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Haematology of Birds and Microbial Analysis of Recycled Poultry Litter Treated with Graded Levels of Aluminium Sulphate (Alum)

The study was carried out at the poultry unit of the Department of Animal Science teaching and research farm, Ahmadu Bello University, Zaria to determine the Chemical and Microbial Analysis of Poultry Litter Treated with Graded Levels of Aluminium sulphate (Alum). The alum used was obtained from the Sabon-garimarket in Zaria, Kaduna State. Aluminium sulphate (alum) was applied to the wood shavings by mixing it with alum thoroughly using hands covered with hand gloves. The rates of alum application was as follows: T1 control (normal poultry litter with no alum), T2 (5% alum by kg weight of poultry litter), T3 (10% alum by kg weight of poultry litter) and T4 (15% alum by kg weight of poultry litter). Five sets of litter samples were obtained fortnightly from each pen from different locations i.e. the four corners and centre from which the microbial load were measured. At the termination of the experiment (day 56), two birds from each pen having representative weights for the group (6 birds per Treatment) were selected and 1.5ml of blood was taken via the wing vein. Haematological parameters were analyzed by an auto haemo-analyser (BC2800 vet auto haemo analyser) at the Clinical Pathology Laboratory, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria. This result shows significant (P<0.05) difference among all the parameters analysed except MCHC. The result shows a significant decrease in the total bacteria, E. coliand Salmonella spp. in the alum treated litter groups (5%, 10% and 15% alum Original Research Article Usman et al.; JALSI, 24(8): 23-30, 2021; Article no.JALSI.77535 24 treated litter) compared to the control group (0% alum treated litter), the mould and yeast load was significantly increased in alum treated litter groups (5%, 10% and 15% alum treated litter) compared to the control group (0% alum treated litter).The study conclude that treating recycled poultry litter with alum can reduce microbial load of the litter. Birds reared on recycled poultry litter have significantly higher haemoglobin and PCV compared to the control.


INTRODUCTION
Chicken litter is a mixture of faeces, wasted feeds, bedding materials, and feathers [1]. Over 14 million tons of chicken litter is produced every year in the US, most of which is usually recycled and spread on arable land as a low cost organic fertilizer [2]. Poultry manure contains significant amounts of nitrogen because of the presence of high levels of protein and amino acids. Owing to its high nutrient content, poultry litter has been considered to be one of the most valuable animal manure as organic fertilizer [3]. Chicken litter is also the source of human pathogens, such as Salmonella, Campylobacter jejuni, and Listeria monocytogenes, which can potentially contaminate fresh produce or the environment and are frequently associated with foodborne outbreaks [4,5]. Although the application of poultry litter for commercial farming has rarely been associated with food borne outbreaks, enhanced consumer awareness of food safety issues has increased the scrutiny of agricultural practices [6]. Microbial concentrations in chicken litter can reach up to 10 10 CFU/g, and Grampositive bacteria, such as Actinomycetes, Clostridia/Eubacteria, and Bacilli/Lactobacilli, account for nearly 90% of the microbial diversity [7]. Pathogens in chicken litter represent the major group of bacteria of special interest to litter processors. A variety of pathogens can be found in chicken litter or chicken litter-based organic fertilizers, such as Actinobacillus, Bordetalla, Campylobacter, Clostridium, Corynebacterium, Escherichia coli, Globicatella, Listeria, Mycobacterium, Salmonella, Staphylococcus, and Streptococcus [7]. Alum treatment has been widely used to reduce pathogens before land application of chicken litter [8], and it can also be applied as an effective way to reduce ammonia volatilization and water-soluble phosphorus runoff from poultry litter in chicken houses [9]. Pathogens in poultry wastes, such as Salmonella and Campylobacter, have been shown to be reduced significantly or eliminated by alum treatment [10]. [8] Used denatured gradient gel electrophoresis (DGGE) and quantitative realtime polymerase chain reaction (PCR) to characterize pathogenic microbial communities in alum-treated poultry litter.

Experimental Site and Location
The study was carried out at the poultry unit of the Department of Animal Science teaching and research farm, Ahmadu Bello University, Zaria. The pen is located in northern guinea savannah zone of Nigeria, latitude 11 0 09' 76'' N and longitude 7 0 38' 20'' E at an altitude of 610 mm above sea level. The climate is relatively dry with a mean annual rainfall of 700-1400mm, occurring between the months of April and September [11].

Experimental Diets and Material
Broiler starter and finisher diets were formulated to meet the nutrient requirement of broilers [12] and used in feeding the experimental birds throughout the period of the study in both experiment one and two. The experimental diets are shown in Table 1. The alum used was obtained from the Sabon-garimarket in Zaria, Kaduna State.

Experimental Animals and their Management
Two hundred and forty (240) day old Marshall Strain broiler chicks of mixed sexes were used for the study. The birds were brooded together using kerosene stoves and electric bulbs in two pens for the first one week due to extremely cold weather conditions. The birds were fed a common diet during this period and were subsequently weighed and randomly assigned to four treatment groups. The treatments were replicated three times with 20 birds per pen. They were housed under a deep litter system with 15kg wood shavings per pen in a completely randomised design. Aluminium sulphate (alum) was applied to the wood shavings by mixing it with alum thoroughly using hands covered with hand gloves. The rates of alum application was as follows: T1 control (normal wood shavings with no alum), T2 (5% alum by kg weight of wood shavings), T3 (10% alum by kg weight of wood shavings) and T4 (15% alum by kg weight of wood shavings). Feed and water were supplied ad libitum throughout the 56 days study period and routine vaccination schedule was administered.

Blood sample collection and haematological analysis
At the termination of the experiment (day 56), two birds from each pen having representative weights for the group (6 birds per Treatment) were selected and 1.5ml of blood was taken via the wing vein. Haematological parameters were analyzed by an auto haemo-analyser (BC2800 vet auto haemo analyser) at the Clinical Pathology Laboratory, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria. The parameters determined were red blood cell (RBC) count, white blood cell (WBC) count, total protein (TP), packed cell volume (PCV), haemoglobin (Hb), differentials, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

Litter sample collection
Five sets of litter samples were obtained fortnightly from each pen from different locations i.e. the four corners and centre from which the microbial load, pH, total nitrogen (N), soluble reactive phosphorus, VFA and NH 4 + concentration were measured. Litter samples were taken by removing the first 10mm of the exposed surface from each location set. The samples from each pen were mixed and homogenized to make one sample and was refrigerated before being taken to the laboratory for analyses.

Microbial analysis of litter
Each homogenous sample mixture from each pen was pooled in one sterile flask as representing sample from one pen and were analysed for microbes in the Department of

Statistical Analyses
All the data collected from the experiment were subjected to analysis of variance (ANOVA) using the general linear model of statistical analysis system [15] software package and the mean separation was done using Duncan multiple range test.

Haematological Parameters of Broiler Chickens Raised on recycled Litter Treated with Graded Levels of Alum
The effect of recycled litter treated with graded levels of on the haematological parameters of broiler chickens was presented in Table 2. This result shows significant (P<0.05) difference among all theparameters analysed except MCHC. The packed cell volume (PCV), haemoglobin (Hb), red blood cell (RBC), lymphocyte, mean cell volume (MCV) and mean corpuscular haemoglobin (MCH) were significantly (P<0.05) higher in all the alum treated litter groups (5%, 10% and 15% alum treated litter) compared to the untreated control group (0% alum treated litter). The total protein (TP), white blood cell (WBC), heterophil, monocytes and eosinophil were significantly (P<0.05) higher in the control group (0% alum treated litter) compared to the alum treated litter groups (5%, 10% and 15% alum treated litter). There is no significant difference in the mean corpuscular haemoglobin concentration (MCHC) across the treatments.

Microbial Load of Recycled Litter Treated with Graded Levels of Alum
The result of the fortnightly effect of alum treated litter on the microbial load of the Litter is presented on Figure 1-4. The result shows a significant decrease in the total bacteria, E. coli and Salmonella spp. in the alum treated litter groups (5%, 10% and 15% alum treated litter) compared to the control group (0% alum treated litter), the mould and yeast load was significantly increased in alum treated litter groups (5%, 10% and 15% alum treated litter) compared to the control group (0% alum treated litter). The Eimeria spp parasite load in the litter remained at below detection level throughout the period of the research.

Haematological Parameters of Broiler Chickens Raised on Recycled Litter Treated with Graded Levels of Alum
Though the PCV, haemoglobin (Hb), red blood cell, lymphocyte, MCV and MCH in the Alum treated litter groups (5%, 10% and 15% alum treated litter) were significantly higher, they were within the normal range reported by [6] and [17] 1.00E+00 .70pg respectively. The significantly higher values obtained in the control treatment (0% alum treated litter) in total protein, white blood cell, heterophil, monocyte, eosinophil and band were above the normal range reported by [16] and [17] who reported normal range for white blood cell, heterophil, monocyte, eosinophil and band to be 2.57-2.72x10 9 /l, 15.83-18.3%, 3.00-4.38%, 3.6-4.2% and 1-2% respectively. The disease condition indicated as higher WBC and differentials blood count observed in the control group (0% alum treated litter) may be due to high microbial load in the litter as shown in Figs. 1-4. This result agrees with the report of [18] who reported better health status of broiler chicken when raised in alum treated litter.

Microbial Load of Recycled Litter Treated with Graded Levels of Alum
The significant decrease in total bacteria, E. coli and Salmonella spp. load of the litter observed at the end of the experiment (week 8) indicates that total bacteria load was reduced by three fold magnitude, E. coli load by two fold magnitude and Salmonella spp. load by one fold magnitude when poultry litter is treated with alum compared to the untreated litter as seen in the work of [19] who reported two fold reduction in the total bacteria of the litter with alum treatment at 16 weeks. This is similar to the work by [20], [10] who all reported significant reduction in microbial load in poultry litter treated with alum. This drastic reduction in the total bacteria load, E. coli and Salmonella spp. can be associated with the low pH in alum treated litter groups (5%, 10% and 15% alum treated litter) as reported by [9] and [19]. The mould and yeast load was seen to be higher in the alum treated litter groups (5%, 10% and 15% alum treated litter) compared to the control (0% alum treated litter) with 6.40 x 10 6 cfu/g, 1.60 x 10 8 cfu/g, 2.03 x 10 8 cfu/g and 3.5 x 10 8 cfu/g in 0%, 5%, 10% and 15% alum treated litter respectively at the end of the experiment (week 8). This is similar to the report by [19], who reported 3.5 x 10 7 cfu/g and 5.5 x 10 4 cfu/g in alum treated and untreated litter respectively, indicating a threefold magnitude higher fungal load in alum treated litter compared to the control untreated litter. This suggests that the addition of alum to poultry litter potentially shifts the microbial loads from bacterially dominated to fungi dominated [8]. The ramifications of this shift in dominance are still unknown, and future work will be aimed at characterizing these fungi and elucidating their role in the acidified litter environment [8].

CONCLUSION
The study concludes that treating recycled poultry litter with alum can reduce microbial load of the litter, hence improve health status of the birds. And tends to shift the microbial load of the litter from bacteria dominant to fungal dominant, hence reducing the risk of most bacterial diseases in broiler chicken as fungi don't convert ammonium into ammonia. Birds reared on recycled poultry litter have significantly higher haemoglobin and PCV compared to the control.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.