Trichothecenes: Diagnostic, Pathology and Influence on Production.
From 1968 with the diagnostic of the X disease in turkeys, the attention of pathologists has focused on aflatoxins. Consequently they have been intervened in this problem by increasing the resistance of seeds at certain fungal infections, through genetic advances, by improving the drying-warehousing methods and with the addition of preservatives. That has caused a decrease in the incidence of aflatoxicosis.
However since mid 80’s, the diagnostic of the presence of mycotoxins is becoming more frequent. Those mycotoxins belong to the chemical Family of Trichothecenes whose biologic origin can be found in several fungal genders, especially in fungus of the Gender Fusarium.
The presence of trichothecenes has been detected in basic cereals for human and animal alimentation, being maize and wheat those which present higher incidence.
We can assert that mycotoxins belonging to the group of trichothecenes constitute nowadays the main toxic problem of fungal origin, characterized by necrotic injuries and important losses of productivity. In the present study, we will describe its chemical structure, biologic origin, effects on animal and public health, effects on animal production, clinical and laboratorial diagnostic, prevention and therapy.
In practice, it is considered that trichothecenes constitute a group formed by around 40 fungical metabolites, biologically actives segregated by fungi of gender of Fusarium so that its pathology is known as fusariotoxicosis. The most common ones are T-2, nivalenol and deoxynivalenol.
From the academic point of view, the denomination of trichothecene corresponds to the common chemical ring.
Around this nucleus there are located the characteristic radicals of every mycotoxin which lead to a diversity of molecules called sesquiterpenes derived from trichothecenes.
To complete this vision of chemical structure, we will mention that in order to reach the chemical structure of sesquiterpenes, producing fungi have complex metabolic pathways. Some of the intermediate metabolites have toxic capacity although, in occasions, they have been described as mycotoxins when in fact are just precursors of mycotoxins.
Hesketh, A. (1990) isolated the metabolite isotrichodol in cultures of Fusarium culmorum. This metabolite contains a chemical ring which will be transformed into trichothecene ring subsequently.
Fungal origin, biosynthesis and incidence
The chemical complexity of the group of trichothecenes corresponds with the complexity of its biological origin.
As we mentioned above, trichothecenes are fungal metabolites biologically actives segregated by species of fungi of the gender Fusarium.
Abramson, D. (1993) isolated 8 species of Fusarium in grain of wheat: Fusarium acuminatum, F. avenaceum, F. crookwellense, F. culmorum, F. equiseti, F. graminearum, F. sporotrichioides and F. poae.
Among all of them, the strains of F. sporotrichioides, F. graminearumand F. poae are the main producers of trichothecenes: Toxin T-2 and HT-2, diacetoxyscirpenol, neosolaniol, nivalenol, deoxynivalenol, 15-acetyldeoxynivalenol, fusarenone.
Luo, Y. (1993) isolated 10 metabolites of Fusarium camptoceras isolated in maize: nivalenol, fusarenona, scirpenetriol, 7-deoxynivalenol, 3-acetoxyscirpenetriol, 15diacetynivalenol, acetoxyscirpenol and dihydroxydiacetoxyscirpenol.
Mirocha, C.J. (1994) isolated in wheat and barley two species of Fusarium capable of producing trichothecenes: F. culmorum and F. graminearum. The mycotoxins produced are nivalenol, deoxynivalenol and 15-acetyldeoxynivalenol.
Biosynthesis of trichothecenes requires the whole existence of genetic and environmental circumstances that can be grouped in the following groups:
1. Genetic capacity: Hohn, T.M. (1993) hast studied the complex Cos1-1- and Cos9-1 carriers of the following gens which are related to the synthesis of toxin T-2 by Fusarium sporotrichioides: Tox1-2, Tox3-1, Tox4-1 and Tox-5. The Tox5-1 gen has been identified by the same author than in Gibberella pulicaris. Proctor,R.H. (1995) has studied F.sporotrichioides and identified the genetic fraction Tri6 whose disappearance involves the incapacity to produce intermediate metabolites of the toxin T-2.
2. Composition of Substratum: The presence of carbohydrates (glucose) is necessary and its contents of leucine stimulate the production of trichothecenes.
3. Environmental conditions: Reddy,G. (1992) has studied the water activity factor (aw) concluding that production of trichothecenes is optimum when aw>0.92 and reduces considerably when aw<0.75. Production of trichothecenes takes place at temperatures within 1.5ºC-28ºC. The sudden drops of temperatures have positive influence in production of trichothecenes.
As treating a problem relatively new and such a numerous group of substances, exhaustive statistics about the presence of trichothecenes do not exist yet.
Hori, Y (1988) appoints that 10 out of 34 samples of wheat flour present contamination by deoxinivalenol (10-81ppb) in Japan.
Borrell, J. (1990) appoints the presence of T-2 toxin in 1.18% of the samples of maize analysed from 1986 to 1990 in Spain.
Smith, T.K. (1990) appoints the necessity of establishing studies to develop techniques for decontamination of contaminated cereals with trichothecenes due to its importance in Canada.
In Argentine (1994) has been appointed the problem of contamination by Fusarium, Toxin T-2 and vomitoxin in wheat destined to human and animal alimentation according the institutions AACREA, IASCAV and INTA.
Absorption of trichothecenes orally is equivalent to 1% of the ingested dose orally. The maximum concentration in tissues is after 3 hours of its ingestion being the liver, kidney and spleen its main goal organs. In liver takes place processes of metabolic hydroxylation. Most disposal is being carried out through excrement due not to have been absorbed or by bile recycling. It is considered that within 80-98% of the ingested dose is disposed after 72 hours of the ingestion. The concentration in egg is 0.17% of the ingested dose, obtaining the largest concentration in yolk.
However, apart from all this data, we need to add the particularity that ingestion in poultry farming is carried out continuously. Thus, in order to interpret toxic phenomena, we have to take into account the circle of absorption and disposal which have been created.
Effects on animal and public health
Fungi of gender Fusarium are microorganisms which cause important agricultural diseases and contaminate crops as well as premises for storaging and processing of cereals. They also can cause mycosis due to its growth in tissues or act as producer agents of toxic metabolites.
Trichothecenes are local irritating substances of the contact epithelium and producers of chromosomal alterations. From these two alterations can be produced other secondary alterations such as low appetite, vomits, blood and nervous alterations …..
It has been demonstrated its toxic synergism with other metabolites such as fusaric acid (Smith 1992), between them (Koshinsky 1992) and with other mycotoxins (Kubena 1989).
In poultry farming it has been demonstrated that DL50 of the following trichothecenes:
Roritoxin B 0.6mg/kpv
Toxin T-2 4mg/kpv
Neosolaniol 24 mg/kpv
Deoxynivalenol 140 mg/kpv
Chicken embryo are highly sensitive to the trichothecenes, even the test for searching the embryotoxicity (Chest) has been established to study the toxicity of the different trichothecenes (Rotter 1992).
Kurmanov, I.A. (1978) indicates that chicks which are fed with trichothecenes show delays on growth, bloody diarrhea and bad plumage.
Kubena, L.F. (1989) describes the appearance of mouth injuries, anemia and reduction of serum content of protein and lactate-dehydrogenise in chicks that consume toxin T-2 in feed. It is determined that exists toxic sinergism for the global administration of ocratoxin A.
Consequently of the necrosis, it is produced diarrhea which causes at the same time dehydration and death. Apart from these actions, it has been observed an increase of vitamin needs on affected birds, as well as injuries by avitaminosis B2 in chicks coming from hens affected by fusariotoxicosis.
Sholsberg,A.S. (1984) indicates that layers feeded with feed contaminated by Toxin T-2 show rejection of feed, cyanosis and decrease of number of lays.
Greenway,J.A. (1976) indicates that geese and ducks reject contaminated feed by toxin T-2 and show diarrhea. This disease has been reproduced experimentally on all avian species (Hoerr,F. 1991).
Xia, Q (1988) indicates that nivalenol, deoxynivalenol, toxin T-2, 15-acetyldeoxynivalenol induce chromosomal aberrations in cells B79 and V79 at concentrations of 1ng/ml. He found some meaningful statistic relation between the incidence of carcinoma of esophagus and stomach in human being groups, and the consume of contaminated maize with the trichothecenes indicated above.
Vidal,D.R. (1990) mentions the immunosuppressant activity of trichothecenes in human beings referencing the alimentary toxic leucopoenia (described by Mayer 1953).
Effects on animal production
In chicks, it has been described the reduction of the daily weight gain and increase of conversion rate from contents of 4ppm. Its mortality is low but the morbidity is very high, which produces an increase on the incidence of other diseases, especially coccidiosis and colibacilosis as well as vaccine failures.
In laying hens it has been described a decrease around 10-20% of the lay at concentrations of 25ppm. On the contrary, at 8ppm we can observe a reduction of feed consume, decrease of lay and alteration on quality of the egg shell.
The first information appoints the possibility that this toxicosis is constituted by the retardation on growth, reduction of consume as well as erratic behavior regarding the consumption of feed.
The necropsy of affected chicken arise irritation of gastrointestinal mucosa, spotted liver, relaxed biliar vesicle, pancreatic atrophy and visceral hemorrhages. The image of the intestinal epithelium is very similar to velvet when is going to be cut off. Frequently we find feathers inside the gizzard and we can observe that feathers of dead animals are taken them away quickly.
In serious cases, necrosis of the mouth mucosa can be observed and due to the necrotizing character, some irritating or vesicular external injuries can be found in toes and legs. Rarely, nervous alterations are produced. On the contrary, symptoms of Avitaminosis B2 in newly-born broilers from affected breeding hens can be detected.
Necropsy in affected laying hens also presents ovarian atrophy.
On the other hand, necropsy in ducks presents necrosis in esophagus, proventricle, gizzard and intestinal epithelium.
The laboratorial confirmation of the clinical diagnostic or the demonstration of mycotoxins in feed must follow several prompts: isolation of tissues and cereals of fungi producer, demonstration of the toxigenic capacity of the isolated strain, chemical detection of trichothecenes and histopathology.
Cultures for Fusarium isolation can be carried out with affected tissues (esophagus, gizzard and intestine) or with cereals in the Czapek’s medium with doxycycline. Incubation will be carried out at 25ºC during 5-7 days and the identification will be based on the macroscopic and microscopic morphology of the colonies (microculture of Kowarsky).
The medium Weet-Bix in liquid or powder is specific to demonstrate the toxigenic capacity of the isolated strains of Fusarium in cereals and feed (Wing, N. 1993).
The chemical detection of trichothecenes is carried out following chromatographic techniques of thin layer, liquids and gases. Its detection is also possible by immune chemical techniques.
Histopathologically necrosis can be observed in epithelium of proventricle, gizzard and hepatocytes. Ito, E. (1993) carried out histological studies with electronic microscopy and he concluded that the consumption of Toxin T-2 and fusarenone X cause exfoliation of the glandular epithelium of the stomach and the intestinal epithelium, bacterial growth in their surface, cellular infiltration of the sub mucosa and flaccidity of the muscular layer.
This flaccidity effect of the muscular layer was also described in aorta for the toxin T-2 and Roridin-A by Kimbrough, T.D. (1994).
The trichothecene ring is very stable and resists storages for a long time so the best prevention will consist of avoiding its synthesis. Therefore we go back to the same measures which were effective for the aflatoxins:
• Improve the genetic resistance of seeds in Fusarium
• Improve the processes of collection, drying and storage of seeds.
• Systematic analysis of fungi and mycotoxins in raw materials and final products.
In case some cases of contamination appear, we can resort to refuse or dilute the affected cereal as well as the addition of metabolizer microorganisms, the extraction of solvents or the incorporation of absorbing mycotoxins.
Swanson. S. (1988) isolated metabolizer microorganisms of the intestinal flora of rats, pigs and cows capable of metabolizing partially the trichothecenes to derived deepxy less toxic than former ones. On the contrary birds, dogs and horses are not capable of that.
Linder, W.(1992) managed to reduce the content of deoxynivalenol from 400 ppb to 30 ppb through an extraction process with water: ethanol (20:1) in grains of maize.
Borrell, J. (1990) mentioned that trichothecenes due to their reactive radicals can be fixed by hydrated silicates of aluminum-sodium-calcium except T-2 and diacetoxyscirpenol which are attackable with acids and oxidant agents.
When the diagnostic has been carried out is necessary to interrupt the supply of contaminated feed and substitute it by another one of easy digestibility based on maize, soy, fish meal and milk powder if possible. It is recommended to supply probiotic germs and a vitamin complex with high content of vitamin A and vitamin group B. There are more specific measures such as the supply of hydrated silicates of aluminum-sodium-calcium and a preservative product that avoids the proliferation of Clostridium, E.Coli and Salmonella over the necrotic epithelia.
• Trichothecenes is a group of biologically active fungal metabolites which have a chemical ring in common.
• The more significant action of trichothecenes consists of the necrosis of contact tissues although their carcinogenetic and immune suppressive actions are also recognized.
• The control of aflatoxins and the proliferation of the plant pathology by Fusarium have produced a significant increase of the pathology by trichothecenes.
• The necrotizing action of the intestinal epithelium causes the loss of the absorbing capacity of the intestine by affecting the efficiency of food, the specific absorption of Active ingredients such as vitamins and the efficacy of some medicines such as antibiotics or coccidiostatics.
• The inhibiting effect on some microorganisms of the intestinal contents affects the biosynthesis of vitamins and digestive enzymes.
• Serious economic losses produced by trichothecenes make necessary the introduction of systematic controls intended for detecting the presence of Fusarium and trichothecenes in cereals, facilities and avian feed.
• The symptomatic treatment of the mycotoxicosis by trichothecenes will be based on recovering the necrotizing mucosa based on treatments with vitamin A, restock the lost intestinal flora and provide vitamin B which absorption has been affected.
• The treatment will consist of applying mycotoxin absorbents (hydrated silicates of aluminum-sodium-calcium) and antifungal preservatives which are active against clostridia, coliforms and salmonellas.