Mycotoxins in layer and breeder feed impact hens, eggs, hatchery, and chicks

White Chickens Farm

By Marisabel Caballero, Global Technical Manager Poultry

As the planet’s climate experiences changes, new patterns affect the microbial communities colonizing crops. Recently, several areas of the planet have experienced extreme temperatures, drought, changes in the humid/dry cycles, and an increase in atmospheric carbon dioxide (1,2). As a response, the fungi affecting the crops have shifted their geographical distribution, and with this, the pattern of mycotoxin occurrence also changed. For instance, in Europe, we are looking at higher frequencies and levels of Aflatoxins (AF), Ochratoxins (OT), and Fumonisins (FUM) than ten or even five years ago (2-4).

This affects animal production, as mycotoxin challenges show increased frequency, quantity, and variety. Mainly long-living animals, such as laying hens and breeders, can have a higher risk. Moreover, mycotoxins can also be carried over to the eggs, potentially risking human health in the case of layers (table eggs) and in the case of breeder hens, hatchery performance and day-old chick (DOC) quality.

Laying hens and breeders: carryover of mycotoxins into eggs

Most mycotoxins are absorbed in the proximal part of the gastrointestinal tract (Table 1). This absorption can be high, as in the case of aflatoxins (~90%), but also very limited, as in the case of fumonisins (<1%), with a significant portion of unabsorbed toxins remaining within the lumen of the gastrointestinal tract (5).

Once mycotoxins are ingested, detoxification and excretion processes are started by the body, and at the same time, organ damage ensues. The detoxification of mycotoxins is mainly carried out by the liver (6), and their accumulation happens primarily in the liver and kidneys. However, accumulation in other tissues, such as the reproductive organs and muscles, has also been found (7-9). The detoxification process’ objective is the final excretion of the toxins, which occurs through urine, feces, and bile; often, the toxins can also reach the eggs (7-20).

Table 1: mycotoxin absorption rates for poultry and their carry-over rate into eggs

Mycotoxin Main absorption sites Absorption rate in poultry Carry-over rate into eggs
Aflatoxins Duodenum, jejunum ≈90% ≈0.55%
DON Duodenum, jejunum ≈20% ≈0.001%
Fumonisins Duodenum, jejunum ≈1% ≈0.001%
Ochratoxin Jejunum ≈40% ≈0.15%
T-2 Duodenum, jejunum ≈20% ≈0.10%
Zearalenone Small & large intestine ≈10% ≈0.30%

(Adapted from 5, 7-17, 19-21)

Table 1 shows carry-over rates of mycotoxins into eggs, resulting from diverse studies (7-10, 14, 16, 19). However, the same studies indicate that results can vary broadly due to different factors, as reviewed by Völkel and collaborators (26). This variability is related to the amount and source of contamination, way of application, period, and the possible co-occurrence of various mycotoxins or several metabolites. Other factors to consider are animal-related, such as species, breed, sex, age group, production level, and health status. Environmental and management factors can play a role in carry-over rates, and finally, detection limits and analytical procedures also influence these results. In summary, highly varying carry-over has been demonstrated, and the risk needs to be considered when animals are exposed.

Mycotoxins in breeder’s feed impact hatchery performance and day-old chick quality

When hens are exposed to mycotoxins, their effects on the intestine, liver, and kidney decrease egg production and quality (10, 14, 27), and, in the case of breeders, consequently, affect hatchery performance, DOC production, and DOC quality (28-30). The main effects of mycotoxins, when we speak about DOC production, are exerted in the gastrointestinal tract, the liver, and the kidneys, affecting embryos and young chicks:

  • Intestine and kidneys: Mycotoxins harm the intestinal epithelium and have nephrotoxic effects, affecting calcium and vitamin D3 absorption and metabolism, necessary for eggshell quality (31). Thin and fragile shells can increase embryonic mortality, lower embryonic weight gain, and hinder hatchability (32).
  • Liver: The liver plays a central role in egg production as it is responsible for vitamin D3 metabolism, the production of nutrient transporters, and the synthesis of the lipids that make up the yolk. Thus, when liver function is impaired, the internal and external quality of the egg declines, which affects DOC production (31-34).
  • Embryo and young chicks: Studies (33-38) have found how mycotoxins affect the embryos. In general, there are two possibilities: the direct one, when the mycotoxin is transferred into the egg, and the indirect one, when the mycotoxin impacts egg quality and, therefore, leads to disease or death of the embryo. The result is a higher embryonic mortality or lower DOC quality. These, among others, result from the lower transfer of antioxidants and antibodies from the hen, low viability of the chick’s immune cells, and higher bacterial contamination. A lower relative weight of the bursa of Fabricio and the thymus is often found.

Qreshi’s team (29) studied the effects on the progeny of broiler breeders consuming feed highly contaminated with AFB1, finding suppression in antibody production and macrophage function in chicks after ten days. Similar results were found by other researchers (36, 37) evaluating the effects of AF and OTA as single and combined contamination. When both mycotoxins are present in the feed, the effect on hatchability and DOC quality are synergistic.

Due to mycotoxin contamination, the reproduction and immune response are impaired, resulting in decreased DOC production and increased early chick mortality, as they are more susceptible to bacterial and viral infections.

Mycotoxins impair table egg production and quality

Studies (22-24) have found mycotoxin contamination in commercial table eggs. A meta-analysis of mycotoxins’ concentration based on 11 published papers was completed recently (22): counting with data from 9509 samples, the meta-analysis reveals an overall presence of mycotoxins in 30% of the samples, being Beauvericin in the first place, followed by DON as well as AF and OTA in third and fourth place, respectively. The risk for humans depends on the intake of contaminated foods in terms of amount and frequency (25), and so far, it has not been estimated in most parts of the world.

Natural contamination in laying hens: a case report

Giancarlo Bozzo’s team (39) reported and published a veterinary case regarding natural mycotoxin contamination in commercial egg production: up to week 47 of age, production parameters were on top of the genetic standards. However, a drop in egg production started at around week 47, and at week 50, egg production was only 68% (figure 1).

Figure
Figure 1: production of laying hens fed naturally contaminated feed with AFB1 and OTA
The house with the reduced performance received feed with linseed. In other houses of the same complex, which did not include linseed in the feed, production was unaffected. Therefore, this raw material was considered a possible cause of the issue. Linseed was removed from the formula, and three weeks after (53 weeks of age), egg production was at 84%. Afterward, linseed got back into the formulation, and the laying rate dropped again to 70% (week 56), this time accompanied by a significant increase in mortality.

Samples were collected at week 56, and AFB1 and OTA were detected in feed and the kidneys and livers of the hens consuming it (table 2). While the levels in the feed were not considered high risk, evidence from necropsy and histopathology suggested either a higher or a prolonged exposure; a synergistic effect of both mycotoxins on hen’s health and productivity can be inferred.

Table 2: mycotoxin analysis results for feed and organs

HPLC analysis results in samples of:
toxin Feed 1
(n=5)
Feed 2
(n=5)
Kidney

(n=10)

Liver

(n=10)

OTA 1.1 ± 0.1 ppb 31 ± 3 ppb 47 ± 3 ppb 24 ± 2 ppb
AFB1 ND 5.6 ± 0.3 ppb 1.4 ± 0.3 ppb 3.6 ± 0.4 ppb

The liver and kidneys were enlarged and showed signs of damage. Furthermore, urate crystals in the peritoneum and the abdominal air sac were observed, indicating renal failure. This limited the excretion of both toxins in the urine, increasing their half-life in the organism and enhancing the effects in target organs, contributing to the synergistic effect observed.

After using mycotoxin-free certified linseed, the problem receded. Though this is the best option to keep animals healthy and productive, it may not be practical in the long term due to the ubiquitous nature of the toxins and the cost and availability constraints of feed raw materials. Moreover, the mycotoxin levels present in the feed were relatively low and fell under recommended guidelines. For these reasons, in-feed toxin mitigation solutions must also be considered to reduce exposure for production animals.

In-feed intervention mitigates the effects of intermittent exposure to multiple mycotoxins

EW Nutrition conducted a study with Hy-Line W-36 layer-breeders intercalating three 10-day cycles of feed with 100ppb AFB1 + 100ppb OTA, with two 21-day cycles of non-challenged feed. An in-feed intervention (Solis Max 2.0, displayed as IFI) containing bentonite, yeast cell wall components, and a mixture of phytogenic components mitigated all effects.

Table 3: experimental groups and mycotoxin challenge

Treatment Group 100 ppb AFB1+ 100 ppb OTA IFI (2 kg/ton)
T-1 Control (C)
T-2 C+IFI X
T-3 Challenge (Ch) X
T-4 Ch+IFI X X

Trial design:

A total of 576 hens (18 replicates per diet, 8 hens each) and 58 roosters were randomly assigned to four diets at 28 weeks of age, as shown in Table 3. The 72-day experimental period included alternating 10-day challenge and 21-day non-challenge intervals (Figure 2). During the challenge intervals, the breeders in T-3 and T-4 were fed the mycotoxin-contaminated feed with and without the IFI.

FigureFigure 2: trial timeline showing challenge and non-challenge intervals and days of data collection and sampling.

Mitigated effects on egg production and egg quality

The challenge decreased overall egg production (Figure 3), egg mass, and shell thickness (Table 4). The first challenge interval did not affect production, but days later, from the first non-challenge period, all parameters were lower for the challenged group.

FigureDifferent letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1) are indicated by (*).

Figure 3: Egg production of hens intermittently challenged with AFB1 and OTA, with and without in-feed Solis Max

The adverse effects on productivity and egg quality started after the first challenged feed was withdrawn and persisted through the following intervals until the end of the experiment. Similar effects in chronic mycotoxin challenges have been previously found (37, 39).

Table 4: Average egg quality parameters of hens intermittently challenged with AFB1+OTA, with and without an in-feed intervention (IFI)

Group Eggshell strength (N) Eggshell thickness (mm) Haugh Units
Control 21,02a 0,3661ab 70,88
IFI 21,16a 0,3702a 71,68
Challenge 20,05b 0,3630b   70,07*
Ch+IFI 21,06a 0,3698a 71,06

Different letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1) are indicated by (*).

Mitigated effects on the progeny in incubation trials

Three incubation trials were performed: after the first challenge and non-challenge interval and at the end of the trial period after the third challenge interval. A significant decrease in fertility and hatchability was observed for the challenged group in all incubation trials. As mycotoxins affect egg quality (22-24) and can be transferred to the eggs (10, 14, 27), the effects were also shown in the case of hatchability and offspring performance. Fertility was affected from the first challenge interval onwards, continuing to be low for the challenge group until the end of the trial. However, the hatchability of fertile eggs dropped after the withdrawal of the contaminated feed and showed the lowest value during the third challenge interval.

The in-feed supplementation of Solis Max 2.0 (IFI) resulted in the consistent recovery of egg production and egg quality throughout the whole experimental period, achieving the same levels of productivity as the non-challenged control.

Figure
Letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1), indicated by (*).

Figure 4: Hatchery parameters of eggs from breeders intermittently challenged with AFB1 and OTA, with and without an in-feed intervention (IFI).

Results in hatch of fertile can be related to egg quality, as the thickness of the eggshell influences the egg’s moisture loss and exchange with the environment during the incubation period. Thinner eggshells lead to higher embryo mortality (31, 32). The group having the challenge with Solis Max showed the same performance as the non-challenged control regarding hatchery performance.

Day-old chick weight was not affected. However, weight gain and mortality after ten days were hindered for the chicks from breeders taking the mycotoxin-contaminated feed (Table 5).

Table 5: Average day- and 10-day-old chick parameters from hens intermittently challenged with AFB1+OTA, with and without an in-feed intervention (IFI)

Parameter Control Challenge Ch + IFI
DOC body weight (g) 36,67 36,24 36,80
10-day body weight (g) 76,30a 75,94b 79,50a
10-day mortality (%) 0,94 1,26 0,97

Letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1) indicated by (*)

At the end of the experiment, oxidative stress biomarkers were measured in the blood serum of 15 hens per treatment, showing significantly lower GPx, and SOD (figure 5) in the challenged group, which indicates a depletion of the mechanisms to fight oxidative stress (40), the hens taking the in-feed product did not show this depletion.

FigureFigure 5: Antioxidants in blood serum, glutathione peroxidase (GPx), and superoxide dismutase (SOD) from breeders intermittently challenged with AFB1 and OTA, with and without an in-feed intervention (IFI).

Intermittent exposure to AFB1 and OTA negatively affected layer breeder productivity, egg quality, and hatchability and promoted oxidative stress in the birds. Intermittent mycotoxin challenges may affect animals even after the contamination is withdrawn. In-feed interventions showed effectiveness in mitigating these effects.

Climate changes bring new mycotoxin challenges – the right in-feed solutions can help

Today’s mycotoxin scenario shows increased frequency, quantity, and variety. Mainly long-living animals, such as laying hens and breeders, can be at more risk. Additionally, the contamination can be carried over to the eggs, potentially risking human health in the case of table eggs and hatchery performance and DOC quality in the case of breeders.

From case reports, we learn the consequences of real challenges and struggles in commercial production; from scientific trials based on possible commercial situations, we realize the advantages of interventions designed to tackle those challenges.

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Organic acids can play a crucial role in zinc oxide replacement

HEADER LOW Shutterstock

Dr. Inge Heinzl, Editor EW Nutrition &
Juan Antonio Mesonero Escuredo, GTM Swine/GPM Organic Acids EW Nutrition

The use of high levels of Zinc Oxide (ZnO) in the EU before 2022 was one of the most common methods to prevent postweaning diarrhea (PWD) in pig production. Pharmacologically high levels of ZnO (2000-3000 ppm) increase growth and reduce the incidence of enteric bacterial diseases such as post-weaning diarrhea (PWD)( Carlson et al., 1999; Hill et al., 2000; Hill et al., 2001; Poulsen & Larsen, 1995; De Mille et al., 2019).

However, ZnO showed adverse effects, such as the accumulation of heavy metal in the environment, the risk for antimicrobial resistance (AMR), and problems of mineral toxicity and adverse growth effects when feeding it longer than 28 days (Jensen et al., 2018; Cavaco et al., 2011; Vahjen, 2015; Romeo et al., 2014; Burrough et al., 2019). To replace ZnO in pig production, let us first look at its positive effects to know what we must compensate for.

ZnO has a multifactorial mode of action

ZnO shows several beneficial characteristics that positively influence gut health, the immune system, digestion, and, therefore, also overall health and growth performance.

FigureFigure 1. Beneficial effects and ZnO mode of action in postweaning piglets

1.   ZnO acts as an antimicrobial

Concerning the antimicrobial effects of ZnO, different possible modes of action are discussed:

  • ZnO in high dosages generates reactive oxygen species (ROS) that can damage the bacterial cell walls (Pasquet et al., 2014)
  • The death of the bacterial cell due to direct contact of the metallic Zn to the cell (Shearier et al., 2016)
  • Intrinsic antimicrobial properties of the ZnO2+ ions after dissociation. The uptake of zinc into cells is regulated by homeostasis. A concentration of the ZnO2+ ions higher than the optimal level of 10-7 to 10-5 M (depending on the microbial strain) allows the invasion of Zn2+ ions into the cell, and the zinc starts to be cytotoxic (Sugarman, 1983; Borovanský et al., 1989).

ZnO shows activity against, e.g., Staphylococcus aureus, Pseudomonas aeruginosa, E. coli, Streptococcus pyogenes, and other enterobacteria (Ann et al., 2014; Vahjen et al., 2016). However, Roselli et al. (2003) did not see a viability-decreasing effect of ZnO on ETEC.

2.   ZnO modulates the immune system

Besides fighting pathogenic organisms as described in the previous chapter and supporting the immune system, ZnO is an essential trace element and has a vital role in the immune system. ZnO improves the innate immune response, increasing phagocytosis and oxidative bursts from macrophages and neutrophils. It also ameliorates the adaptative immune response by increasing the number of T lymphocytes (T cells) in general and regulatory T lymphocytes (T-regs) in particular. These cells control the immune response and inflammation (Kloubert et al., 2018). Macrophage capacity for phagocytosis (Ercan and Bor, 1991) and to kill parasites (Wirth et al., 1989), and also the killing activity of natural killer cells depends on Zn (Rolles et al., 2018). By reducing bacterial adhesion and blocking bacterial invasion, ZnO disburdens the immune system (Roselli et al., 2003).

ZnO reduces the expression of several proinflammatory cytokines induced by ETEC (Roselli et al., 2003). Several studies have also shown a modulation effect on intestinal inflammation, decreasing levels of IFN-γ, TNF-α, IL-1ß and IL-6, all pro-inflammatory, in piglets supplemented with ZnO (Zhu et al., 2017; Grilli et al., 2015).

3.   ZnO improves digestion and promotes growth

Besides protecting young piglets against diarrhea, the goal is to make them grow optimally. For this target, an efficient digestion and a high absorption of nutrients is essential. Stimulating diverse pancreatic enzymes such as amylase, carboxypeptidase A, trypsin, chymotrypsin, and lipase increases digestibility (Hedemann et al., 2006; Pieper et al., 2015). However, Pieper et al. (2015) also showed that a long-term supply of very high dietary zinc triggers oxidative stress in the pancreas of piglets.

By stimulating the secretion of ghrelin at the stomach level and thereby promoting the release of insulin-like growth factor (IGF-1) and cholecystokinin (CCK), ZnO enhances muscle protein synthesis, cell proliferation, and feed intake (Yin et al., 2009; MacDonald et al., 2000)).

The result of improved digestion is increased body weight and average daily gain, which can be seen, e.g., in a study by Zhu et al. (2017).

4.   ZnO protects the intestinal morphology

ZnO prevents the decrease of the trans-endothelial electrical resistance (TEER), usually occurring in the case of inflammation, by downregulating TNF-α and IFN-γ. TNF-α, as well as IFN-γ, increase the permeability of the epithelial tight junctions and, therefore, the intestinal barrier (Al-Sadi et al., 2009).

The enterotrophic and anti-apoptotic effect of ZnO is reflected by a higher number of proliferating and PCNA-positive cells and an increased mucosa surface in the ileum (higher villi, higher villi/crypt ratio)(Grilli et al., 2015). Zhu et al. (2017) also saw an increase in villus height in the duodenum and ileum and a decrease in crypt depth in the duodenum due to the application of 3000 mg of ZnO/kg. Additionally, they could notice a significant (P<0.05) upregulation of the mRNA expression of the zonula occludens-1 and occluding in the mucosa of the jejunum of weaned piglets.

In a trial conducted by Roselli et al. (2003), the supplementation of 0.2 mmol/L ZnO prevented the disruption of the membrane integrity when human Caco-2 enterocytes were challenged with ETEC.

5.   ZnO acts antioxidant

The antioxidant effect of ZnO was shown in a study conducted by Zhu et al., 2017. They could demonstrate that the concentration of malondialdehyde (MDA), a marker for lipid peroxidation, decreased on day 14 or 28, and the total concentration of superoxide dismutase (SOD), comprising enzymes that transform harmful superoxide anions into hydrogen peroxide, increased on day 14 (P<0.05). Additionally, Zn is an essential ion for the catalytic action of these enzymes.

Which positive effects of ZnO can be covered by organic acids (OAs)?

1.   OAs act antimicrobial

OAs, on the one hand, lower the pH in the gastrointestinal tract. Some pathogenic bacteria are susceptible to low pH. At a pH<5, the proliferation of, e.g., Salmonella, E. coli, and Clostridium is minimized. The good thing is that some beneficial bacteria, such as lactobacilli or bifidobacteria, survive as they are acid-tolerant. The lactobacilli, on their side, can produce hydrogen peroxide, which inhibits, e.g., Staphylococcus aureus or Pseudomonas spp. (Juven and Pierson, 1996).

Besides this more indirect mode of action, a more direct one is also possible: Owing to their lipophilic character, the undissociated form of OAs can pass the bacterial membrane (Partanen and Mroz, 1999). The lower the external pH, the more undissociated acid is available for invading the microbial cells. Inside the cell, the pH is higher than outside, and the OA dissociates. The release of hydrogen ions leads to a decrease in the internal pH of the cell and to a depressed cell metabolism. To get back to “normal conditions”, the cell expels protons. However, this is an energy-consuming process; longer exposure to OAs leads to cell death. The anion remaining in the cell, when removing the protons, disturbs the cell’s metabolic processes and participates in killing the bacterium.

These theoretical effects could be shown in a practical trial by Ahmed et al. (2014). He fed citric acid (0.5 %) and a blend of acidifiers composed of formic, propionic, lactic, and phosphoric acid + SiO2 (0.4 %) and saw a reduction in fecal counts of Salmonella and E. coli for both groups.

2.   OAs modulate the immune system

The immune system is essential in the pig’s life, especially around weaning. Organic acids have been shown to support or stimulate the immune system. Citric acid (0.5%), as well as the blend of acidifiers mentioned before (Ahmed et al., 2014), significantly increased the level of serum IgG. IgG is part of the humoral immune system. They mark foreign substances to be eliminated by other defense systems.

Ren et al. (2019) could demonstrate a decrease in plasma tumor necrosis factor-α that regulates the activity of diverse immune cells. He also found lower interferon-γ and interleukin (Il)-1ß values in the OA group than in the control group after the challenge with ETEC. This trial shows that inflammatory response can be mitigated through the addition of organic acids.

3.   OAs improve digestion and promote growth

In piglets, the acidity in the stomach is responsible for the activation and stimulation of certain enzymes. Additionally, it keeps the feed in the stomach for a longer time. Both effects lead to better digestion of the feed.

In the stomach, the conversion of pepsinogen to pepsin, which is responsible for protein digestion, is catalyzed under acid conditions (Sanny et al., 1975)group. Pepsin works optimally at two pH levels: pH 2 and pH 3.5 (Taylor, 1959). With increasing pH, the activity decreases; at pH 6, it stops. Therefore, a high pH can lead to poor digestion and undigested protein arriving in the intestine.

These final products of pepsin protein digestion are needed in the lower parts of the GIT to stimulate the secretion of pancreatic proteolytic enzymes. If they do not arrive, the enzymes are not activated, and the inadequate protein digestion continues. Additionally, gastric acid is the primary stimulant for bicarbonate secretion in the pancreas, neutralizing gastric acid and providing an optimal pH environment for the digestive enzymes working in the duodenum.

As already mentioned, the pH in the stomach influences the transport of digesta. The amount of digesta being transferred from the stomach to the small intestine is related to the acidity of the chyme leaving the stomach and arriving in the small intestine. Emptying of the stomach can only take place when the duodenal chyme can be neutralized by pancreatic or other secretions (Pohl et al., 2008); so, acid-sensitive receptors provide feedback regulation and a higher pH in the stomach leads to a faster transport of the digesta and a worse feed digestion.

4.   OAs protect the intestinal morphology

Maintaining an intact gut mucosa with a high surface area is crucial for optimal nutrient absorption. Research suggests organic acids play a significant role in improving mucosal health:

Butyric acid promotes epithelial cell proliferation, as demonstrated in an in vitro pig hindgut mucosa study (Sakata et al., 1995). Fumaric acid, serving as an energy source, may locally enhance small intestinal mucosal growth, aiding in post-weaning epithelial cells’ recovery and increasing absorptive surface and digestive capacity (Blank et al., 1999). Sodium butyrate supplementation at low doses influences gastric morphology and function, thickening the stomach mucosa and enhancing mucosal maturation and differentiation (Mazzoni et al., 2008).

Studies show that organic acids affect gut morphology, with a mixture of short-chain and mid-chain fatty acids leading to longer villi (Ferrara et al., 2016) and Na-butyrate supplementation increasing crypt depth and villi length in the distal jejunum and ileum (Kotunia et al., 2004). However, the villi length and mucosa thickness in the duodenum were reduced. Dietary sodium butyrate has been linked to increased microvilli length and cecal crypt depth in pigs (Gálfi and Bokori, 1990).

5.   OAs show antioxidant activity

The last characteristic, the antioxidant effect, cannot be provided at the same level as with ZnO; however, Zhang et al. (2019) attest to OAs a certain antioxidant activity. Oxalic, citric, acetic, malic, and succinic acids, which were extracted from Camellia oleifera, also showed good antioxidant activity in a trial conducted by Zhang et al. (2020).

Organic acids are an excellent tool to compensate for the ban on ZnO

The article shows that organic acids have similar positive effects as zinc oxide. They act antimicrobial, modulate the immune system, maintain the gut morphology, fight pathogenic microbes, and also act – slightly – antioxidant. Additionally, they have a significant advantage: they are not harmful to the environment. Organic acids used in the proper pH range and combination are good tools for replacing zinc oxide.

References on request




Salmonella in pigs: a threat for humans and a challenge for pig producers

Header SWINE Fotolia

By Dr. Inge Heinzl, Editor, EW Nutrition

Salmonellosis is third among foodborne diseases leading to death (Ferrari, 2019). More than 91,000 human cases of Salmonellosis are reported by the EU each year, generating overall costs of up to €3 billion a year (EFSA, 2023), 10-20% of which are attributed to pork consumption (Soumet, 2022). The annual costs arising from the resulting human health losses in 2010 were about €90 million (FCC Consortium, 2010). Take the example of Ireland, where a high prevalence of Salmonella in lymph nodes still shows a severe issue pre-slaughter and a big challenge for slaughterhouses to stick to the process hygiene requirements (Deane, 2022).

Several governments already have monitoring programs in place, and the farms are categorized according to the salmonella contamination of their pigs. In some countries, e.g., Denmark, an economic penalty of 2% of the carcass value must be paid if the farm has level 2 (intermediate seroprevalence) and 4-8% if the level is 3. Other countries, e.g., Germany, the UK, Ireland, or the Netherlands, use quality assurance schemes. The farmers can only sell their carcasses under this label if their farm has a certain level.

Let’s take a quick look at the genus of Salmonella

Salmonellas are rod-shaped gram-negative bacteria of the family of enterobacteria that use flagella for their movement. They were named after the American vet Daniel Elmer Salmon. The genus of Salmonella consists of two species (S. bongori and S. enterica with seven subspecies) with in total more than 2500 serovars (see Figure 1). The effects of the different serovars can range from asymptomatic carriage to severe invasive systemic disease (Gal-Mor, 2014). All Salmonella serovars generally can cause disease in humans; the rosa-marked ones already showed infections.

Figure Genus Salmonella For ART PIGFigure 1: the genus of Salmonella with Salmonella serovars relevant for pigs (according to Bonardi, 2017: Salmonella in the pork production chain and its impact on human health in the European Union)

Within the group of Salmonella, some serovars can only reside in one or few species, e.g., S. enterica spp. enterica Serovar Dublin (S. Dublin) in bovines (Waldron, 2018) or S. Cholerasuis in pigs (Chiu, 2004). An infection in humans with these pathogens is often invasive and life-threatening (WHO, 2018). On the contrary, serovars like S. Typhimurium and S. Enteritidis are not host-specific and can cause disease in various species.

The serotypes S. Typhi and S. Paratyphi A, B, or C are highly adapted to humans and only for them pathogenic; they are responsible for the occurrence of typhus.

Serovars occurring in pigs and relevant for humans are, for example, S. Typhimurium (Hendriksen, 2004), S. Serotype 4,[5],12:I (Hauser et al., 2010), S. Cholerasuis (Chiu, 2004), S. Derby (Gonzalez-Santamarina, 2021), S. Agona (Brenner Michael, 2006) and S. Rissen (Elbediwi, 2021).

Transmission of Salmonella mostly happens via contaminated food

The way of transmission to humans depends on the serovar:
Human-specific and, therefore, only in humans and higher primates residing serovars S. Typhi and Paratyphi A, B, or C (typhoidal) are excreted via feces or urine. Therefore, any food or water contaminated with the feces or urine of infected people can transmit this disease (Government of South Australia, 2023). Typhoid and paratyphoid Salmonellosis occur endemic in developing countries with the lack of clean water and, therefore, inadequate hygiene (Gal-Mor, 2014).

Serovars which can cause disease in humans and animals (non-typhoidal), can be transmitted by
– animal products such as milk, eggs, meat
– contact with infected persons/animals (pigs, cows, pets, reptiles…) or
– other feces- or urine-contaminated products such as sprouts, vegetables, fruits….

Farm animals take salmonellas from their fellows, contaminated feed or water, rodents, or pests.

Symptoms of Salmonellosis can be severe

In the case of typhoid or paratyphoid Salmonellosis, the onset of illness is gradual. People can suffer from sustained high fever, unwellness, severe headache, and decreased appetite, but also from an enlarged spleen irritating the abdomen and dry cough.

A study conducted in Thailand with children suffering from enteric fever caused by the typhoid serovars S. Typhi and Paratyphi showed a sudden onset of fever and gastrointestinal issues (diarrhea), rose spots, bronchitis, and pneumonia (Thisyakorn et al., 1987)

The non-typhoid Salmonellosis is typically characterized by an acute onset of fever, nausea, abdominal pain with diarrhea, and sometimes vomiting (WHO, 2018). However, 5% of the persons – children with underlying conditions, e.g., babies, or people who have AIDS, malignancies, inflammatory bowel disease, gastrointestinal illness caused by non-typhoid serovars, and hemolytic anemia, or receiving an immunosuppressive therapy can be susceptible to bacteremia. Additionally, serovars like S. Cholerasuis or S. Dublin are apt to develop bacteremia by entering the bloodstream with little or no involvement of the gut (Chiu, 1999). In these cases, consequences can be septic arthritis, pneumonia, peritonitis, cutaneous abscess, mycotic aneurysm, and sometimes death (Chen et al., 2007; Chiu, 2004, Wang et al., 1996).

In pigs, S. Cholerasuis causes high fever, purple discolorations of the skin, and thereinafter diarrhea. The mortality rate in pigs suffering from this type of Salmonellosis is high. Barrows orally challenged with S. Typhimurium showed elevated rectal temperature by 12h, remaining elevated until the end of the study. Feed intake decreased with a peak at 48h after the challenge and remained up to 120h after the challenge. Daily gain reduced during the following two weeks after infection. A higher plasma cortisol level and a lower IGF-I level could also be noticed. All these effects indicate significant changes in the endocrine stress and the somatotropic axis, also without significant alterations in the systemic pro-inflammatory mediators (Balaji et al., 2000)

To protect humans, Salmonella in pork must be restraint

There are three main steps to keep the contamination of pork as low as possible:

  1. Keeping Salmonella out of the pig farm
  2. Minimizing spreading if Salmonella is already on the farm
  3. Minimizing contamination in the slaughterhouse

1. How to keep Salmonella out of the pig farm?

To answer this question, we must look at how the pathogen can be transported to the farm. According to the Code of Practice for the Prevention and Control of Salmonella on Pig Farms (Ministry of Agriculture, Fisheries and Food and the Scottish Executive Rural Affairs Department), there are several possibilities to infiltrate the pathogen into the farm:

  • Diseased pigs or pigs which are ill but don’t show any symptoms
  • Feeding stuff or bedding contaminated with dung
  • Pets, rodents, wild birds, or animals
  • Farm personnel or visitors
  • Equipment or vehicles

Caution with purchased animals!

To minimize/prevent the entry of Salmonella into the livestock, bought-in animals must come from reputable breeding farms with a salmonella monitoring system in place. As possible carrier animals are more likely to excrete Salmonella when stressed; they should be kept in isolation after purchasing. Additionally, the animals must go through a disinfectant foot bath before entering the farm.

Keep rodents, wild animals, and vermin in check!

Generally, the production site must be kept clean and as unattractive as possible for all these animals. Rests of feed must be removed, and dead animals and afterbirths must be promptly and carefully disposed of. A well-planned baiting and trapping policy should be in place to effectively control rodents.

Only selected people should enter the hog houses

In any case, the number of persons entering the hog house must be kept as low as possible. Farmworkers should be trained in the principles of hygiene. They should wear adequate clothing (waterproof boots and protective overalls) that can be easily cleaned/laundered and disinfected. The clothes/shoes should always be used only at this site. Thorough hand washing and the disinfection of the boots when entering and leaving the pig unit are a must.

If visits are necessary, the visitors should take the same measures as the farm workers. And, of course, they should not have had contact with another pig farm during the last 48 hours.

Keep pens, farm equipment, and vehicles clean!

Farm equipment should not be shared with other farms. If this cannot be avoided, it must be cleaned and disinfected before re-entering the farm. Also, the vehicles for the transport of the animals must be cleaned and disinfected as soon as possible after usage, as contaminated transporters always pose the risk of infection.

Feed should be Salmonella-free!

To get high feed quality, the feed should be purchased from feed mills/sources with a well-functioning bacterial control to guarantee the absence of Salmonella. It is essential that birds, domestic and wild animals cannot enter the feed stores.
It is also advised to keep dry feed dry as possibly contaminating Salmonella can multiply in such humid conditions. Additionally, all feed bins and delivery pipes for dry and wet feed must be consciously cleaned, and the damp feed pipes also disinfected.
The change from pellets to mash could be helpful as the pellets facilitate Salmonella colonization by stimulating the secretion of mucins (Hedemann et al., 2005).

For sanitation of the feed, we offer organic acids (Acidomix product range) or mixtures of organic acids and formaldehyde in countries where formaldehyde products are allowed (Formycine) to decrease the pathogenic load of the feed materials. In vitro trials show the effectiveness of the products:

 

For the in vitro trial with Formycine, autoclaved feed samples were inoculated with Salmonella enteritidis serovar Typhimurium DSM 19587 strain to reach a Salmonella contamination of 106 CFU/g of feed. After incubating at room temperature for three hours, Formycine Liquido was added to the contaminated feed samples at 0, 500, 1000, and 2000 ppm. The control and inoculated feed samples were further incubated at room temperature, and Salmonella counts (CFU/g) were carried out at 24, 48, 72 hours and on day 15. The limit of Salmonella detection was set at 100 CFU/g (102). Results are shown in figure 2.

Figure FormycineFig. 2: Effect of treatment time and different inclusion levels of Formycine Liquido on the Salmonella count in feed

As important as uncontaminated feed is clean water for drinking. It can be achieved by taking the water from a main or a bacteriologically controlled water borehole. Regular cleaning/disinfection of the tanks, pipes, and drinkers is essential.

Bedding should be Salmonella-free

Straw material containing feces of other animals (rodents, pets) always carries the risk of Salmonella contamination. Also, wet or moldy bedding is not recommended because it is an additional challenge for the animal. To optimize the quality of bedding, the straw should be bought from reliable and as few as possible sources. The material must be stored dry and as far as practicable from the pig buildings (Ministry of Agriculture, Fisheries and Food & Scottish Executive Rural Affairs Department, 2000).

Vaccination is a beneficial measure

For the control of Salmonella in swine herds, vaccination is an effective tool. De Ridder et al. (2013) showed that an attenuated vaccine reduced the transmission of Salmonella Typhimurium in pigs. The vaccination with an attenuated S. Typhimurium strain, followed by a booster vaccination with inactivated S. Cholerasuis, showed better effects than an inactivated S. Cholerasuis vaccine alone (Alborali et al., 2017). Bearson et al. (2017) could delimitate transmission through less shedding and protect the animals against systemic disease.
To achieve the best effects, the producer must understand the diversity of Salmonella serovars to choose the most promising vaccination strategy (FSIS, 2023).

2. How to minimize the spreading of Salmonella on the farm?

If there are already cases of Salmonella on the farm, infected animals must be separated from the rest of the herd. Small batch sizes are beneficial, as well as not mixing different litters after weaning. If feasible, separate units for different production phases with an all-in/all-out system could break the reinfection cycle and help reduce Salmonella contamination on the farm. And also in this case, vaccination is helpful.

Salmonella doesn’t like acid conditions

An effective tool is acidifying the feed with organic acids, as Salmonella doesn’t like acid conditions. A trial was conducted with Acidomix AFG and Acidomix AFL to show their effects against Salmonella. For the test, 105 CFU/g of Salmonella enterica ser. Typhimurium was added to feed containing 1000 ppm, 2000 ppm, and 3000 ppm of Acidomix AFG or AFL. The stomach and intestine were simulated in vitro by adjusting the pH with HCl and NaHCO3 as follows:
Stomach              2.8
Intestine              6.8-7.0

After the respective incubation, the microorganisms were recovered from feed and plated on an appropriate medium for CFU counting. The results are shown in figures 3 and 4.

Combi
Combi

Figures 3 + 4: Effects of different concentrations of Acidomix AFG and Acidomix AFL against Salmonella enterica ser. Typhimurium in feed

Phytomolecules can support pigs against Salmonella

Plant compounds or phytomolecules can also be used against Salmonella in pigs. Some examples of phytomolecules to be used are Piperine, Allicin, Eugenol, and Carvacrol. Eugenol, e.g., increases the permeability of the Salmonella membrane, disrupts the cytoplasmic membrane, and inhibits the production of bacterial virulence factors (Keita et al., 2022; Mak et al., 2019). Thymol and Carvacrol interact with the cell membrane by H bonding, also resulting in a higher permeability.

An already published in vitro trial conducted with our product Ventar D also showed excellent effects against Salmonella while sparing the beneficial gut flora. A further trial once more demonstrated the susceptibility of Salmonella to Ventar D. It showed that Ventar D controls Salmonella by suppressing their motility and, at higher concentrations, inactivating the cells (see figures 5 + 6):

Figure Motility TestFigure 5: S. enterica motility test: on the left side – control; on the right side – motility medium containing.750 µg/mL of Ventar
Figure Disk DiffusionFig 6 . Disk diffusion assay employing S. enterica. upper left side – disk containing 10 µL of Ventar; upper right – 5 µL; lower left – control; lower right – 1µL.

In addition to the direct Salmonella-reducing effect, essential oils / secondary plant compounds / phytomolecules improve digestive enzyme activity and digestion, leading to increased nutrient absorption and better feed conversion (Windisch et al., 2008).

3. How can the farmer keep Salmonella contamination low in the slaughterhouse?

In general, the slaughterhouse personnel is responsible for adequate hygiene management to prevent contamination of carcasses and meat. However, also the farmer can make his contribution to maintain the risk of contamination in the slaughterhouse as low as possible. A study by Vieira-Pinto (2006) revealed that one Salmonella-positive pig can contaminate several other carcasses.

According to a trial conducted by Hurd et al. (2002), infection and, therefore, “contamination” of other pigs can rapidly occur, meaning that cross-contamination is a topic during transport to the slaughterhouse and in the lairages when the pigs come together with animals from other farms. The stress to which the pigs are exposed influences physiological and biochemical processes. The microbiome and animal’s immunity are affected, leading to higher excretion of Salmonella during transport and in the lairages. So, the animals should not be stressed during loading and unloading or transportation. The trailer poses a further risk of infection if it was not cleaned and disinfected before. So, reliable people who treat the animals well and keep their trailers clean should be chosen for transportation.

Pig producers are obliged to keep Salmonella in check – phytomolecules can help

At least in the EU, pig producers have the big duty to keep Salmonella low in their herds; otherwise, they will have financial losses. They are not only responsible for their farm, but also the slaughterhouses count on them. Besides the standard strict hygiene management and vaccination, farmers can use products provided by the industry to sanitize feed but also to support their animals directly with phytomolecules acting against pathogens and supporting gut health.

All these measures together should be a solution to the immense challenge of Salmonella, to protect people and prevent economic losses.

References:

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Hauser, Elisabeth, Erhard Tietze, Reiner Helmuth, Ernst Junker, Kathrin Blank, Rita Prager, Wolfgang Rabsch, Bernd Appel, Angelika Fruth, and Burkhard Malorny. “Pork Contaminated with            Salmonella            Enterica            Serovar 4,[5],12:I:−, an Emerging Health Risk for Humans.” Applied and Environmental Microbiology 76, no. 14 (2010): 4601–10. https://doi.org/10.1128/aem.02991-09.

Health and Wellbeing; address=11 Hindmarsh Square, Adelaide scheme=AGLSTERMS.AglsAgent; corporateName=Department for. “Sa Health.” Typhoid and paratyphoid – including symptoms, treatment, and prevention, April 3, 2022. https://www.sahealth.sa.gov.au/wps/wcm/connect/public+content/sa+health+internet/conditions/infectious+diseases/typhoid+and+paratyphoid/typhoid+and+paratyphoid+-+including+symptoms+treatment+and+prevention.

Hedemann, M. S., L. L. Mikkelsen, P. J. Naughton, and B. B. Jensen. “Effect of Feed Particle Size and Feed Processing on Morphological Characteristics in the Small and Large Intestine of Pigs and on Adhesion of Salmonella Enterica Serovar Typhimurium DT12 in the Ileum in Vitro1.” Journal of Animal Science 83, no. 7 (2005): 1554–62. https://doi.org/10.2527/2005.8371554x.

Hendriksen, Susan W.M., Karin Orsel, Jaap A. Wagenaar, Angelika Miko, and Engeline van Duijkeren. “Animal-to-Human Transmission ofSalmonellaTyphimurium DT104A Variant.” Emerging Infectious Diseases 10, no. 12 (2004): 2225–27. https://doi.org/10.3201/eid1012.040286.

Keita, Kadiatou, Charles Darkoh, and Florence Okafor. “Secondary Plant Metabolites as Potent Drug Candidates against Antimicrobial-Resistant Pathogens.” SN Applied Sciences 4, no. 8 (2022). https://doi.org/10.1007/s42452-022-05084-y.

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Morrow, W.E. Morgan, and Julie Funk. Ms. Salmonella as a Foodborne Pathogen in Pork. North Carolina State University Animal Science, n.d.

Soumet, C., A. Kerouanton, A. Bridier, N. Rose, M. Denis, I. Attig, N. Haddache, and C. Fablet. Report, Salmonella excretion level in pig farms and impact of quaternary ammonium compounds based disinfectants on Escherichia coli antibiotic resistance § (2022).

Thisyakorn, Usa. “Typhoid and Paratyphoid Fever in 192 Hospitalized Children in Thailand.” Archives of Pediatrics &amp; Adolescent Medicine 141, no. 8 (1987): 862. https://doi.org/10.1001/archpedi.1987.04460080048025.

Ung, Aymeric, Amrish Y. Baidjoe, Dieter Van Cauteren, Nizar Fawal, Laetitia Fabre, Caroline Guerrisi, Kostas Danis, et al. “Disentangling a Complex Nationwide Salmonella Dublin Outbreak Associated with Raw-Milk Cheese Consumption, France, 2015 to 2016.” Eurosurveillance 24, no. 3 (2019). https://doi.org/10.2807/1560-7917.es.2019.24.3.1700703.

Vieira-Pinto, M, R Tenreiro, and C Martins. “Unveiling Contamination Sources and Dissemination Routes of Salmonella Sp. in Pigs at a Portuguese Slaughterhouse through Macrorestriction Profiling by Pulsed-Field Gel Electrophoresis.” International Journal of Food Microbiology 110, no. 1 (2006): 77–84. https://doi.org/10.1016/j.ijfoodmicro.2006.01.046.

Waldron, P. “Keeping Cows and Humans Safe from Salmonella Dublin.” Cornell University College of Veterinary Medicine, December 25, 2018. https://www.vet.cornell.edu/news/20181218/keeping-cows-and-humans-safe-salmonella-dublin.

Wang, J.-H., Y.-C. Liu, M.-Y. Yen, J.-H. Wang, Y.-S. Chen, S.-R. Wann, and D.-L. Cheng. “Mycotic Aneurysm Due to Non-Typhi Salmonella: Report of 16 Cases.” Clinical Infectious Diseases 23, no. 4 (1996): 743–47. https://doi.org/10.1093/clinids/23.4.743.

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Windisch, W., K. Schedle, C. Plitzner, and A. Kroismayr. “Use of Phytogenic Products as Feed Additives for Swine and Poultry1.” Journal of Animal Science 86, no. suppl_14 (2008). https://doi.org/10.2527/jas.2007-0459.

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Masked mycotoxins – particularly dangerous for dairy cows

Cows

By Si-Trung Tran, SEAP Regional Technical Manager, EW Nutrition

Marisabel Caballero, Global Technical Manager Poultry, EW Nutrition, and
Inge Heinzl, Editor, EW Nutrition

Mycotoxins are secondary metabolites of fungi, commonly found as contaminants in agricultural products. In some cases, these compounds are used in medicine or industry, such as penicillin and patulin. In most cases, however, they are considered xenobiotics that are toxic to animals and humans, causing the disease collectively known as mycotoxicosis. The adverse effects of mycotoxins on human and animal health have been documented in many publications. Aflatoxins (AFs) and deoxynivalenol (DON, vomitoxin) are amongst the most critical mycotoxins affecting milk production and -quality.

Aflatoxins do not only affect cows

Aflatoxins (AFs) are highly oxygenated, heterocyclic difuranocoumarin compounds produced by Aspergillus flavus and Aspergillus parasiticus. They colonize crops, including many staple foods and feed ingredients. Within a group of over 20 AFs and derivatives, aflatoxin B1 (AFB1), B2, G1, and G2 are the most important naturally occurring compounds.

Among the aflatoxins, AFB1 is the most widespread and most toxic to humans and animals. Concern about mycotoxin contamination in dairy products began in the 1960s with the first reported cases of contamination by aflatoxin M1 (AFM1), a metabolite of AFB1 formed in the liver of animals and excreted in the milk.

There is ample evidence that lactating cows exhibit a significant reduction in feed efficiency and milk yield within a few days of consuming aflatoxin-contaminated feed. At the cellular level, aflatoxins cause degranulation of endoplasmic membranes, loss of ribosomes from the endoplasmic reticulum, loss of nuclear chromatin material, and altered nuclear shapes. The liver, as the organ mainly dealing with the decontamination of the organism, gets damaged, and performance drops. Immune cells are also affected, reducing immune competence and vaccination success (Arnold and Gaskill, 2023).

DON reduces cows’ performance

Another mycotoxin that can also reduce milk quality and affect metabolic parameters, as well as the immune function of dairy cows, is DON. DON is produced by different fungi of the Fusarium genus that infect plants. DON synthesis is associated with rainy weather from crop flowering to harvest. Whitlow and co-workers (1994) reported the association between DON and poor performance in dairy herds and showed decreased milk production in dairy cows fed 2.5 mg DON/kg. However, in cows fed 6 to 12 mg DON/kg dry matter for 10 weeks, no DON or its metabolite DOM-1 residues were detected in milk.

Masked mycotoxins hide themselves during analysis

Plants suffering from fungal infestations and thus confronted with mycotoxins convert the harmful forms of mycotoxins into less harmful or harmless ones for themselves by conjugation to sulfates, organic acids, or sugars. Conjugated mycotoxins cannot always be detected by standard analytical methods. However, in animals, these forms can be released and transformed into parent compounds by enzymes and microorganisms in the gastrointestinal tract. Thus, the feed may show a concentration of mycotoxins that is still below the limit value, but in the animal, this concentration is suddenly much higher. In dairy cows, the release of free mycotoxins from conjugates during digestion may play an important role in understanding the silent effects of mycotoxins.

Fusarium toxins, in particular, frequently occur in this “masked form”. They represent a serious health risk for animals and humans.

Aflatoxins first show up in the milk

Masked aflatoxins may also play a role in total aflatoxin contamination of feed materials. Research has harvested little information on masked aflatoxins that may be present in TMR ingredients. So far, metabolites such as Aflatoxin M2 have been identified (Righetti, 2021), which may reappear later in milk as AFM1.

DON-related symptoms without DON?

Sometimes, animals show DON-related symptoms, with low levels detected in the feed or raw materials. Besides sampling errors, this enigma could be due to conjugated or masked DON, which is structurally altered DON bound to various compounds such as glucose, fatty acids, and amino acids. These compounds escape conventional feed analysis techniques because of their modified chemical properties but can be released as their toxic precursors after acid hydrolysis.

Masked DON was first described in 1984 by Young and co-workers, who found that the DON content of yeast-fermented foods was higher than that of the contaminated wheat flour used in their production. The most plausible reason for this apparent increase was that the toxin from the wheat had been converted to a compound other than DON, which could be converted back to DON under certain conditions. Since this report, there has been much interest in conjugated or masked DON.

Silage: masked DON is a challenge for dairy producers

Silage is an essential feed for dairy cows, supporting milk production. Most silage is made from corn and other grains. The whole green plant is used, which can be infected by fungi. Since infection of corn with Fusarium spp. and subsequent DON contamination is usually a major problem in the field worldwide, a relatively high occurrence of this toxin in silage must be expected. The ensiling process may reduce the amount of Fusarium fungi, but the DON formed before ensiling is very stable.

Corn Silage

Silage samples show DON levels of concern

It is reasonable to assume that the DON biosynthesized by the fungi was metabolized by the plants to a new compound and thus masked DON. Under ensiling conditions, masked DON can be hydrolyzed, producing free DON again. Therefore, the level of free DON in the silage may not reflect the concentration measured in the plants before ensiling.

A study analyzed 50 silage samples from different farms in Ontario, Canada. Free DON was found in all samples, with levels ranging from 0.38 to 1.72 µg/g silage (unpublished data). Eighty-six percent of the samples contained DON at concentrations higher than 0.5 µg/g. Together with masked DON, it poses a potential threat to dairy cattle.

Specific hydrolysis conditions allow detection

However, in the natural ensiling process, the conditions for hydrolysis of masked DON are not optimal. The conditions that allow improved analysis of masked DON were recently described. This method detected masked DON in 32 of 50 silage samples (64%) along with free DON, increasing DON concentration by 23% in some cases (unpublished data).

Mycotoxins impact humans and animals

Aflatoxins, as well as DON, have adverse effects. In the case of DON, the impact on the animal is significant; in the case of aflatoxin, the possible long-term effects on humans are of higher relevance.

DON has more adverse effects on the animal and its performance

Unlike AFs, DON may be found in milk at low or trace concentrations. It is more associated with negative effects in the animal, altered rumen fermentation, and reduced flow of usable protein into the duodenum. For example, milk fat content was significantly reduced when cows were fed 6 µg DON/kg. However, the presence of DON also indicates that the feed probably contains other mycotoxins, such as zearalenone (ZEA) (estrogenic mycotoxin) and fusaric acid (pharmacologically active compound). All these mycotoxins may interact to cause symptoms that are different or more severe than expected, considering their individual effects. DON and related compounds also have immunosuppressive effects, resulting in increased somatic cell counts in milk. The U.S. FDA has established an action level for DON in wheat and wheat-derived products intended for cows, which is 5µg DON/g feed and the contaminated ingredient must not exceed 40% of the ration.

Aflatoxins decrease milk quality and pose a risk to humans

Aflatoxins are poorly degraded in the rumen, with aflatoxicol being the main metabolite that can be reconverted to AFB1. Most AFs are absorbed and extensively metabolized/hydrolyzed by enzymes found mainly in the liver. This results in the formation of AFM1, a part of which is conjugated to glucuronic acid and subsequently excreted in the bile. The other part enters the systemic circulation. It is either excreted in urine or milk. AFM1 appears within 12-48 hours after ingestion in cow’s milk. The excreted amount of AFM1 in milk from dairy cows usually ranges from 0.17% to 3% of the ingested AFB1. However, this carryover rate may vary from day to day and from one milking to the next in individual animals, as it is influenced by various factors, such as feeding regime, health status, individual biotransformation capacity, and, of course, by actual milk production. Carryover rates of up to 6.2% have been reported in high-yielding dairy cows producing up to 40 liters of milk per day.

In various experiments, AFM1 showed both carcinogenic and immunosuppressive effects. Accordingly, the International Agency for Research on Cancer (IARC) classified AFM1 as being in Group 2B and, thus, possibly carcinogenic in humans. The action level of 0.50 ppb and 0.05 ppb for AFM1 in milk is strictly adhered to by the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), respectively.

Trials show the high adsorption capacity of Solis Max

A trial was conducted at an independent laboratory located in Spain. The evaluation of the performance of Solis Max was executed with the following inclusion levels:

  • 0.10% equivalent to 1.0 kg of Solis Max per ton of feed
  • 0.20% equivalent to 2.0 kg of Solis Max per ton of feed

A phosphate buffer solution at pH 7 was prepared for the trial to simulate rumen conditions. Each mycotoxin was tested separately, preparing solutions with known contamination (final concentration described in the table below). The contaminated solutions were divided into 3 parts: A positive control, 0.10% Solis Max and 0.20% Solis Max. All samples were incubated at 41°C for 1 hour, centrifuged, and the supernatant was analyzed for the mycotoxin added to determine the binding efficacy. All analyses were carried out by high-performance liquid chromatography (HPLC) with standard detectors.

Mycotoxin Contamination Level (ppb)
Aflatoxin B1 800
DON 800
Fumonisin B1 2000
ZEA 1200

Results:
The higher concentration of Solis max showed a higher adsorption rate for most mycotoxins. The high dose of Solis Max adsorbed 99% of the AFB1 contamination. In the case of DON, more than 70% was bound. For fumonisin B1 and zearalenone, Solis max showed excellent binding rates of 87.7% and 78.9%, respectively (Figure 1).

FigureFigure 1: Solis Max showed a high binding capacity for the most relevant mycotoxins

Another trial was conducted at an independent laboratory serving the food and feed industry and located in Valladolid, Spain.

All tests were carried out as duplicates and using a standard liquid chromatography/mass spectrometry (LC/MS/MS) quantification. Interpretation and data analysis were carried out with the corresponding software. The used pH was 3.0, toxin concentrations and anti-mycotoxin agent application rates were set as follows (Table 1):

TableTable 1: Trial set-up testing the binding capacity of Solis Plus 2.0 for several mycotoxins in different contamination levels

Results:

Under acidic conditions (pH3), Solis Plus 2.0 effectively adsorbs the three tested mycotoxins at low and high levels. 100% binding of aflatoxin was achieved at a level of 150ppb and 98% at 1500ppb.In the case of fumonisin, 87% adsorption could be reached at 500ppb and 86 for a challenge with 5000ppb. 43% ochratoxin was adsorbed at the contamination level of 150ppb and 52% at 1500ppb.

FigureFigure 2: The adsorption capacity of Solis Plus 2.0 for three different mycotoxins at two challenge levels

Mycotoxins – Effective risk management is of paramount importance

Although the rumen microflora may be responsible for conferring some mycotoxin resistance to ruminants compared to monogastric animals, there are still effects of mycotoxins on rumen fermentation and milk quality. In addition, masked mycotoxins in feed present an additional challenge for dairy farms because they are not readily detectable by standard analyses.

Feeding dairy cows with feed contaminated with mycotoxins can lead to a reduction in milk production. Milk quality may also deteriorate due to an adverse change in milk composition and mycotoxin residues, threatening the innocuousness of dairy products. Dairy farmers should therefore have feed tested regularly, consider masked mycotoxins, and take action. EW Nutrition’s MasterRisk tool provides a risk evaluation and corresponding recommendations for the use of products that mitigate the effects of mycotoxin contamination and, in the end, guarantee the safety of all of us.

 




Toxin Mitigation 101: Essentials for Animal Production

Fusarium Mycotoxins

By Monish Raj, Assistant Manager-Technical Services, EW Nutrition
Inge Heinzl, Editor, EW Nutrition  

Mycotoxins, toxic secondary metabolites produced by fungi, are a constant and severe threat to animal production. They can contaminate grains used for animal feed and are highly stable, invisible, and resistant to high temperatures and normal feed manufacturing processes. Mycotoxin-producing fungi can be found during plant growth and in stored grains; the prevalence of fungi species depends on environmental conditions, though in grains, we find mainly three genera: Aspergillus, Penicillium, and Fusarium. The most critical mycotoxins for poultry production and the fungi that produce them are detailed in Fig 1.

FigureFigure 1: Fungi species and their mycotoxins of worldwide importance for poultry production (adapted from Bryden, 2012).

The effects of mycotoxins on the animal are manifold

When, usually, more than one mycotoxin enters the animal, they “cooperate” with each other, which means that they combine their effects in different ways. Also, not all mycotoxins have the same targets.

The synergistic effect: When 1+1 ≥3

Even at low concentrations, mycotoxins can display synergistic effects, which means that the toxicological consequences of two or more mycotoxins present in the same sample will be higher than the sum of the toxicological effects of the individual mycotoxins. So, disregarded mycotoxins can suddenly get important due to their additive or synergistic effect.

Table 1: Synergistic effects of mycotoxins in poultry

Synergistic interactions
DON ZEN T-2 DAS
FUM * * *
NIV * * *
AFL * *

Table 2: Additive effects of mycotoxins in poultry

Additive interactions
AFL T2 DAS MON
FUM + + + +
DON + +
OTA + +

Recognize the effects of mycotoxins in animals is not easy

The mode of action of mycotoxins in animals is complex and has many implications. Research so far could identify the main target organs and effects of high levels of individual mycotoxins. However, the impact of low contamination levels and interactions are not entirely understood, as they are subtle, and their identification requires diverse analytical methods and closer observation.

With regard to the gastrointestinal tract, mycotoxins can inhibit the absorption of nutrients vital for maintaining health, growth, productivity, and reproduction. The nutrients affected include amino acids, lipid-soluble vitamins (vitamins A, D, E, and K), and minerals, especially Ca and P (Devegowda and Murthy, 2005). As a result of improper absorption of nutrients, egg production, eggshell formation, fertility, and hatchability are also negatively influenced.

Most mycotoxins also have a negative impact on the immune system, causing a higher susceptibility to disease and compromising the success of vaccinations. Besides that, organs like kidneys, the liver, and lungs, but also reproduction, endocrine, and nervous systems get battered.

Mycotoxins have specific targets

Aflatoxins, fumonisins, and ochratoxin impair the liver and thus the physiological processes modulated and performed by it:

  • lipid and carbohydrate metabolism and storage
  • synthesis of functional proteins such as hormones, enzymes, and nutrient transporters
  • metabolism of proteins, vitamins, and minerals.

For trichothecenes, the gastrointestinal tract is the main target. There, they hamper digestion, absorption, and intestinal integrity. T-2 can even produce necrosis in the oral cavity and esophagus.

Figure Main Targets Of Important MycotoxinsFigure 2: Main target organs of important mycotoxins

How to reduce mycotoxicosis?

There are two main paths of action, depending on whether you are placed along the crop production, feed production, or animal production cycle. Essentially, you can either prevent the formation of mycotoxins on the plant on the field during harvest and storage or, if placed at a further point along the chain, mitigate their impact.

Preventing mycotoxin production means preventing mold growth

To minimize the production of mycotoxins, the development of molds must be inhibited already during the cultivation of the plants and later on throughout storage. For this purpose, different measures can be taken:

Selection of the suitable crop variety, good practices, and optimal harvesting conditions are half of the battle

Already before and during the production of the grains, actions can be taken to minimize mold growth as far as possible:

  • Choose varieties of grain that are area-specific and resistant to insects and fungal attacks.
  • Practice crop rotation
  • Harvest proper and timely
  • Avoid damage to kernels by maintaining the proper condition of harvesting equipment.

Optimal moisture of the grains and the best hygienic conditions are essential

The next step is storage. Here too, try to provide the best conditions.

  • Dry properly: grains should be stored at <13% of moisture
  • Control moisture: minimize chances of moisture to increase due to condensation, and rain-water leakage
  • Biosecurity: clean the bins and silos routinely.
  • Prevent mold growth: organic acids can help prevent mold growth and increase storage life.

Mold production does not mean that the war is lost

Even if molds and, therefore, mycotoxins occur, there is still the possibility to change tack with several actions. There are measures to improve feed and support the animal when it has already ingested the contaminated feed.

1.    Feed can sometimes be decontaminated

If a high level of mycotoxin contamination is detected, removing, replacing, or diluting contaminated raw materials is possible. However, this is not very practical, economically costly, and not always very effective, as many molds cannot be seen. Also, heat treatment does not have the desired effect, as mycotoxins are highly heat stable.

2.    Effects of mycotoxins can be mitigated

Even when mycotoxins are already present in raw materials or finished feed, you still can act. Adding products adsorbing the mycotoxins or mitigating the effects of mycotoxins in the organism has been considered a highly-effective measure to protect the animals (Galvano et al., 2001).

This type of mycotoxin mitigation happens at the animal production stage and consists of suppressing or reducing the absorption of mycotoxins in the animal. Suppose the mycotoxins get absorbed in the animal to a certain degree. In that case, mycotoxin mitigation agents help by promoting the excretion of mycotoxins, modifying their mode of action, or reducing their effects. As toxin-mitigating agents, the following are very common:

Aluminosilicates: inorganic compounds widely found in nature that are the most common agents used to mitigate the impact of mycotoxins in animals. Their layered (phyllosilicates) or porous (tectosilicates) structure helps “trap” mycotoxins and adsorbs them.

  • Bentonite / Montmorillonite: classified as phyllosilicate, originated from volcanic ash. This absorbent clay is known to bind multiple toxins in vivo. Incidentally, its name derives from the Benton Shale in the USA, where large formations were discovered 150 years ago.
    Bentonite mainly consists of smectite minerals, especially montmorillonite (a layered silicate with a larger surface area and laminar structure).
  • Zeolites: porous crystalline tectosilicates, consisting of aluminum, oxygen, and silicon. They have a framework structure with channels that fit cations and small molecules. The name “zeolite” means “boiling stone” in Greek, alluding to the steam this type of mineral can give off in the heat). The large pores of this material help to trap toxins.

Activated charcoal: the charcoal is “activated” when heated at very high temperatures together with gas. Afterward, it is submitted to chemical processes to remove impurities and expand the surface area. This porous, powdered, non-soluble organic compound is sometimes used as a binder, including in cases of treating acute poisoning with certain substances.

Yeast cell wall: derived from Saccharomyces cerevisiae. Yeast cell walls are widely used as adsorbing agents. Esterified glucomannan polymer extracted from the yeast cell wall was shown to bind to aflatoxin, ochratoxin, and T-2 toxin, individually and combined (Raju and Devegowda 2000).

Bacteria: In some studies, Lactic Acid Bacteria (LAB), particularly Lactobacillus rhamnosus, were found to have the ability to reduce mycotoxin contamination.

Which characteristics are crucial for an effective toxin-mitigating solution

If you are looking for an effective solution to mitigate the adverse effects of mycotoxins, you should keep some essential requirements:

  1. The product must be safe to use:
    1. safe for the feed-mill workers.
    2. does not have any adverse effect on the animal
    3. does not leave residues in the animal
    4. does not bind with nutrients in the feed.
  2. It must show the following effects:
    1. effectively adsorbs the toxins relevant to your operation.
    2. helps the animals to cope with the consequences of non-bound toxins.
  3. It must be practical to use:
    1. cost-effective
    2. easy to store and add to the feed.

Depending on

  • the challenge (one mycotoxin or several, aflatoxin or another mycotoxin),
  • the animals (short-cycle or long-living animals), and
  • the economical resources that can be invested,

different solutions are available on the market. The more cost-effective solutions mainly contain clay to adsorb the toxins. Higher-in-price products often additionally contain substances such as phytogenics supporting the animal to cope with the consequences of non-bound mycotoxins.

Solis – the cost-effective solution

In the case of contamination with only aflatoxin, the cost-effective solution Solis is recommended. Solis consists of well-selected superior silicates with high surface area due to its layered structure. Solis shows high adsorption of aflatoxin B1, which was proven in a trial:

FigureFigure 3: Binding capacity of Solis for Aflatoxin

Even at a low inclusion rate, Solis effectively binds the tested mycotoxin at a very high rate of nearly 100%. It is a high-efficient, cost-effective solution for aflatoxin contamination.

Solis Max 2.0: The effective mycotoxin solution for sustainable profitability

Solis Max 2.0 has a synergistic combination of ingredients that acts by chemi- and physisorption to prevent toxic fungal metabolites from damaging the animal’s gastrointestinal tract and entering the bloodstream.

Figure

Figure 4: Composition and effects of Solis Max 2.0

Solis Max 2.0 is suitable for more complex challenges and longer-living animals: in addition to the pure mycotoxin adsorption, Solis Max 2.0 also effectively supports the liver and, thus, the animal in its fight against mycotoxins.

In an in vitro trial, the adsorption capacity of Solis Max 2.0 for the most relevant mycotoxins was tested. For the test, the concentrations of Solis Max 2.0 in the test solutions equated to 1kg/t and 2kg/t of feed.

FigureFigure 5: Efficacy of Solis Max 2.0 against different mycotoxins relevant in poultry production

The test showed a high adsorption capacity: between 80% and 90% for Aflatoxin B1, T-2 Toxin (2kg/t), and Fumonisin B1. For OTA, DON, and Zearalenone, adsorption rates between 40% and 80% could be achieved at both concentrations (Figure 5). This test demonstrated that Solis Max 2.0 could be considered a valuable tool to mitigate the effects of mycotoxins in poultry.

Broiler trial shows improved performance in broilers

Protected and, therefore, healthier animals can use their resources for growing/laying eggs. A trial showed improved liver health and performance in broilers challenged with two different mycotoxins but supported with Solis Max 2.0.

For the trial, 480 Ross-308 broilers were divided into three groups of 160 birds each. Each group was placed in 8 pens of 20 birds in a single house. Nutrition and management were the same for all groups. If the birds were challenged, they received feed contaminated with 30 ppb of Aflatoxin B1 (AFB1) and 500 ppb of Ochratoxin Alpha (OTA).

Negative control: no challenge no mycotoxin-mitigating product
Challenged group: challenge no mycotoxin-mitigating product
Challenge + Solis Max 2.0 challenge Solis Max 2.0, 1kg/t

The body weight and FCR performance parameters were measured, as well as the blood parameters of alanine aminotransferase and aspartate aminotransferase, both related to liver damage when increased.

Concerning performance as well as liver health, the trial showed partly even better results for the challenged group fed with Solis Max 2.0 than for the negative, unchallenged control (Figures 6 and 7):

  • 6% higher body weight than the negative control and 18.5% higher body weight than the challenged group
  • 12 points and 49 points better FCR than the negative control and the challenged group, respectively
  • Lower levels of AST and ALT compared to the challenged group, showing a better liver health

The values for body weight, FCR, and AST, even better than the negative control, may be owed to the content of different gut and liver health-supporting phytomolecules.

FigureFigure 6: Better performance data due to the addition of Solis Max 2.0

FigureFigure 7: Healthier liver shown by lower values of AST and ALT

Effective toxin risk management: staying power is required

Mycotoxin mitigation requires many different approaches. Mycotoxin mitigation starts with sewing the appropriate plants and continues up to the post-ingestion moment. From various studies and field experience, we find that besides the right decisions about grain crops, storage management, and hygiene, the use of effective products which mitigate the adverse effects of mycotoxins is the most practical and effective way to maintain animals healthy and well-performing. According to Eskola and co-workers (2020), the worldwide contamination of crops with mycotoxins can be up to 80% due to the impact of climate change and the availability of sensitive technologies for analysis and detection. Using a proper mycotoxin mitigation program as a precautionary measure is, therefore, always recommended in animal production.

Toxin Risk ManagementFigure

EW Nutrition’s Toxin Risk Management Program supports farmers by offering a tool (MasterRisk) that helps identify and evaluate the risk and gives recommendations concerning using toxin solutions.




Feed hygiene protects animals and humans

Img

By Vaibhav Gawande, Assistant Manager Technical Services, Dr. Inge Heinzl, Editor, and Marisabel Caballero, Global Technical Manager Poultry, EW Nutrition

The utility value of feed consists of the nutritional value and the quality. The first covers all characteristics concerning the essential nutrients and is important for feed formulation and the adequate supply of the animals.

Feed quality comprises all characteristics of a feed influenced by treatment, storage, conservation, hygiene, and its content of specific substances. For many factors, guidance and threshold values are available which should be met to guarantee animal health and welfare, as well as to protect public health, since some undesirable substances can be transferred to animal products such as meat, eggs, and milk.

In this article, we will focus on feed hygiene. We will talk about the consequences of low feed quality, how to understand it, its causes, and possible solutions.

What are the effects of deficient feed hygiene?

The consequences of deficient feed hygiene can be divided into two parts, impurities and spoilage.

Impurities comprise:

  • the presence of soil, sand, or dust
  • contamination with or residues of heavy metals, PCB, dioxins, pesticides, fertilizers, disinfectants, toxic plants, or banned feed ingredients

In the case of spoilage, we see:

  • degradation of organic components by the action of molds and bacteria
  • growth of pathogens such as E. coli, salmonella, etc.
  • accumulation of toxins such as mycotoxins or bacterial toxins (Hoffmann, 2021)

Bad feed hygiene can also negatively impact the feed’s nutritional value by leading to a loss of energy as well as decreasing the bioavailability of vitamins A, D3, E, K, and B1.

But, how can all signs of deficient feed hygiene be recognized? Soil, sand, and probably dust can be seen in well-taken samples and impurities can be analyzed. But is it possible to spot spoilage? In this case, agglutinated particles, rancid odor, moisture, and discoloration are indicators. Sometimes, also the temperature of the feed or ingredient increases. However, spoilage is not always obvious and an analysis of the feed can give more information about the spoilage-related organisms present. It also helps to decide if the feed is safe for the animals or not. In the case of obvious alterations, the feed should not be consumed by any animal.

Different organisms decrease feed quality and impact health

Several organisms can be responsible for a decrease in feed quality. Besides the visible pests such as rats, mice, or beetles, which can easily be noticed and combatted, there are organisms whose mastering is much more difficult. In the following part, the different harmful organisms and substances are described and solutions are presented.

Enteropathogens can cause diarrhea and production losses

In poultry, different bacteria responsible for high production losses can be transferred via the feed. The most relevant of them are Clostridium perfringens, Escherichia coli, and some strains of Salmonella.

Clostridium perfringens, the cause of necrotic enteritis

Clostridium perfringens is a Gram-positive, anaerobic bacterium that is extremely resistant to environmental influences and can survive in soil, feed, and litter for several years and even reproduce. Clostridium perfringens causes necrotic enteritis mainly in 2-16 weeks old chickens and turkeys, being more critical in 3-6 weeks old chicks.

There is a clinical and a subclinical form of necrotic enteritis. The clinical form can be detected very well due to clear symptoms and mortality rates up to 50%. The subclinical form, while harder to detect, also raises production costs due to a significant decrease in performance. The best prophylaxis against clostridia is the maintenance of gut health, including feed hygiene.

Clostridia can be found in animal by-products, as can be seen in table 1.

Sr. No. Sample details Clostridium perfringens contamination Total number of samples Positivity %
Positive Negative
1 Meat and bone meal 39 52 91 42.86
2 Soya meal 0 3 3 0
3 Rape seed meal 0 1 1 0
4 Fish meal 21 17 38 55.26
5 Layer Feed 21 71 93 22.58
6 Dry fish 5 8 13 38.46
7 De-oiled rice bran 0 2 2 0
8 Maize 0 2 2 0
9 Bone meal 13 16 29 44.83

Table 1: Isolation of Clostridium perfringens from various poultry feed ingredients in Tamil Nadu, India (Udhayavel et al., 2017)

Salmonella is harmful to animals and humans

Salmonella is a gram-negative enterobacterium and can occur in feed. There are only two species – S. enterica and S. bongori (Lin-Hui and Cheng-Hsun, 2007), but almost 2700 serotypes. The most known poultry-specific Salmonella serotypes are S. pullorum affecting chicks and S. gallinarum affecting adult birds. The other two well-known serotypes, S. enteritidis and S. typhimurium are the most economically important ones because they can also infect humans.

Salmonella enteritidis, in particular, can be transferred via table eggs to humans. The egg content can be infected vertically as a result of a colonization of the reproductive tract of the hen (De Reu, 2015). The other possibility is a horizontal infection, as some can penetrate through the eggshell from a contaminated environment or poor egg handling.

Salmonella can also be transferred through meat. However, as there are more production steps where contamination can happen (breeder and broiler farm, slaughterhouse, processing plants, food storage…), traceability is more complicated. As feed can be vector, feed hygiene is crucial.

Moreover, different studies have found that the same Salmonella types found in feed are also detected – weeks later – in poultry farms and even further in the food chain, as reviewed by Ricke and collaborators (2019). Other researches even imply that Salmonella contamination of carcasses and eggs could be significantly reduced by minimizing the incidence of Salmonella in the feed (Shirota et al., 2000).

E. coli – some are pathogenic

E. coli is a gram-negative, not acid-resistant bacterium and most strains are inhabitants of the gut flora of birds, warm-blooded animals, and humans. Only some strains cause disease. To be infectious, the bacteria must have fimbriae to attach to the gut wall or the host must have an immune deficiency, perhaps due to stress. E. coli can be transmitted via contaminated feed or water as well as by fecal-contaminated dust.

Escherichia coli infections can be found in poultry of all ages and categories and nearly everywhere in the bird. E. coli affects the navel of chicks, the reproductive organs of hens, several parts of the gut, the respiratory tract, the bones and joints, and the skin and are part of the standard control.

The feed microbiome can contribute to a balanced gut microbial community. The origins of pathogenic E. coli in a flock can also be traced to feed contamination (Stanley & Bajagai, 2022). Especially in pre-starter/starter feeds, E. coli contamination can be critical as the day-old chick’s gut is starting to be colonized. Especially in this phase, maintaining a low microbial count in feed is crucial.

Molds cause feed spoilage and reduce nutritional value

Molds contaminate grains, both in the field and during storage, and can also grow in stored feed and even in feed stored or accumulated in storage facilities in animal production farms.

The contamination of feed by molds and their rapid growth can cause heating of the feed. As molds also need nutrients, their growth results in a reduction of energy and the availability of vitamins A, D3, E, K, and B1, thus decreasing the feed’s nutritional value. This heating occurs in most feeds with a moisture content higher than 15 /16%. Additionally, mold-contaminated feed tends to be dusty and has a bad taste impacting palatability and, as a consequence, feed intake and performance.

Molds produce spores that can, when inhaled, cause chronic respiratory disease or even death if the animals are exposed to contaminated feed for a longer time. Another consequence of mold contamination is the production of mycotoxins by several mold species. These mycotoxins can affect the animal in several ways, from decreasing performance to severe disease (Esmail, 2021; Government of Manitoba, 2023).

With effective feed hygiene management, we want to stop and prevent mold growth, as well as all its negative consequences.

Prevention is better than treatment

It is clear that when the feed is spoiled, it must be removed, and animal health supporting measures should take place. However, it is better to prevent the consequences of low feed hygiene on animals. Proper harvest and adequate storage of the feed are basic measures to stop mold growth. Additionally, different tools are available to protect the animals from feed bacterial load and other risk factors.

Solutions are available to support feed hygiene

There are several solutions to fight the organisms which decrease feed quality. Some directly act against the harmful substances / pathogens, and others act indirectly, meaning that they change the environment to a non-comfortable one for the organism.

Formaldehyde and propionic acid – an unbeatable team against bacteria

A combination of formaldehyde and propionic acid is perfect to sanitize feed. Formaldehyde results in bacterial DNA and protein damage, and propionic acid is active against bacteria and molds. Together, they improve the microbiological quality of the feed and reduce the risk of secondary diseases such as necrotic enteritis or dysbiosis on the farm. In addition to the pure hygienic aspect, organic acids support digestion.

An in-vitro trial was conducted to evaluate the effect of such a combination (Formycine Gold Px) against common poultry pathogens. Poultry feed was spiked with three different bacteria, achieving very high initial contamination of 1,000,000 CFU/g per pathogen. One batch of the contaminated feed served as a control (no additive). To the other contaminated batches, 1, 2, or 4 kg of Formycine per ton of feed were added. The results (means of triplicates) are shown in figures 1 a-c.

Figure A Salmonella

Figure B E

Figure C Clostridium PerfringensFigures 1 a-c: Reduction of bacterial count due to the addition of Formycine

Formycine Gold Px significantly reduced the bacterial counts in all three cases. A clear dose-response-effect can be seen and by using 2 kg of Formycine / t of feed, pathogens could not be detected anymore in the feed.

A further trial showed the positive effects of feeding Formycine Gold Px treated feed to the animals. Also here, the feed for both groups was contaminated with 1,000,000 CFU of Clostridium/g. The feed of the control group was not treated and to the treatment group, 2 kg of Formycine per t was added.

Figure Preventive EffectFigure 2: Preventive effect of Formycine Gold Px concerning necrotic enteritis gut lesions

Figure A Daily GainFigure 3a and 3b: Performance-maintaining effect of Formycine Gold Px

The trial showed that Formycine Gold Px reduced the ingestion of the pathogen, and thus could prevent the lesions caused by necrotic enteritis (Fig. 2). The consequence of this improved gut health is a better feed conversion and higher average daily gain (Fig.3a and 3b).

Products containing formaldehyde may represent a risk for humans, however, the adequate protection equipment helps to reduce/avoid exposure.

A combination of free acids and acid salts provides optimal hygienic effects

Additionally, another blend of organic acids (Acidomix AFG) shows the best effects against representatives of relevant feed-borne pathogens in poultry. In a test, 50 µl solution containing different microorganisms (reference strains of S. enterica, E. coli, C. perfringens, C. albicans, and A. niger; concentration 105 CFU/ml, respectively) were pipetted into microdilution plates together with 50 µl of increasing concentrations of a mixture of organic acids (Acidomix) After incubation, the MIC and MBC of each pathogen were calculated.

The test results show (figure 4, Minimal Bactericidal Concentration) that 0.5% of Acidomix AFG in the medium (≙ 5kg/t of feed) is sufficient to kill S. enterica, C. albicans, and A. niger and even only 2.5kg/t in the case of E. coli. If the pathogens should only be prevented to proliferate, even a lower amount of product is requested (figure 5, Minimal Inhibitory Concentration – MIC)

Figure MbcFigure 4: MBC of Acidomix AFG against different pathogens (%)

Figure MicFigure 5: MIC of Acidomix AFG against different pathogens (%)

In addition to the direct antimicrobial effect, this product decreases the pH of the feed and reduces its buffering capacity. The combination of free acids and acid salts provides prompt and long-lasting effects.

Feed hygiene: a critical path to animal performance

Feed accounts for 65-70% of broiler and 75-80% of layer production costs. Therefore, it is essential to use the available feed to the utmost. The quality of the feed is one decisive factor for the health and performance of the animals. Proper harvesting and storage are in the hands of the farmers and the feed millers. The industry offers products to control the pathogens causing diseases and the molds producing toxins and, therefore, helps farmers save feed AND protect the health and performance of their animals.

References:

Dinev, Ivan. Diseases of Poultry: A Colour Atlas. Stara Zagora: Ceva Sante Animal, 2007.

Esmail, Salah Hamed. “Moulds and Their Effect on Animal Health and Performance.” All About Feed, June 17, 2021. https://www.allaboutfeed.net/all-about/mycotoxins/moulds-and-their-effect-on-animal-health-and-performance/.

Government of Manitoba. “Spoiled Feeds, Molds, Mycotoxins and Animal Health.” Province of Manitoba – Agriculture. Accessed March 16, 2023. https://www.gov.mb.ca/agriculture/livestock/production/beef/spoiled-feeds-molds-mycotoxins-and-animal-health.html.

Hoffmann, M. “Tierwohl Und Fütterung.” LKV Sachsen: Tierwohl und Fütterung. Sächsischer Landeskontrollverband e.V., January 25, 2021. https://www.lkvsachsen.de/fuetterungsberater/blogbeitrag/artikel/tierwohl-und-fuetterung/.

Ricke, Steven C., Kurt Richardson, and Dana K. Dittoe. “Formaldehydes in Feed and Their Potential Interaction with the Poultry Gastrointestinal Tract Microbial Community–A Review.” Frontiers in Veterinary Science 6 (2019). https://doi.org/10.3389/fvets.2019.00188.

Shirota, Kazutoshi, Hiromitsu Katoh, Toshihiro Ito, and Koichi Otsuki. “Salmonella Contamination in Commercial Layer Feed in Japan.” Journal of Veterinary Medical Science 62, no. 7 (2000): 789–91. https://doi.org/10.1292/jvms.62.789.

Stanley, Dragana, and Yadav Sharma Bajagai. “Feed Safety and the Development of Poultry Intestinal Microbiota.” Animals 12, no. 20 (2022): 2890. https://doi.org/10.3390/ani12202890.

Su, Lin-Hui, and Cheng-Hsun Chiu. “Salmonella: Clinical Importance and Evolution of Nomenclature.” Chang Gung Med J 30, no. 3 (2007): 210–19.

Udhayavel, Shanmugasundaram, Gopalakrishnamurthy Thippichettypalayam Ramasamy, Vasudevan Gowthaman, Shanmugasamy Malmarugan, and Kandasamy Senthilvel. “Occurrence of Clostridium Perfringens Contamination in Poultry Feed Ingredients: Isolation, Identification and Its Antibiotic Sensitivity Pattern.” Animal Nutrition 3, no. 3 (2017): 309–12. https://doi.org/10.1016/j.aninu.2017.05.006.




Rancidity in fats and oils: Considerations for analytical testing

enzymes feed pellets

By Dr. Ajay Bhoyar, Global Technical Manager – Poultry, EW Nutrition

 

Rancidity testing is essential in the feed industry, as a key indicator of product quality and shelf life. It is conducted to determine the level of oxidation in samples of feed or feed ingredients and it can be performed through a number of analytical methods.

Rancidity is the process by which fats and oils in food become degraded, resulting into off-odor/flavor, taste, and texture. This process is caused by the oxidation of unsaturated fatty acids and can be accelerated by factors such as exposure to light, heat, and air. Rancidity can occur naturally over time, but it can also be accelerated by improper storage or processing of animal products. Fats are highly susceptible to degradation due to their chemical nature.

How does oxidative rancidity occur?

Oxidation occurs when an oxygen ion replaces a hydrogen ion within a fatty acid molecule and higher numbers of double bonds within the fatty acid increase the possibility of autoxidation. Oxidative rancidity results from the breakdown of unsaturated fatty acids in the presence of oxygen. Light and heat promote this reaction, which results in the generation of aldehydes and ketones – compounds which impart off-odors and flavors to food products. Pork and chicken fat demonstrate a higher degree of unsaturated fatty acids compared with beef fat and are therefore more prone for rancidity.

Oxidation: a three-step process

Fat/oil oxidation is a three-step process (Initiation, Propagation and Termination). Therefore, the oxidation products depend on the time. In the first phase, called Initiation, the formation of free radicals begins and accelerates.

Once the initial radicals have formed, the formation of other radicals proceeds rapidly in this second phase called Propagation. In this part of the process, a chain reaction of high energy molecules, which are variations of free radicals and oxygen, are formed and can react with other fatty acids. These reactions can proceed exponentially, if not controlled. Also in this phase, the rate of peroxide radical formation will reach equilibrium with the rate of decomposition to form a bell-shaped curve.

In the final phase, called Termination, the starting material has been consumed, and the peroxide radicals, as well as other radicals decompose into secondary oxidation by-products such as esters, short chain fatty acids, polymers, alcohols, ketones and aldehydes. It is these secondary oxidation by-products, which can negatively affect the growth and performance of animals.

Three Phase
Fig. 1: Oxidation: a three-phase series of reactions

Antioxidants preserve the quality of rendered products

Chemical antioxidants are used in the rendering industry to help preserve the quality of animal by-products. Synthetic antioxidants, such as BHA, BHT, and ethoxyquin, can help prevent the oxidation of these by-products, which can cause them to become rancid. These chemical antioxidants are added in small amounts to the raw materials prior to rendering or can be incorporated into the finished products to help extend their shelf life and maintain their nutritional value. It is important to note that the use of antioxidants in the rendering industry must be done in compliance with regulations and guidelines set forth by the FDA and other governing bodies.

Natural antioxidants like tocopherols, rosemary extract, ascorbyl palmitate, etc. are also used to prevent oxidation and maintain the freshness of rendered products, if the chemical antioxidants cannot be used.

Rancidity testing

Rancidity testing is the process of determining the level of rancidity in a product. Testing for level of rancidity is used widely as an indication of product quality and stability.

There are several methods used for rancidity testing, including:

Organoleptic rancidity testing

Oxidation of fats and oils leads to a change in taste, smell, and appearance. Organoleptic testing involves using the senses (sight, smell, taste) to determine the level of rancidity. Trained testers will examine the product for visual signs of spoilage, such as discoloration or the presence of crystals, and will also smell and taste the product to detect any off-flavors or odors.

Chemical & instrumental rancidity testing

Chemical testing involves using chemical methods to measure the level of rancidity. One common method is the peroxide value test, which measures the amount of peroxides (indicators of rancidity) in the product. Another method is the p-anisidine test, which measures the level of aldehydes (another indicator of rancidity) in the product.

Peroxide value

Peroxide Value (PV) testing determines the amount of peroxides in the lipid portion of a sample through an iodine titration reaction targeting peroxide formations. Peroxides are the initial indicators of lipid oxidation and react further to produce secondary products such as aldehydes. Because peroxide formation increases rapidly during the early stages of rancidification but subsequently diminishes over time, it is best to pair PV testing with p-Anisidine Value to obtain a fuller picture of product quality.

Three Phase Graph
Fig.2: Oxidation products changes with time

p-Anisidine (p-AV)

p-AV is a determination of the amount of reactive aldehydes and ketones in the lipid portion of a sample. Both compounds can produce strong objectionable flavors and odors at relatively low levels. The compound used for this analysis (p-Anisidine) reacts readily with aldehydes and ketones and the reaction product can be measured using a colorimeter. Samples that are particularly dark may not be the most applicable for this analysis as the colorimeter may not be able to adequately measure the wavelength required.

TBARS

Thiobarbituric acid reactive substances (TBARS) are a byproduct of lipid peroxidation (i.e. as degradation products of fats). This can be detected by the TBARS assay using thiobarbituric acid as a reagent. TBA Rancidity (TBAR) also measures aldehydes (primarily malondialdehyde) created during the oxidation of lipids. This analysis is primarily useful for low-fat samples, as the whole sample can be analyzed rather than just the extracted lipids.

The Instrumental testing involves using instruments to measure the level of rancidity.

Gas chromatography

One common method is the use of a gas chromatograph, which can detect the presence of volatile compounds that indicate rancidity.

Fourier-transform infrared spectrophotometer (FTIR)

FTIR method can detect changes in the chemical makeup of the product that indicate rancidity.

Free Fatty Acids (FFA)

FFA testing determines the fatty acids that have been liberated from their triglyceride structure. A titration is performed on the extracted fat from a specific sample. The FFA content is then determined through a calculation of the amount of titrant used to reach the final result. Knowing what type of fat or fat containing product is being tested is important for this analysis to ensure that the appropriate calculation is applied. As the test does not differentiate between fatty acid types, samples with high palmitic or lauric fatty acid composition should have a different calculation factor applied so as to accurately represent the free fatty acid result.

Oxidative Stability Index (OSI)

OSI indicates how resistant a sample is to oxidation. Samples are subjected to heat while air is injected – a process which accelerates oxidation reactions. The samples are monitored, and the time required for the sample to reach an inflection point is determined. This test is useful when testing the efficacy of an antioxidant added to a product. Antioxidants should inhibit free radical propagation and thus increase a samples ability to hold up under the stressing conditions imposed by the OSI analysis. The measuring instrument, the Rancimat.

Analytical testing considerations in rendering operations

It is common to perform regular analytical testing in a rendering operation as a part of quality control and quality assurance program. There are several methods for testing rancidity in rendering operations. It is important to choose the appropriate method based on the type of product and the desired level of accuracy.

The results of rancidity testing are used to monitor and control the rendering process to prevent or minimize rancidity. This may involve adjusting processing conditions, using antioxidants, or implementing other measures to reduce oxidation.

Test objective Analysis Remarks
Current state of oxidation
  1. Peroxide Value (PV)
  2. Secondary Oxidatives (p-Anisidine, TBARS)
  1. PV:< 5 meq/kg
  2. 50 ppm
Potential for future oxidation Oxidative Stability Index (OSI) Analyze the stability of oil/fats
Residual antioxidant Gas chromatography Value decreases as the antioxidant gets sacrificed

Table. 1: Analytical testing considerations for rendering

Conclusion

Rancidity is a common problem in rendered animal products. It can have detrimental effects on both the quality and safety of the product. It is caused by the oxidation of fats and oils, leading to the formation of harmful compounds such as free radicals and hydroperoxides. The best way to prevent rancidity is through proper storage, packaging, and handling techniques, as well as the use of antioxidants to slow down the oxidation process. It is important for manufacturers and consumers to be aware of the potential for rancidity in rendered animal products and take the necessary precautions to ensure the safety and quality of the product. 




Salmonella in poultry: What are the most effective natural solutions?

layer imgp1242 scaled

By Dr. Inge Heinzl, Editor, EW Nutrition

Salmonella infection in poultry is a problem for the producer because of the performance losses of his flock. At the same time, products of salmonella-contaminated animals pose a severe risk to human health. In the USA, Salmonellosis in poultry is estimated to cost $ 11.6 billion each year (Wernicki et al., 2017) and more than € 3 billion in the EU (Ehuwa, 2021). As the use of antibiotics needs to be reduced to keep them effective, Salmonella control in poultry requires new solutions. This article shows how organic acids and phy­tomolecules can help to fight this problematic disease.

Salmonellosis: what it is, how it works, and why it’s such a problem

 

Salmonellosis is a zoonosis, meaning that it can be easily transferred from animals to humans. The transfer can occur via different routes:

  • Direct contact with an infected animal
  • Handling or consuming contaminated animal products such as eggs or raw meat from pigs, turkeys, and chicken
  • Contact with infected vectors (insects or pets) or contaminated equipment

Frozen or raw chicken products, as well as the eggs of backyard hens, are the most frequent causes of animal-mediated Salmonella infections in humans. The following graphic shows a clear relationship between the occurrence of Salmonella in layer flocks and the event of disease in humans:

Salmonella Infection Populations Chart
(Source: Koutsoumanis et al., 2019)

The impact of Salmonella on poultry depends on the bird’s age

Within the poultry flock, there are two ways of spreading: the fecal-oral way (horizontal infection) or the infection of the progeny in the egg (vertical infection). The effects of the disease depend on the age of the birds: the younger the animals, the more severe the impact.

If the brood eggs already carry salmonellae, the hatchability dwindles. During their first month of life, infected chicks show ruffled downs and higher temperatures. Diarrhea leads to fluid losses and frequently to the chicks’ death.

Adult animals usually do not die from Salmonellosis; often, the infection remains unnoticed. During a substantial acute salmonella outbreak, the animals show weakness and diarrhea. They lose weight, resulting in decreased egg production in layers and worse growth performance in broilers. The birds need more water to compensate for the fluid losses, and their crowns and jowls appear pale.

Salmonella protects itself through an intelligent infection style

Salmonellae have developed a clever way to protect themselves. After they arrive in the gut, they attach to the epithelial cells and form small molecular “syringes” to inject divers substances into the gut cells (Type-3-injection system). These signaling substances make the gut cells bulge their membranes and enclose the bacterium. Finally, the manipulated gut cell absorbs the Salmonella, the host “allows” the bacterium to enter, and it can proliferate in the gut cells (Fischer, 2018).

When an antibiotic is attacking the bacterium, Salmonellae stop their cell division. Since many antibiotics are only effective against bacteria during cell division and growth, Salmonellae survive the attack by staying as dormant variants or persisters until the treatment stops (Fischer, 2018).

Salmonellae – a big “family”

The genus of Salmonella consists of more than 2600 serovars (Ranieri et al., 2015), of which less than 100 are relevant for humans (CDC, 2020). More than 1500 serovars belong to the Salmonella enterica subspecies that colonize the intestinal tract of warm-blooded animals. These serovars are responsible for 99 % of salmonella infections (Mendes Maciel et al., 2017). The main serovars relevant for poultry are S. Gallinarum and S. Pullorum, but also S. Enteritidis, Typhimurium, and in recent years, S. Kentucky, S. Heidelberg, S. Livingstone, and S. Mbandaka (Guillén et al., 2020).

(Source: Mkangara et al., 2020)

The zoonosis Salmonellosis must be controlled

Several Salmonella serovars are critical for animals and humans. Since more than 91,000 salmonellosis cases are reported for Europe and more than 1.35 million for the USA every year (EFSA, 2022; FDA, 2020), their spread must be prevented by all means. Governments have enacted some laws to curtail this disease. The EU, for example, implemented extended control programs for zoonotic diseases, with Salmonella set as a priority. These programs include the provision of scientific advice, targets for reducing Salmonella in poultry flocks, and restrictions on the trade of products from infected flocks.

For farmers and vets, this means the obligation to notify the occurrence of the disease to the authorities. Depending on the country, it also entails compulsory vaccination and the documentation of hygienic measures. In the EU, due to the risk of developing resistances, the EFSA recommends limiting the use of antimicrobials to individual cases, e.g., to prevent inordinate suffering of animals.

Prevention of Salmonella infection is the key

The best strategy for salmonella control is prevention based on three key points (Visscher, 2014):

  • Preventing the introduction of Salmonella into the farm/flock through effective hygiene measures
  • Preventing the spread of the pathogens within a flock/farm
  • Prophylactic measures to recover immune resistance of the animals against Salmonella infection

For this purpose, the following steps are requested/recommended:

1.    Keeping the litter dry

The use of well-absorptive material such as wood shavings, straw pellets, or straw granulates and regular removal of the used litter is recommended. The animals must be controlled for diarrhea to avoid wet droppings. The water supply must be adequate; an excessive water supply wets the litter.

2.    Providing a clean environment

To keep the poultry house clean, broken eggs and dead animals (potential sources of infection) must be removed. In general, the houses should be cleaned and disinfected before every restocking.

Clean feed and water are essential; therefore, feed should not be stored outside but be kept dry and protected from pests and rodents. The feeding of the animals should take place inside to avoid contamination by wild birds. Concerning the water for drinking, the flow rate must be high enough to provide the birds with sufficient water but not too high that the floor gets wet. The troughs must be clean from droppings.

3.    Limiting contacts

To limit the spread of Salmonella, only a restricted number of persons can have access to the flocks. They must wear clothes, and instruments should be exclusively used for the respective poultry house.

Knowing the optimal growth conditions for Salmonella facilitates control

Salmonellae are a genus in the family of Enterobacteriaceae. They are gram-negative, rod-shaped (size: approx. 2 µm), glucose-fermenting facultative anaerobes that are motile due to peritrichous flagella. Since Salmonellae do not form spores, they can be easily destroyed by heating them to 60°C for 15-20 min (Forsythe, 2001), especially in food/feed with higher water content.

Salmonella facilitates control

For the storage of food, Bell and Kyriakis (2002) found that most serovars of Salmonella will not grow at temperatures lower than 7°C and a pH lower than 4.5. Wessels et al. (2021) showed optimal growth conditions for Salmonella: temperatures between 5 and 46°C (optimum 38°C), a water activity of 0.94-0.99, and a pH of 3.8-9.5.

A high fat content in the feed or food increases the likelihood of infection with Salmonella because the fat protects the bacteria during the passage through the stomach. Doses of 10 to 100 Salmonella cells can already pose a severe risk (University of Georgia, 2015).

Natural alternatives to antibiotics: effective Salmonella control?

To reduce the incidence of Salmonella while simultaneously lowering the use of antibiotics in animal production, there are different possibilities. On the one hand, veterinary medicine offers vaccines. On the other hand, the feed industry provides additives that strengthen the immune system, improve gut health, or support the animals in another manner. Other than pro- and prebiotics, the main active ingredient categories for such additives are organic acids and phy­tomolecules.

Organic acids worsen the conditions for Salmonella

Already in ancient Egypt, the method of fermentation and the generated acids have been used for the conservation of food (Ohmomo et al., 2002). Nowadays, it is a standard tool to protect feed  (silage) and food from spoilage. Also for animals, organic acids added to the feed or the water have proven helpful against pathogens. These modes of action can be combined against Salmonella: reducing the pathogen load in the feed to limit the intake of bacteria and fighting against these pathogens in the animal.

Organic acids reduce Salmonella in feed materials

In general, the antimicrobial activity of organic acids in feed is based on lowering the pH (Pearlin et al., 2019). pH-sensitive bacteria such as Salmonella minimize their proliferation at a pH <5. Additionally, the organic acids attack bacteria directly. The acid’s undissociated and more lipophilic form penetrates the bacterial cell membrane. At the neutral pH within the cell, the acid dissociates, releases protons, and lowers the pH, leading to the impediment of metabolic processes in the cell. The cell spends a lot of energy trying to get the pH back to neutral (Mroz et al., 2006). Additionally, the anions become toxic for the cell metabolites and disrupt the membrane (Russel, 1992).

What do organic acids do in the bird?

According to Hernández and co-workers (2006) and Thompson and Hinton (1997), the addition of organic acids to the feed does not change the pH in the various digestive tract segments. Still, literature shows a clear reduction of Salmonella in the gut or litter when using propionic or/and formic acid (McHan and Emmett, 1992; Hinton and Linton, 1988; Humphrey & Lanning, 1988). A likely mode of action is described by Van Immerseel et al. (2004). He asserts that SCFAs such as propionic and formic acid as well as MCFAs can inhibit Salmonella’s penetration of the intestinal epithelium and, therefore, can control these invasive phenotypes of Salmonella (S. Typhimurium and S. Enteritidis).

Different acids show different efficacy

Depending on the acid, the efficacy against Salmonella varies (see figure 3). Formic acid shows the highest effect, followed by fumaric acid. Then, lactic, butyric, and citric acid follow, showing lower efficacy.

Efficacy of different organic acids against Salmonella
Figure 3: Efficacy of different organic acids against Salmonella in feed

Trials prove the efficacy of organic acids

An in-vitro trial was conducted at a commercial research facility in the US to test the efficacy of Acidomix AFL, a liquid mixture of propionic and formic acid, against Salmonella. The bacterial strain used in these studies was nalidixic acid-resistant Salmonella typhimurium. The bacteria were maintained in broth cultures of tryptic soy broth.

They were added to 5 g of dry feed in a 50 ml tube to a final concentration of 40,000 CFU/g. Next, Acidomix AFL was added to the desired inclusion rate, and the samples were incubated at room temperature. After 18 to 72 hours of incubation, viable bacteria were counted using the plate count method.

Results: As shown in figure 4, the trial found that at an inclusion rate of 2.0 %, Salmonella inhibition was nearly 100 %. Already at a 0.4 % inclusion rate, Salmonella could be reduced by 45-60 %, showing a clear dose dependency.

Efficacy of Acidomix AFL (liquid) on Salmonella Typhimurium in dry feed
Figure 4: Efficacy of Acidomix AFL (liquid) on Salmonella Typhimurium in dry feed

Phytomolecules combat Salmonella through complex modes of action

Plants produce phytogenic substances to protect themselves from molds, yeasts, and bacteria, among others. After several purification steps, these phy­tomolecules can be used to fight Salmonella in poultry. They work through different modes of action, from attacking the cell wall (terpenoids and phenols) to influencing the genetic material of the pathogenic cells or changing the whole morphology of the cell.

Due to the different modes of action, it was long thought that there would be no resistance development. Still, Khan et al. (2009) found some microorganisms such as multidrug-resistant E. coli, Klebsiella pneumoniae, S. aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Salmonella typhimurium can show a certain – perhaps natural – resistance to some components of herbal medicines.

Gram-negative bacteria such as Salmonella are usually less attackable by phytomolecules because the cell wall only allows small hydrophilic solutes to pass; however, phy­tomolecules are hydrophobic. However, mixing the phytomolecules with an emulsifier facilitates the invasion into the cell. Their efficacy depends on their chemical composition. It is also decisive if single substances or blends (possible positive or negative synergies) are used.

The best-clarified mode of action is the one of thymol and carvacrol, the major components of the oils of thyme and oregano. They can get into the bacterial membrane and disrupt its integrity. The permeability of the cell membrane for ions and other small molecules such as ATP increases, decreasing the electrochemical gradient above the cell membrane and the loss of energy equivalents.

Trials show the efficacy of phy­tomolecules against Salmonella

Two different phytogenic compositions were tested for their efficacy against Salmonella.

Trial 1: Blend of phy­tomolecules and organic acids shows best results in an in-vitro assay

To evaluate its potential as a tool for antibiotic reduction, a trial was conducted to test the antimicrobial properties of Activo Liquid, a mixture of selected phy­tomolecules and an organic acid designed for application in water. The laboratory test was carried out at the Veterinary Diagnosis Department of Kasetsart University in Thailand. Standardized suspensions [1×104 CFU/ml] of three poultry-relevant Salmonella strains were incubated in LB medium, either without or with Activo Liquid. The tests were run at concentrations of 0.05%; 0.1%; 0.2% and 0.4%. After incubation at 37°C for 6-7 hours, serial dilutions of the cell suspensions were transferred onto LB agar plates and incubated for 18-22h at 37°C. Subsequently, colonies (CFU/ml) were determined.

Results: Activo Liquid was found to be growth-inhibiting to all Salmonella strains from a concentration of 0.1% onwards. At 0.2%, Activo Liquid already exhibited bactericidal efficacy against all tested Salmonella isolates, which was confirmed at a concentration of 0.4%.

Inhibiting effect of Activo Liquid against three different Salmonella serovars
Table 1: Inhibiting effect of Activo Liquid against three different Salmonella serovars

Trial 2: Blend of nature-identical phy­tomolecules inhibits Salmonella

On Mueller Hinton agar plates where Salmonella enterica were spread uniformly, small disks containing 0 (control, only methanol), 1, 5, and 10 µl of Ventar D were placed and incubated at 37 °C for 16 hours. The presence of clearing zones indicates antimicrobial activity.

Additionally, a motility test was performed in tubes with a motility test medium containing 0 (control) and 750 µL Ventar D. For this purpose, one colony of Salmonella enterica grown on the agar was stuck in the middle of the medium and incubated at 37 °C for 12-16 hours. Growth can be visualized through the formation of red color.

Result: Ventar D inhibited S. enterica in a dose-dependent manner. Clearing zones were visible within the lowest tested concentration. At its inhibitory concentration, Ventar D suppressed S. enterica motility (figures 5 and 6).

S. enterica motility test
Figure 5: S. enterica motility test

Disk diffusion assay employing S. enterica
Figure 6: Disk diffusion assay employing S. enterica

Let’s fight Salmonella through effective and sustainable natural tools

The zoonosis Salmonella generates high costs in the poultry industry. As Salmonellosis can be transferred to humans, it must be kept under control by all means. Antibiotics are one tool to fight Salmonella, but they have their “side effects”: they are no longer well respected by the consumer, and, even more critically, they create resistance. To help keep antibiotics effective, poultry producers seek to use effective but not resistance-creating natural solutions against Salmonella.

As shown with the reviewed trials, organic acids and phytomolecules are highly active against diverse Salmonella serovars. Accordingly, feed additives based on these active ingredients offer effective tools for controlling Salmonella in poultry while also contributing to the overarching aim of reducing antibiotic use in poultry production.


References

Bell, Chris, and Alec Kyriakides. Salmonella: A Practical Approach to the Organism and Its Control in Foods. Oxford: Blackwell Science, 2002.

Castro-Vargas, Rafael Enrique, María Paula Herrera-Sánchez, Roy Rodríguez-Hernández, and Iang Schroniltgen Rondón-Barragán. “Antibiotic Resistance in Salmonella Spp. Isolated from Poultry: A Global Overview.” October-2020 13, no. 10 (October 3, 2020): 2070–84. https://doi.org/10.14202/vetworld.2020.2070-2084.

CDC. “Serotypes and the Importance of Serotyping Salmonella.” Centers for Disease Control and Prevention, February 21, 2020. https://www.cdc.gov/salmonella/reportspubs/salmonella-atlas/serotyping-importance.html.

EFSA. “Salmonella.” European Food Safety Authority. Accessed February 1, 2022. https://www.efsa.europa.eu/en/topics/topic/salmonella.

Ehuwa, Olugbenga, Amit K. Jaiswal, and Swarna Jaiswal. “Salmonella, Food Safety and Food Handling Practices.” Foods 10, no. 5 (2021): 907. https://doi.org/10.3390/foods10050907.

FDA. “Get the Facts about Salmonella.” U.S. Food and Drug Administration, July 28, 2020. https://www.fda.gov/animal-veterinary/animal-health-literacy/get-facts-about-salmonella.

Fischer, Andreas. “Clever Infiziert – Die Tricks Der Bakterien.” HZI – Helmholtz Zentrum für Infektionsforschung, August 19, 2021. https://www.helmholtz-hzi.de/de/aktuelles/thema/clever-infiziert-die-tricks-der-bakterien/.

Forsythe, Steve J. The Microbiology of Safe Food. Hoboken, NJ: Wiley-Blackwell, 2020.

Gheisari, A.A., M. Heidari, R.K. Kermanshahi, M. Togani, and S. Saraeian. “Effect of Dietary Supplementation of Protected Organic …” WPSA, 2007. https://www.cabi.org/Uploads/animal-science/worlds-poultry-science-association/WPSA-france-2007/74.pdf.

Guillén, Silvia, María Marcén, Ignacio Álvarez, Pilar Mañas, and Guillermo Cebrián. “Stress Resistance of Emerging Poultry-Associated Salmonella Serovars.” International Journal of Food Microbiology 335 (2020): 108884. https://doi.org/10.1016/j.ijfoodmicro.2020.108884.

Hernández, F., V. García, J. Madrid, J. Orengo, P. Catalá, and M.D. Megías. “Effect of Formic Acid on Performance, Digestibility, Intestinal Histomorphology and Plasma Metabolite Levels of Broiler Chickens.” British Poultry Science 47, no. 1 (2006): 50–56. https://doi.org/10.1080/00071660500475574.

Hinton, M. “Antibacterial Activity of Short-Chain Fatty Acids.” The Veterinary Record 126 (n.d.): 416–21.

Hinton, M., and A. Linton. “Control of Salmonella Infections in Broiler Chickens by the Acid Treatment of Their Feed.” Veterinary Record 123, no. 16 (1988): 416–21. https://doi.org/10.1136/vr.123.16.416.

Humphrey, T. J., and D. G. Lanning. “The Vertical Transmission of Salmonellas and Formic Acid Treatment of Chicken Feed: A Possible Strategy for Control.” Epidemiology and Infection 100, no. 1 (1988): 43–49. https://doi.org/10.1017/s0950268800065547.

Khan, Rosina, Barira Islam, Mohd Akram, Shazi Shakil, Anis Ahmad Ahmad, S. Manazir Ali, Mashiatullah Siddiqui, and Asad Khan. “Antimicrobial Activity of Five Herbal Extracts against Multi Drug Resistant (MDR) Strains of Bacteria and Fungus of Clinical Origin.” Molecules 14, no. 2 (2009): 586–97. https://doi.org/10.3390/molecules14020586.

Koutsoumanis, Kostas, Ana Allende, Avelino Alvarez‐Ordóñez, Declan Bolton, Sara Bover‐Cid, Marianne Chemaly, Alessandra De Cesare, et al. “Salmonella Control in Poultry Flocks and Its Public Health Impact.” EFSA Journal 17, no. 2 (2019). https://doi.org/10.2903/j.efsa.2019.5596.

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Thompson, J. L., and M. Hinton. “Antibacterial Activity of Formic and Propionic Acids in the Diet of Hens on Salmonellas in the Crop.” British Poultry Science 38, no. 1 (1997): 59–65. https://doi.org/10.1080/00071669708417941.

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Water Hygiene: The missing ingredient for successful ABF poultry

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By T.J. Gaydos

Water quality is a frequently overlooked part of animal production and it becomes even more important when producing animals in an antibiotic-free (ABF) system. Chickens drink almost twice as much water as they consume feed, and water hygiene is often a second-level priority. Microbes present in water can be primary or secondary pathogens or non-pathogenic. Consuming impure water can add a challenge to the immune system, negatively impacting performance. 

Water hygiene is essential

Water hygiene is essential for achieving antibiotic-free poultry production

Significant resources are spent on the correct nutrients in the diet and the correct additives for bird health. Water quality should be a priority, and a water quality monitoring program is essential for success in an ABF program. All things being equal, animals will perform better if they have access to high-quality water.

The variability of water quality in the grow-out region should determine how many water quality samples are taken. In highly variable areas, water quality should be measured at every season change on enough farms in every region to know if the solutes are changing. If the water quality is good and consistent, monitoring may be reduced significantly. Water quality should be a part of a “problem farm” work up or related to otherwise unexplained poor performance.

Water-soluble additives: Prevent biofilm

The use of water-soluble products is common in ABF production systems and their frequent use may provide a carbon source for bacteria. This, along with warm temperatures and slow water flow in enclosed water systems, makes the perfect environment for biofilm development.

It is important to frequently flush lines, give birds access to fresh water between additives, and sanitize water lines after using a product that can provide nutrients to bacteria in the line. The biofilm is a perfect location to harbor and protect pathogens from acids and mild or under-dosed disinfectants.

Designing a water quality program

Sample collection

The first step to building a water quality program is to understand the challenge on every farm. Correct sample collection is critical to achieving good results. Take a water sample from as close to the well as possible and submit for water quality analysis: pH, hardness, and minerals. This sample should also be submitted for bacterial load: total aerobic plate count (CFU) per mL and total coliforms per mL.

Monitor bacterial load

A drip sample should be collected from the end of the line for bacterial load analysis as well. This will help determine if the bacterial challenge begins at the source or is limited to the house. Additionally, a swab from the inside of the end of the water line should be taken to determine the level of biofilm. The total bacterial count should be less than 1,000 CFU/mL without fecal coliforms in a free-flowing sample, and total bacteria should be less than 10,000 CFU/mL on a biofilm swab.

Monitor water pH

Water should have a pH between 5 and 8. Water with a pH consistently lower than 5 can be damaging to equipment, while a pH over 8 reduces the efficacy of many disinfectants and can have a bitter taste to birds. Hard water can increase scaling of lines and equipment, leading to leaking seals. Scale also provides a matrix for biofilm formation, making cleaning and disinfection more difficult.

Clean and disinfect water lines

Cleaning water lines between flocks is the minimum program for ABF production. Stabilized hydrogen peroxide products are excellent for disinfecting water lines between flocks. The levels needed for proper disinfection of lines are generally too strong for birds, and the lines must be flushed prior to bird placement.

Water lines are often only cleaned in the house; it is important to periodically clean the lines that transport water from the well or water source to the poultry house as this may be a significant reservoir for bacteria. If the well is identified as a source of contamination, it is essential to seek the help of a qualified technician before adding any sanitizing product to a wellhead.

Designing a water quality program poultry farm

Continuous disinfection

Ideally, water should be continuously disinfected with a product that is approved for poultry consumption. One of the best products for continuous disinfection is chlorine dioxide, which is effective at reducing bacteria and also reducing the concentrations of some mineral components. High levels of iron in the water can create a favorable environment for E. coli and other bacteria such as C. perfringens.

In addition to disinfection, chlorine dioxide is an effective treatment to reduce soluble iron levels. High sodium and chloride levels can lead to flushing and promote the growth of some bacteria. If high levels of sodium and chloride are consistent across a grow-out region, it may be possible to decrease the levels in the feed to reduce flushing. If the levels of sodium and chloride are considerably high, reverse osmosis should be considered to improve water quality.

Bottom line: invest in high-quality water

Another effective product is stabilized hydrogen peroxide at an appropriate residual level for bird consumption. There are other options for water line sanitation that can be explored on a case-by-case basis.

There are excellent online resources [link] for poultry water quality. The important message remains, in any case, that investment in high-quality water is a critical step for success in ABF poultry production.

 

References

Austin, B.J., J. Payne, S.E. Watkins, M. Daniels, and B.E. Haggard. 2016. How to Collect Your Water Sample and Interpret the Results for the Poultry Analytical Package. Arkansas Water Resources Center, Fayetteville, AR, FS-2017-01: 8 pp.

Scantling, M. and Watkins, S. 2013. Identify Poultry Water System Contamination Challenges. FSA8011. University of Arkansas Division of Agriculture Research and Extension.

Watkins, S. 2008. Water: Identifying and correcting challenges. Avian Advice 10(3):10-15. University of Arkansas Cooperative Extension Service, Fayetteville, AR




Want to reduce antibiotic use? Biosecurity and sanitation are crucial

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By T.J. Gaydos

Biosecurity may not sound like an exciting topic at first, but it is a critical component of responsible poultry production. It is not enough to devise a strong biosecurity program; that program must also be followed by all people that interact within the system. It only takes one dirty boot or tire to ruin months of hard work.

Achieving good results with a flock largely depends on protecting the birds from biosecurity risks

Antibiotic reduction in poultry requires biosecurity

In a poultry operation, feed, people, and equipment constantly need to go in and out of farms and mills. Thus, no biosecurity program can be perfect. The intensity of the program needs to balance the realities of farming and the current disease pressure. The best program takes all of those into account, additionally considers local weather, availability of supplies, and company/farm staff. It is simple enough to be done even when no one is watching and should be easily scalable in case of increased disease pressure.

The rigorousness of a program must be in due proportion to the local circumstances. Having a biosecurity program that is too strict for the perceived disease pressure may result in people taking the path of least resistance. They probably will not follow instructions, especially if there is not enough monitoring and training to reinforce the value of biosecurity. On the other hand, a program with too lax guidelines will not have the desired effect.

The discrepancy between care requirements and separation

Unfortunately, the most valuable animals in an operation are often the most frequently visited by the most people. Pullets need closely monitored feedings, vaccines, and deworming. Breeders need eggs collected and shipped. Hatcheries require a labor force and maintenance. The feed mill and hatchery are central and overlapping points for all areas of the operation. The human and vehicle traffic at these locations must be closely monitored to reduce the risk of rapid disease transmission.

Feed mills are critical sites for biosecurity measures in poultry production

A physical barrier or sign indicating a biosecurity area on a farm or building entrance can help remind people of the program. Of course, these signs will not stop a disease from entering, nor a person determined to enter a site, but they will cause well-trained people to pause and reflect if they are making a sound decision.

Hygiene is a critical factor

It is well documented that hands and feet are significant transmitters of human and animal pathogens. Several studies have shown that hand washing can reduce absenteeism in school-aged children by 29-57%, thanks to a decrease in gastrointestinal diseases (Wang et al., 2017). Hand washing also reduces the incidence of respiratory illness in human populations by up to 21% (Aiello et al., 2008). Mycoplasmas can survive for one day in a person’s nose, for up to three days in hair, and up to 3-5 days on cotton or feathers (Christensen et al., 1994). Influenza viruses endure 1-2 days on hard surfaces (Bean et al., 1982) and more than a month in pond water (Domanska-Blicharz et al., 2010).

When building a biosecurity program, it is essential to consider the relevant pathogens of concern and the practical ways to reduce their risk of transmission.

How to establish an effective biosecurity program

Generally, biosecurity comprises two important parts:

  • Physical biosecurity, being the combination of all the physical barriers such as boot washes, signs, and disinfection
  • Operational biosecurity, covering the processes that protect an operation. This includes downtime, visiting birds in age order, time out for birds from people visiting sick flocks, and respect for physical biosecurity measures. Operational biosecurity starts with training, not only regarding the tasks required to be secure, but also the importance of disease prevention.

Establish several zones

When designing a program, consider four zones of increasing cleanliness: off-farm, on-farm, transition zone, and the animal housing area (Figure 1). Each zone should have a control point to reduce the pathogen load coming in, with exact measures depending on current disease status and bird value. These measures include vehicle sanitation and movement restrictions, footwear cleaning and disinfection, and use of personal protective equipment (PPE).

Figure 1: the four “cleanliness zones” in a farm

Increasing cleanliness from off-farm (red) to on-farm (orange) separated by a physical barrier. The entrance to the facility (transition zone; yellow) and the animal housing area (green).

Cleaning and disinfection are two of the core measures

As hands and feet are the main transmitters of pathogens, washing and sanitizing them is a priority. The outside of the house must be left outside, meaning that hands should be washed frequently and shoes sanitized between sites. Shoe covers should be put on when entering the house.

Cleanliness of the cell phone is often overlooked as a source of disease transmission (Olsen et al., 2020). It is a powerful tool: camera, notebook, light… and notoriously hard to clean. Cleaning and disinfection also apply to all shared tools and equipment that enter farms.

Prevent undesired “cohabitants”

Another critical point in biosecurity is the control of undesired pests and farm animals. Baits must be rotated, available where rodents are frequent, appropriately spaced, and secured from non-target animals. Habitats for pests need to be removed, the perimeter of the buildings must be clear of vegetation and debris, feed and grain spills picked up, and equipment stored away from the facilities. Pets and other farm animals should be kept away from the perimeter of the house and should under no circumstance be allowed to enter the facilities.

Tailored biosecurity programs keep your flock healthy

It is impossible to design a blanket biosecurity program for every operation. Understanding microbiology and disease transmission along with the risk points in a production system will allow a comprehensive plan to be developed. It is important to consider biosecurity as an investment in health and not an optional expense. No program is perfect, but small changes can significantly reduce the risk of pathogens entering the system and leading to major economic and animal welfare issues.

References

Aiello, Allison E., Rebecca M. Coulborn, Vanessa Perez, and Elaine L. Larson. “Effect of Hand Hygiene on Infectious Disease Risk in the Community Setting: A Meta-Analysis.” American Journal of Public Health 98, no. 8 (2008): 1372–81. https://doi.org/10.2105/ajph.2007.124610

Bean, B., B. M. Moore, B. Sterner, L. R. Peterson, D. N. Gerding, and H. H. Balfour. “Survival of Influenza Viruses on Environmental Surfaces.” Journal of Infectious Diseases 146, no. 1 (1982): 47–51. https://doi.org/10.1093/infdis/146.1.47.

Christensen, N. H., Christine A. Yavari, A. J. McBain, and Janet M. Bradbury. “Investigations into the Survival of MYCOPLASMA GALLISEPTICUM, Mycoplasma Synoviae And Mycoplasma Iowae on Materials Found in the Poultry House Environment.” Avian Pathology 23, no. 1 (1994): 127–43. https://doi.org/10.1080/03079459408418980.

Domanska-Blicharz, Katarzyna, Zenon Minta, Krzysztof Smietanka, Sylvie Marché, and Thierry van den Berg. “H5n1 High Pathogenicity Avian Influenza Virus Survival in Different Types of Water.” Avian Diseases 54, no. s1 (2010): 734–37. https://doi.org/10.1637/8786-040109-resnote.1.

Olsen, Matthew, Mariana Campos, Anna Lohning, Peter Jones, John Legget, Alexandra Bannach-Brown, Simon McKirdy, Rashed Alghafri, and Lotti Tajouri. “Mobile Phones Represent a Pathway for Microbial Transmission: A Scoping Review.” Travel Medicine and Infectious Disease 35 (2020): 101704. https://doi.org/10.1016/j.tmaid.2020.101704.

Wang, Zhangqi, Maria Lapinski, Elizabeth Quilliam, Lee-Ann Jaykus, and Angela Fraser. “The Effect of Hand-Hygiene Interventions on Infectious Disease-Associated Absenteeism in Elementary Schools: A Systematic Literature Review.” American Journal of Infection Control, 2017. https://doi.org/10.1016/j.ajic.2017.01.018.