Salmonellosis is a perilous threat to livestock and public health, caused by Salmonella spp. These organisms are motile (exclude S. pullorum and S. gallinarum), gram-negative, rod-shaped, non-spore forming, non-capsulated, facultative anaerobic bacteria belongs to family Enterobacteriaceae (Agbaje et al., 2011). Being that, Salmonella is consistently among the leading birthplace of food-borne illness throughout the world. More than 36,000 serotypes were described and named are considered potentially pathogenic (Jackson et al., 2013). Although this may be true that some serotypes are host-specific, but the majority can affect multitudinous hosts (Uzzau et al., 2000). The primary reservoir of Salmonella is the intestinal tract of humans and animals, particularly in poultry and swine. Contaminated meats, mainly from avian and livestock origins are the prospective source of human salmonellosis therefore the most important fountainhead of meat-borne public health hazard (Buncic et al., 2014, Kabir, 2010). Previous study in Nepal by Dhakal and Manandhar (2005) isolated 12 percent Salmonella from the chicken litter, food and water on poultry farm in Chitwan District. Among them, 59 percent isolates were found Salmonella spprelevant to public health importance. However, the full extent of the social and economic impact is hard to measure due to under reporting of cases (Omurtag et al., 2013). Analogously, Nepal is also caught in the same web of under reporting system even of increasing cases of food-borne illness. On the other hand unhygienic shed, farm, slaughter spot, meat shop, equipments, environment, skin and viscera utilization habits seem to be more likely responsible for Salmonella contamination (Betancor et al., 2010). Although plenty of research on Salmonella has been reported, after all most of the authors were focused on the prevalence of Salmonellosis. Yet, there is a dearth of information regarding the prevalence, types of serovars and antimicrobial susceptibility of Salmonella from retail meats in Nepal. Therefore, this study was focused on the degree of contamination of livestock (chevon, pork, buff) and chicken meat with Salmonella, distribution of serotype and anti-microbial resistance in the Pokhara valley.

 

The study was carried out under the Lekhanath Municipality and Pokhara sub-Metropolitan city of Pokhara Valley in Nepal. In Lekhanath Municipality and Pokhara sub-Metropolitan city, altogether, there are 425 meat shops in the valley of pork (n=23), chicken (n=271), chevon (n=50) and buff (n=81). A total of 320 meat samples were collected from the mentioned sources (80 from each of four animal species). The sample size was calculated by using the formula mentioned by C. R. Khothari (Kothari, 2004).

 

About 25 gm raw meat sample was randomly collected from different slaughter area. The sample was collected in sterile plastic container at morning, and shifted immediately to the Regional Veterinary Laboratory for further investigation.

 

Immediately after arrival of sample at the laboratory, it was reweighed 25 ± 0.5 gm, and kept in the sterile blender jar to mix with 225 ml of buffer peptone water (BPW). Subsequently about 10 ml of each blended sample was incubated at 37?C for 24 hours and then 1 ml of this pre-enriched suspension was mixed with 10 ml Tetrathionate (TT) (Hi Media Laboratories Pvt. Ltd, India) broth and incubated at 42?C for 24 hours.

 

A loop full of culture from TT broth was inoculated both onto Xylose-Lysine Tergitol- 4 (XLT4) (Hi Media Laboratories Pvt. Ltd, India) and sulfa supplemented Brilliant Green (BG) agar (Hi Media Laboratories Pvt. Ltd, India) plates then incubated at 37?C for 24 hours. After the recommended incubation intervals, the selective differential agar plates were examined for the presence of typical colonies as per manufacturer instruction. The typical Salmonella colonies were again inoculated onto nutrient agar (NA) (Hi Media Laboratories Pvt. Ltd, India) plates for getting homogenous pure colonies. Purity of the isolates were confirmed my Gram’s staining.

 

Eight different biochemical tests (Triple Sugar Iron, Lysine Iron Agar, Simmons Citrate utilization, Urea utilization, Methyl red, Voges–Proskauer, Catalase, and oxidase tests) were performed for the identification of the Salmonella (Edwing, 1986). The isolates having Salmonella positive biochemical profile were again tested for sugar fermentation to identify different species. For Sugar fermentation tests, eight different sugars were used: Maltose (mal), Rhamnose (Rha), Arabinose (Ara), Inositol (Ino), Dulcitol (dul), Salicin (Sal), Glucose (Glu) and Mannitol (Man).

 

Pure colonies from NA plates were transferred to a para well glass slide with the help of sterile stick. Nearly 1-2 drops of Poly ‘O’ Sera (Difco^TM, USA) were dropped in Para wail glass slide then colony was emulsified properly and observed for agglutination. The result was noted according to the manufacturer instructions.

 

All positive isolates (n=26) were tested to identify the distribution of serotype in meat in the study area. Serotyping was performed at Walter Reed/Afrim’s Research Unit, Nepal (WARUN-USA) by using Salmonella O polyvalent and VI antisera (S &A reagents lab Ltd., Thailand). Serotyping test sequence for isolates is given in Table 1.

All isolates were tested for susceptibility to antimicrobial agents on Mueller-Hinton agar by modified Kirby-Bauer disk diffusion method as recommended by the Clinical & Laboratory Standards Institute (CLSI, 2007). The diameters of the zone of inhibition were measured, including the diameter of disc by using HI Antibiotics Zone Scale-c. Antibiotic resistance results were classified according to their inhibition zone Salmonella relative chart of zone sizes (Inhibited zone in mm), chart reproduced from sixth report of a WHO expert committee.

 

Data were entered to the spreadsheet of Microsoft office Excel 2007. Descriptive statistics were used to describe the result of prevalence rate and self evaluation model. Results of sensitivity test of different antibiotics on tests meat type were analysed by M stat-C uses single factor CRD. The comparative study of media was tested by using χ² test.

 

In this study, 320 meat samples from livestock and poultry (broiler) were collected from different slaughter spot of Pokhara valley, Kaski District, Nepal. Equal number of sample (80) was collected from each species. All slaughter spot’s name and address are presented in appendages as coding symbols (Appendix 6, Supporting file 1).

 

The XLT4 and BGA were used for the isolation of Salmonella from meat samples. The S. typhisuis on XLT-4 shows yellow colonies with black centre but S. choleroisuis shows red colonies only. Similarly, all other Salmonella spp., except S. typhisuis and S. cholerosuis shows red colony on BGA and red colony with black centre on XLT-4. The BGA rejected a significant number of samples in chicken as compared to XLT4 (Table 3) but, highly significant rejection of samples by BGA was found in chevon, buff, pork and also in total samples. A total of, 194 samples were conformed as positive based on the colonial morphology (Table 2).

Out of 320 samples 26 was found to be positive for Salmonella organism that is accounted by 8.13% prevalence (Table 5). Species wise prevalence was 7.5% (6 of 74), 10% (8 of 72), 5% (4 of 76) and 10% (8 of 72) in chevon, buff, pork, and chicken meat respectively. Comparison of meat with Salmonella spp., test on Salmonella O polyvalent and antisera shows that S. typhi had a higher prevalence in the buffalo, goat and chicken meat followed by S. enteritidis which has a significant role to cause human Salmonellosis (Table 4, 5, 6 and 7)

Out of 26 Salmonella positive isolates, 24 were susceptible to chloramphenicol, 23 gentamicin and ceftriaxone, 13 cotrimoxazole, 5 erythromycin, and only 3 were to oxytetracycline. The number of isolates resistant to erythromycin was 20, oxytetracycline (19), cotrimoxazole (7), gentamicin (3), chloramphenicol (2), cetriaxone (1) and none of the isolates were resistant to Ciprofloxacin (Table 8). The detail antibiotic susceptibility pattern is demonstrated in Table 9. Single factors complete randomized block design analysis showed that ciprofloxacin is the most susceptible antibiotic (Table 10). Ceftriaxone is equally susceptible to the isolates from buff, chevon and pork but not chicken. In case of pork, ciprofloxacin, ceftriaxone, chloramphenicol, gentamicin and cotrimoxazole have no significant difference. Erythromycin and oxytetracycline are less sensitive compared to others.

Management practiced in slaughterhouse influences greatly on microbial contamination as well as their concentration in meat. We consider nine important point (Floor type, Water supply, Slaughter species, Cleanness, freezing, House management, Chop board, Store, Viscera) during the regular observation of slaughterhouse (Table 11). Floor types have three categories, where the number of cemented and mud floor are more than tile floor. Daily cleaning of chop board was rare. In case of water used, they use drum water by 50%, which is a major source of microbial contamination. Freezing also lacking in some shop, which is a compulsory tool for meat preservation. Likewise, 50% of meat shop had no glass or mesh cover and situated near by dusty and busy road. Where, sources of contamination might be the dust, flies and possibly others many objects. Half of the observed meat shops were in same building.

Meat is an essential part of the human diet, and it also contains a generous supply of nutrients conducive to the growth of bacteria (Dave and Ghaly, 2011). The contamination of livestock and poultry meat is very much dependent on the status of the animals and birds prior to slaughter and on operational hygiene during meat processing. During processing, meat is exposed to contamination from the outside of the animal and bird, potentially the intestinal contents. The load of contamination at a given time depends on the handling, storage time and temperature (Fuzihara et al., 2000).

 

The overall prevalence of Salmonella was 8.13%, and specifically, the prevalence was 7.5%, 10%, 5%and 10%in goat, buffalo, pig and chicken meat respectively. The lowest prevalence was found in samples collected from pig meat and highest in buffalo and poultry meat. A similar higher prevalence of Salmonella was stated in buffalo meat (13.5%) and chicken meat (14.5%), whereas lower in goat meat (3.2%) at Kathmandu (Maharjan et al., 2006). In China, chicken (54%), pork (31%), beef (17%)and lamb (20%) samples were reported positive to Salmonella (Yang et al., 2010). Likewise, a high prevalence (35.83%) of Salmonella was reported from retail chicken meat in Spain, where, predominant serovars were S. enteritidis (47.88%) and S. hadar (25.35%) (Dominguez et al., 2002). Highest prevalence of Salmonella in chicken and buffalo meat might be due to poor quality water used during dressing from nearby contaminated water tank.

 

From the Greater Washington, D.C., area the prevalence was recorded in chicken, pork and beef as 4.2%, 3.3% and 1.9% respectively. One study on Irish retail pork shop showed the prevalence of 2.6% in pork meat (Prendergast et al., 2009), whether our findings were nearly double may be due to the poor condition of slaughterhouse. These differences in prevalence of Salmonella might be due to several factors such as differences in origin, variation in sample processing, sampling procedure, contamination level of animals, husbandry, slaughterhouse sanitation, cross contamination of the products, and differences in methodology applied to detect the pathogen.

 

In this study, two selective agars were used to isolate Salmonella. Out of 640 (320 in XLT-4 and 320 in the BGA) totals collected and processed samples, only 463 were analyzed because 177 were rejected due to either failure to grow on media or beyond the character of Salmonella, each sample was inoculated in both media. Among the 177 rejected samples, 131 were from BGA and 46 from XLT-4 media. Overgrowth of nuisance or contaminating organisms can be a major problem when recovery of a specific organism or species is desired. This is particularly true of Salmonella isolation media where overgrowth of Proteus, Providencia and Psudomonas can dramatically interfere with the detection of Salmonella (Miller et al., 1991). Moreover, the Pseudomonas aeruginosa strain can give false-positive results on BGA agar (Schönenbrücher et al., 2008).

 

Food contamination with antibiotic-resistant bacteria can be a major threat to public health, as the antibiotic resistance determinants can be transferred to other bacteria of human clinical significance. The prevalence of antimicrobial resistance among food-borne pathogens has increasing from decades to decades (Khabbaz et al., 2014). The use of antimicrobials for prophylaxis in food producing animals has been an important factor in the emergence of strains with resistance to certain antimicrobials (Andersson and Hughes, 2014).

 

In this study, majority of the isolates were sensitive to ciprofloxacine followed by chloramphenical, gentamicin and cetriaxone. Oxytetracycline and erythromycin have a greater resistance than that of remaining antibiotics. Resistance to these antimicrobials may be due to frequent and traditional use of these antibiotics in animal for treatment of infections, for prophylaxis and use as growth promoters in sub therapeutic doses in animal feed. A high percentage of Salmonella isolates were reported resistant to antibiotics, including nalidixic acid (82%), tetracycline (69%), trimethoprim (63%) and streptomycin (52%) from chicken and beef samples of Tehran, Iran (Dallal et al., 2010). In Northern Vietnam, resistance to at least one antibiotic agent was detected in 78.4% of isolates and the most frequent resistance were to tetracycline (58.5%), sulphonamides (58.1%), streptomycin (47.3%), ampicillin (39.8%), chloramphenicol (37.3%), trimethoprim (34.0%) and nalidixic acid (27.8%) and 100% sensitivity was found to ceftazidime from the chicken and pork meat (Thai et al., 2012). In Seoul, South Korea the highest antibiotic resistance was observed to erythromycin (100%) followed by streptomycin (22.2%), tetracycline and chloramphenicol (16.7%), samples from chicken meat, beef, and pork collected from wholesale markets, retail stores, and traditional markets (Hyeon et al., 2011). These trends of increasing multi-drug resistance constitutes a potential source of transmission of resistant strains to human and pose a problem of public health issue.

 

In a study carried out in Katmandu, Nepal reported that environmental sanitary conditions of meat shops were not satisfactory and standards (Maharjan et al., 2006). Out of 70 slaughtering places, the environmental sanitary conditions of 14.35% were good whereas 70% and 15.7% were average and poor respectively. In this study, we surveyed 33 slaughter spots. Based on their management, none of them were fulfilled their minimum requirement of sanitation. Lack of knowledge of butchers, lack of water and bad sanitary conditions in slaughter places are the plausible reasons for contamination of Salmonella in livestock and poultry meat of Pokhara valley. Similarly confirmation of the circulation of antibiotic resistant and biofilm forming pathogens were detected in raw meat and its environment in retail shops of Pakistan (Ali et al., 2010). Therefore, professional training for butchers is an essential requirement for achieving global food safety goals (Gomes-Neves et al., 2011).

 

The present study revealed that XLT-4 is an excellent culture medium for isolation of Salmonella. Out of 26 positive samples 4 serotypes of Salmonella (A, B, D and E) were revealed. The only D serotype was obtained from buff and chicken meat, however serotypes D and E from chevon and serotype A, B and D was revealed from pork. The presence of S. typhi and S. enteritidis is a great threat to human health. The S. typhi, S. enteritidis, and S. pullorum having the 30.77%, 23.10% and 19.23% prevalence respectively. The S. dublin, S. anatum, S. chleraesuis, S. typhisuis S. paratyphi S. derby and S. gallinarum were showed their presence only in 3.85%, but majority of them are also equally important for human illness. Ciprofloxacin was having 100% susceptibility of all the isolates of Salmonella in different species of meat samples. From the study, it can be concluded that the retail poultry meat of Pokhara valley poses a great risk for consumers’ health. A basic requirement of slaughter spots were not maintained properly. Mix with two or more meat species in a single stall would be the cause of presence of host adopted Salmonella in others species meat. However, condition could be improved by implementing a HACCP system along with Good Hygienic Practices (GHP) and Good Manufacturing Practices (GMP) from the production sector to consumption table.

 

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Appendices