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Research Article
 

Litter Treatment with Aluminum Sulfate (Alum) Produced an Inconsistent Reduction in Horizontal Transmission of Campylobacter in Chickens



K. Arsi, P.A. Moore Jr., A.M. Donoghue, M.L. Dirain and D.J. Donoghue
 
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ABSTRACT

Background and Objective: Campylobacteriosis is a significant health problem worldwide and poultry are considered as one of the main vehicles of transmission. This study was conducted to determine whether alum reduces Campylobacter colonization in broilers by reducing horizontal transmission between birds or by reducing Campylobacter counts in birds already colonized (Therapeutic efficacy). Materials and Methods: Two replicate experiments were conducted and in each experiment, day of hatch broiler chicks (n = 295) were divided into 7 treatment groups including controls. Each treatment was reared in either no (0 kg), low (0.78 kg m–2) or high (1.58 kg m–2) concentrations of aluminum sulfate (alum; Al+ Clear). During days 7, 14, 28 and 42, ten birds from each treatment were analyzed for Campylobacter counts in the ceca. To evaluate whether alum inhibits horizontal transmission between birds, Campylobacter negative birds were reared with seeder birds that served as carriers. Results: Alum reduced (p<0.05) horizontal transmission of Campylobacter at 14 and 28 days in experiment 1 and only with the highest concentration of alum at 42 days in experiment 2. To evaluate the therapeutic efficacy of alum, all birds were inoculated with Campylobacter (5.2×106 CFU mL–1) prior to placement in pens. Infected birds reared on low or high alum had lower (p<0.05) Campylobacter counts at 14 and 28 days in only 1 of 2 experiments. At 42 days, there were no differences in cecal Campylobacter counts between alum treated and untreated controls in experiment 1 and for only the highest concentration in experiment 2. Conclusion: It appears treating litter with alum is not a consistent way to reduce enteric Campylobacter counts.

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  How to cite this article:

K. Arsi, P.A. Moore Jr., A.M. Donoghue, M.L. Dirain and D.J. Donoghue, 2017. Litter Treatment with Aluminum Sulfate (Alum) Produced an Inconsistent Reduction in Horizontal Transmission of Campylobacter in Chickens. International Journal of Poultry Science, 16: 31-36.

DOI: 10.3923/ijps.2017.31.36

URL: https://scialert.net/abstract/?doi=ijps.2017.31.36
 
Received: December 02, 2016; Accepted: January 02, 2017; Published: January 15, 2017


Copyright: © 2017. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Campylobacter has been recognized as the leading cause of bacterial foodborne illness in the world and poultry products are reported to be the prevailing vehicles of campylobacteriosis transmission1-3. The colonization of Campylobacter in the bird’s gut and the subsequent contamination of the meat during mechanized processing is the contributing factor in the prevalence of this pathogen on poultry carcasses4. Effective pre-harvest intervention strategies are therefore needed to reduce enteric colonization and the incidence of campylobacteriosis in humans5-8.

To this end, Line9 supplemented poultry litter with aluminum sulfate (alum) [Al2(SO4)3.14H2O] and reported a decrease in cecal Campylobacter frequency and counts and corresponding reduction in whole carcass contamination. Alum may be added to poultry litter to precipitate soluble phosphorous and consequently minimize phosphorous runoff10-12. Alum is also used to reduce ammonia concentrations and thus, promotes poultry health and well being13,14. It appears that litter application of alum may also help control the prevalence of Campylobacter, however, it is unknown how alum exerts its effects on Campylobacter colonization in the intestinal tract of birds.

The ability of alum to lessen Campylobacter contaminations in chickens may be related to alum’s effect on litter pH. Litter is acidified upon alum application9,15 and a reduced pH can be lethal to Campylobacter16-19. This reduction in litter pH may lessen horizontal transmission of Campylobacter within flock. Although horizontal transmission was not directly determined by Line9, other mechanisms may play a role for the reduction in this foodborne pathogen following alum administration. Because of litter consumption by chickens and corresponding alum ingestion, this compound may also act as a therapeutic agent against Campylobacter to limit colonization in the gut of chickens. To investigate the manner in which alum exerts its effects, this study was conducted to determine whether alum effects Campylobacter colonization in the ceca of broilers by reducing horizontal transmission between birds or by reducing Campylobacter counts in birds already colonized (Therapeutic efficacy). This study may provide an understanding of the mechanisms of action of alum and how it may be used to control the colonization of Campylobacter in chickens.

MATERIALS AND METHODS

Animal care and alum administration: At the beginning of each of two replicate experiments, fresh pine shaving were used and served as litter in seven isolation pens of about 3.06 m2 (33 ft2) equipped with feeders and bell-shaped water drinkers. Of the 7 and 4 pens were top dressed with commercially available aluminum sulfate (Al+ Clear, General Chemical, Parsippany, N.J.), treated with either low (0.78 kg m–2) or high (1.58 kg m–2) concentrations of alum as per the procedure used by Line9. The litter was then sprayed with 1 L of water to provide adequate moisture for activation of the product. Although untreated control pens received no litter treatment, water (1L) was sprayed to maintain moisture as the rest of the pens. Water application in all pens (1 L pen–1) was repeated every week throughout the experiment to maintain moisture concentrations near commercial conditions. The birds were reared for 6 weeks, feed and water were available ad libitum throughout the study. Alum was re-applied at 5 weeks as practiced from the previous alum study9.

Experimental treatments: Two separate replicate experiments were conducted in this study. A total of 590 chicks were used in this study (n = 295 replicate trials–1). Birds were divided into 7 treatment groups. These were: (1) Negative controls (NC, birds not inoculated with C. jejuni, no alum), (2) Horizontal transmission controls (HC, negative control birds reared together with C. jejuni infected seeders, no alum), (3) Horizontal transmission/low alum (HL, birds not inoculated with C. jejuni but reared with C. jejuni infected seeders on a low concentrations of alum), (4) Horizontal transmission/high alum (HH, birds not inoculated with C. jejuni but reared with C. jejuni infected seeders on high concentrations of alum, (5) Therapeutic efficacy controls (TC, all birds inoculated with C. jejuni, no alum), (6) Therapeutic efficacy/low alum (TL, all birds inoculated with C. jejuni and raised on low concentrations of alum) and (7) Therapeutic efficacy/high alum (TH, all birds inoculated with C. jejuni and raised on high concentrations of alum).

Of the 295 total newly hatched chicks used in each trial, 160 were released into pens (NC, HC, HL, HH, 40 birds pen–1), whereas another 135 birds were orally gavaged with 0.2 mL of C. jejuni (approx. 5×106 CFU mL–1) and released in TC (n = 55), TL (n = 40), TH (n = 40) treatment pens. After 7 days, a total of 15 birds from TC were transferred to HC, HL and HH pens to serve as Campylobacter seeders (n = 5 group–1) in these treatment groups. Seeders used to evaluate horizontal transmission were fitted with leg bands to distinguish them from the rest of the birds in the pen and were not included in the data analysis. Seven strains of Campylobacter jejuni previously isolated from chickens were used to colonize the birds in this study following the procedure described previously20.

Data collection: Birds from each treatment group (n = 10) were analyzed at days 7, 14, 28 and 42 (n = 10 day–1) for C. jejuni counts in the ceca as previously described by Cole et al.20. Briefly, chickens were euthanized by CO2 and the ceca of each bird were aseptically dissected and diluted in Butterfield’s Phosphate Diluent (BPD, 68 g L–1 KH2PO4, pH 7.2) and vortexed. The homogenous mixture was serially diluted (1:10) with BPD and inoculated on Campy Line Agar (CLA) plates20. Labeled CLA plates were incubated for 48 h at 42̊C under microaerophilic conditions. Counts were recorded and direct bacterial counts were converted to CFU g–1 of the cecal content. Campylobacter colonies were confirmed by latex agglutination test (PANBIO, Inc. Columbia, MD) and further identified as C. jejuni isolates using API® Campy (Biomėrieux® Durham, NC). In addition, about 20 g of litter sample were obtained weekly from each treatment group, which were used to monitor litter pH levels. Litter pH levels were analyzed by diluting (1:2) the litter samples with de-ionized water and pH was determined by a pH meter.

Statistical analysis: The numbers of Campylobacter colonies were logarithmically transformed (log10 CFU g–1) prior to analysis to achieve homogeneity of variance21. Data were subjected to ANOVA using the GLM procedure of SAS22. Treatment means were partitioned by LSMEANS analysis. A probability of p<0.05 was required for statistical significance.

RESULTS

Cecal Campylobacter counts for the alum treatments are presented in Table 1 and 2. In birds evaluated for horizontal transmission, Campylobacter was not detected in non-inoculated groups (HC, HL, HH) at 7 days of age in both replicate experiments. However, after transfer (7 days) of Campylobacter inoculated seeder chicks in groups HC, HL and HH, birds in these groups became Campylobacter positive by 14 days of age. In experiment 1, HH demonstrated reduced Campylobacter counts compared to HL and HC at 14 days (p<0.05). On day 28, both HL and HH had lower (p<0.05) Campylobacter counts compared to HC. By 42 days, no difference in Campylobacter counts were observed between treatments. In experiment 2, no differences were observed in Campylobacter counts at 14 and 28 days for horizontal treatments. However, at 42 days, a reduction (p<0.05) in Campylobacter counts was observed in HH compared to HC. None of the negative controls (NC, not inoculated, no seeder birds) had Campylobacter counts during the entire study. Birds inoculated with Campylobacter were evaluated for therapeutic efficacy of alum. All chicks inoculated at day of hatch with Campylobacter were colonized by 7 days (groups TC, TL, TH) in both replicate experiments. In experiment 1, both TL and TH reduced (p<0.05) Campylobacter counts compared to TC at 14 and 28 days.

Table 1: Mean counts of Campylobacter jejuni recovered from ceca of broilers reared on litter with or without alum at 7, 14, 28 and 42 days (Experiment 1)1
a,b,c,dDifferent letter superscripts in the same column indicates significant difference (p<0.05), 1All data were log10 transformed for statistical analysis. For clarity of presentation, arithmetic means are presented, ND: Not detected

Table 2: Mean counts of Campylobacter jejuni recovered from ceca of broilers reared on litter with or without alum at 7, 14, 28 and 42 days (Experiment 2)1
a,bDifferent letter superscripts in the same column indicates significant difference (p<0.05), 1All data were log10 transformed for statistical analysis. For clarity of presentation, arithmetic means are presented, ND: Not detected

Fig. 1:
Effect of alum on litter pH, litter was treated with low (2.4-3.06 kg m–2) or high (4.8-3.06 kg m–2) concentrations of alum. Controls received no litter treatment. Alum was re-applied at 5th week

By 42 days, no reduction in Campylobacter counts were observed. In experiment 2, the therapeutic efficacy of alum to reduce Campylobacter counts in inoculated birds were not evident. In both replicate experiments, birds in the therapeutic groups (TC, TL, TH) had higher cecal Campylobacter counts at 14 days of age when compared with the horizontal group (HC, HL, HH). This is possibly due to the fact that the therapeutic groups were inoculated with Campylobacter directly as opposed to horizontal group being indirectly exposed to Campylobacter infected seeders.

Top dressing the litter with alum reduced (p<0.05) litter pH when evaluated weekly during the 6 weeks study (Fig. 1). The highest alum concentrations yielded the greatest reduction (p<0.05) in litter pH. There were no differences in litter pH for pens treated with alum and inoculated with Campylobacter compared to those pens treated with alum but not inoculated (Fig. 1).

DISCUSSION

In a previous study, alum applied on chicken litter reduced Campylobacter colonization frequency and populations in the ceca9. Line9 suggested that the decrease in pH of the litter caused by alum treatment may have reduced Campylobacter populations in the litter, thus reducing Campylobacter transmission in poultry flocks. As reported from other studies, Campylobacter cells cannot survive under acidic conditions, e.g., below 4.018,23. The appropriate pH range for growth of Campylobacter is reported to be around 5.5-7.523.

In this study, two potential mechanisms (reduced horizontal transmission or therapeutic efficacy) on how alum might reduce Campylobacter counts in chickens were evaluated. In both replicate experiments, litter treatments with alum reduced cecal Campylobacter counts in both the birds tested for horizontal or therapeutic efficacy. The decrease in Campylobacter counts in the horizontal group in both replicate trials may be due to the acidifying effect of alum in litter (Fig. 1). Litter application of alum decreased litter pH compared to the non-acidified litters, however, the reductions in Campylobacter counts in treated litters are minimal and inconsistent in both replicate experiments. This suggests that although alum lowers the pH of the litter, the acidifying effect of alum to lyse Campylobacter cells is either limited or conversely did not have enough contact with Campylobacter contained within fecal dropping sitting on top of the alum treated litter. The difference in the outcome between this study and Line’s study9 may be explained by the experimental design employed by that researcher. In this study, researchers utilized used chicken litter contaminated with Campylobacter followed by alum treatment prior to bird placement9. The alum litter treatment may have reduced or eliminated Campylobacter counts, reducing the potential for Campylobacter colonization in placed birds. In the present study, to evaluate whether alum inhibits horizontal transmission between birds, Campylobacter inoculated chicks (seeders) were used as the source of Campylobacter infection. The experimental design used in this study might be more representative of a commercial broiler setting because uninfected birds would be exposed to the constant shedding from Campylobacter contaminated birds. The results obtained in the present study are consistent with a follow-up study conducted by Line9 and Bailey15 who reported that alum application in litter in a commercial broiler farm reduced the onset of Campylobacter colonization in broilers but did not reduce final Campylobacter counts in 6 weeks old market age birds.

Because of the coprophagous nature of chickens, it is proposed that alum may have therapeutic efficacy to reduce Campylobacter counts in the ceca. Interestingly, the therapeutic efficacy of alum was observed during 14 and 28 days of the first experiment. It is possible that birds may have ingested alum particles through litter pecking, acidifying their gut and consequently reduced Campylobacter counts in the ceca. Alternatively, the application of alum may have caused litter aggregation making the Campylobacter contaminated litter unavailable for ingestion by the birds.

In this study, the effect of alum on cecal Campylobacter counts declined over time. Unfortunately, re-application (35 days) of alum elicited little effect on reducing Campylobacter counts towards the end of this study. Nonetheless, this study demonstrated that alum has some potential to reduce Campylobacter loads at specific times during the rearing period. Further research is necessary to optimize the efficacy of alum as a tool to reduce Campylobacter colonization during broiler production.

SIGNIFICANCE STATEMENT

Previous study has shown that litter amendments, mainly acidification of litter, using alum can reduce ammonia levels and improve the health and performance of birds
This study explored the potential application of alum to reduce Campylobacter colonization or horizontal transmission of Campylobacter in poultry
Alum demonstrated some potential to reduce both Campylobacter colonization and horizontal transmission to some extent, however, the effect of alum on cecal Campylobacter counts declined over time
Further study is aimed at combing litter amendments with other pre-harvest invention strategies for reducing Campylobacter colonization in broiler chickens
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