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nutrition ANIMAL

SWINE DIARRHEA : A WORLDWIDE PROBLEM

Image: Scott Olson, Getty Images

Introduction - Diarrhea is one of the most common occurrences and probably the most important challenge for swine producers. Common causes of diarrhea are viruses (e.g., rotavirus, coronavirus), bacteria (e.g., Escherichia coli, Clostridium difficile and Clostridium perfringens), and even protozoans. Diarrhea (i.e., disease outbreak) are often times complex and multi-factorial in nature1. In many cases, diarrhea may be caused by multiple disease agents (e.g. co- or mixed-infections)2–4, making proper diagnostic and treatment difficult. Other conditions such as oversupply of nutrients and poor nutrient digestibility can disrupt the balance of gut microbiota, which in turn encourage the proliferation of pathogenic bacteria. Untreated animals often experience high morbidity and mortality due to dehydration or complication with other diseases (e.g., secondary infections and necrotizing lesions). This eventually leads to the spread of diseases in the herd, resulting in drop in animal growth performance and economical loss. Clostridial species and swine diarrhea - Diarrhea caused by enteric pathogens, especially clostridial species is common in commercial farms. Two clostridial species namely Clostridium difficile and Clostridium perfringens are well known causative agents of swine diarrhea and neonatal enteritis, with high isolation rate of these two bacterium from many pig farms in the Asia Pacific regions, i.e, Australia5, Thailand6, Taiwan7, Japan8. Some studies reported as many as >50% of fecal and rectal samples collected from animals were tested positive for either one of the two clostridial species5,8; with up to 90% of these isolates being toxigenic strains5, indicating that the two clostridial species including their toxigenic subtypes may have high prevalence

worldwide. The most common C. perfringens serotypes isolated from animals belong to serotype A and C4,6. C. perfringens type C often causes high morbidity and high mortality in neonates and suckling piglets, with syndromes ranging from bloody diarrhea to necrotic enteritis, and sometimes even sudden deaths. Contrary, animals infected by C. perfringens type A usually experience moderate morbidity and lower mortality compared to infections by C. perfringens type C. Outbreaks of C. perfringens type C are acute and occur in piglets less than one week old, chronic cases have been recognized with increasing frequency in pigs up to several weeks after birth. For C. difficile, the most common isolates in most parts of the world are ribotype RT014 and RT0788,9. Infections by C. difficile is often characterized by diarrhea with variable morbidity and mortality. The pathogenicity of these two clostridial species are largely attributed to their capability to produce toxins which can be detrimental to the intestinal lining. Animals, especially neonatal and suckling piglets which lack immunity against infections are susceptible to infections; though, infected animals can also become asymptotic without showing symptoms of infection10,11. Risk factors for infection of young animals include contact through farm environments12,13 and transmission from infected sow4, but vector of transmission may vary from farms to farms. Sick and asymptotic but otherwise healthy animals can shed the disease agents through their feces, increasing transmission rate in the litter and herd14. The implication of managing clostridial species in farms and animals go beyond animal health and welfare as transmission to human, i.e., through meat and direct contact is possible7.

More worrying is the fact that hypervirulent strains of C. difficile isolated from animals and human (identical genotypes), as well as C. perfringens which only infects animals, have become resistant to many antibiotics6. Effective management of disease agents such as clostridial species in farm environments and animals is not only important to maintain healthy herd, but also crucial to prevent the spreading of this disease agent14,9. Probiotic for diarrhea management - Current approach for swine producers to control diarrhea is palliative and antidotal, i.e. the use of antibiotics at therapeutic dose. Antibiotics are also often used in sub-therapeutic doses for prophylaxis. The use of antibiotics at sub-therapeutic doses is not the desired approach as it creates bacterial resistances. Other means for the prevention of post-weaning diarrhea is through diet supplementation of zinc oxide15; though a more recent study has highlighted that high dosage (i.e., 2.5g/ kg feed) in diet for over two weeks may promote the development of bacterial resistance to some antibiotics such as tetracycline and sulfonamide16. Another effective alternative for diarrhea management relies on a holistic approach to biosecurity which also necessitates diet intervention. Diet is an important aspect in maintaining gut health as excess supply of nutrition may promote the growth of pathogens. Precision nutrition and good quality raw materials are important to improve digestibility and reduce wastage. Precision nutrition requires the accurate use of proper ingredients and nutrients for optimal animal performance. Using the right additives such as probiotic, organic acids, essential oils and prebiotic (Figure 1) may reduce diarrhea incidence and reliance on antibiotics17.

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bacteriocins including surfactin, difficin, bacilysin, mersacidin and macrolactin, etc. The ability of strain PB6 in producing different types of bacteriocins is noteworthy, as each bacteriocin works differently to cause cell lysis and death. Mersacidin, for example, targets peptidoglycan biosynthesis pathway and inhibits cell wall synthesis, which can be detrimental to many Gram positive pathogens18. On the other hand, other bacteriocins such as surfactin forms pores on the cell membrane, leading to cystoplasm leakage and cell death. Together, these different bacteriocins act synergistically in destabilising the cell membrane of pathogens, thereby achieving killing, illustrated in Figure 2. III.B for C. perfringens. Figure 2. Antagonistic effect of Bacillus subtilis PB6 against Clostridium perfringens isolated from pig farms in Australia, determined using (I) streak line assay. Horizontal streak line represents culture of strain PB6, whereas vertical line represents culture of Clostridium perfringens. A clear zone of

CLOSTATTM stands out to be one of the most effective antibiotic alternatives for diarrhea management through early diet intervention. CLOSTATTM, which is one of Kemin’s product in the Antibiotic Alternative platform (ABA; Figure 1) contains an active probiotic Bacillus subtilis PB6, isolated from the gut of healthy animal. Bacillus subtilis PB6 is a Gram positive bacterium and it is capable of forming endospore - a dormant and tough structure capable of withstanding extreme environment. Spore-forming is an important attribute as this allows the organism to survive the low acidic conditions in the stomach, as well as pelleting conditions (e.g. 90°C for 5 – 10 mins) used in manufacturing pelleted feed by the feed industry. Upon ingested by the animals and passage into the intestinal compartment, the endospore of strain PB6 will germinate into vegetative cells where the bacterium exerts its antagonistic effects against potential pathogens through competitive exclusion and production of antimicrobial peptides.

Figure 1: Kemin’s product portfolio supporting the Antibiotic Alternative platform (ABA). Kemin provides a series of solutions to replace Antimicrobial Growth Promoters (AGPs) in a progressive manner. Products in the ABA platform can be used individually or in combination to replace AGP in animal feeds to maintain healthy herd and promote animal performance. CLOSTATTM belongs to the category of Probiotics and contains an active probiotic Bacillus subtilis PB6 that is effective against many Gram positive pathogens (e.g., Clostridium perfringens and Clostridium difficile) known to be the causative agent of swine diarrhea. The in vivo study presented in this article shows that supplementing diet with CLOSTATTM can be an effective way to manage diarrhea occurrence for nursey piglets. Bacillus subtilis PB6 (in CLOSTATTM) is effective at destroying C. difficile and C. perfringens. Figures 2.I and 2.II illustrate the antagonistic effect of strain PB6 against C. perfringens isolated from pig farms in Australia. This antagonistic effect is partly attributed to the actions of antimicrobial peptides, also known as bacteriocins (cell free supernatant as in Figure 2.II) produced by strain PB6. Strain PB6 is not only effective against Clostridial species but also other Gram positive pathogens including Staphylococcus aureus and Streptococcus suis, although the latter was mostly associated with causing respiratory diseases. The mechanisms of strain PB6 in causing cell death of many Gram positive pathogens has been deciphered through whole genome sequencing and peptide identification using MALDI-TOF; with evidence showing that strain PB6 can produce multiple

inhibition was observed for C. perfringens, indicating inhibition for growth by strain PB6. (II) well diffusion assay using culture supernatant of an overnight culture of strain PB6. Clear zone of inhibition was visible around a lawn of C. perfringens, indicating that the secretions of PB6 contains antimicrobial factors that are capable of inhibiting the growth of C. perfringens. (III) Transmission electron micrograph (x29500): action of B. subtilis PB6 on C. perfringens cell lysis. A: 1 hour after adding PB6 supernatant. B: 8 hours after adding PB6 supernatant. Animal trial - An animal trial was

conducted at the University of the Philippines Los Baños to investigate the interactive effects of Bacillus subtilis PB6 (in CLOSTATTM) with and without in-feed antibiotics or zinc oxide on the diarrhea occurrence in nursery pigs. A total of 144 weanling pigs were divided into 6 treatment groups (4 pigs/pen for 6 pens per treatment) receiving different diets in a randomized complete block trial (Table 1). Diets with high protein content to induce diarrheic conditions were fed from day 42 to 70 post-weaning, and diarrhea score and frequency were collected on day 0, 7, 14, 21 and 28 post-weaning. Overall, pigs fed with diet supplemented with zinc oxide experienced fewer diarrhea days and frequency, and had the lowest diarrhea score compared to piglets fed with only basal diet (Figure 3). Zinc oxide is routinely used to prevent diarrhea (e.g., post-weaning diarrhea) in piglets, with evidence showing improved animal performance15. However, this benefit of zinc oxide is even more apparent when used in combination with CLOSTATTM. Overall, supplementation of diets with CLOSTATTM resulted in litter having significantly lower diarrhea frequency and diarrhea days (p-value<0.05) compared to animals fed with diet not supplemented with CLOSTATTM.

Table 1: Details of experimental groupExperimental DietGroupT1 Basal feedT2 Basal feed + 3000 ppm zinc oxideT3 Basal feed + 500 g/MT CLOSTAT™ 11 DryT4 Basal feed + 3000 ppm zinc oxide + 500 g/MT CLOSTAT™ 11 Dry

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Figure 4: Effects of treatments on diarrhea day in weaning pigs. All treatments supplemented with (w) CLOSTATTM

showed significant reduction (p-value <0.05) in diarrhea frequency compared to treatments without (w/o) CLOSTATTM. Conclusion - Managing diarrhea occurrence in swine production can be challenging due to complexity of infections. Young animals can be exposed to pathogens through contacts with the environment or transmission from infected animals. Antimicrobial growth promoters, i.e. antibiotic or other means (e.g., zinc oxide) are used to control the disease in farms, but tighter regulations requires effective alternatives to manage diarrhea occurrence. CLOSTATTM is an obvious alternative that can be used to prevent and reduce diarrhea occurrence in pigs, especially for weaning and nursery piglets where growth rate at this stage is critical. CLOSTATTM, when used at the recommended dosage, effectively and significantly reduces diarrhea occurrence, helping farmers to maintain healthy herd and reduce treatment cost.

- TAN BOON FEI, Ph. D and CHAN POH SOON, DVM., Kemin Animal Nutrition and Health, Asia Pacific.

CHAN POH SOONTAN BOON FEI

- THE WRITERS -

Figure 3: Effects of treatments on diarrhea frequency in weaning pigs. All treatments supplemented with (w) CLOSTATTM showed significant reduction (p-value <0.05) in diarrhea frequency compared to treatments without (w/o)