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Reducing Salmonella Effect in Poultry

Probiotics are one class of alternatives being considered to fill the gap created with the removal of AGP. The efficacy of some of these additives has been clearly demonstrated, this efficacy is explain by the capacity of working in various modes of action such as: direct pathogen inhibition, Immunomodulation and modification of the gut environment and microbiota.

oguz kiyak salmonella evonik - Reducing Salmonella Effect in PoultryOğuz Kıyak
Business Manager Animal Nutrition/ Hayvan Besleme İş Birimi Müdürü
Evonik, Turkey & Azerbaijan
Poultry production plays a very important role in meeting the increasing demand for animal protein in the growing world population. However, this increased pressure comes at a cost to poultry producers – to increase production in a safe, efficient, and sustainable manner, primarily due to public health concern of antibiotic resistance. In a 2014 report, FAO estimated in 25,000 human deaths per year due to these pathogens resistant to antibiotics in Europe, the cost to society generated by these conditions was estimated as €1,500 million per year (sources: ECDC 2007, Pumart et al 2012, US CDC 2013). This public health concern has led to changes in the sub-therapeutic use of antibiotics for growth promotion (AGP) in many countries. Euro¬pean Union banned use of AGPs in 2006 (Cogliani et al., 2011) due to concerns about antimicrobial resistance in humans. In the USA, the Food and Drug Administration (FDA) recently mandated limited use of medically-important antibiotics to therapeutic applications in food-producing animals under veterinary supervision and as prescribed by a veterinary feed directive. As a result of these changes, there is a need to develop and use alternatives to AGP, especially to control enteric pathogens. Among the various classes of products being used, probiotics, also known as direct-fed microbials (DFM), are becoming increasingly successful. The Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) defined Probiotics as “live micro-organisms which when administered in adequate amounts confer a health benefit on the host” (FAO/ WHO, 2001).

As the demand for growth rate and feed efficiency of the modern broiler increase, the birds’ nutritional and health status provided through “gut health” need to be managed efficiently. Probiotics can alter the dynamics of the gut microflora and, thus, improve animal performance through the combination of different mechanisms of actions. Spore-forming Bacillus based probiotics are tolerant to heat, pressure, and harsh pH environments, and, therefore, can survive feed processing and digestion in the stomach (Cartman et al., 2008). Not all strains of Bacillus spp. are the same. Strains differ in their mode of action, characteristics such as the capacity to tolerate an acidic environment and bile, and mechanisms by which they exert their beneficial effect on the GI microflora (Fioramonti et al., 2003).

Bacillus amyloliquefaciens CECT 5940 was developed using several in-vitro and in-vivo tests that defined the strain’s ability to inhibit pathogenic bacteria colonizing the gut. Major mechanism of action of Bacillus amyloliquefaciens CECT 5940 include production of secondary metabolites, quorum quenching and production of lactic acid. These mechanisms promote beneficial flora, play an important role in the development and maintaining the structural integrity of the intestinal epithelium and positively modulate the immune response.

Quorum quenching (disruption of quorum sensing) is a mechanism that is getting more and more attention in recent days as it conjugates pathogens’ control with a non resistance promoting therapy.

But what is quorum sensing? The first indication of bacterial cell-cell communication was introduced in 1965, when Tomasz suggested that the regulation of competence in Streptococcus pneumoniae was aided by a hormone-like extracellular product (Tomasz and Beiser, 1965). However, cell-cell signaling and coordinated microbial group behavior was officially ascertained by Nealson and co-workers, who reported that the bioluminescence developed by the marine bacterium Vibrio fischeri (formerly Photobacterium fischeri) in its symbiotic relationship with the Hawaiian squid Euprymna scolopes was controlled by one or more signaling molecules accumulating in the extracellular milieu as a function of cell growth (Nealson et al., 1970a). V. fischeri colonize the light organ of the squid, where the cell density reaches 1010-1011 cells/ml, then the signal molecules can accumulate to an adequate concentration to trigger the transcription of genes encoding luminescence enzymes.

evonik salmonella table1 - Reducing Salmonella Effect in PoultryMicrobial cell-cell signalling is known as “quorum sensing” (QS), and this system allows microorganisms to sense its own population density. When the external signals (known as auto inducers) reaches a threshold or “quorum” a number of target genes are activated or repressed in order to synchronize processes such as bioluminescence, antibiotic production, conjugative DNA transfer, sporulation, virulence, biofilm formation, etc.

The best-known auto inducer is N-acylhomoserine lactone (AHL). It has been widely accepted that Gram-negative bacteria utilize various AHLs to regulate the mechanisms that help them to adapt to changes in the environment. AHL signals appear to be dedicated molecules produced with the sole purpose of mediating specific quorum sensing processes. Different AHLs are usually characterized by acyl chains with variable length, saturation level and oxidation state. In AHL-dependent quorum sensing systems, the specificity of the transcriptional activator protein for its cognate AHL depends on both the length of the acyl side chain and chemical modification at the ß-position of the HSL ring. It is also well known, that this auto inducers regulate the expression of virulence genes responsible for the production of extracellular proteases, hemolysin and other extracellular factors contributing to cytotoxic activity.

Some compounds are able to hindering the communication, disrupting the quorum sensing, this effect is known as quorum quenching (QQ).

evonik salmonella table2 - Reducing Salmonella Effect in PoultryThe interference with the quorum sensing system is a potential strategy for replacing traditional antibiotics because quorum quenching does not aim to kill the pathogen or limit cell growth but to shut down the expression of the pathogenic genes. Therefore they do not create a selection pressure on the organisms and as result do not promote the appearance of resistances. QQ can be developed as a technique for disrupting the ability of a pathogen to sense its cell density and disable or diminish the capability of triggering the virulent expression. This capability ensures that the host has time to eradicate the pathogens naturally through normal immune system function. Additionally, AHL-mediated signalling mechanisms are widespread and highly conserved in many pathogenic bacteria, being an attractive target for novel anti-infective therapies.

Due to its capacity to produce violacein (a violet pigment) when QS is activated, Chromobacterium violaceum CECT 5999 (CV026) is used in QS assays. In these tests, QS is activated by applying in the culture medium AHLs with acyl chains of 4 to 8 carbons resulting in the apparition of violacein.

In the detection assay (based in the methodology described by Romero et al, 2011) the appearance (+) or absence (-) of violet color was controlled in C. violaceum seeded plates when adding the following test compounds: sterile distilled water(-), AHL(+), probiotic sterile culture medium(-), probiotic sterile culture medium + AHL(+), sterile medium + probiotic culture supernatant(-), AHL + probiotic culture supernatant(-)
The violet pigment was detected in the plates whenever AHL was applied. The combination of AHL with B. amyloliquefaciens CECT 5940 culture supernatant resulted in an inhibition of the production of the pigment violacein, indicating that Quorum Sensing was blocked by the probiotic culture supernatant, and subsequently demonstrate the capacity of ECOBIOL to potentially inhibit the QS-mediated virulence genes.

Probiotics has also demonstrated its capacity to directly inhibit the growth of typical poultry production pathogens. It is described in the scientific literature that some probiotics are able to produce inhibitory compounds in order to occupy an ecological niche. Thanks to these molecules, pathogenic bacteria such as Salmonella and E. coli are ousted and its space is rapidly colonized by beneficial microorganisms. In order to determine the bactericide effect of Ecobiol® (Bacillus amyloliquefaciens CECT 5940) a growth inhibition test was performed following the methodology described by Cintas et al., 1995; consisting on measuring diameter of inhibition of growth of the pathogenic bacteria around a well that contains metabolites produced by Ecobiol®. To obtain the B. amyloliquefaciens supernatant the microorganism was grown in industrial conditions and different samples were taken. Cells were eliminated by centrifugation at 4000 rpm for 20 minutes and by passing the supernatant through a 0.22 µm pore size filter. 50 µl of the treated supernatant were placed in the well for the test. Clear inhibition halos were obtained (>10 mm) with the supernatants for the following strains: E. coli CECT 35218, E. coli CECT 501, S. enterica CECT 722, S. enterica CECT 443, S. enterica CECT 7161, S. typhimurium 301/99 (Table 1). We can conclude that Ecobiol® has a clear bactericide effect against the listed E. coli and Salmonella strains.

The control of Salmonella occurrence and spread is essential to ensure the safety of chicken products. Biosecurity practices are the most important mechanism to prevent Salmonella in broiler flocks, associated with other tools like probiotics, aiming to control Salmonella infections by competitive exclusion and immune modulation effects. An experiment was carried out to evaluate the effect of probiotics on Salmonella enteritidis (SE) contamination and mucosa immune response of broiler challenged orally with 108cfu/mL of SE at 7 days. Twenty five one-day-old male broilers (Cobb 500) were housed from 1 to 35 days in 2 groups: a Positive Control (PC, birds challenged by SE); and Probiotic (PRO, birds supplemented with 100 g/ton of Bacillus amyloliquefaciens CECT 5940: Ecobiol Plus®, and challenged by SE). The birds were housed on litter, with water and feed ad libitum. At 21 days of age, five litter samples were collected from each treatment for Salmonella quantitative analysis. At 35 days, 10 animals/treatment were aseptically euthanized and necropsied for the collection of the cecum used in the Salmonella analysis. The data was evaluated by a Kruskal-Wallis test at 5% probability. The use of probiotic significantly reduced Salmonella counts in the litter samples compared to the PC. At 35 days, the microbiological analysis showed significant reduction in cecum between PRO and the PC groups. Bacillus amyloliquefaciens CECT 5940-based probiotic in broilers challenged with Salmonella enteritidis allows a significant reduction on the incidence of Salmonella contamination on litter and cecum.

Antibiotic-free production needs a holistic approach. In order to be able to be successful in this new production paradigm we need to take into account every single variable involved, a Multifactorial Model must be applied:

• Farm Management: biosecurity, humidity control, ventilation, vaccination programs, water quality, litter quality, etc.
• Veterinarians: Consultation. Important role in responsible use of antibiotics.
• Nutrition and Feed: digestibility and absorption of nutrients, prevention and y maintenance of health through raw material quality, additives, etc.

Probiotics are one class of alternatives being considered to fill the gap created with the removal of AGP (Denev, 1996; Kabir, 2009; and Tellez et al.,2012). The efficacy of some of these additives has been clearly demonstrated, this efficacy is explain by the capacity of working in various modes of action such as: direct pathogen inhibition, Immunomodulation and modification of the gut environment and microbiota.

Ecobiol due to its capacity to exert all of this benefits might be a very useful tool to cope with the nowadays poultry production challenges.

References are available from the author on request.

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