Aflatoxicosis: Lessons from Toxicity and Responses to Aflatoxin B 1 in Poultry

This review is a comprehensive introduction to the effects of poultry exposure to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1). The relationship between AFB1 sensitivity and metabolism, major direct and indirect effects of AFB1, recent studies of gene expression and transcriptome responses to exposure, and mitigation strategies to reduce toxicity are discussed. Exposure to AFB1 primarily occurs by consumption of contaminated corn, grain or other feed components. Low levels of residual AFB1 in poultry feeds can cause reduction in growth, feed conversion, egg production, and compromised immune functions, resulting in significant economic costs to producers. Thus, AFB1 acts as a " force multiplier " synergizing the adverse effects of microbial pathogens and other agents, and factors detrimental to poultry health. Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1 due, in large part, to a combination of efficient hepatic bioactivation by cytochromes P450 1A5 and 3A37, and deficient hepatic glutathione-S-transferase (GST)-mediated detoxification. Because of their sensitivity, turkeys are a good model to investigate chemopreventive treatments and feed additives for their ability to reduce AFB1 toxicity. Transcriptome analysis (RNA-seq) of turkey poults (liver and spleen) has identified AFB1-induced gene expression changes in pathways of apoptosis, carcinogenesis, lipid regulation, antimicrobial activity, cytotoxicity and antigen presentation. Current research focuses on further identifying the molecular mechanisms 743 underlying AFB1 toxicity with the goal of reducing aflatoxicosis and improving poultry health.


Introduction
Dietary exposure to aflatoxins can have severe toxic and carcinogenic effects in humans and animals.The production and metabolism of aflatoxin, symptoms and biomarkers of exposure, and methods to reduce aflatoxicosis have been extensively investigated over the past 50 years [1][2][3][4][5][6][7][8][9].Most studies have focused on humans, laboratory model species, or agricultural animals, and have identified conserved and species-specific aspects of aflatoxicosis [2,[10][11][12][13][14][15].This review specifically examines the responses of poultry, a particularly sensitive group, to aflatoxin B1.Aflatoxin metabolism, toxicity, and expression responses in poultry are discussed, along with potential mitigation strategies.
Livestock, including poultry, are exposed to AFB1 and other aflatoxins by consuming contaminated feed.Many agricultural feed commodities (corn, cottonseed, peanuts and sorghum) and other foods (figs, tree nuts and spices) are at especially high risk [21,23].Stress from drought or insect damage can reduce crop resistance to Aspergilli and lead to aflatoxin contamination prior to harvest [21,24].Warm and humid conditions during maturation, harvest, transport or storage, promote Aspergillus colonization and subsequent aflatoxin production [21,24].Temperatures near 30 °C and water activity of 0.99 provide ideal conditions for AFB1 biosynthesis, although substrate, time, CO2 levels, and other environmental factors are also important [25][26][27][28][29][30].Along with primary contamination of crops, aflatoxins can transfer to milk, meat and eggs of livestock and poultry fed the toxins [23,[31][32][33][34][35][36][37][38].Therefore, AFB1 is a human food safety risk in both plant and animal products.Animal susceptibility to the acute effects of aflatoxicosis varies widely (Table 1).Domestic turkeys and ducks (Anas platyrhynchos) are highly sensitive to both the acute and chronic toxicity of AFB1 [21,39,40].Chickens (Gallus gallus) are more resistant to acute aflatoxicosis than other poultry species, except during embryonic development (Table 1).Even when exposure does not cause mortality or morbidity, aflatoxicosis contributes directly and indirectly to losses for the poultry industry.While precise numbers are not available, it has been estimated that aflatoxins inflict a loss of at least $143 million each year due to AFB1-induced hepatotoxicity, reduced performance and secondary infections [23,41].

Sensitivity: Poultry
Poultry are sensitive to even low levels of AFB1, and among species of agricultural importance, the order of sensitivity is ducks > turkeys > Japanese quail (Coturnix japonica) > chickens [7,21,[72][73][74][75].Therefore, lower concentrations of AFB1 are lethal to turkeys and ducks and more adversely affect production and health in these species (Table 2).In domestic turkey, efficient production of AFBO contributes to sensitivity.We have conducted considerable research to determine the P450 enzymes responsible for AFB1 bioactivation and metabolism in turkey livers.This work revealed two turkey P450 enzymes, encoded by CYP1A5 and CYP3A37, predominantly responsible for converting AFB1 into AFBO in vitro and in vivo [76][77][78][79].P450 1A5 has high-affinity (high Vmax, Kcat; low Km) and catalyzes the production of both exo-AFBO and the detoxified metabolite AFM1 according to traditional Michaelis-Menten kinetics [76,77].P450 3A37 is the lower affinity catalyst, exhibiting apparent subunit allostery conforming to Hill enzyme kinetics and producing exo-AFBO and AFQ1 [76,77].
We used polyclonal anti-peptide antibodies in a series of immunoinhibition experiments to determine the relative importance of these P450 enzymes in AFB1 bioactivation in turkey livers and liver microsomes.Turkey P450 1A5 is the predominant catalyst (>98%) at low (<50 μM) pharmacologically-relevant AFB1 concentrations commonly seen in the livers of exposed turkeys, while P450 3A37 predominates at much higher AFB1 concentrations not likely to be achieved in tissues under in vivo conditions [78].Another P450 enzyme, likely orthologous to mammalian P450 2A6, also converts AFB1 to AFBO in turkeys, chickens, ducks and quail [80][81][82].However, the specific gene encoding this protein has not been identified in any poultry species [80][81][82][83].These same studies implicated a P450 1A1 orthologue in AFBO production, which is likely encoded by CYP1A5 in turkeys [78,[80][81][82].The activity of P450 enzymes in the turkey, and therefore AFBO production, is inversely related to age, with extreme sensitivity to AFB1 in young poults [39,77,84,85].A similar age-dependent sensitivity occurs in chickens [86][87][88].Hepatic microsomal P450 enzymes from turkeys and quail produce 2 to 4 times more AFBO than ducks or chickens [74].A later study confirmed the highest AFBO production in turkey, with intermediate levels in duck and quail, and the lowest in chicken [83].AFBO production correlates with the sensitivity of poultry, except for the duck.In addition, the levels of AFL produced by liver cytosol are highest in turkey, followed by ducks, chickens and quail [74].The sensitivity of ducks may be more linked to AFL production and toxicity or to oxidative stress [74,83,89].
Like the cytochrome P450 enzymes, hepatic tGSTA enzymes contribute to the severity of aflatoxicosis in the turkey.Six tGSTA genes have been amplified, cloned and heterologously expressed turkey liver (GSTA1.1,GSTA1.2,GSTA1.3,GSTA2, GSTA3, and GSTA4) [90,91].Hepatic cytosolic GST enzymes from domestic turkey have essentially no AFBO-conjugating activity [40,91].However, tGSTA enzymes can conjugate AFBO to GSH in vitro using recombinant proteins in an E. coli expression system, suggesting that gene silencing mechanisms or post-transcriptional modifications are likely responsible for their lack of function in vivo [91].The high sensitivity of the domestic turkey to AFB1 appears to be due to an unfortunate combination of efficient P450 enzymes and dysfunctional GST enzymes that allow accumulation of AFB1 adducts in the liver.
The effects of AFB1 exposure on North American wild turkeys are similar to, but less severe than those seen in domestic poultry [92].This difference in response may be the result of genetic changes that occurred during domestic selection, but could also be due to the separate genetic background of the domestic turkey.Although members of the same species, domestic turkeys were originally derived from the Mexican subspecies (Meleagris gallopavo gallopavo) of wild turkey native to Central America; the Eastern subspecies from North America (Meleagris gallopavo silvestris) was likely involved in later crosses [93].Wild turkeys were originally exported to Europe from Mexico in the 1500's and then reintroduced to North America in the 1600's.Reintroduced turkeys were selectively bred, first forming the heritage breeds, and then developed into the modern commercial breeds.Current commercial production predominately utilizes the Broad Breasted White.Selection for production traits in poultry is known to increase metabolic disorders [94] and decrease immune functions [95-98].Likewise, detoxification capabilities could be reduced as an unintended side-effect of selection in domestic birds.
Eastern wild turkeys are more resistant to aflatoxicosis than their domestic relatives [92].AFBO detoxification is likely a main contributor to the differences seen among turkeys.Consistent with this hypothesis, hepatic cytosolic GST enzymes from both the Eastern and Rio Grande (Meleagris gallopavo intermedia) subspecies of wild turkey have activity against AFBO [99], unlike their domestic counterparts.Wild turkey GSTA enzymes heterologously expressed in vitro also had AFBO-conjugating activity [99].Like wild strains, the Royal Palm turkey, a heritage breed, retains hepatic and in vitro activity [99].tGSTM enzymes were also amplified, cloned and heterologously expressed from the livers of wild and domestic turkeys, but had no measurable affinity toward AFBO, and therefore are not thought to be involved in AFB1 detoxification [100].

Mutagenicity
Binding of AFBO to DNA introduces G-T transversion mutations in hepatic DNA [4,65,105].The high incidence of G-T transversions results from AFB1-FAPY adducts predisposing bypass DNA synthesis machinery to make G-T changes [101].A G-T transversion in codon 249 of the p53 tumor suppressor has been identified in many human liver cancers and may mechanistically contribute to cancer formation [7,65,71,101,[105][106][107].Chronic AFB1 exposure, especially in combination with hepatitis-B infections, severely increases the risk of hepatocellular carcinoma in humans [1,[3][4][5][6]9,21,105,108].Dietary AFB1 is known to have hepatocarcinogenic effects in other mammals, especially the sensitive rat [4,[109][110][111].Based on this evidence from humans and other mammals, AFB1 is classified as a group I carcinogen by the International Agency for Research on Cancer [4,105].AFBO is highly mutagenic in poultry, although adenoma and hepatocellular carcinoma have only been reported in ducks [82,[112][113][114].The potential synergistic effect of hepatitis-B virus and AFB1 has not been consistently reproduced in these studies [112][113][114].
Exposure to aflatoxins lowers reproductive performance in poultry.In layers fed AFB1, age to maturity is increased [34] and egg production is reduced [34,121,125,[128][129][130][131].Egg quality parameters such as total weight, shape, albumin or yolk percentage, and shell thickness in chickens and quail can be adversely affected by AFB1, although the effects were variable among studies [34,125,126,128,[131][132][133].The declines in poultry production traits are often indirect effects of AFB1 reducing the metabolic potential of the liver.For example, impaired hepatic protein production likely contributes to AFB1-induced changes within eggs, as the liver is the chief site of synthesis of proteins and lipids incorporated into the egg yolk.
Both acute and chronic AFB1 consumption by poultry cause other hepatic lesions.Common histopathological signs of AFB1-induced liver damage include focal necrotic hepatocytes or hemorrhages [10,34,39,84,116,121,137]. Acute damage initiates inflammatory responses and leads to leukocyte infiltration and proliferation in the liver [10,34,84,112,116,138].Short-term exposure to higher dose can cause morbidity and mortality from extensive liver damage [3,21].In poultry, chronic AFB1 consumption is mutagenic and leads to remodeling of liver tissues.Hyperplasia of bile duct epithelial cells or oval cells develops first, followed by periportal fibrosis and nodular tissue regeneration [10,34,39,84,112,116,121,126,134,139].
AFB1 adducts with biomolecules cause damage to hepatocytes that impairs metabolic functions of the liver during AFB1 exposure.This is exemplified by AFB1-reduced total serum protein levels, as the liver is responsible for production of most circulating proteins [88,115,[117][118][119]123,127,129,140,141].Aflatoxicosis negatively affects albumin, globulin, cholesterol, and triglyceride levels in serum [88,115,117,119,127,129,136,[140][141][142].Multiple blood coagulation factors are produced in the liver; the activity of these clotting factors and the serum levels of fibrinogen are diminished by AFB1 in both chickens and turkeys [142][143][144][145][146]. Hepatic protein concentrations also decrease in AFB1-fed chickens [88].Protein content likely declines because AFB1-DNA adducts inhibit transcription or translation and AFB1-lysine adducts result in protein degradation or excretion.Reduced synthesis of enzymes in the liver would have systemic effects on poultry metabolism.For example, the decreased hepatic fatty acid synthesis observed in AFB1-exposed chickens [147] could be responsible for lower production of serum cholesterol and triglycerides [129,140].

Immunotoxicity
The avian immune system relies on the bursa of Fabricius, thymus and spleen to produce mature or active leukocytes.Even at low dietary concentrations, AFB1 can damage these immune tissues and suppress innate and adaptive immune responses [3,7,148].AFB1 consumption during growth can lead to immune tissue atrophy, reducing relative weights of the bursa, spleen and thymus [120,122,134,139,[149][150][151][152].Increases in relative spleen weight have also been observed during aflatoxicosis [117,120,[153][154][155][156].In young chickens, tissue changes are concomitant to the development of histopathological lesions.AFB1 exposure causes visible congestion in the spleen and thymus, while nuclear debris accumulates in the thymus and bursa [139,149,150,156].In AFB1-exposed chicks, vacuoles increase in the lymphoid follicles of the bursa and the white pulp of the spleen, especially the T-cell rich periarteriolar lymphoid sheaths [139,150,156].
At the cellular level, innate and adaptive cell-mediated immune functions are impaired by AFB1 exposure in poultry [148].In vivo exposure to AFB1 and other aflatoxins has been shown to decrease phagocytic activity in chicken leukocytes, including heterophils [157,158], macrophages [159][160][161], and monocytes [162].Reduced phagocytosis was demonstrated in vitro for peritoneal macrophages isolated from both chickens and turkeys, although microsomally activated AFB1 was required [163,164].In contrast, the phagocytic activity of thrombocytes was not affected in chickens fed an aflatoxin-contaminated diet [165].Dietary AFB1 inhibits T lymphocyte activation in both chickens and turkeys as evidenced by delayed hypersensitive skin tests and graft-versus-host response tests [39,84,116,122,159,166,167].
AFB1 can induce circulating lymphocytopenia [34,159,166,168] and cause lymphoid depletion in the bursa, spleen and thymus [34,134,139,149,150,159,166].This likely results from increased apoptosis of splenocytes, thymocytes and bursal B-cells as seen in young chickens during aflatoxicosis [139,149,156,169,170].In the chicken, both CD4 + and CD8 + T lymphocytes in the spleen, thymus, peripheral blood, and even the ileum can be affected by AFB1 exposure [149,150,156,168,171,172]. Oxidative stress and DNA damage from AFB1 are likely responsible for initiating apoptotic processes in lymphocytes [169,170].Together these losses reduce the adaptive immune potential of poultry fed AFB1.
Although some studies only observed effects on cell-mediated immunity [39,84,116], others found that AFB1 can similarly diminish innate and adaptive humoral immune capabilities.Total serum complement is decreased by feeding AFB1 to chickens, ducks and turkeys [115,127,[173][174][175].Antibody titers are often reduced, whether measured as total serum levels of IgA, IgG and IgM [139,141,167], as production of specific antibodies in response to sheep red blood cells [122,152,159] or as exposure response to infectious bronchitis virus, infectious bursal disease virus, Newcastle disease virus or Pasteurella multocida [128,176,177].AFB1 can also impair the effectiveness of vaccination for these poultry diseases [128,158,[176][177][178].

Intestinal Toxicity
Nutrients are primarily absorbed by epithelial cells in the small intestine and these cells facilitate uptake of AFB1.Most AFB1 is transferred directly into the blood stream; however, as in mammals [43], some may be metabolized in intestinal tissues.Therefore in poultry, both local and systemic effects of AFB1 exposure likely occur in the small intestine, but these are not well characterized.Dietary exposure to AFB1 can lower the unit weight (length/weight) of the duodenum and jejunum [194,195] and affect tissue morphology.In chickens, AFB1 has been shown to raise crypt depth in the jejunum [196], decrease villus height in the duodenum [197], and lower the ratio between villus height/crypt depth in all three sections of the small intestine [197].However, these histopathological effects may not be pervasive [198].
The severity of aflatoxicosis in poultry can be affected by nutrition [7].Deficiencies in some dietary vitamins raised AFB1 sensitivity in chickens, although feeding vitamins in excess was not protective [199,200].Dietary supplementation with tryptophan also increased hepatotoxicity of AFB1 [201].Conversely, diets high in fat or protein may be beneficial to chickens and turkeys fed AFB1 [202][203][204].
Direct investigations of AFB1-effects on absorption or retention of individual nutrients have had variable results [34,194,196,[205][206][207][208].Exposure to AFB1 decreases the apparent metabolizable energy poultry can obtain from their diet [34,195,196,[205][206][207].Therefore, increased dietary nutrients are needed to compensate for impaired uptake.It is currently unclear how much reduced nutrient uptake in the intestine contributes to AFB1 effects on growth and feed efficiency in poultry.

Embryotoxicity
Exposure during development recapitulates many of signs of aflatoxicosis seen in hatched chicks and poults.Although most studies are carried out by in ovo AFB1 injections, embryonic exposure to the toxin is a known risk to poultry.AFB1 and its metabolites can be transferred from the laying hen into the albumin and yolk of the egg [31,34,37,128,131,133,209].AFM1 is a common metabolite detected in eggs and, while not as carcinogenic as AFB1, is acutely toxic [4,37,133].Contamination of unfertilized shell eggs is therefore a food safety risk when used for human consumption.
Transfer of aflatoxins into embryonated eggs is also a concern for poultry producers.In experimental settings, in ovo exposure of chickens or turkeys to AFB1 caused DNA damage in the embryonic liver and increased embryo mortality [131,161,[210][211][212][213][214][215].When introduced into the maternal diet to simulate the natural route of embryonic exposure, AFB1 caused reduced hatchability [130,131,161].Consistent with studies in hatched poultry, turkeys may be more sensitive to embryotoxic effects than chickens [215].Since embryonic liver has an active protein from the CYP1A family [216], hepatotoxicity in turkey embryos is likely mediated by the same P450 1A5 as in poults.
Embryonic AFB1 exposure can lead to morphological defects [212], such as abnormal area opaca cells [210,214], skeletal defects in the tibia growth plate [213], and inhibition of bursal follicle development [210,214].These mutagenic effects can reduce embryo viability and adversely affect hatched progeny.In ovo AFB1-driven immunosuppression has the potential to increase the incidence of infectious disease in young poultry and negatively affect their health and productivity.Whether in ovo injection or maternal feeding, chickens exposed to AFB1 during embryogenesis have compromised cellular and humoral immune functions post hatch [160,161,211,217,218].

P450 and GST Enzymes
Gene expression can improve our understanding of responses to AFB1 and provide targets to modulate the mechanisms of toxicity.Both cellular responses to toxicity and AFB1 inhibition of transcription and translation will affect gene expression [102].For example, AFB1 down-regulates p53 expression in human cells [67] and in tissue from hepatocellular carcinomas in rats [219], likely due to mutations introduced by AFB1-DNA adducts.Expression of AFB1-metabolizing genes can also be affected by AFB1.Exposure in the rat liver to AFB1 can increase expression of P450 (CYP3A, CYP4F1), and rGSTM2 genes (Yb2) [220].Another study in rats identified effects on multiple P450 and GST genes, with greatest up-regulation in rGSTA5 and pi-class GST (GSTP) rGSTP1 [221].In chickens, dietary AFB1 up-regulated hepatic expression of CYP1A1 and CYP2H1, and down-regulated expression of epoxide hydrolase (EH) and GSTA, although the specific GSTA gene target was not identified [123,124].Other P450 family members were significantly down-regulated [124].Interestingly, a recent study in broilers observed the opposite changes in the liver, down-regulation of CYP1A1 and up-regulation of EH and GST [115].

Moving towards Transcriptomics
Few studies have examined gene expression changes across the entire transcriptome.Although the specific genes affected by AFB1 vary between species, exposure to the toxin has an up-regulatory effect on expression of damage responses, or when those fail, pathways of carcinogenesis.In rats, AFB1 enhanced hepatic expression of genes involved in xenobiotic detoxification, cell cycle regulation, oxidative stress, DNA damage repair, tumor development and amino acid metabolism [221].For example, E2F transcription factor 1 (E2F1) and many genes downstream of E2F1 were up-regulated by AFB1 exposure; E2F1 regulates DNA replication and apoptosis, and could contribute to carcinogenesis [221].Aflatoxicosis in pigs affects expression of many of the same pathways, with greatest effects on metabolism, cell cycle, DNA damage responses and apoptotic processes in one study [239] and on protein degradation, metabolism, apoptosis, and immune responses in another [227].Hepatocellular carcinomas from AFB1-exposed rainbow trout showed changes in cell cycle, metabolism, immune and acute phase response genes when compared to adjacent non-cancerous liver tissue [228].Two of these studies utilized high throughput RNA sequencing (RNA-seq), rather than microarrays, to characterize transcriptome changes after AFB1 exposure [221,239].

Poultry Transcriptomics and AFB1
AFB1 effects on the transcriptome have been investigated in chickens using microarray [123].Exposure to AFB1 affected hepatic expression of genes associated with fatty acid metabolism, development, detoxification, coagulation, immunity and cell proliferation [123].Among these, the greatest proportions of differentially expressed genes were involved in cell proliferation and metabolism.For example, aflatoxicosis had the greatest effect on expression of the cell signaling inhibitor Dickkopf homolog 3 (DKK3) (down-regulated) and the metabolic glycogen synthase 1 (GYS1) (up-regulated).
More recently, RNA-seq has been applied to the domestic turkey transcriptome to elucidate hepatic and splenic responses to dietary AFB1 [227,238,240].Similar to expression changes in other species, transcriptome analysis in the turkey found that more genes were up-regulated than down-regulated by AFB1 [227,238,240].In one study of the liver, more than 80 genes belonging to cancer or focal adhesion pathways were up-regulated during aflatoxicosis, whereas smaller numbers of genes involved in cell signaling, cytoskeleton, and cell cycle were also up-regulated [227].Conversely, the greatest numbers of down-regulated genes were involved in complement and coagulation.
Our investigation of AFB1-hepatotoxicity in the turkey by RNA-seq found 313 transcripts significantly affected by AFB1 exposure, with up-regulation of genes involved in apoptosis, cancer, and cell cycle regulation, and down-regulation of lipid metabolism [240].Greatest up-regulation was observed for keratin 20 (KRT20), cell-death activator CIDE-3 (CIDEC), and E3 ubiquitin-protein ligase Mdm2 (MDM2).Alpha-2-macroglobulin (A2M) was the most down-regulated gene.Exposure to AFB1 also turned on expression of HEPACAM family member 2 (HEPACAM2), a protein important in modulating cell adhesion and migration, and S-adenosylmethionine synthase isoform type-2 (MAT2A), important in methylation pathways.
Utilizing spleen samples from the same AFB1-challenge trial, immunotoxicity in domestic turkey was also examined through RNA-seq [238].Exposure to AFB1 induced significant expression changes in 391 de novo assembled transcripts; the greatest up-regulation was seen in E3 ubiquitin-protein ligase CBL-B (CBLB) and ubiquitin specific peptidase 40 (USP40).Of the significantly altered transcripts, 27.6% encoded proteins with known immune functions representing both innate and adaptive responses.Antimicrobial genes, including beta-defensin 1 (THP1) and 2 (THP2), were down-regulated, while cytotoxic and antigen presentation genes, such as granzyme A (GZMA), perforin 1 (PRF1), MHC class IA and class IIB, were up-regulated in AFB1-exposed tissue.
These studies were also designed to evaluate the ability of a Lactobacillus-based oral probiotic to reduce AFB1-effects on the liver and spleen [238,240].In the same challenge trial, domestic turkeys were exposed to probiotics alone or in combination with AFB1.Addition of probiotics during AFB1 exposure mitigated AFB1-induced expression changes in genes such as serine/arginine repetitive matrix protein 1 (SRRM1), 28S ribosomal RNA (RNA28S), and ISG12-2 protein-like (ISG12-2) (Figure 3A) [240].In the spleen, probiotics had greater ameliorating properties, significantly reducing AFB1-effects on multiple immune genes, including THP2, GZMA, and PRF1 (Figure 3B) [238].However, probiotics were unable to reverse most AFB1-induced expression changes and even had synergistic effects with AFB1.For example, combined treatment increased differential expression in apolipoprotein A-IV (APOA4) in the liver and interestingly both RNA28S and ISG12-2 in the spleen (Figure 3).Therefore, oral probiotics modulated expression in both tissues, but did not restore normal transcriptome profiles.

Chemical Detoxification
Since prevention of AFB1 contamination is often impractical, methods to chemically detoxify this mycotoxin have been intensely investigated [7].Several candidate chemicals have been examined for their ability to detoxify AFB1 in crops such as grain, rice, corn, and cottonseed.These include ammonium hydroxide [241][242][243][244][245][246], calcium hydroxide [243,247], hydrogen peroxide [243,248], sodium hydroxide [243,249], and sodium hypochlorite [243,250] all of which reduce AFB1 concentrations through hydrolysis and produce a degraded form with reduced or no toxicity.However, most of these chemicals are of themselves, hazardous and safe use is often expensive and may decrease the nutrient value of feed components.In addition, it is not possible to fully preclude low levels of AFB1 in feed production, especially in crops that are heavily contaminated, severely reducing returns for producers.
Strains LGG and LC-705 can sequester up to 80% of AFB1 introduced into growth media [272,280].AFB1 interacts with the thick peptidoglycan layer characteristic of the Gram-positive bacterial cell wall [282,285].When bound to AFB1 in vitro, LGG and a mixture of LC-705 and PJS interacted less with intestinal mucus [276].Incubation of LGG with AFB1 reduced LGG adhesion to a Caco-2 intestinal cell monolayer [284] and decreased transport of AFB1 across the monolayer [278].These in vitro models suggest the probiotic and the toxin would be excreted together in vivo and thereby decrease the effective dose of AFB1.
Reduced AFB1 toxicity has been demonstrated in both mice [270] and in rats [277] after addition of dietary LGG.In humans, probiotics enhanced excretion of AFB1 [289,290].Furthermore, addition of LGG, LC-705 or PJS ex vivo through injection of AFB1 into the lumen of the chicken duodenum significantly reduced AFB1 uptake (by 74%, 63% and 37%, respectively) [274].A mixture of LC-705 and PJS caused a 40% reduction in AFB1 absorption into chicken duodenal tissue in a repeat experiment [42].Therefore, a probiotic mixture including LGG or LC-705 could be an effective preventative for aflatoxicosis if added to poultry feeds.

Selection for Resistance
Another option to minimize the adverse effects of AFB1 is to increase the resistance of domestic poultry [7].Selection for AFB1 resistant lines of chicken [291][292][293][294][295] and quail [296][297][298][299] has been investigated; however, selection studies have not been performed in the far more sensitive domestic turkey.Improvement in AFB1 resistance is highly dependent on starting population [299] and selection is most effective during AFB1 challenge since correlations of phenotypic measures like weight gain or blood parameters are most informative during exposure [291,292,298].The requirement for concurrent aflatoxicosis makes phenotypic selection difficult to implement in a commercial setting.However, understanding the molecular mechanism of aflatoxicosis in domestic turkey and identifying the genetic differences underlying decreased sensitivity in wild turkeys could allow targeted genetic selection for resistant alleles without constant AFB1-exposure.Due to the similarity of symptoms of aflatoxicosis across species, potential genetic targets may be translatable to other poultry species.

Conclusions: Suggested Areas for Further Research
Future gene expression analyzes can provide insight into the mechanisms of aflatoxicosis and methods to reduce its effects.Our characterization of AFB1-induced changes in the turkey liver and spleen transcriptomes identified genes responding to aflatoxicosis and host responses to toxicity.Since both proliferation and apoptosis occur during aflatoxicosis, the expression of cell cycle genes in the liver needs to be quantified alongside measures of apoptotic and mitotic cells.Similarly, gene expression in immune tissues such as the spleen should be measured concurrent with lymphocyte numbers, activation or apoptotic state to better clarify gene functions.Investigation of individual cell types could also detect cell-specific gene modulation.For example, the effects of AFB1 on expression of immune genes could be measured in heterophils or T lymphocyte subsets (e.g., CD4 + verses CD8 + T cells).Gene expression in cells of the bile duct, known targets of AFB1 mutagenesis, could be used to characterize hyperplasia.Expression patterns, as determined from mRNA, do not always directly correlate with protein levels or stability; therefore, proteomics could confirm effects on cell cycle regulators or immune mediators and provide a measure for AFB1 inhibition of protein synthesis.
Analyses of systemic responses to AFB1 in other tissues, such as the bursa, thymus, kidney or small intestine are needed to fully elucidate AFB1 effects.Intestinal epithelial cells are directly exposed to AFB1 during absorption and the potential prevention of AFB1 uptake by feed additives also occurs within the small intestine.Therefore, investigation of intestinal transcriptome responses to AFB1 is a priority.
The utility of a Lactobacillus-based probiotics as preventative for aflatoxicosis requires further examination in vivo.Higher concentrations or different compositions of dietary probiotics should be examined for their ability to protect poultry from the development of hepatic lesions and in restoring gene expression profiles.Furthermore, no definitive conclusions can be made regarding the effects of probiotics without characterizing the intestinal microbiota.Shifts in bacterial population structure of the microbiome could be investigated by 16S NGS sequencing for direct comparison to the host effects shown by RNA-seq.
Comparative analysis of transcriptome responses of poultry to AFB1 could help resolve differences in their sensitivity.RNA-seq studies investigating both domestic and wild turkey using an in ovo exposure model [300] and dietary challenge of poults [301] are currently underway.Preliminary data from embryonic exposures illustrates conserved effects on cell cycle regulators and variation in metabolic and anti-oxidant enzymes [300].Genes and pathways identified in these studies will provide targets for selection efforts to improve resistance to aflatoxicosis in domestic poultry.85.Klein

Figure 1 .
Figure 1.Molecular structures of the four primary aflatoxins.

Figure 2 .
Figure 2. Metabolites and enzymes involved in aflatoxin B1 (AFB1) metabolism in the turkey liver.Arrow width shows reaction efficiency (wide > narrow), while a dashed line indicates that a reaction does not occur in all birds.Cytochrome P450 (CYP) enzymes effectively interact with AFB1.Domestic turkey glutathione S-transferase (GST) enzymes cannot conjugate exo-AFB1-8,9-epoxide (AFBO), although wild and heritage turkey GST enzymes can have activity.

Table 1 .
Comparative acute toxicity of a single oral dose of aflatoxin B1 (AFB1).

Table 2 .
Minimum AFB1 concentrations with major effects in poultry.