PJZ_57_5_2009-2016

INTRODUCTION

The term poultry can be used for wide range of birds but in Pakistan, poultry generally refers to chickens (Krnjaja et al., 2008). Rapid population expansion in the 20th century resulted in increased demand of food that leads to the industrialization of different food sectors. About 40-45% of meat requirements in Pakistan are fulfilled from chicken. In Pakistan, poultry industry business first started in the 1960s and in 2018, it became the most organized branched of Agro-business, producing 18000 million eggs and 2250-million-kilogram chicken meat annually (Hussain et al., 2015).

With the development of the poultry industry, the production of poultry feed increased significantly and the industries offered different types of poultry feed either in the form of pellets or mash. Poultry feeds are formulated to meet the dietary requirements of chicken and can be categorized as chick feed, grower feed, broiler feed and layer feed. However, the safe food chain can be interrupted by the presence of microorganisms. Contaminated feed is a major source of transfer of these pathogens to poultry. These pathogens may become a source of diseases that include fowl typhoid, Newcastle disease, fowl pox diseases and other respiratory, or gastrointestinal diseases resulting in mortality of chickens. It is reported that human can become ill after consuming the chickens that were fed on contaminated feed (Yunus et al., 2009).

Different factors contribute to the contamination of feed. Different type of feed ingredients and animal products used in the poultry feed are a source of contamination (Lemme et al., 2004). Drying activity and storage of grains in poorly ventilated rooms also contribute to the contamination of poultry feed (Habib et al., 2015). Different microbes associated with poultry feed contamination include Pseudomonas, Yersinia (Waldroup, 1996), Listeria, Streptococcus, Clostridium, Staphylococcus, Enterobacter, Proteus, Bacillus (Bryan and Doyle, 1995; Obi and Ozugbo, 2007; Uwaezuoke and Ogbulie, 2010), Salmonella (Alshawabkeh, 2010; Uwaezuoke and Ogbulie, 2010), Shigella and Escherichia (Obi and Ozugbo, 2007; Chowdhury et al., 2011). Different fungi have also been associated with poultry feed i.e., Aspergillus (Cegielska-Radziejewska et al., 2013; Aliyu et al., 2016), Rhizopus, Mucor (Cegielska-Radziejewska et al., 2013; Okoli et al., 2007), Penicillium, Fusarium, Stachybotrys and Claviceps (Sobczak et al., 2016; Anifowose and Bakre, 2021).

Rapid identification of pathogens involve identification at the molecular level (Mandal et al., 2011). 16S ribosomal RNA gene amplification is used to identify the isolates, as it is present in all bacteria as a multigene family, its size is suitable for different kind of studies (Janda and Abbott, 2007) and it marks evolutionary distance and relatedness of organisms (Lagatolla et al., 1996). The present study was designed to check the poultry feed contamination in Pakistan with respect to viable, coliform, Staphylococcus, Salmonella and Shigella count and characterization and identification of the isolates by morphological (colony and cell characteristics), biochemical (Gram staining, Catalase, oxidase, carbohydrate fermentation test) and molecular means (16S rRNA gene sequencing). The feed samples were also checked for fungal contamination.

MATERIALS AND METHODS

Sampling

Total twenty-four samples (Chick feed, Grower feed, Layer feed, Broiler feed) were collected from Tollinton market (Lahore) and Pakpattan market (Pakpattan), factory areas in Lahore and rearing places of poultry in Pakpattan, Pakistan in sterilized bags and were processed in the laboratory.

Culturing

Different media; Nutrient agar (NA), Mannitol salt agar (MSA), Salmonella-Shigella agar (SSA), MacConkey Agar (MCA), Eosin Methylene Blue agar (EMBA) and Potato Dextrose Agar (PDA) were used for isolation of microbes. Samples were processed in duplicates. Samples (0.1 mL) were cultured from 10-4 dilution. Colonies were counted from 24 h incubated plates at 37oC and their colony forming unit (CFU/g) were calculated by using the formula:

CFU/g= Colony number × dilution factor/ volume plated (mL)

Morphological characterization

Morphological characteristics (color, shape, form, elevation, and margin) of isolated bacterial colonies were noted from 24 h incubated plates at 37 oC. Morphological characteristics of the fungal colony (color, surface, and texture) were noted from 3-5 days incubated plates of potato dextrose agar at 30 oC.

Biochemical characterization

Bacterial isolates were subjected to biochemical characterization using Gram staining, catalase and oxidase test. Sugar fermentation tests were performed with respect to glucose, maltose, sucrose and xylose (Holt et al.,1994).

Molecular characterization

DNA was isolated by using the method of Kronstad et al. (1983) and 16S rRNA gene was amplified by using the primers 27F; 5’-AGAGTTTGATCCTGGCTCAG 3’

and 1492R; 5’ GGTTACCTTGTTACGACTT 3’. PCR reaction mixture contained RNase treated genomic DNA (1.5 µL), 2.5 mM dNTPs (2.5 µL), 10 µM forward and reverse primer (1.25 µL each), 25 mM magnesium chloride (2.5 µL), IX PCR buffer (2.5 µL) and 2.5 units of Taq DNA polymerase (1 µL) in a 25 µL reaction mixture. The reaction mixture was incubated at 94oC for 5 min and then subjected to 35 cycles of 94oC for 30 sec, 54oC for 30 sec and 72oC for 90 sec; followed by one final cycle of 72oC for 10 min. PCR products were analyzed by using 1% agarose gel electrophoresis. The amplified gene was sequenced commercially and Blast tool of NCBI was used to find the highest similarity of 16S rRNA gene with related strains. The phylogenetic analysis was carried out using Mega X with bootstrap values (%) based on 1000 replications (Jukes and Cantor, 1969).

Statistical analysis

Total viable count (TVC), total coliform count (TCC), Total Staphylococcus count and total Salmonella and Shigella were statistically analysed using ANOVA and Duncan’s multiple range tests using Costat (Snedecor and Cochran, 1980).

RESULTS

Among the twenty-four samples tested, highest TVC (7.07 ± 0.17 log CFU/g) was obtained for chick feed while the layer feed had the least contamination (6.22±0.06 log CFU/g). Grower feed had TVC value (6.93 ± 0.06 log CFU/g) (Table I). Highest TCC (6.19 ± 0.18 log CFU/g) was found for broiler feed on EMBA while lowest count (4.30 ± 0.45 log CFU/g) was recorded for grower feed (Table I). Staphylococcus contamination was higher in broiler and chick feed with mean log CFU/g values of 6.89 ± 0.65 and 6.56 ± 0.64, respectively while in grower feed, mean log CFU/g value was 5.56 ± 0.04 (Table II). Three samples of layer feed were found positive for Salmonella (5.53 ± 0.36 mean log CFU/g). Shigella was found only in broiler feed (33% samples) with average mean log CFU/g value of 6.54 ± 0.78 (Table II).

Table I. Total viable and coliform counts.

Sample	Total samples	Total viable count		Total coliform count
		Total viable count		EMB agar		MacConkey agar
		Positive samples (%)	log Mean CFU/g ± SD	Positive samples (%)	Mean log CFU/g ± SD	Positive samples (%)	Mean log CFU/g ± SD
Chick feed	6	100	7.07 ± 0.17b	100	5.79 ± 0.73b	66	5.40± 0.37b
Grower feed	6	100	6.93 ± 0.06b	100	4.30 ± 0.45a	100	6.06 ±0.14c
Layer feed	6	100	6.22 ± 0.06a	100	5.99 ± 0.45b	100	5.25±0.56b
Broiler feed	6	100	6.34 ± 0.86a	100	6.19 ± 0.18c	83	4.53±0.74a

± indicates standard deviation among samples. Values carrying the same alphabet are not significantly different at (P < 0.05).

Table II. Total Staphylococcus, Salmonella and Shigella counts.

Sample	Total samples	Staphylococcus count		Total Salmonella count		Total Shigella count
Sample	Total samples	Positive samples (%)	Mean log CFU/g ± SD	Positive samples (%)	Mean log CFU/g ± SD	Positive samples (%)	Mean log CFU/g ± SD
Chick feed	6	100	6.56 ± 0.64b	0	-	0	-
Grower feed	6	100	5.56 ± 0.04a	0	-	0	-
Layer feed	6	100	6.25 ± 0.82b	50	5.53 ± 0.36	0	-
Broiler feed	6	100	6.89 ± 0.65b	0	-	33	6.54 ± 0.78

± indicates standard deviation among samples. Values carrying the same alphabet are not significantly different at (P < 0.05).

Morphological characterization results (color, shape, form, elevation and margin) of thirty-two different bacterial colonies isolated from twenty-four feed samples on different media (NA, MCA, MSA, EMBA, SSA) showed that colonies obtained on mannitol salt agar were yellow, circular, gelatinous, convex and entire; on eosin methylene blue agar, black circular, gelatinous, convex and entire colonies with green sheen were observed. On MacConkey agar, two types of colonies were found, one type being pink, circular, gelatinous, convex, entire and the second were light pink, punctiform, gelatinous, convex and entire. Two types of colonies were observed on Salmonella-Shigella agar, colorless colonies and colorless colonies with black centers (Supplementary Table I). Regarding biochemical characterization, isolates pfs4, pfs10, pfs16, pfs18, pfs29, pfs34 and pfs37 were catalase positive, oxidase positive, ferment glucose, maltose, sucrose and xylose but without gas production. Isolates pfs13, pfs17, pfs23, pfs30 and pfs38 were found catalase positive, oxidase negative, ferment glucose, maltose, sucrose and xylose but were negative for gas production. Isolates pfs5 and pfs35 were found positive for all biochemical tests except gas production. Isolates pfs6 and pfs36 were found oxidase negative, catalase positive, gave positive reaction for glucose, maltose, sucrose and xylose fermentation without gas production. Isolate pfs24 was found negative for gas production and for glucose fermentation while all other tests were positive. Isolates pfs72 and pfs65 were found catalase positive, oxidase negative and negative for xylose fermentation and gas production. Gram negative isolates pfs41, pfs46, pfs53 and pfs77 were oxidase negative, catalase positive and ferment glucose, maltose, sucrose and xylose with gas production, however isolate pfs103 was negative for sucrose fermentation and isolate pfs107 was found negative for gas production and did not ferment glucose and sucrose (Supplementary Table II).

16S rRNA gene was amplified for thirty-two isolates and was sequenced commercially. Sequence analysis results showed that feed samples were contaminated with different gram positive and gram-negative bacteria (Table III). Phylogenetic tree showed that isolates pfs5, pfs6, pfs10, pfs13, pfs16, pfs17, pfs18, pfs24, pfs29, pfs34, and pfs37 were closely related to Bacillus sp., isolate pfs65 showed highest similarity with Staphylococcus aureus, isolate pfs41 was related to Enterobacter cloacae, isolate pfs77 was closely related to Escherichia vulneris, isolates pfs46 and pfs53 were associated with Ralstonia sp. Isolates pfs103 and pfs107 were identified as Salmonella enterica and Shigella sp., respectively (Supplementary Fig. I).

Fungal isolates were obtained on PDA after 3-5 days of incubation at 30oC. Isolate PFF1 gave white colored colony which turned black on maturation; the colony was round and powdery while the back side of colony was pale yellow. Isolate PFF2 had yellowish green circular colony

Table III. Similarity between 16S rRNA gene sequence of isolated isolates with related isolates established on Mega Blast.

S. No.	Isolate	Accession No.	Related isolate	% age similarity
1	pfs2	MK033494	Bacillus methylotrophicus NR 116240	95
2	pfs4	MK170135	Bacillus subtilis NR 112116	100
3	pfs5	MK170134	Bacillus tequilensis NR 104919	99
4	pfs6	MK170133	Bacillus idriensis NR 043268	98
5	pfs8	MK170132	Bacillus humi NR 025626	96
6	pfs10	MK170131	Bacillus subtilis NR 112116	99
7	pfs13	MK170130	Bacillus haynesii NR 157609	99
8	pfs14	MK170129	Bacillus wiedmannii NR 152692	95
9	pfs15	MK170128	Bacillus wiedmannii NR 152692	96
10	pfs16	MK170127	Bacillus subtilis NR 112116	99
11	pfs17	MK170126	Bacillus haynesii NR 157609	99
12	pfs18	MK170125	Bacillus subtilis NR 112116	99
13	pfs23	MK170124	Bacillus haynesii NR 157609	99
14	pfs24	MK170123	Bacillus methylotrophicus NR 116240	99
15	pfs25	MK170122	Bacillus nakamurai NR 151897	97
16	pfs27	MK170121	Bacillus wiedmannii NR 152692	96
27	pfs28	MK170120	Bacillus wiedmannii NR 152692	98
18	pfs29	MK170119	Bacillus subtilis NR 112116	99
19	pfs30	MK170118	Bacillus haynesii NR 1576091	97
20	pfs34	MK170117	Bacillus subtilis NR 112116	99
21	pfs35	MK170116	Bacillus tequilensis NR 104919	99
22	pfs36	MK170115	Bacillus mojavensis NR 024693	97
23	pfs37	MK170114	Bacillus subtilis NR 112116	99
24	pfs38	MK170113	Bacillus haynesii NR 157609	97
25	pfs41	MK170112	Enterobacter cloacae NR 118568	100
26	pfs46	MK170111	Ralstonia syzygii NR 134150	99
27	pfs53	MK170110	Ralstonia pickettii NR 114126	99
28	pfs65	MK170109	Staphylococcus warneri NR 025922	100
29	pfs72	MK170108	Staphylococcus aureus NR 037007	99
30	pfs77	MK170107	Escherichia vulnaris NR 119109	99
31	pfs103	MK170106	Salmonella enterica NR 074910	98
32	pfs107	MK170105	Shigella dysenteriae NR 026332	97

that turned green on maturation. Isolates PFF1 and PFF2 had septate hyphae and globose spores with blackish brown and green color, respectively. Based on colony morphology and microscopic characteristics (Supplementary Table III), the isolated fungi PFF1 and PFF2 were identified as Aspergillus niger and Aspergillus flavus, respectively.

DISCUSSION

In the present study, commercially available poultry feed samples, collected from different locations of Pakistan, were analyzed using different media to determine TVC, TCC, total Salmonella count, total Shigella count and total Staphylococcus count. TVC in poultry feed ranged from 6.22-7.07 log CFU/g. Contamination of poultry feed was found in the range of 5.0 × 103 to 1.76 × 106 CFU/g in Ilorin, Nigeria (Sule and Ilori, 2017). Sultana et al. (2017) found the TVC of poultry feed (9.5×105 CFU/g) in Dhaka region, Bangladesh and in another study in Bangladesh, it was reported as 5.45×106 CFU/g (Chowdhury et al., 2011). Pavlovic et al. (2019) reported average bacterial contamination (1.95×104 CFU/g) in feed for laying hens from the Serbian market.

EMBA medium gave efficient growth of coliforms. The coliform count was found in the range of 4.30 – 6.19 log CFU/g. Previously reported coliform counts ranged from 0 - 3.0×105 CFU/g (Sule and Ilori, 2017), 0 - 6.75×104 CFU/g (Chowdhury et al., 2011) and 2.68×103 - 3.15x104 CFU/g (Sultana et al., 2017). Staphylococcus count in the poultry feeds ranged from 5.56-6.89 log CFU/g. Salmonella and Shigella contamination was in the range of 0-5.53±0.36 log CFU/g and 0-6.54±0.78 log CFU/g, respectively. Chowdhury et al. (2011) found the Salmonella contamination rate from 0 to 3.05×104 CFU/g in poultry feed samples collected from Dhaka, Bangladesh. In contrast, Salmonella and Shigella were not reported in the poultry samples collected from Nigeria (Sule et al., 2017).

According to the Bergey’s manual, Bacillus subtilis strains have biochemical characteristics similar to those obtained for isolates pfs4, pfs10, pfs16, pfs18, pfs29, pfs34 and pfs37. Biochemical properties of isolates (pfs13, pfs17, pfs23, pfs30 and pfs38) were found similar to the biochemical characteristics reported for B. haynesi by Dunlap et al. (2017). Biochemical characteristics of isolates (pfs5 and pfs35) matches with B. tequliensis (Gatson et al., 2006). Isolate pfs8 gave biochemical properties similar to B. humi (Heyrman et al., 2005). Results of biochemical characteristics of isolates pfs6 and pfs36 were in accordance to the results of Ko et al. (2006) and Roberts et al. (1994), who reported similar properties for B. idriensis and B. mojavensis. Biochemical characteristics of isolate pfs24 matches to B. methylotropicus (Madhaiyan et al., 2010). Isolates (pfs72 and pfs65) gave similar biochemical results as reported by Olugbojo and Ayoola (2015) for Staphylococcus. Gram negative isolates (pfs41, pfs46, pfs53, pfs77, pfs107) gave biochemical characteristics similar to the gram-negative isolates (E. coli, Salmonella sp. and Shigella sp.) obtained by Olugbojo and Ayoola (2015). Phylogenetic analysis based on 16S rRNA gene sequences showed that poultry feed samples were contaminated with bacteria belonging to Salmonella enterica, Staphylococcus aureus, Enterobacter cloacae, Bacillus haynesii, Bacillus subtilis, Shigella sp., Escherichia sp. and Ralstonia sp.

More contamination was found in chick and grower feed. These results are in accordance with Xiulan et al. (2006) who stated that chick feed and grower feed had high contamination due to high protein content. These findings are also in accordance with Yunus et al. (2009) who found that diseases were more common in chicks which were fed on commercially available feed as compared to home mixed feed. 100% samples were found positive for Bacillus and Staphylococcus. The results are in accordance to the results of Roy et al. (2017), who reported Staphylococcus as the most abundant pathogenic bacteria found in poultry feed samples collected from Dhaka city, Bangladesh. Staphylococcus has been found to be associated with bumblefoot disease and arthritis in poultry (Nazia et al., 2015). Salmonella was found in layer feed samples. This is in accordance to Shirota et al. (2000) who stated that layer and broiler feed were more susceptible to Salmonella. Salmonella is responsible for salmonellosis in poultry. Salmonella serotypes have great ability to adapt in number of host environments and diverse routes of transmission so can cause chronic and acute infections in almost all kind of birds and animals (Kwiatek and Kukier, 2008). Shigella was found in 8.3% samples. It is reported to be associated with gastrointestinal infections in poultry and humans (Baker and The, 2018). Enterobacter is normal inhabitant of gut flora but it can become opportunistic pathogen in immune compressed human and can cause different kinds of respiratory and gastrointestinal infections (Keller et al., 1998). Enterobacter cloacae was the most common bacterial species identified (54.5%) followed by Bacillus cereus (27. 3%) and then Klebsiella pneumoniae (18.2%) (Mahami et al., 2019). Ngai et al. (2021) reported E. coli and Salmonella in the poultry feed samples from Kenya. Gram-negative bacteria, Escherichia vulneris is closely related isolate of E. coli. It can cause gastrointestinal, respiratory and urinary infections (Jain et al., 2016). Ralstonia is soil-inhabiting bacteria and its occurrence in poultry feed samples indicates contamination of feed with soil particles (Ryan et al., 2007).

Fungal contamination was comparatively less than bacterial contamination. Only 8.3% samples were positive for fungal contamination. Observed colony and microscopic characteristics of fungal isolates were also supported by the characteristics studied by Pathak and Narulu (2013) and Hedayati et al. (2007). Contamination of poultry feed samples with Aspergillus had also been reported in Nigeria (Uwaezuoke and Ogbulie, 2010; Anifowose and Bakre, 2021), Bangladesh (Saleemi et al., 2020), Poland (Cegielska-Radziejewska et al., 2013), Egypt (Laban et al., 2014), Kaduna state (Habib et al., 2015), Skoto (Aliyu et al., 2016), Iraq (Alkhursan et al., 2021) and Saudia Arabia (Gherbawy et al., 2020). Presence of Aspergillus is a great threat to poultry as well as other consumers of poultry. Aflatoxins produced by fungi are found to be associated with sinusitis, keratitis, cutaneous aspergillosis, and systemic infections in individuals having weak immune system. Aflatoxins can also cause cutaneous Aspergillosis in other vertebrates (Hedayati et al., 2007). These aflatoxins also contribute to the economic loss of poultry industry as they weaken the immune system of chicks and interfere with the reproductive power of chickens (Habib et al., 2015).

CONCLUSION

Commercialization of the poultry industry has increased risks to human health in the absence of adequate supervision and care taken during the production of poultry feeds. Contamination of poultry feed with microbes not only causes animal health problems but can also pose health risks to humans. Poultry feeds must therefore be introduced into the market after adequate experimental analysis and certification to avoid health risks associated with poultry products.

DECLARATIONS

Acknowledgement

Authors acknowledge Department of Biological Sciences, Virtual University of Pakistan for providing the resources and laboratory facilities.

Funding

The study received no external funding.

Ethical statement

The study was conducted adhering to all the lab safety protocols.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20231129053939

Statement of conflict of interest

The authors have declared no conflict of interest.

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