600. HRPO conjugate for cattle, buffaloes, and goats was used at 1:4000. For porcine, conjugate was applied at 1:20,000.

Two hundred serum samples collected from cattle, buffaloes, goats, and pigs were screened by WC- and OMP-based indirect ELISA. 95% exact confidence intervals (CI) were considered to express the seroprevalence percentages. Based on the outcome, the overall seroprevalence of Aeromonas spp. using both antigens was 56% (41–70%) in cattle, 36% (23–51%) in buffaloes, 52% (37–66%) in goats, and 20% (10–34%) in pigs. The observations on serodiagnosis employing WC- and OMP-based ELISA were compared as illustrated in Table 2. The results of studies on cross-reactions with other organisms using anti-WC (1:3400) revealed negative reactions at 10⁷ cells/mL concentration.

Table 2.

Aeromonas spp. Seroprevalence in Serum Samples from Food Animals in Nagpur, India

		WC-based ELISA		OMP-based ELISA		WC- and OMP-based ELISA
Source	Total samples processed	Positive	Percentage (95% CI)	Positive	Percentage (95% CI)	Positive	Percentage (95% CI)
Cattle	50	34	68 (53–80)	28	56 (41–70)	28	56 (41–70)
Buffaloes	50	22	44 (30–59)	24	48 (34–63)	18	36 (23–51)
Goats	50	30	60 (45–74)	26	52 (37–66)	26	52 (37–66)
Pigs	50	15	30 (18–45)	11	22 (12–36)	10	20 (10–34)

WC, whole cell; ELISA, enzyme-linked immunosorbent assay; OMP, outer membrane protein.

Spearman rank correlation coefficient for the association between WC and OMP equaled 0.75 (p<0.001). This implies a significant association between the results obtained by WC and OMP.

Discussion

The genus Aeromonas is widely distributed both in aquatic and terrestrial environments. Recent years have witnessed motile aeromonads emerging as an important foodborne pathogen worldwide (Arora et al., 2006). Consumption of undercooked or raw meat or meat products is an important route of human infection with Aeromonas species (Igbinosa et al., 2012). In the present study, prevalence and seroprevalence of Aeromonas spp. were evaluated among commonly slaughtered food animals in Nagpur, India.

We obtained an overall prevalence of Aeromonas spp. of 22% in raw meat samples. These results are in accordance with other studies conducted in this region. Kolhe et al. (2004), Shinde et al. (2005), and Khan et al. (2008) reported 26%, 24%, and 19% prevalence among chevon, chicken, and milk samples, respectively. However, these results are lower than those of Hanninen et al. (1997) and Jaulkar et al. (2008), who reported 93% and 76% prevalence from fish samples. This can be attributed to the fact that Aeromonas spp. are more prevalent in aquatic environment than nonaquatic; water and aquatic animals are the main sources of human infection. Furthermore, the analysis of a small amount of raw meat may have reduced the probability of isolation of Aeromonas spp., and therefore, the prevalence of the pathogen may have been underestimated.

The investigation revealed that A. sobria was the dominant species followed by A. hydrophila and A. caviae with overall prevalences of 8.5%, 7.5%, and 6%, respectively. A higher prevalence of A. sobria (53%), A. hydrophila (11.5%), and lower prevalence of A. caviae (2.3%) were reported by Radu et al. (2003) among market fish samples. A variation in the prevalence is expected considering different geographical area, origin of the samples, sampling period, and methodology of analysis.

Serodiagnosis of an infection by employing specific antigen is a technique of choice for quick diagnosis and to check future spread of pathogen. WC-based assay as followed in the present investigation has also been reported by Yoshimizu et al. (1991). The authors applied the WC-based ELISA to detect antibodies against A. salmonicida in immunized fish as well as among wild matured salmonid fish in hatcheries in Japan. They reported it to be a practical and sensitive tool for screening of large number of wild fish with low antibody titers. Using the test, they reported seropositivity of 4–90% among fishes. The efficiency of the WC antigen by employing Dot-ELISA has also been mentioned by Shome et al. (2005) wherein the assay was used to screen 58 samples collected from 20 infected Indian major carps. The authors reported 32.7% samples positive.

Use of OMP as an antigen in the present study can be compared with Sachan and Agarwal (2002), who exploited antigenicity of OMP to raise antisera for its application in ELISA to detect Aeromonas spp. among spiked food samples. Furthermore, the high titer of antiserum against OMP was suggested to have an advantage of avoiding cross-reactions. In another study, Arora et al. (2006) proved OMP-based assay to have the ability to detect Aeromonas irrespective of type of strain involved.

The literature on direct application of the WC- and OMP-based ELISA to detect antibodies against Aeromonas in food animals are largely lacking, though the previous works of Sachan and Agarwal (2002) and Arora et al. (2006) exploited these antigens indirectly to detect Aeromonas pathogens in experimentally spiked samples. The present investigation in this direction seems to be systematic study with respect to the application of WC and OMP as antigen for detection of antibodies from animals under field conditions. The inclusion of large number of samples will be needed to revalidate the concept.

Association between WC and OMP was tested by Spearman rank order correlation coefficient, showing a significant association between results obtained by WC and OMP. OMP-based ELISA and WC-based ELISA were consonant with one another. However, revalidation requires inclusion of samples from clinical cases. Observation on the similar efficacy of OMP and WC directly as antigen to study seroconversions in host as observed in the present investigation could not be compared, as the studies in this area are lacking. However, their indirect application as antigen to raise hyperimmune sera for application in detection of experimentally spiked food samples proved to be equally useful (Sachan and Agarwal, 2002; Arora et al., 2006).

In order to study the role of antibodies and detection of pathogen in host, the isolation pattern of the Aeromonas was compared with that of detection of antibodies against the pathogen. Out of 200 food animals analyzed for the prevalence and detection of antibodies against Aeromonas, 14 cattle (28%), 10 buffaloes (20%), 8 (16%) goats, and 10 (20%) pigs exhibited positivity for both cultural isolation and presence of antibodies against Aeromonas spp.

The simultaneous positivity among both cultural isolation and antibodies can be explained on the basis of detection of antibodies from the fish in the symptomatic carrier phase of infection (Weber and Zwicker, 1979). In the present investigation, as the samples were from food animals that were apparently healthy without any clinical symptoms, the animals might have been in an asymptomatic carrier phase. However, a single animal showing positivity for isolation from pork, blood, and antibodies against OMP as well as WC confirms a bacteremic stage of the animal. The stage of active infection in this animal cannot be denied. However, infected animals presented for slaughter cannot be adequately handled by traditional inspection methods, as there are no pathognomic lesions associated with this organism.

The revalidation of simultaneous cultural and seropositivity status demands implementation of this parameter on clinical cases of Aeromonas infection. Nevertheless, the study confirms equal or similar efficacy of serodetection with that of isolation. Thus, WC- and OMP-based indirect ELISA will prove useful to analyze large numbers of samples for a quicker and better estimation of Aeromonas prevalence.

Conclusions

Our findings demonstrate the presence of Aeromonas spp. and the presence of antibodies against Aeromonas spp. in slaughtered food animals intended for consumption in Nagpur, India. The application of both WC and OMP as antigen for detection of antibodies from animals under field conditions showed that both antigens were consonant with each other. All culture-positive food animals were antibody positive, indicating that serology may be an alternative screening method to increase the capacity to analyze a large number of samples. Occurrence of these organisms in food animals should be considered an important threat to public health.

Footnotes

Acknowledgments

We would like to thank the Head, Department of Veterinary Public Health, Maharashtra Animal and Fishery Science University, Seminary Hills, Nagpur for providing necessary laboratory facilities.

Disclosure Statement

No competing financial interests exist.

References

Abbott

, Cheung

, Janda

. The genus Aeromonas: Biochemical characteristics, atypical reactions, and phenotypic identification schemes. J Clin Microbiol, 2003; 41:2348–2357.

Agarwal

, Bhilegaonkar

, Singh

, Kumar

, Rathore

. Laboratory Manual for the Isolation and Identification of Food Borne Pathogen. 1 ^st ed. Bareilly, UP, India: Indian Veterinary Research Institute Izatnagar, 2003, pp. 30–31.

Arora

, Agarwal

, Bist

. Comparison of ELISA and PCR vis-a vis cultural methods for detecting Aeromonas spp. in foods of animal origin. Int J Food Microbiol, 2006; 106:177–183.

Engvall

, Perlmann

. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunohistochemistry, 1971; 8:871–874.

Hanninen

, Oivanen

, Hirvela-Koshi

. Aeromonas species in fish, fish-eggs, shrimp and freshwater. Int J Food Microbiol, 1997; 34:17–26.

Hochedez

, Rapp

, Olive

, Nicolas

, Beaucaire

, Cabie

. Bacteremia caused by Aeromonas hydrophila complex in the Caribbean Islands of Martinique and Guadeloupe. Am Soc Trop Med Hyg, 2010; 83:1123–1127.

Ibrahim

, MacRae

. Incidence of Aeromonas and Listeria spp. in red meat and milk samples in Brisbane, Australia. Int J Food Microbiol, 1991; 12:263–269.

Igbinosa

, Igumbor

, Aghdasi

, Tom

, Okoh

. Emerging Aeromonas species infections and their significance in public health. J Scint Wld, 2012; 2012:1–13.

Janda

, Abbott

. Evolving concepts regarding the genus Aeromonas: An expanding panorama of species, disease presentations, and unanswered questions. Clin Infect Dis, 1998; 27:332–344.

10.

Jaulkar

, Katre

, Karikkathil

, Shinde

, Khan

, Chaudhari

, Zade

. Prevalence and antibiogram study of Aeromonas species from fishes. In: 7th Annual Conference of Indian Association of Veterinary Public Health Specialists and International Symposium on Quality Assurance & Global Trade: Concerns and Strategies, November, 7- 9, 2008, Pantnagar, India, p. 107.

11.

Khan

, Zade

, Shinde

. Prevalence of Aeromonas species in milk and milk products. R Vet J, 2008; 4:32–36.

12.

Kolhe

, Zade

, Shinde

, Karpe

. Prevalence of Aeromonas species in chevon and fresh water fishes. J Vet Pub Hlth, 2004; 2:55–58.

13.

Laemmli

. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (Lond), 1970; 227:680–685.

14.

Merino

, Rubires

, Knochel

, Tomas

. Emerging pathogens: Aeromonas spp. Int J Food Microbiol, 1995; 28:157–168.

15.

Nagar

, Shashidhar

, Bandekar

. Prevalence, characterization, and antimicrobial resistance of Aeromonas strains from various retail food products in Mumbai, India. J Food Sci, 2011; 76:486–492.

16.

Neyts

, Huys

, Uyttendaele

, Swing

, Debevere

. Incidence and identification of mesophilic Aeromonas spp. from retail foods. Lett Appl Microbiol, 2000; 31:359–366.

17.

, Marín

, García

, Tormo

, Selgas

, Casas

. Incidence of motile Aeromonas spp. Food Microbiol, 1994; 10:257–262.

18.

Radu

, Ahmad

, Ling

, Reezal

. Prevalence, and resistance to antibiotics for Aeromonas species from retail fish in Malaysia. Int J Food Microbiol, 2003; 81:261–266.

19.

Sachan

, Agarwal

. A simple enzyme-linked immunosorbent assay for the detection of Aeromonas spp. Veterinarski Arhiv, 2002; 72:327–334.

20.

Schacterle

, Pollack

. A simplified method for the quantitative assay of small amount of protein in biological material. Anal Biochem, 1973; 51:654–655.

21.

Shinde

, Zade

, Kolhe

, Karpe

. Prevalence of multidrug resistance Aeromonas species from chicken. J Bombay Vet Coll, 2005; 13:36–39.

22.

Shome

, Shome

, Gaur

. Rapid detection of Aeromonas hydrophila (Chester, 1901) infection in freshwater fish by Dot ELISA. Indian J Fish, 2005; 52:269–273.

23.

Subashkumar

, Thangavelu

, Govindhasamy

, Perumalsamy

. Occurrence of Aeromonas hydrophila in acute gastroenteritis among children. Indian J Med Res, 2006; 123:61–66.

24.

Thuvander

, Wichardt

, Reitan

. Humoral antibody response of brown trout Salmotrutta vaccinated against furunculosis. Dis Aquat Org, 1993; 17:17–23.

25.

Vivekanandhan

, Hatha

AAM

, Lakshmanaperumalsamy

. Prevalence of Aeromonas hydrophila in fish and prawns from the seafood market of Coimbatore, South India. Int J Food Microbiol, 2005; 22:133–137.

26.

Weber

, Zwicker

. Aeromonas salmonicida in Atlantic salmon (Salmosalmar). J Fish Res Board Can, 1979; 36:1102–1111.

27.

Yadav

, Kumar

. Prevalence of enterotoxigenic motile aeromonads in chicken, fish, milk and ice cream and their public health significance. S E Asian J Trop Med Public Hlth, 2000; 31:153–156.

28.

Yoshimizu

, Direkbusarakom

, Nomura

, Ezura

, Kimura

. Detection of antibody against Aeromonas salmonicida in the serum of salmonid fish by the enzyme linked immunosorbent assay. Fish Path, 1991; 27:73–82.