Bacterial Profile of Livestock Farms in South-East Nigeria

The unregulated practice of livestock production has endangered the public health sector through the multiplication and spread of bacterial pathogens. This study investigated the bacterial profiles of livestock farms in Aba, Umuahia, Okigwe and Mbaise in the Southeast - ern part of Nigeria. Air was sampled with passive sedimentation technique; water samples were collected randomly from the farm water sources while hand swabs from the farmers and feeds were collected with sterile swab sticks and containers respectively. Total heterotrophic bacterial count (THBC) was analyzed by pour plate method; total coliform count (TCC) was determined by membrane filter technique while total potential pathogenic bac terial count (TPPBC) was examined by growing the samples in some selective agar media. Of the four cities studied, Aba had the highest THBC (28.43±0.3×10 5 , 26.70±0.7×10 5 , 26.26±0.5×10 5 CFU/ml), TPPBC (17.47±0.5×10 5 CFU/ml and 20.02±0.5×10 5 CFU/ml) and TCC (24.06±0.4×10 5 , 17.93±0.6×10 5 and 22.36±0.4×10 5 CFU/ml) for pig, cow and poultry farms respectively while Mbaise had the least value. A total of thirteen (13) bacterial species were isolated in the study but, only Escherichia coli, Staphylococcus aureus, Klebsiella sp., Salmonella sp., Proteus mirabilis and Bacillus subtilis were commonly distributed in the four cities . Bacillus subtilis, Salmonella sp. and Staphylococcus aureus were isolated more in Okigwe, Aba and Umuahia respectively than Mbaise. Salmonella sp . (60.00%) had the highest occurrence followed by Staphylococcus aureus (55.33%) while Proteus mirabilis (4.50%) had the lowest occurrence. High bacterial loads were obtained in the study especially in Aba. Livestock farmers should consider proper hygienic measures in order to limit the spread of pathogenic bacteria among surrounding communities.


INTRODUCTION
Livestock farming is currently one of the leading agricultural practices in developing countries that is economically viable and performed both in urban and rural areas (Thornton, 2010). Urban livestock is a farming activity that is practiced within the urban centers. It is an important aspect that develops the urban areas. Apart from the economic impact on cities, urban livestock also produces negative effects for instance, increased health failure, environmental contamination and spread of diseases (Asadu et al., 2021). Livestock farming contributes to climate change which consequently affects the distribution of bacteria and other unhealthy chemical substances (Grossi et al., 2015). Livestock farms are agents of bacterial transmission and animal-related pathogens, especially the antibiotic-resistant strains. Livestock diseases are very significant worldwide based on their effects on the environment and inhabitants. These diseases produce direct effects on human and animal health and negatively affect the economy and food supply (Thornton, 2010;Gebreyes et al., 2020). Nigeria is geopolitically grouped into six zones including the South-East which is made up of Enugu, Anambra, Imo, Abia, and Ebonyi States. The occupation of the southeasterners is mainly trading, crop production and livestock farming (Nwanta et al., 2011). The zone has so many urban towns with growing populations such as Aba, Umuahia, Okigwe and Mbaise. A lot of urban agricultural activities take place in these towns especially rearing of sheep, goats and pig (Asadu et al., 2021). They are managed in both intensive and semi-intensive systems. Livestock farming generate animal protein for consumers and revenue to the sellers (Nwanta et al., 2011). Most livestock farmers largely venture into pig farming while 65% included poultry, and 31% indulge in goat and sheep production (Nwanta et al., 2011). These animals are sources of direct and indirect disease spread. Microbial pathogens are transmitted via excreta, urine and flesh of animal hosts. They can also be isolated from feeds, drinking water, rain splashes, feeding troughs and hands of farmers (Alegbeleye et al., 2018). Microorganisms are microscopic living organisms that survive either in their natural environment or in the body of both animals and humans. They are an important part of atmospheric particulate matter, water bodies, soil and are closely associated with human health. The growth and spread of these microbes are dependent on the available nutrients, hosts and locations (Köhl et al., 2019). Bacteria are the most highly bountiful microbes in livestock farms. Their ubiquity and survival mechanisms within and outside of the hosts make them most successful in disease transmission among other pathogens. They are distributed through mediums such as urine, faeces and hides of livestock and from their aerosols (Klous et al., 2016). Several strains of pathogenic bacteria such as Escherichia coli, Vibrio sp., Shigella sp. and Salmonella sp and https://journals.e-palli.com/home/index.php/ajaset Am. J. Agric. Sci. Eng. Technol. 6(3) [16][17][18][19][20][21][22][23]2022 Soil Quality Sampling Briefly, 2 kg of soil samples were collected in sterile polythene bags using soil auger at 30 cm depth (Bhat et al., 2011). The soil samples were collected from different locations in the pig farm, poultry farm and cow farm. Soil samples were placed on ice in a cooler box immediately after collection and transported to the laboratory for analysis.

Hand Swab Sampling
Hand swab samples were collected with sterile swab sticks from the hands of the livestock farmers and properly labeled. In the laboratory, 5 ml of normal saline was transferred into the swab sticks and allowed to stand for 10 min. Thereafter, ten-fold serial dilution (10 -1 -10 -4 ) was performed with the solution, and appropriate dilution inoculated on agar plates and incubated (Sampson et al., 2019).

Heterotrophic Bacteria
After ten-fold serial dilution, 1 ml from 10 -4 was pipetted onto NA plates in triplicates. The discrete colonies in each NA plate were counted and recorded in CFU/ml for water, soil, hand swabs and feed samples and CFU/ plate/hour for air sample. Plates with colonies between the ranges of 30-300 were counted. The heterotrophic bacterial count was recorded as total heterotrophic bacterial count (THBC).

Total Coliform Bacterial Count
Water TCC was performed using membrane filter technique according to method described by Harley and Prescott (2002) with slight modification. Briefly, after serial dilution, 100 ml of the water sample from 10 -4 dilution was transferred onto a membrane filter with pore size of 0.45 µm. After filtration, the absorbent paper was laid carefully on the MacConkey agar plate with sterile tweezers. The plates were incubated at 30 o C for 24 h. After incubation, total coliform bacterial colonies were enumerated with the help of a magnifying glass.

Soil
Ten-fold serial dilution (10 -1 -10 -7 ) was performed according to the method described by Adhikari et al. (2007) with slight modifications. Briefly, 10 g of soil was suspended into 90 ml of distilled water and mixture shaken properly. After serial dilution, 50 ml filtrate from 10 -4 was transferred onto a membrane filter (0.45 µm). The absorbent paper after filtration was transferred onto MacConkey agar plate and incubated for 24 h at 30 o C. For confirmation, inoculums from the MacConkey agar plate were inoculated in tubes containing 10 ml of lactose bile broth. The mixture was incubated for 24 h at 30 o C for fermentation to occur.

Hand Swab
A ten-fold serial dilution (10 -1 -10 -4 ) was conducted, some non-pathogenic bacteria have been isolated from air, water and soil (Ugbogu et al., 2016). Through human and animal activities, these bacteria are spread thereby resulting in infectious diseases (Ali et al., 2021). This study is aimed at determining the bacterial species that are predominant in the livestock farms in four cities of the southeast.

Study Area
The study was conducted in the four most populated cities in Nigeria's South-East region, namely Aba, Umuahia (Abia State) and Okigwe, Mbaise (Imo State). The selection of the cities was based on random sampling where livestock activities are predominant. The samples were collected from urban areas where livestock farming is vigorously practiced. These four cities stretch from latitude 4 o 50' to 7 o 20' N and longitude 6 o 51' to 8 o 20' E. It has common boundary with Benue State in the North, in the East it is bounded by Cross River and Akwa Ibom States, in the West by Delta State and River Niger (Kalu & Zakiora, 2019). The zone has diverse ecological variations and land mass of 22,525 km 2 (Madu, 2006). Its annual rainfall is between March and October while the dry season starts from November and ends in February (Kalu & Zakiora, 2019). The study was carried out from December 2019 to April, 2022. A total of 600 samples (air, water, soil, feeds) were collected from pig, poultry and cow farms including hand swabs of their keepers. Feeds were obtained only from poultry and pig farms in Aba, Umuahia, Okigwe and Mbaise while air, water and soil samples were collected from pig, cow and poultry farms in the four cities of the two states using scientific standards. Hand swabs of the livestock workers were also collected with sterile swab sticks in all the farms.

Air Quality Sampling
Passive air sampling was performed using settle plates. Freshly prepared nutrient agar (NA), blood agar (BA), Salmonella-Shigella agar (SSA), MacConkey (MCA) and Thiosulphate citrate bile salt sucrose (TCBS) plates were allowed to solidify and dry. The plates were exposed at the height of 1.5 m above the ground for 60 min at various locations in the poultry farm, cow ranches and pig farms. The samples were sealed, labeled appropriately, put inside sterile polythene bags, transported to the Laboratory, and incubated at 30 o C for 24 h for bacterial growth. The experiment was repeated in triplicate and expressed as CFU/plate/hour.

Water quality sampling
Water samples were collected in 2.5 litre plastic containers and transported to the laboratory for analysis (Ugbogu et al., 2016). Ten-fold serial dilution of the water samples collected from Aba, Umuahia, Okigwe and Mbaise were performed according to method described by Harley and Prescott (2002). thereafter 1 ml from the 10 -4 dilution was inoculated onto MAC plate using spread plate technique for 24 h at 37 o C.

Total Potential Pathogenic Bacteria
TPPB was enumerated with selective media selected for potential pathogenic bacteria. SSA for Salmonella sp. and Shigella sp.; TCBS for Vibrio cholera and Vibrio parahaemolyticus; EMB for Escherichia coli and Enterobacter aerogenes; MSA for Staphyloccocus aureus; Blood agar for Streptococcus pyogenes and MCA for Pseudomonas aeruginosa. For air samples, each plate was exposed to air for 60 min; aliquots from water, soil and feeds were seeded onto the media plates while hand swabs were inoculated onto the plates. After incubation for 24 h at 30 o C, the bacterial species were identified based on their colony appearances (Lama et al., 2013). The colonies on each plate were counted with magnifying glass.

Feed
Total heterotrophic bacterial count, total potential pathogenic bacterial count and total coliform counts were performed according to the method described by Onyeagba (2015) and Adhikari et al. (2007) with slight modifications.

Characterization and Identification of Bacterial Isolates
The bacterial isolates were characterized based on their colonial/cultural characteristics, macroscopic and microscopic appearances including elevation, margin, colour, size and surface texture and afterwards, biochemical reactions.
The isolates were further confirmed by culturing them in selective media. These approaches were done according to Onyeagba (2015). The results were compared with standard reference of Bergey's Manual of Determinative Bacteriology (Buchanan and Gibons, 1974).

Isolation and Maintenance of Pure Culture
The representative bacterial colonies were sub-cultured on freshly prepared nutrient agar plates by streaking. The pure cultures were sub-cultured onto nutrient agar slants in bijou bottles and incubated at 30 o C. After 24h, the slants were kept in the refrigerator at -4 o C for storage until further processing.

Statistical Analysis
The results were expressed as mean± SD using graph pad prism graphical statistical package version 5. The student t-test at p<0.05 was applied to assess the difference between the mean for variables in triplicates and twoway analysis of variance (ANOVA) for more than two variables followed by Bonferreni post hoc test.

Bacteria Profile of the Poultry Farm in the Four Southeastern Cities
From Table 3, the THBC varied between 9.53±0.8×10 5 and 26.26±0.5×10 5 CFU/ml; TPPBC was within the range of 9.86±0.4×10 5 to 20.20±0×10 5 CFU/ml while the values 9.97±0.8×10 5 to 22.36±0.4×10 5 CFU/ml were for TCC. The highest counts of THBC (26.26±0.5×10 5 CFU/ml) were obtained in Aba and Mbaise. TPPBC and TCC were more only in Aba (20.20±0.5×10 5 and 22.36±0.4×10 5 CFU/ml) respectively. The hand swabs of workers produced the highest count for TPPBC while highest amount of THBC and TCC appeared more in soil samples. Result for the enumeration of bacteria in poultry farm is shown in Table 3.

Distribution of Bacteria in Aba, Umuahia, Okigwe and Mbaise
Of the thirteen different bacterial strains isolated from the four cities, only six, namely Escherichia coli, Staphylococcus aureus, Klebsiella sp., Salmonella sp., Proteus mirabilis and Bacillus subtilis were commonly distributed among the four cities (Table 4) Percentage Bacterial Occurrence in Aba, Umuahia, Okigwe and Mbaise Salmonella spp (60.00%) obtained from Aba were significantly higher (p<0.01) than those obtained from Umuahia (25.00%), Okigwe (18.33%) and Mbaise (32.33%). Staphylococcus aureus (55.33%) in Umuahia was appreciably higher (p<0.05) than others (50.00%, 15.67% and 8.33%) obtained. In Okigwe, Bacillus subtilis (38.33%) obtained was significantly (p<0.01) higher than the other three cities 15.00% (Aba), 24.00% (Umuahia) and 19.00% (Mbaise). From the result, the livestock in Mbaise had the lowest percentage bacterial occurrence while Aba had the highest. Proteus mirabilis was the least isolated bacteria in all the cities while Salmonella sp., Klebsiella sp. and Escherichia coli were predominantly isolated. Result for the percentage bacterial occurrence of bacteria commonly distributed in Aba, Umuahia, Okigwe and Mbaise is presented in Figure  1. Values with different numbers as superscripts within a row for the same parameter are significantly different

DISCUSSION
Livestock farming is a major contributor to small and medium scale enterprises and has been advocated for at both the state and federal levels. The spread of bacteria in livestock farms has been a major challenge in public Total heterotrophic bacteria are the most multifaceted group of microorganisms with different nutritional and survival requirements. Some are primary colonizers while others are secondary invaders directly deriving their nutrients from the primary colonizers. The compositions of these microbes differ based on the location and nutrient available (Serves et al., 1996). According to World Health Organization (WHO, 2018), the standard bacterial count stipulated for drinking water is approximately 100 CFU/ml. Infectious diseases result when that quantity is exceeded. From this study, THBC was very significant in pig farm (Table 1) cow farm (Table 2) and poultry farm (Table 3) (28.43±0.5×10 5 ; 26.70±0.7×10 5 and 26.26±0.5×10 5 CFU/ml) respectively. The high values obtained could be attributed to water and soil pollution and improper disposal of waste generated from homes and industries (Ali et al., 2021). These wastes serve as https://journals.e-palli.com/home/index.php/ajaset Am. J. Agric. Sci. Eng. Technol. 6(3) [16][17][18][19][20][21][22][23]2022 breeding sites for bacteria (Agodo et al., 2016). During rainfall, splashes of water from these wastes percolate through underground water and affect the streams while the surrounding air become polluted. THB, TC as well as the hand swabs of livestock keepers were assessed from Air, water, soil and livestock. THBC and TCC were appreciably higher in soil followed by water than air, feeds and hands of keepers. Soil and water support the multiplication of these microbes due to availability of nutrients. High values of THBC and TCC in soil samples is as a result of their ability to tap the available nutrients in soil sediments more than water (Adhikari et al., 2007). According to Adhikari et al. (2007), the availability of nutrient and water retaining capacity of soil bacteria tend to increase their survival rate. Air particles do not have enough resources to sustain microbes; the chemical preservatives used in producing feeds inhibit the survival of bacteria (Soriano, 2020). The TPPBC were seen to increase among hand swabs (26.23±0.4×10 5 and 20.20±0.5×10 5 CFU/ml) of pig and poultry keepers respectively while soil sample (17.47±0.5×10 5 CFU/ml) had the highest TPPBC for cow farm. These increased values could result from improper handling of the excreta, feeds and drinking water of the animals (McAllister and Toppt, 2012) and due to frequent close contact of livestock farmers with their livestock in our area (Klous et al., 2016). Most pathogenic bacteria which colonize the skins and excreta of host animals can be transmitted as zoonotic pathogens to humans (Klous et al., 2016). Aba, from the results had the highest isolated THB, TC and TPPB. The increase in THBC, TCC and TPPBC in Aba can be attributed to the location of the pig and cow farms and the activities being performed. Aba River, where the research was conducted is a tributary of Imo River and all abattoir activities take place there. The river runs through two local governments in the State, and serve as sites for washing slaughtered animals and burning of hides (Ngozi and Humphrey, 2019). Of the sixteen bacteria isolated from the study (Table 4), only Escherichia coli, Staphylococcus aureus, Klebsiella sp. Salmonella sp., Proteus mirabilis and Bacillus subtilis were distributed in the four cities. The bacterial species isolated from the farms in the four cities were in agreement with Adogo et al. (2016). Percentage occurrence of Escherichia coli, Salmonella sp., Klebsiella sp., Staphylococcus aureus, Bacillus subtilis and Proteus mirabilis is shown in Figure 1. Salmonella sp. have the highest occurrence at 60.00% while the least occurring bacteria was Proteus mirabilis (4.50%). Bacillus subtilis appeared more in Okigwe than in all the other cities. Salmonella spp are Gram negative rods in the family of Enterobacteriacea and more than 2,500 serovars have been identified. They are ubiquitous bacteria that survive in dry environments and water bodies for a long time (WHO, 2018). The strains that inhabit the colons of pig and cow according to WHO, are invasive and life threatening to humans and become major concerns to public health. From the study, Salmonella species have the highest percentage occurrence especially in Aba. Due to lack of potable drinking water and good roads to assess it, the dwellers depend on the Aba River. They drink, bathe, cook and wash clothes with the water without proper treatment. These activities lead to increase in spread of Salmonella spp and Escherichia coli which subsequently cause waterborne diseases (Ali et al., 2021). The ability to survive in low moisture environments contributes to their widespread. They have high osmo-tolerant membranes, filamentous cells and strong metabolic process (Finn et al., 2013). In contrast, Proteus mirabilis recorded the lowest percentage occurrence. Proteus mirabilis is a motile bacterium that survives better in alkaline and urea-rich environments. It causes urinary tract infections, wound infections and kidney stones in humans (Zafar et al., 2019). Their low percentage values in all the four cities can be attributed to the fact that P. mirabilis is a human pathogen that is rarely transmitted and isolated in livestock except when a person comes in contact with poultry flesh and droppings (Nahar et al., 2014). The microbe has been reported to be transmitted through person-to-person contacts and food (Zafar et al., 2019). Bacillus subtilis is a spore forming bacterium that is air-borne. The formation of spores is a survival mechanism utilized by B. subtilis to thrive even in a challenging environment (Ravine, 2019). Only few literatures attributed B. subtilis to causing human diseases. They are reported to be beneficial in medicine as they can be used in producing probiotics, vaccines and enzymes (Piewngam and Otto, 2019;Sun et al., 2018). From the study, Okigwe had the highest percentage of B. subtilis. This could result from the economic activities carried out in the city. Livestock farming and crop production are major occupation of Fulani occupants; deliberate disposal of animal dungs, farm waste and pesticides could increase the spread of spores of B. subtilis (Jorgensen et al., 2015). Comparing the percentage of occurrence of bacteria in the four cities, it can be seen that Aba had the highest bacterial loads compared with Mbaise that had the least percentage. This could be attributed to the type of economic activity predominant in these two areas https://journals.e-palli.com/home/index.php/ajaset Am. J. Agric. Sci. Eng. Technol. 6(3) 16-23, 2022 (Okoro and Ibe, 2017). Aba is a metropolitan city with large population of residents having diverse culture and business inclinations. It is the center for commercial activity in the South-east. As a center for Small and Medium Scale Enterprise (SME) in Nigeria (Agu et al., 2019), small businesses that involve waste generation and degradation of ecosystem usually take place which lead to increase in the spread of bacteria and infectious diseases (Wizor, 2019).

CONCLUSION
Livestock farming has remained one of the veritable means of improving the livelihood of the Southeasterners. The challenge often experienced is the spread of pathogenic bacteria directly from the farm animals or the agents surrounding them such as air, water and soil. Of all the cities studied, Aba had the highest loads of bacteria. This calls for concerted effort by all stakeholders residing in the city and by extension the entire Southeast as the disease resulting from the spread of these microbes from Aba could reach to other South-east States. Holistic hygienic practices should be promoted by livestock keepers as health workers should routinely conduct inspection on those farms and markets where these animals are reared and consumed respectively.