Feed quality and poultry gut colonisation

Q: What are the negative effects of pesticides and herbicides in feed?

Pesticides and herbicides that readily accumulate in feed can have negative effects. The most susceptible poultry feedstuffs include cereal samples such as wheat, rye, barley, oats, maize, buckwheat, and others (Walorczyk and Drozdzynski, 2012). Additionally, the runoff into the waterways ensures a high presence and accumulation of both pesticides and herbicides in fish (Yang et al., 2020).

I. Introduction

Immediately after birth, or hatch in birds, the initial inoculum shapes the gut microbiota for life. The first bacteria to settle in the intestine can attach to epithelial cells with no competition, rapidly establish, grow, and set the intestinal environment to suit their own needs (Stecher and Hardt, 2011; Edwards, 2017). The first bacterial settlers have the most substantial influence on developing the host's immune system and overall ability to thrive (Stecher and Hardt, 2011; Edwards, 2017). While in humans gut microbial community takes around two years to mature, the timeframe to maturity is significantly reduced in chickens. Studies report that chicken microbiota stabilises by day 3 (Apajalahti et al., 2004). The maturity of gut microbiota assumes the ability to resist change to a certain level. Studies on humans report that any early adversities, from mild, such as nutritional imbalance, to major like antibiotic administration, prior to the establishment of a mature intestinal microflora can leave permanent consequences that lead to obesity, asthma, allergic diseases and diabetes (Kaplan and Walker, 2012; Wallace et al., 2016).

Poultry research invested decades in optimising bird nutrition to achieve maximum health and performance. The early nutritional needs of hatchlings are well defined. However, advances in molecular microbiology and microbiota research have shed new light on the role of early chick feed, not just in terms of providing the nutrition to the host, but also providing the nutrition to beneficial microorganisms and restricting the essential nutrients to pathogenic microorganisms in the first days post-hatch. This way, the early feed can contribute to the formation of a balanced gut microbial community. This review will concentrate on the role of feed in gut colonisation, focusing on early feed safety.

II. Biological contaminants in feed

a) Microbial contamination of feed

The most critical requirement for early post-hatch feed is biosecurity since providing early pathogen access to the naïve gut could lead to mortality, lifelong colonisation, and permanent pathogen shedding. However, it is well established that feed can get contaminated with biological pollutants at any production stage. Salmonella, Campylobacter, Clostridium perfringens, and Escherichia coli feed contamination have been at the centre of feed safety research in chickens and other livestock. Shirota et al., (2000) acknowledged that it is generally presumed that baby chicks bring Salmonella sp. to the farm, implying hatchery contamination, while only a limited number of studies look at the feed as a probable source of contamination. It was reported that only trace levels of Salmonella could lead to young chick mortality (Henderson et al., 1960). Shirota et al., (2000) analysed 4418 samples of finished layer feed in Japan and found 46 Salmonella strains in 143 feed samples. The isolates belonged to a minimum of 32 serovars, with the most abundant being S. Enteritidis, S. Livingstone, S. Bareilly, and S. Derby. The authors concluded that the contamination was often limited to the same mills, and although the source of salmonellae was identified, the mills were persistently contaminated.

Gosling et al., (Gosling et al., 2021) summarised the literature on wide contamination of feed mills with Salmonella, concluding that the ingredient intake pits were Salmonella hot spots extending to all stages of growing, shipping, processing, storage, and finished feed. The authors continued to suggest less toxic organic acids for decontamination of Salmonella and E. coli instead of widely used formaldehyde-based treatments. Many authors investigated improved ways to remove Salmonella and other pathogens from the fed mills. Common methods of disinfection of feed mill food contact surfaces were based on "sequencing" of raw feed ingredients so that those most likely to carry pathogens are left for last, followed by "flushing" of equipment with a pulse of animal food such as chemically treated rice hulls (Gebhardt et al., 2018) to clean the equipment and minimise leftover pathogens. The critical issue was the breaking of biofilms formed on the mill equipment. Muckey et al., (2020) investigated methods of sanitation following controlled contamination with Salmonella using a commercially available essential oil blend or rice hulls treated with medium-chain fatty acids, finding that both treatments can reduce contamination compared to the control. The authors suggested that feed sequencing can reduce Salmonella contamination on manufacturing surfaces, particularly when flushing is combined with chemical treatments.

Sauli (2005) investigated data on Salmonella contamination of pig feed in Switzerland to conclude that the probability that finishing pig feed contains Salmonella ranged from 34% (no decontamination step) to 0% (with organic acids and heat treatment decontamination step). Another study from China (Yang et al., 2017) investigated the contamination of 1077 feed samples, including raw ingredients and finished feeds, collected from feed mills, farms, and feed sales between 2009 and 2012. Salmonella contamination ranged from 4.7 % in 2009 to the lowest of 0.66 % in 2011. Salmonella contamination came from animal protein material such as meat meal, meat and bone meal, feather meal, blood meal, and fish meal but was not identified in microbial protein, rapeseed, and soybean meal. Salmonella contamination was found in mills, farms and feed wholesale (Yang et al., 2017).

Despite no positive samples in Chinese soybean, others (Wierup and Kristoffersen, 2014) reported frequent Salmonella contamination in soybean imported to Norway mostly from South America. This study covered data from 19 years of testing, finding that 34% of samples were positive to Salmonella; with variation from 12-62% each year. Additionally, the dust samples from all shiploads from South America yielded Salmonella. This study reported 94 Salmonella serovars in soybeans over 19 years of import, including 9 of 10 top serovars isolated from clinical cases of salmonellosis.

The data of feed and bird carriage of Salmonella differ between the studies and countries. Shirota et al., (2000) pointed out the issues with sample collection and analysis, emphasising that each feed sample tested is usually a single sample of 30-100g taken from a massive batch of tonnes of feed and suggested that better sampling methods and strategies should be introduced.

Feed contamination with other pathogens is comparable to Salmonella contamination, which remains the most investigated feed contaminating pathogen. Similarly to Salmonella, contamination of poultry feed with Clostridium perfringens comes mostly from fish meal followed by bone meal, meat and bone meal and dry fish (Udhayavel et al., 2017). In a controlled experiment with a feed mill contaminated with Salmonella and E. coli, E.coli was reported as less resilient and faster to die off than persistent Salmonella (Gosling et al., 2021). In addition to bacterial pathogens, the feed can also carry antimicrobial resistance within pathogens, devastating viruses (Schumacher et al., 2018) or mycotoxin producing mycobiota (Pereyra et al., 2011; Namulawa et al., 2020).

b) Feed microbiota

Instead of investigating feed and raw feed ingredients using classic PCR and other methods that target specific species or genus, a recent study (Haberecht et al., 2020) performed amplicon sequencing of raw and finished poultry feed to find that each feed source carried a rich microbial community. Investigated raw ingredients included meat and bone meal, wheat, corn, canola, barley, soybean, millrun, sorghum, poultry oil, oats, limestone and bloodmeal from four geographically distinct feedstuff suppliers. In agreement with pathogen tracing to high protein raw feedstuffs, the authors reported that the meat and bone meal and bloodmeal samples contained the most complex microbial communities, very distinct from one another. Additionally, unique and dissimilar microbial communities were reported in poultry oil and limestone, distinct from highly overlapping microbiota found in the grain and seed samples: barley, canola, corn, millrun, oats, sorghum, soybean meal and wheat.

Feed microbial composition contained four phyla, in order of abundance: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes and 50 genera that included both beneficial like Bacillus, Bifidobacterium, Lactobacillus and Ruminococcus, as well as pathogenic Clostridium, Enterobacter, Staphylococcus and Streptococcus. No Salmonella and Escherichia were detected in this study. The authors followed the feed microbiota through the intestinal sections to find that different taxa from feed persisted in different gut sections investigated, including the caecum, ileum and excreta. Additionally, the feed mill source of raw and finished feed had a substantial influence on microbial communities in feed and the geographic location of the feed mill also played a role.

Despite the fact that people used bacteria contained within the grain throughout history to start sourdough fermentation, there is not much literature on microbial communities in grains. Cereal grains are composed mainly of starch, and it was reasonable to expect that they would carry beneficial fibre/starch loving probiotic bacterial strains and could be a good source of starch degrading enzymes. It was reported that whole-grain oats carry probiotic lactic acid bacteria (Wu et al., 2018). However, Carrizo et al. (2016) investigated lactic acid bacterial microbiota of quinoa grains and spontaneous quinoa sourdough to isolate and identify a range of Lactobacillus species, including multiple strains of L. plantarum, L. rhamnosus, L. sakei, Pediococcus pentosaceus, Leuconostoc mesenteroides, Enterococcus casseliflavus, E. mundtii, E. hirae, E. gallinarum, Enterococcus sp., and E. hermanniensis. They continued to investigate the enzymatic and nutritional benefits of these strains to conclude that rich probiotic microbiota present in quinoa carries a potent starter culture able to increase the nutritional value of grains.

While investigating rumen starch-hydrolysing bacteria (SHB) possessing active cellsurface-associated alpha-amylase activity, using fluorescence in situ hybridization, Xia et al., (2015) discovered that 19-23% of the total rumen bacterial cells were attached to particles of four cultivars of barley and corn used for feed. The vast majority of these bacteria were members of the Ruminococcaceae. By microscopical inspection of whole and crushed corn and barley cells wash, the authors identified cocci of different sizes, single or in chains, and rods of different morphology in all samples. The proportion of barley grain in the diet had a large impact on the percentage abundance of total SHB and Ruminococcaceae SHB in these animals. Pan et al., (2015) investigated the ways to reduce Fusarium graminearum in wheat to control Fusarium head blight and subsequent contamination of grain with mycotoxins. They evaluated bacterial endophytes isolated from wheat grain for antagonistic ability against F. graminearum under field conditions. They identified a range of grain endophytes with one isolate of Bacillus megaterium and three of Bacillus subtilis, significantly inhibiting growth of Fusarium on grain.

Bacterial and fungal endophytes are well-reviewed and documented in grains (Abdallah et al., 2018; Ahlawat et al., 2021; Makar et al., 2021) and in legumes (Ruiz Mostacero et al., 2021), thus adding more evidence to the observation of grain microbiota. High prevalence of probiotics in grains and as discussed above the high prevalence of pathogens in protein-rich feedstuffs, indicate that the first feed offered to hatchings, selected and formulated to promote the growth of probiotics and inhibit pathogens, should be grain-based and rely on grains and cereals as a protein source for the first three days of gut microbiota establishment.

III. Chemical contaminants in feed

c) Mycotoxins

Mycotoxins are secondary metabolites of filamentous fungi that are causing massive losses to agriculture worldwide. Aflatoxins, ochratoxin A, deoxynivalenol patulin, fumonisins, zearalenone, trichothecenes, fumonisins and ergot alkaloids are presently the most important for food and feed safety (Abrunhosa et al., 2016). Furthermore, the range of fungal species that produce these toxins is broad, including Fusarium, Aspergillus, Penicillium, and Claviceps species. Fungi are widely distributed in nature and foods and feedstuffs from all parts of the world, especially in high rain and high humidity tropical climates. In a comprehensive study from tropical Malaysia, the authors report an abundance of mycotoxins in peanuts, cereals, cocoa, spices, feeds and nuts consumed in Malaysia. Moreover, spices, oilseeds, milk, eggs, and herbal medicine products were also contaminated. Malaysian rice, oat, barley, maize meal, and wheat were contaminated with some of the most toxic mycotoxins (Afsah-Hejri et al., 2013). Mycotoxins in food and feed constitute a significant issue for animal and human safety, and they are comprehensively reviewed by many, including Pleadin et al. (2019) and specifically in pig and poultry feed (Guerre, 2016).

Mycotoxins are carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic, and immunosuppressant (Abrunhosa et al., 2016), and they affect gut microbiota most negatively by increasing the abundance of pathogens and reducing or eliminating beneficial bacteria (reviewed in Liew and Mohd-Redzwan, 2018). Gao (2020) reviewed the effects of mycotoxins on the leaky gut and intestinal barrier that include compromised intestinal integrity, thinned mucus layer and imbalance of inflammatory markers in addition to the disturbed microbial community. Others have also reviewed new targets of mycotoxins – gut mucus layer and microbiota (Robert et al., 2017). Based on the above, even traces of mycotoxins in early hatchling feed would disturb bird heath and colonisation with the beneficial microbial community with likely consequences for the bird's long-term health and performance.

d) Heavy metals

Maximum allowed concentrations of heavy metals in livestock feed are recognised as a concern and are tightly regulated in countries worldwide. Heavy metals are the fourth most often notified hazard in Rapid Alert System for Food and Feed (RASFF) (Piglowski, 2018). In some countries like the European Union, firm actions are taken to standardise proficiency tests for the determination of heavy metals in feed (Guntinas et al., 2011). Testing for heavy metals in feed is often performed together with mycotoxin testing. Heavy metal contamination often differs from country to country and depend on levels of heavy metal pollution in nature. For example, all of the tested 40 feed samples in the Iran study (Eskandari and Pakfetrat, 2014) had acceptable Pb concentrations, while a high portion of feed samples had As, Cd and Hg above the maximum limits.

The consequences of poor testing in livestock feed can translate to human health. For example, in the Pakistan study, Kabeer et al., (2021) tested Ni, Pb, Zn, Mn, Cr, Cu and Se concentrations in poultry eggs to find that concentrations of Pb, Cr and Se in egg white, egg yolk and both feed and water were above permissible limits in tested farms and backyard birds (Kabeer et al., 2021). In India, heavy metals (Cu, Zn, Cr, Pb, and Cd) originating from feed were found in milk in excessive concentrations (Yasotha et al., 2021). The main concern is the effect that heavy metal contaminated milk and eggs could have on young children whose diets are often rich in milk and eggs.

In addition to various methods developed to remove heavy metal contamination from the feed, novel approaches to this significant feed issue are desperately needed. Recently Yang et al., (2021) tested 11 maize varieties in experimentally polluted soil Cd, As, and Pb to identify cultivars with low seed uptake of heavy metals. The hypothesis was that, in some varieties, the heavy metals might be accumulated in non-edible parts of the plant. Major differences between the varieties were identified, providing a new perception in dealing with soil pollution with heavy metals and pointing towards the development of improved varieties.

Due to the ability of fish to concentrate heavy metals from polluted waters, heavy metal concentrations in fishmeal are of concern (Jia et al., 2006), especially in fish collected in the proximity or downstream from industrial waste disposal sites (Utomo et al., 2021). Furthermore, some heavy metals are highly accumulated in black soldier fly (Wu et al., 2020), indicating different feed source susceptibility. Heavy metals are known for promoting antimicrobial resistance in a similar way to antibiotic addition (Holzel et al., 2012; Yu et al., 2017; Rilstone et al., 2021) and for a range of negative effects on the host-microbiota (Richardson et al., 2018), general toxicity to microorganisms (Giller et al., 1988), plants and humans (Islam et al., 2007) and their ability to concentrate in both chicken meat (Mondal, 2020) and plant feed products (Islam et al., 2007) calling for a cautious approach to heavy metals in feed used in early bird's life.

e) Pesticides and herbicides

Other common feed contaminants include pesticides and herbicides that readily accumulate in feed. A range of highly sensitive methods is developed for screening of over 100 pesticides in feed (van der Lee et al., 2008). The most susceptible poultry feedstuffs include cereal samples such as wheat, rye, barley, oats, maize, buckwheat and others (Walorczyk and Drozdzynski, 2012). Additionally, the runoff into the waterways ensures a high presence and accumulation of both pesticides and herbicides in fish (Yang et al., 2020). Pesticides are highly toxic and, in sufficient concentrations, fatal for humans (Moebus and Boedeker, 2021), while in lower concentrations, they disrupt microbiota and cause serious health problems (de Boer, 2021; Utembe and Kamng'ona, 2021).

Glyphosate is the most highly used herbicide in agriculture, with just recently identified carcinogenic effects (Rueda-Ruzafa et al., 2019). Glyphosate is the most challenging herbicide accumulated in feedstuffs and livestock feed. The consequences of glyphosate in feed for livestock health and productivity were recently reviewed (Vicini et al., 2019; Sorensen et al., 2021), summarising detrimental effects on animal health, including neurological damage and microbiota impairment (Rueda-Ruzafa et al., 2019). Surprisingly, Clostridium and Salmonella are highly resistant to glyphosate resulting in an imbalance between beneficial and pathogenic microorganisms. Also, glyphosate-induced clostridia overgrowth is linked to neurological toxicity (Rueda-Ruzafa et al., 2019).

f) Other feed contaminants

There are many more chemical feed contaminants that include residual chemicals such as antibiotics introduced by cross-contamination via lack of equipment cleaning between the batches of feed (Peeters et al., 2016; Przenioslo-Siwczynska et al., 2020) to radiation toxicity (Iammarino et al., 2015) accelerated after Chernobyl and other more recent nuclear disasters, industrial waste products (Torres et al., 2013) and oestrogenic polychlorinated biphenyls (Pinto et al., 2008).

IV. Conclusions

The present literature review summarised in Figure 1, strongly implies the need for highly internationally regulated global livestock feed testing due to the high import and export of final livestock food products. From the gut microbiota establishment point of view, the feed offered to hatchings during the first three days of microbiota formation should be immaculate in terms of both biological and chemical contaminants and, if possible, enriched with beneficial and free of pathogenic bacteria with nutritional composition highly supportive of fibre and other prebiotics loving bacteria.

Figure 1 - Possible feed contaminants that can disrupt early gut colonisation. Created with BioRender.com