The Common Clinical Signs and Pathological Lesions of Mycotoxicoses in Poultry
Poultry feed production and cost are major issues faced by poultry industries in many countries of the world. Grains, such as corn, wheat, soybean, rice and their by-products used for the production of poultry feed are shared by humans and animals. Due to the competitive pricing of raw material and their availability on the market, sometimes lower quality feed ingredients are used in poultry feed production. These materials may be stored for longer periods and due to poor storage conditions, they may be contaminated with fungi, especially in humid and hotter countries, such as Indonesia. Some fungi may produce mycotoxins as secondary metabolites that further contaminate these grains used for feed ingredients.
Mycotoxicoses is a general term for toxic and/or carcinogenic diseases caused by ingestion, inhalation, or direct contact of feed contaminated with one or more mycotoxins. These diseases may vary on the severity, target sites, and mechanism of toxicity. The severity of the disease condition varies in type and dose of mycotoxins as well as the duration and frequency of contact with mycotoxin/mycotoxins. Poultry ingesting mycotoxin in high concentration may likely suffer from acute or sub-acute disease where toxic effects directly affect specific tissues or organs. However, mycotoxicoses in lower levels with continued introduction of mycotoxins may cause chronic disease, which may further worsen by secondary infections due to their immune suppressive effects (Naehrer, 2012). Specific mycotoxins affect various organs and/or tissues, such as the liver, kidneys, brain, mucous membrane of the gastrointestinal, respiratory tract, reproductive and urogenital system, and skin (Devegowda and Murthy, 2005).
Mycotoxicoses should be differentiated from mycoses which are systemic diseases caused by invasion of growing fungi into living tissues, initiating mechanical destruction.
Mycotoxins are a group of structurally diverse toxic and/or carcinogenic chemical compounds produced by certain fungi as secondary metabolites as a result of their organic processes. They are synthesized and excreted during the maximum growth of certain fungi under favorable conditions (Naehrer, 2012). Most mycotoxins are known to hazardously contaminate crops and consequently poultry feeds and poultry products, causing significant economic losses associated with their impact on poultry and human health, poultry performance, and domestic as well as international trade.
This paper presents the common clinical signs and pathological lesions caused by the most important mycotoxins in the poultry industry in Indonesia.
Mycotoxin producing fungi
The process of mycotoxin production by fungi is not well known. Higher mycotoxin contamination can be found in crops subjected to stress, such as drought, poor fertilization or excess of water. A possible explanation is that mycotoxins are produced so that fungi win a competitive advantage on other organisms (Rankin and Grau, 2002). Despite the fact that proper conditions for growth of fungi can occur at all times during crop growth, harvest, and storage, fungal species can be divided into field fungi, which infect crops as parasites, and storage fungi which grow in feedstuffs stored under suboptimal conditions. Field fungi are those, which in general require higher moisture to grow and produce mycotoxins, infecting seeds and plants in the field, such as Fusarium sp. Storage fungi are those, which require lower water activity, thus being more prominent after harvest and during storage, such as Aspergillus sp. and Penicillium sp. (Naehrer, 2012).
In general, the production of these mycotoxins is ubiquitous and more prevalent in warm and moderate climates; however, trichothecenes and zearalenone may equally be produced at lower temperatures. The optimum temperature range for mycotoxin formation should be 25°-33 °C for Aspergillus sp., 15°-30 °C for Fusarium sp., and 20°-25 °C for Penicillium sp.; relative humidity of 85%; high moisture content in grains; and optimum water activity (aw) around 0.99 (CAST, 2003, Ribeiro et al., 2006).
Grains stored under high moisture/humidity (>14%) at warm temperatures (>20 °C) or/and inadequately dried can potentially become contaminated with fungi. Grains must be kept dry, free of damage and free of insects; these conditions can result in mold “hot spots”. Initial growth of fungi in grains can form sufficient moisture from metabolism to allow for further growth and mycotoxin formation. Finished feed should not be stored for long periods of time as mycotoxin contamination can occur in these products (Richard, 2012).
Species of fungi which often infect crops/grains in the field, during storage, shipment, and food processing are Aspergillus sp., Fusarium sp., Penicillium sp., Alternaria sp., Claviceps sp. Each fungus species can produce more than one type of mycotoxin, such as Aspergillus sp. can produce aflatoxins, ochratoxin, patulin, cylopiazonic acid; Fusarium sp. can produce trichothecenes toxins (T-2 toxin, diacetoxyscirpenol, DAS, deoxynivalenol, DON), fumonisins, zearalenone, moniliformin; Penicillium sp., can produce ochratoxin, citrinin, cyclopiazonic acid, patulin (Sweeney and Dobson, 1998). Therefore, the probability of contamination of certain feed ingredients or finished feed with multi-mycotoxins is very high (Speijers and Speijers, 2004).
Characteristics of mycotoxins
Mycotoxins are chemical compounds of low molecular weight and low immunogenic capacity. They are chemically stable due to their chemical structure and low molecular weight. They resist high temperatures, remain stable during storage and during feed processing conditions (Bullerman and Bianchini, 2007). Mycotoxins carried over at low levels, such as aflatoxins, ochratoxins, T-2 toxins can be found in the liver, kidney, muscle, and eggs CAST, 2003, Volkel et al., 2011). At some extent, they may be a potential risk to consumers of food products of poultry origin (Naehrer, 2012).
Mycotoxin contamination and the severity of the problem caused by these compounds vary from year to year and also from one geographic region to the other. Many fungi contaminate the crop during the growing season and others are seed borne and grow along with the plants, whereas others infect commodities during storage. Contamination and subsequent mycotoxin production may be influenced by the environmental conditions at specific times during the crop development or storage (Sanchis, 2004). Therefore, the formation of mycotoxins in the field is difficult to control. As stated by Park and Stoloff (1989) “Mycotoxin contamination is unavoidable and unpredictable, which makes it a unique challenge to food and feed safety”. The FAO has estimated that up to 25% of the world’s food are significantly contaminated with mycotoxins (Adams and Motarjemi, 1999). However, genetic and breeding studies may eventually provide for obtaining resistant or non-susceptible varieties of certain commodities for fungal contamination and/or mycotoxin formation.
The only proven way to determine, if grains contain mycotoxins or not, is to test for them in the laboratory. However, contamination with mycotoxins can be suspected if certain signs or characteristics appear in the raw materials for feed. Such signs may be visually discolored kernels, musty odor, lighter weight than usually kernels, or with wrinkle kernels in grain (Richard, 2012). Several hundreds of mycotoxins have been reported and isolated, as many as approximately 400 varieties, with their target sites and toxicity, with varying chemical structures of each one. In the poultry industry, mycotoxins that cause the most economic impacts on poultry production are afltoxins (Afla), trichothecenes (namely T-2 toxin, T-2; deoxynivalenol, DON), ochratoxin A (OTA), fumonisins (FUM), zearalenone (ZEN), and ergot alkaloids (Ergots) (Jelinek et al., 1989, CAST, 2003, Naehrer, 2012).
Masked mycotoxins are mycotoxins that experienced changes in their chemical structures. Protein and glucosides, as an example, can be bound to mycotoxins by growing plants in the field to protect themselves from foreign compounds or by microorganisms which may change the mycotoxin structure during storage. In rare cases, some mycotoxins conjugates can be excreted directly by fungi, such as 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol by Fusarium sp. or by mammals (Berthiller et al., 2009). Alteration of mycotoxins structure my also occur during feed processing, as in the case of fumonisin reaction with reducing sugars (Lu et al., 2002).
More than 50% of the amount of free mycotoxins, especially zearalenone and deoxynivalenol is considered to exist in commodities in a masked form (Vendl et al., 2010). Unfortunately these conjugate mycotoxins cannot be detected by most routine analysis. However, during digestion the mycotoxin-ligand bond can be released and the mycotoxin act as a toxin, thus causing its hazardous effects on animals (Berthiller et al., 2003).
Interaction between multi mycotoxins
In most cases, more than a single mycotoxin may simultaneously contaminate feed ingredients/finished feed. When they are simultaneously present, their interactive effects can be classified as additive, synergistic, or antagonistic. The co-exposure of two mycotoxins led to more severe total effect than each individual toxin, even in cases categorized less than additive or antagonistic. The interaction between mycotoxins often leads to synergistic effects, when the negative effects of one mycotoxin are amplified the presence of another. In case of poultry, synergistic effects were frequently described in instances where aflatoxins were involved, with the same for ochratoxin A, T-2 toxin, and fumonisin B1. Afaltoxin B1, which is known to be a hepatotoxin and ochratoxin A, a nephrotoxin, acted synergistically when fed simultaneously to broiler chicks (Huff et al., 1988a). Synergistic effects were also seen in broilers fed aflatoxin B1 and T-2 toxin (Huff et al., 1988b), or T-2 toxin and deoxynivalenol (Rottinghaus, 1989), whereas T2 toxin and ochratoxin A caused additive effects in broilers (Wang et al., 2009).
Problems with mycotoxins
Mycotoxins producing fungi are ubiquitous in nature and under ideal conditions, often contaminate economically important crops in the fields, as well as during their harvest, storage, shipment, and processing. It is known that mycotoxins exist worldwide where their formation are no longer restricted to hot and humid climates and being widely distributed due to international trade in various agricultural raw materials (Naehrer, 2012). Since mycotoxins are invisible, odorless, and tasteless, it is difficult to prove if grains/finished feeds are contaminated with mycotoxins without testing samples in the laboratory (Richard, 2012).
Mycotoxins are most often found at low levels in raw materials/finished feed which are not detected at routine laboratory test. However, these low levels of mycotoxins may induce subclinical diseases in poultry, which indicated that there is no existing save level for mycotoxins (Borutova, 2010). Effects of low levels of mycotoxins will be more severe if they are found as multi-mycotoxins that have synergistic or additive effects. Another common problem with mycotoxins is the contamination of commodities with masked mycotoxins which cannot be detected by routine laboratory analysis (Vendl et al., 2010).
Mycotoxicoses are difficult to diagnose because of a great variation in possible symptoms and target organs as well as pathological lesions. Toxic effects of mycotoxins can occur at toxin concentration below detection limits. Their toxic effects in poultry are very diverse, varying from immune suppression to death in severe cases, depending on toxin related (type of mycotoxins, level and duration of intake), animal-related (species, sex, age, breed, general health, immune status, nutritional condition), and environmental factors (farm management, biosecurity, temperature, humidity) (Naehrer, 2012).
Mechanisms of mycotoxins toxicity
The mechanisms of mycotoxins toxicity are not fully understood due to the diversity in their chemical structures and target organs. Ochratoxin A, T-2 toxin, aflatoxin B1 may inhibit the synthesis of DNA, RNA, and protein and may damage DNA. Most mycotoxins can cause lipid peroxidation, damage of membrane structures and their functions, induces apoptosis (programmed cell death) leading to cellular necrosis (Surai, 2002, Fink-Gremmels, 2008). The mycotoxins may cause immune suppression (impairment of immune system), hepatotoxicity (damage to the liver), nephrotoxicity (damage to the kidneys), neurotoxicity (damage to the central and peripheral nervous systems), genotoxicity (damage to DNA leading to cell transformation, abnormal cellular proliferation and finally tumor formation) (Devegowda and Murthy, 2005, Tabbu, 2015).
Effects of mycotoxins in poultry
Poultry are farm animals with heterogeneous sensitivity to mycotoxins, as different species suffer from different toxic effects. Ducks, geese, and turkeys seem to be more sensitive to mycotoxicoses than chickens and quails. Young chickens are more sensitive to the effects of mycotoxins. Chickens placed in a hostile environment, such as high temperatures and humidity, poor ventilation, high density, and challenges of other diseases are more susceptible to mycotoxins (Naehrer, 2012).
The effects of mycotoxins in poultry are very complex and varies greatly according to their mechanism of toxicity and primary target organs. When mycotoxins are present simultaneously in feed, they may have synergistic or additive effects. Their effects are diverse, varying from immune suppression to death. A low level of mycotoxins in feed even below its restricted levels when exposed for long periods can impair the immune system leading to the immune suppressive conditions. Aflatoxins, ochratoxin, trichothecenes, and fumonisins are known to induce immune suppressive effects in chickens, rendering them more susceptible to diseases (Singh et al., 1990, Ghosh et al., 1991). In addition, low level of mycotoxins can have an antimicrobial effect and can cause feed passage (Devegowda and Murthy, 2005).
Mycotoxins can inhibit the absorption of vital nutrients for maintaining health condition, growth, productivity, and reproductive, include amino acids, lipid soluble vitamins (vitamin A, D, E, and K), and minerals, especially Ca, P (Devegowda and Murthy, 2005). The impact of impaired absorption of vital nutrient on the performance of poultry are poor growth/weight gain, decreased egg production, poor egg shell and interior egg quality, decreased fertility and hatchability in parent stocks, and immune suppression (CAST, 2003, Bryden, 2012, Tabbu, 2015).
Diagnosis of mycotoxicoses
Suspicion on mycotoxicoses can be made if a group of chicken in a farm which consumed the same feed/raw materials are affected, there is no signs of disease transmission between chickens, antibiotic treatment has little or even no effect on the disease, field outbreaks are seasonal and associated with specific feedstuffs, and examination of the suspected feed reveals signs of fungal activity (Richard, 2012).
Even though the effects of mycotoxins are very complex and there is a great variation in possible symptoms, target organs, and pathological lesions from one mycotoxin to the other (Naehrer, 2012), presumptive diagnosis can be based on clinical signs, pathological lesions on target organs, especially when moldy ingredients or feed are evident. Definitive diagnosis should be based on isolation, identification, and quantification of the specific mycotoxin/mycotoxins in feed ingredients or finished feed. Samples of feed and ingredients should be collected and promptly submitted for laboratory analysis. Multiple samples should be collected from different sites of mycotoxin formation zone (“hot spots”) (Whitaker et al., 2005, Krska and Schuhmacher, 2012).
Clinical signs and pathological lesions related to different mycotoxins
Clinical signs and pathological lesion related to mycotoxins are closely related to poultry species, type of mycotoxins, dose ingested, and duration of exposure. In the field, poultry, such as chickens are exposed to multi-mycotoxins and subjected to a broader variety of stressing factors. Therefore, chickens may exhibit signs and lesions of mycotoxicoses, even at apparent low level of mycotoxins present in the feed (Naehrer, 2012). Symptoms and lesions can be very general and vary greatly between mycotoxins; usually as a secondary effect of mycotoxicoses. In the field, disease process related to mycotoxicoses tend to be chronic, even though acute or subacute diseases may occurred rarely. They may have systemic or local effects, specific or often non-specific target organ.
The following section presents the common clinical signs and pathological lesions related to the most frequent mycotoxins found in poultry operations, especially chicken farms in Indonesia, include aflatoxins, trichotecenes (T-2 toxin, DON), ochratoxin, fumonisins, and zearalenone. Field trials, scientific, and laboratory research have been performed leading to different results and conclusions. Nevertheless, it must not be forgotten that effects of mycotoxins are very complex and it is possible that symptoms and pathological lesions differ to the ones presented here may also occur (Naehrer, 2012).
Aflatoxin is known to have a hepatotoxic effect in chickens (Dalvi, 1986, Espada et al., 1992) and also known to have hepatocarcinogenic effect in exposed animals (CAST, 2003).
The common clinical signs related to aflatoxicosis in chickens, include decreased feed intake, poor growth and inhomogeneous flocks (Figure 1), increased mortality, abnormal pigmentation (shank, feet), feed passage, and higher feed conversion rate (FCR). The most consistent findings in chickens suffer from aflatoxicosis was immune suppressive effects, include more susceptible to diseases, decreased responds to vaccination and antibiotic treatment, and decreased resistance to environment stress. In many cases of aflatoxicosis there was an increase in leg problems, leg weakness, reduced bone strength, short shank, and leg deformity. In some cases, there were anemia and abnormal blood clotting, increased incidence of bruising and down grading, and nervous syndrome (abnormal behavior).
The most frequent effects of aflatoxicosis in layers and parent stocks were decreased egg production, reduced egg size, poor (thin) egg shell, pale egg shell and egg yolk. In parent stocks, there was a reduction in fertility and hatchability, increased embryo mortality in the hatchery, and lower semen volume in male parent stocks.
The most common pathological lesions associated with aflatoxicosis in chickens were found in liver, lymphoid organs, and testes (male parent stocks) and the process of this disease was commonly chronic. In acute-subacute aflatoxicosis, the liver appeared enlarged, pale yellow in color, friable, and usually the gall bladder was enlarged and filled with bile. The pancreas was usually small and depigmented and there were hemorrhages on subcutaneous tissues and muscles. In chronic aflatoxicosis, the liver was small, firm, and rounded (Figure 2). Sometimes this organ was very small, rounded, and rubbery which very often complicates with ascites and hydropericardium. The other consistent lesions in aflatoxicosis were found in bursa Fabricius, thymus, and spleen which appeared smaller than normal (Figure 3); in male parent stocks, the size of testes was significantly reduced (Figure 4).
Trichothecenes mycotoxins affect actively dividing cells, such as those lining the gastrointestinal tracts, skin, lymphoid and erythroid cells. They have caustic and irritant effects on the skin and mucous membrane (Devegowda and Murthy, 2005).
Trichothecenes mycotoxins which are commonly found in the field, include T-2 toxin, diacetoxyscirpenol (DAS), and deoxynivalenol (DON). The common clinical signs related to these mycotoxins were decreased feed intake, included feed refusal or prolonged feed finish, growth depression, inhomogeneous flocks, impaired FCR, immune suppression, poor or abnormal feathering (Figure 5), dermal and oral lesions (crust on the beak, ulcers in oral cavity) (Figure 6). Additional symptoms which were commonly found were diarrhea, abnormal pigmentation, anemia, and rickets effects, including severe fragility and bending of long bones and shanks, soft and bending of beak. In layers and parent stocks, there was a sharp decrease in egg production, poor egg shell, increased number of cracked eggs, reduced egg size; cyanosis of the comb and wattle; decreased fertility and hatchability (parent stocks). Sometimes there were neural disturbances, such as abnormal wing positioning or lack of reflexes.
The common pathological lesions related to trichothecenes mycotoxicoses were crust on the beak, yellow caseous ulcers at the margin of the beak, mucous of the hard palate and the angle between mouth and tongue (Figure 7); ulcers may extend to the esophagus and larynx, gizzard erosion (GE) (Figure 8), atrophy of thymus, bursa Fabricius, and spleen. Other lesions commonly found were hemorrhages on the intestinal mucosa, pale or yellow discoloration of the bone marrow, dermal necrosis, vesicular lesions on the feet and legs, and enlargement of costo-chondral joints (related to rachitic rosary). In layers and parent stocks, there was regression of ovaries and atrophy of oviduct.
Ochratoxin type mycotoxin which is most commonly found in the field is ochratoxin A (OTA), which has a primary target organ on the kidneys as it is known to be nephrotoxic (Pfohl-Leszkowic and Manderville, 2007). Residues of OTA may be found in liver, kidneys, muscle, and eggs (CAST, 2003) and possess carcinogenic effects, which may be harmful when consumed by humans (Pfohl-Leszkowic and Manderville, 2007).
The common clinical signs related to OTA were decreased feed consumption, decreased weight gain, retarded growth (Figure 9), poor feathering, higher mortality rate, higher FCR, abnormal pigmentation (shank, feet), increased water consumption, and immune suppressive effects. In layers and parent stocks, observations were decreased egg production, egg size, and egg weight; poor egg shell; eggs with blood/meat spots in the yolk or albumin; delay in sexual maturity (Figure 10). In parent stocks, there was a decrease in hatchability and poor progeny performance.
The common pathological lesions related to OTA were seen in the kidneys, which were extremely swollen and pale in color with very distended ureters due to the accumulation of urates (Figure 11); atrophy of bursa Fabricius, thymus, and spleen; and soft or severely fragile of bones. Other common changes were found in eggs, which had watery thin albumin and blood/meat spots on the thick albumin or yolk (Figure 12).
Fumonisin which is most frequently found in the field is fumonisin B1. Poultry are less sensitive to this compound. Fumonisins disrupt sphingolipid metabolism and block the synthesis of complex sphingolipids from sphinganine (Sa) and sphingosine (So). As a consequences, Sa and So accumulate in tissues and their accumulation can be used as a biomarker to indicate fumonisin contamination (Starkl and Naehrer, 2015). Fumonisin B1 is classified as a possible human carcinogen (Group 2B) (IARC, 1993).
The common clinical signs related to fumonisins were severe reduction in feed intake, especially in layers, lowered average daily weight gain, reduced body weight, impaired FCR, increased mortality (sometimes spiking mortality), diarrhea (sometimes dark and sticky excreta were seen) (Figure 13), abnormal pigmentation, immune suppression, and rickets effects. In layers and parent stocks, there were decreases in egg production, lower egg weight, poor egg shell (more brittle), an increased percentage of eggs with small and disintegrated yolks. In parent stocks a decrease in fertility and hatchability were observed.
The common pathological lesions associated with fumonisins were enlargement of proventriculus and ventriculus (Figure 14), gizzard erosion (Figure 15), watery accumulations in the lumen of intestine, sometimes enlargement of pancreas; increased weight of liver, kidneys, and heart; atrophy of bursa Fabricius, thymus, and spleen. Sometimes there were indications of rickets effects, such as enlargement of costo-chondral joints, severe fragility or bending of long bones (Figure 16); soft and bending of beaks and claws.
Since zearalenone is known to be primarily an estrogenic mycotoxin, this toxin appears to bind to estrogenic receptors and results in hormonal changes. Chickens are highly tolerant to zearalenone; therefore high doses are required to cause reproductive disorders. In the field, zearalenone and DON were found simultaneously in feed or raw materials (Richard, 2012), and may have synergistic interaction (Naehrer, 2012).
The common clinical signs associated with zearalenon in layers were decreased egg production, reduced egg specific gravity, poor egg shell and interior egg quality. In parent stocks, reduction in egg production, enlarged abdomen due to cystic oviduct, enhanced secondary sex characteristics and lowered serum progesterone occurred. In broilers, enlargement of comb and wattles (Figure 17), prolapsus of cloaca (Figure 18) were observed.
The common pathological lesions related to zearalenon in layers and parent stocks included the prolapsus of oviduct and the enlargement of cloaca. In layer chicks, an enlargement (due to local edema) of bursa Fabricius (Figure 19), cystic on the peritoneal surface (Figure 20) or within the oviduct were identified. In male broilers, there were decreased comb and testes weights, whereas in female broilers, there were increased comb, bursa Faricius, and ovary weights, and enlargement of oviduct.
Field observations have suggested that the most common mycotoxins found in poultry operations in Indonesia, include aflatoxins, trichothecenes, ochratoxins, fumonisins and zearalenone, which all can reduce performance and increase disease incidence in chicken farm operations. They exert their effects through alteration in nutrient content, absorption, and metabolism; changes in the endocrine function; and suppression of the immune system.
Clinical signs are closely related to pathological lesions on primary target organs which subsequently altered functions of other related vital organs. There are no pathognomonic symptoms or pathological lesions related to particular mycotoxicosis due to a great variation in possible clinical signs, target organs, and pathological lesions from one mycotoxin to the other.
Clinical signs and pathological lesions on primary target organs can be used as an early warning system (EWS) for mycotoxin contamination in feed/raw materials. Final diagnosis should be based on isolation, identification, and quantification of the specific mycotoxin/mycotoxins in finished feed/ ingredients.