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AFLATOXICOSIS
A. Rajan, M. Krishnan Nair and T. Gopal

Courtesy : Festschrift - Dr. S. Ramachandran

Mycotoxins are a group of relatively small molecular weight metabolites produced by fungi which are capable of growing on foods, animal feeds or raw materials used in their manufacture. Mycotoxicosis was recognized many centuries ago with the identification of ergotism, which was popularly known as St. Anthony’s fire. The discovery of aflatoxicosis as a distinct disease syndrome in the 1960’s and the subsequent implication of aflatoxins as potential carcinogens in the human food chain resulted in a great concern to scientists and others interested in the production, manufacture and handling of food and food products and to livestock and poultry producers. The severe economic loss caused as a result of aflatoxicosis in the livestock sector has been a real nightmare for the farmers. The range of food and feedstuffs affected by atoxic fungi and the number of microbial species has been extensively documented in recent years.

Among the many mycotoxins, (1,2) the natural occurrence of 12 mycotoxins other than aflatoxin are known to affect animal and human health. Although they have been implicated in human illness, more information is available on their role in animal disease. The losses remain undetected. The economic loss due to damaged feed commodities because of the mycotoxic contamination and loss of production in livestock due to chronic mycotoxicosis remains unknown for want of proper mycotoxin surveillance. In fact, the problems draw attention, when the export market is reduced or lost forever.

In 1960, severe mortality of turkey poults and ducklings occurred in certain farms in Great Britain and because of its unknown aetiology, the disease was designated as “Turkey disease”. An in-depth investigation was undertaken and it was identified that the cause of death was due to feeding of imported groundnut meal from Brazil, which was found contaminated with aflatoxins (3). Subsequently extensive investigations were made on the problem (4). The toxigenic fungal strain was identified as Apergillus flavus Link ex Fries and the toxin produced by it named as aflatoxin. 

Studies on aflatoxicosis were initiated in India by Nair and his associates at the Madras Veterinary College (5). The naturally occurring aflatoxicosis in cattle and in poultry was reported in Andhra Pradesh and Karnataka. Recognizing its importance in the livestock and poultry production, Indian Council of Agricultural Research sponsored an All India Co-ordinated Research Project to carry out in-depth investigation on various aspects of mycotoxicosis in several university research institutions in the country. The Centre of Excellence in Pathology, College of Veterinary Sciences, Mannuthy, Kerala as the central co-ordinating institute and in institutions at Hisar, Ludhiana, Andhra Pradesh and Tamil Nadu as subsidiary centres. Presently the work is going on in various states and national institutions of the country.

Nature and sources of aflatoxins 
Aflatoxins are a closely related group of polysubstituted bifurano-coumarins which are secondary metabolites mainly produced by certain strains of Aspergillus sp., particularly A. parasiticus and A. flavus. The aflatoxin producing fungi are ubiquitous in nature and grow on most of the feed commodities when optimum conditions of temperature and humidity prevail in the environment. A temperature of 33-35°C and relative humidity of over 12% are ideal for the growth and toxin production. Because of the climate, poor post-harvest technology and storage conditions the problem is more serious in tropical developing countries. High levels of aflatoxin contamination occur due to fungal contamination of a variety of grains, nuts and nut products, cotton seed and a host of others which are extensively used as livestock feed. In fact there is no feed that is free from aflatoxin. Reddy et al. (6) observed that 59% of chilli samples were found contaminated with aflatoxins. Apart, from the natural occurrence of the aflatoxins in grains and other feedstuff, their metabolite residues present in milk, and other body tissues of animals pose potential health hazards for humans. 

The isolation and characterization of the four closely related toxins, aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) were reported by Hartley et al. (7). Their chemical structure was defined by Buche et al. (8). Subsequently as many as 12 fractions have been identified. The most important and toxic fraction of aflatoxin is B1. The susceptibility to aflatoxin varies in different species as shown below. 

Acute toxicity of aflatoxin B1 Level of aflatoxin B1 resulting in liver lesions

Species  mg/kg bd wt  Ducklings 0.3 - 0.5  Rabbit  0.3 - 0.5  Pig 0.5  Dog 0.62  Sheep 1 Monkey 1-12  Chicken 6-16  Rat (male) 7  (Source: TDRI, London, Mycotoxin Manual)

Species  rbp of B1 in diet Duckling 30 Turkey Poult 300 Chicken 500 Beef cattle 700 Pig 800 Sheep 1000 Monkey 2000 Dairy cattle 2300 Mouse 4500 No effect (Source: TDRI, London)

Besides this, other factors like the age, breed, sex, duration of exposure, dose, presence of other mycotoxins, nutrition, stress and other chemical exposures also influence the toxicity (9).

These toxins may be acutely toxic when given in large doses, chronically toxic in sub-acute doses and low-levels result in carcinogenic response. In general, young animals of any animal species are more susceptible than old animals. Among the avian species ducklings are the most susceptible followed by turkeys, chicken and quails in that order. Among mammals, pigs are the most susceptible followed by rabbits, cattle, sheep, goats and monkeys. The presence of other mycotoxins in the same feed will have a synergistic toxic effect.

Besides this, the exotic breeds and their crosses introduced for intensive livestock and poultry development in our country were found to be more susceptible to mycotoxicosis than the indigenous population.

Aflatoxicosis in livestock and poultry
Aflatoxicosis in ducks: The ducklings are the most sensitive species to aflatoxin. In fact, because of this, day-old ducklings were identified as the species for the biological assay of aflatoxin. The LD 50 is as low as 0.3 mg/kg body wt. Asplin and Carnagahan (10) reported that first signs of aflatoxicosis were inappetence and poor growth and at the onset of mortality two weeks after the commencement of the feeding, ataxia, ophisthotonus, shivering and convulsions are seen. In acute cases subcutaneous and muscular haemorrhages are characteristic, the liver is enlarged, pale yellow with focal areas of haemorrhage and in chronic cases it is firm and shrunken. Multiple greyish white nodules in the liver are common in birds surviving eight weeks. Ascites, hydropericardium and emaciation characterized by gelatinisation of fat are seen in chronic cases. High doses kill day-old ducklings and low doses over a period of time lead to development of hepatic tumours.
Aflatoxicosis in cattle: Mycotoxicosis was suspected as the aetiology by Loosemore and Harding (11) when large number of calves fed ration containing Brazilian groundnut meal died and subsequently the feed was found to contain aflatoxin. Loosemore et al. (12) reported aflatoxicosis in dairy cows which were fed cotton seed cake contaminated with aflatoxin. Subsequently several reports have appeared from different parts of the world describing aflatoxicosis in cattle both in experimental and field situations. 

The majority of field cases were non-febrile, with progressive loss of appetite and reduced milk yield, and death among the young ones. The disease precipitated under stress of transportation, vaccination, change of feed and management or adverse climatic situations. Anorexia syndrome in cattle in an epidemic form has been reported. Investigations revealed that the disease occurred consequent to groundnut cake ingestion. PM changes included gelatinization of fat subcutaneous haemorrhage, hepatomegaly, shrunken livers and ascites with icterus. The liver showed progressive hepatic necrosis with fibrosis and biliary epithelial hyperplasia. Veno-occlusive changes were noticed rarely (13).

Aflatoxicosis in pigs: Loosemore and Harding (11) first described a disease syndrome in pigs, which was demonstrated to be caused by feeding peanut meal contaminated with aflatoxin. Based on the clinico-pathological changes seen in aflatoxicosis and the similarity of these lesions to those seen in moldy corn poisoning in pigs reported earlier it was concluded that moldy corn poisoning was in fact aflatoxicosis. Since then, many reports have appeared describing the natural outbreaks and experimental reproduction of aflatoxicosis in pigs. In India an outbreak of aflatoxicosis was recorded in pigs, when large scale mortalities attributable to aflatoxin toxicity were encountered in Kerala, in 1965 (14). The affected pigs showed varying degree of hepatopathy, atrophic rhinitis and sinus tumors. The ration contained 30% groundnut cake. Nair et al. (15) studied the pathological effect of low doses of aflatoxin (0.1 and 0.2 mg/kg) for 3 months. Toxin fed animals showed decreased growth rate, feed intake, icterus, ascites, hydro-pericardium and hepatic necrosis with fibrosis, biliary hyperplasia, oedema of gall bladder, haemorrhage and lymphoid depletion in the lymph nodes along with dissociation and degeneration of Hassall’s corpuscles in thymus. Aflatoxin treated group picked up many infections due to immunosuppression. 

Chronic aflatoxicosis of clinical / subclinical form has been reported. Clinical manifestation of chronic aflatoxicosis is characterized by rough hairy coat, growth retardation, varying degree of icterus, ophisthotonus and epistaxis. PM lesions included cirrhosis with nodular hyperplasia. Pigs on longer duration of feeding with aflatoxin containing feed revealed hepatocellular carcinomas.

Aflatoxicosis in goats: Detailed studies on the pathological features of aflatoxicosis were carried out by Anilkumar and Rajan (16). Anorexia, icterus, emaciation and diarrhoea were the common symptoms. Abortion was also reported in goats due to consumption of aflatoxin-contaminated feed (17). They described incidence of pneumonia as a result of immunosuppression. Maryamma et al. (17) reported abortion and neonatal deaths in goats on ingestion of aflatoxin contaminated groundnut cake. Enlarged friable liver, haemorrhage and icterus were the common lesions. In chronic cases gelatinisation of the fat and gastro-enteritis were seen.

Aflatoxicosis in chicken: Blount (3) reported many outbreaks of aflatoxicosis in chicken. Later aflatoxicosis in chicken was described by Hamilton (18) and Paul Gupta et al. (19). Subsequently many reports have appeared. It is one of the important problems, which hampers poultry production. Embryo mortality, teratogenic defects, reduced feed intake, growth retardation and decreased eggs production by aflatoxin, cause serious economic loss to poultry industry. However, it should be remembered that there is a lot of variation in the susceptibility of different breeds of chicken to aflatoxin. 

Acute aflatoxicosis causes haemorrhages in various organs and the sub-cutaneous tissue resulting in death. At autopsy the liver is found enlarged, pale yellow and fragile with occasional ascites. In more chronic cases the liver is shrunken and the kidney is enlarged and pale. While 0.5 ppm of AFB1 caused reduced feed consumption, 0.6 ppm caused reduced resistance to infections, 1.55 ppm lead to loss of weight and 0.75 to 2.00 ppm caused decreased egg production.

The disease assumes a serious threat to poultry industry in that the sub-clinical doses of aflatoxin content in the feed obscure the characteristic clinical signs of toxicity, but may show a progressive weight-loss among broilers, deranged fat metabolism characterized by hydropericardium, ascites, increased susceptibility to diseases and vaccination failures. Earlier, when dietary protein for poultry consisted of groundnut cake as the primary source of protein, the mortality among young stock within two weeks used to be quite high, but in the layers the effects were always characterized by reduced weight gain, smaller size and reduced number of eggs. Subsequently, soya bean as a source of protein substitute has considerably reduced the incidence. The presence of aflatoxin even below the permissible limits combined with other interacting infectious / noninfectious causes leading to production losses need thorough understanding of the interplay of these factors.

Biochemical effects of aflatoxin
De Recondo et al. (20) demonstrated that aflatoxin acts directly on the DNA molecule and inhibits its ability to act as a primer for DNA synthesis. A single dose (3 mg/kg) of the toxin caused marked reduction in the mitosis and the DNA synthesis. Aflatoxin was shown to inhibit RNA polymerase activity and prevented DNA dependent RNA synthesis. The inhibition of RNA synthesis leads to inhibition of protein synthesis.

Extensive studies have been carried out on the metabolism of aflatoxin. The toxin has to be bioactivated before it manifests its biological effects. Epoxidation of AFB1 by liver microsomal enzymes is an important step and this epoxide is carcinogenic. The type of metabolic product varies with the species. In lactating cows 90% of oral dose resulted in excretion of toxin as M1 and M2 within 48h, and also as the major residue in milk and urine. Routine pasteurization temperatures do not destroy the toxin.

At the nuclear level the induction of nuclear segregation is followed by the depression of nucleic acid synthesis. Such morphological and biochemical alterations are caused by the binding of active AFB1 to protein components. It has been reported that AFB1 forms covalently linked adducts with protein in vivo and in vitro possibly via oxidative phosphorylation to the reactive form. AFB1 is bio-transformed into the reactive epoxide by the microsomal and nuclear cytochrome p-450 system. AFB1 is a potent carcinogen and this prior activation is essential for the modification of gene expression. Aflatoxin in general is primarily a hepatotoxin. It is also an immunosuppressant, teratogen and a carcinogen. It is one of the most potent naturally occurring carcinogens.

Lipid peroxidation is one of the main manifestations of oxidative damage by AFB1 and has been found to play a significant role in the toxicity and carcinogenesis (21).

Histopathological changes
Since the aflatoxins are primary hepatotoxins, the lesions are significant in the liver in all the species. In acute cases, massive necrosis and focal or diffuse haemorrhage are seen. Hepatosis characterized by extensive fatty change is a feature. Hepatocytomegaly and prominent biliary hyperplasia are generally seen. The biliary hyperplasia may be very extensive as in the case of ducks. The bile ductule formation and fibrosis occurs either periportally, centrally or uniformly throughout the lobules depending on the degree of involvement in cattle. Centrilobular degeneration and necrosis and diffuse fibrosis disrupting the lobular architecture are features in sub-acute and chronic cases. Obliterating endophlebitis of the centrilobular and hepatic veins have been reported in cattle. Cirrhosis may later lead on to hepatomas and hepatocellular carcinomas.

In the kidney, focal non-suppurative nephrosis, chronic pyelitis and varying degree of degeneration of the tubular epithelial cells are seen. High degree of sperm abnormalities have been reported in buffaloes and catarrhal enteritis and gastritis may also be seen (22,23,24). Detailed histopathogical features in pigs have been reported (15,25,26) 

In the ducks, diffuse degenerative changes of hepatocytes, rapid and extensive proliferation of the bile duct epithelium leading to the formation of cords of deeply staining basophilic tubules are very characteristic features and this was recommended by Carnaghan (27) as a marker in the bioassay of aflatoxin. Survival over three weeks showed small islands of hepatic tissue surrounded by dense masses of bile duct and mild fibrosis. The changes seen in the chicken were similar to those seen in the ducklings with less bile duct hyperplasia. Jayakumar et al. (28) described the detailed histopathology and changes in the reproductive organs in aflatoxicosis. Salem et al. (29) observed that there was decrease in the ejaculate volume, sperm concentration, total sperm output and sperm mortality index in rabbits dosed with AFB1.

Ultrastructural changes in aflatoxicosis
In general, the ultrastructural alterations showed a wide spectrum of retrograde changes to carcinogenic manifestation. The changes described are based on hepatic cell alterations. The ultrastructural changes within the hepatocytes are clearly dependent on the location of the cells within the liver lobules. Periportal hepatocytes are very rapidly damaged while centrilobular parenchymal cells which are located at the most peripheral part of the hepatic microcirculation are affected the least. There is disorganization of RER with dilatation, cisternal irregularity and loss of ribosomes attached to the membrane surface. Later there is fragmentation of RER. Glycogen particles are also seen decreased. There is varying amount of proliferation of smooth endoplasmic reticulum depending on the dose of AFB1.

Progressive degenerative changes in centrioles were occasionally encountered. Focal membrane degeneration and rupture of the hepatocytes were often observed. In some of the cells there is a paucity of specialized cytoplasmic organelles and a decrease in the electron density of the cytoplasm giving the cells a pale appearance. The nucleus show changes from focal condensation of chromatin to partial lysis.

Degenerative changes of ER seem to suggest that the membranes per se are the major targets of AFB1 injury. It is clear that disorganization of RER is consistent with the reported decrease in protein synthesis in AFB1 administration.

During carcinogenic transformation the hepatocytes assume a productive appearance. Ultrastructurally the ER-hyperplastic hepatocytes would represent the first adaptation of AFB1 metabolism. These cells are characterized by increased amount of RER and SER. The nucleolus, nucleoplasm, Golgi apparatus, mitochondria, microbodies and cell membranes are apparently normal. Another type of productive hepatocyte is equivalent to the transitional basophilic cells of the megalocytes of light microscopy. The ultrastructural features of basophilic hepatocytes of light microscopy are prominent nucleoli, very prominent RER, presence of intracisternae free ribosomes, increased SER, conspicuous Golgi complex and normal mitochondria, microbodies and cell membranes. These cells display the ultramicroscopic features associated with high rates of ribosomal and protein synthesis and indicate that there is an increased functional demand when the cell passes through the G1 phase of the cell cycle, synthesize DNA and divide.

Some of the above hepatocytes may show degenerative changes. Hepatocytes with certain signs of dedifferentiation, as recognized by loss of organized endoplasmic reticulum, glycogen, Golgi apparatus and surface specialization are also seen. They are characterized by progressive lack of RER agglomerates, concentration of toxic substances in paranuclear location as indicated by complete dissolution of cytoplasmic-material around the nucleus, which frequently becomes similar to that observed in mitotic prophase. These indicate that degeneration occurs after completion of DNA synthesis. The S/G2 degenerating hepatocytes show an inverted nucleocytoplasmic gradient of nucleophilic centres (decrease of RNA in the cytoplasm, increase of DNA in the nucleus).

Immunosuppressive effects of aflatoxin
Immunosuppression is an important effect of aflatoxin. Aflatoxin causes impaired immunogenesis in livestock and leads to various disease outbreaks, some times even after vaccination. The adverse biological effects of aflatoxin on the immune system was brought to light within the first decade of the discovery of aflatoxin in the 1960s (30). Gallikev et al. (31) demonstrated that antibody formation against typhoid was affected. Thaxton et al. (32) observed depression of agglutinin titre to sheep erythrocytes in chicks dosed with aflatoxin. Complement activity was found to be decreased in chicken, pigs and cattle (33). Aflatoxin affects CMI also. Macrophages were found to be less phagocytic in aflatoxicosis (34,35). There was also reduction in the graft-versus-host response in chicken given aflatoxin (36). Generally, aflatoxin causes suppression of humoral and CMI response, inhibits phagocytosis by macrophages and induces thymic aplasia (37). It affects more severely the CMI and the complement system. Embryonic exposure to aflatoxin can occur and this can affect the immune system of neonatal animals. Experimental data indicated that aflatoxin causes defective protein synthesis, atrophy of the bursa of Fabricius, lymphocytopaenia and depression of the complements (38,39). Reduced antibody production to Newcastle disease virus was observed in chicken when the feed contained AFB1 even at the level of 0.2 ppm. Significant reduction in the humoral immunity was reported in pigs, goats and cattle. Aflatoxin was shown to have suppressive effect on the human NK cell activity (40). The aflatoxin induced immunosuppression leads to outbreaks of diseases in spontaneous and experimental situations in goats, ducks and chicken.

Carcinogenic effect of aflatoxin
Rajan (41) reviewed the carcinogenic effect of aflatoxin. Aflatoxin B1 is the most potent active hepatocarcinogen. The hepatocarcinogenic effect of aflatoxin has been demonstrated in various species like the trout, ferret, rat, pig, sheep, duck and monkey. Besides the liver, AFB1 has also been demonstrated to cause cancer of the stomach, kidney, intestine, bone and skin. The first report of the carcinogenic effect of aflatoxin contaminated peanut meal was that of Lancaster et al. (42).

Experimental studies indicated that carcinogenesis depended on dose level and duration. When the diet of rats contained 1 ppm, the males developed carcinomas in 35 weeks while in the females it developed only after 64 weeks. The earliest histologic lesion was a well-differentiated microscopic focus of hypochromatic, hyperplastic parenchymal cells with basophilic cytoplasm and later foci of large eosinophilic cells. The former foci developed into poorly differentiated carcinoma and the latter into well-differentiated carcinoma (43).
Japanese researchers recorded 148 hepatomas out of 1113 ducks examined, which had been fed aflatoxin contaminated groundnut meal. Subsequently spontaneous hepatomas induced by aflatoxin were reported by many workers. Hepatic tumors were produced in ducks by feeding aflatoxin contaminated groundnut meal (44). Rajan et al. (45) recorded 40.5% incidence of hepatosis and 4.83% of hepatic tumors out of 1034 ducks examined during the period 1986-89 at the KAU duck farm. Subsequent extensive studies conducted at the Department of Pathology of Kerala Veterinary College showed a high incidence of hepatic tumors in ducks associated with ingestion of aflatoxin contaminated feed. The high sensitivity of Pekin ducks to the toxic effects of AFB1 was not paralleled by the carcinogenic effect. The higher level of AFB1-DNA adduct formation in the duck correlated with acute toxic effect but not with carcinogenic effect. Therefore, caution should be exercised in relating DNA binding and carcinogenic potential. The induction of transforming mutagenic lesion occurs at a lower frequency. The carcinogenicity of aflatoxin results from its conversion to a more reactive intermediate. The ingested aflatoxin is metabolized primarily by the microsomal mixed function oxidase system of the liver. Hydroxylated intermediates aflatoxin Q-1, M1, B2a that are water soluble are eliminated through urine or bile and certain AFB1 intermediates are also formed such as 8,9 epoxide that react with DNA, RNA and protein and form adducts. The major adduct formed both in-vivo and in-vitro in this reaction is 8,9 dihydro-8-N7 guanyl 9 hydroxy AFB1. These adducts caused mutagenecity and carcinogenecity. It should be emphasized that man cannot be expected to have an immunity to the fungal hepatotoxins since the closely related nonhuman primates have been shown to develop hepatic tumours on administration of AFB1. The International Agency for Research in Cancer (IARC) had made several reports on the carcinogenicity of aflatoxin. In 1987, IARC concluded that there was significant evidence that aflatoxin is a probable human carcinogen. The human population is exposed to the risk of aflatoxin carcinogenesis mostly by ingestion of aflatoxin contaminated food items including milk. Chronic hepatitis B virus infection and exposure to aflatoxins are considered as high risk factors.

Chauhan et al. (46) recorded hepatic tumours in chronic aflatoxicosis. Rajan (47) recorded hepatic tumours in experimental pigs after feeding aflatoxin daily at the dose rate of 25 mg/kg body weight for one year.
Rajan (41) made extensive studies on the association of aflatoxin and ethmoid carcinoma in Kerala and indicated the role of aflatoxin in the causation of ethmoid carcinoma in cattle and pigs. The bovine olfactory mucosa has a high AFB1 bio activating capacity, which can be related to the potent DNA damaging and mutagenic effects (48). These results supported the assumption that AFB1 plays a role in the aetiology of ethmoid carcinoma in cattle (49).

Detoxification of aflatoxins
Mycotoxin contaminated feeds and feed ingredients in which the toxin remains for longer period are difficult to get rid off. Their impact could be reduced by proper post harvest management of crops and storage technology to prevent fungal contamination and production of mycotoxins. Fumigation and improvement in storage practices would suppress growth and activity of fungi. Buffered organic acids and gentian violet added to feed were found useful in this regard, but they did not destroy any mycotoxin that had already been formed. 

Exposure to sun, U.V. rays and moist heat has been tried for detoxification at high cost. Chemical detoxification such as use of hydrogen peroxide (50), calcium hydroxide, sodium hydroxide and ammonification have been tried. Ammonia treatment was found to be the most effective and practical method for use in large scale feed processing plants (51) with 95% of successful detoxification.

In recent years, the mycotoxin binding capacity of specific clays like zeolites, activated charcoal and other clays such as bentonite manoterenorillonine, sepilone and kaolin are being used for detoxification. The use of hydrated sodium-calcium-alumino silicate (HSCAS) crystals, was found to be effective both in vitro and in vivo (52). Aflatoxin binds HSCAS by chemisorption in vitro and relatively stable over a wide range of pH and temperatures. However, use of large quantities at 1 to 2% and possible binding of vitamins, minerals and amino acids were major drawbacks.

A new class of natural mycotoxin binder is emerging. Addition of yeast culture Saccharomyces cerviciae to the feed protected poultry from deleterious effect of mycotoxin. The protective component is identified as esterified glucomannon (EGM) (53). Modification of EGM enhanced the mycotoxin binding capacity and strongly binds mycotoxins that are common in livestock, poultry and other animal feeds.

Advances in the development of newer antibiotics and other antimicrobial agents, and immunologicals have brought many infectious diseases under control. However, the deliterious effects of mycotoxin will continue to affect animal and human health. The mycotoxin story, particularly that of aflatoxins will still remain incomplete and needs further critical study to eliminate the wide spread problem in the developing areas of the world.


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Authors Corresponding address: 

Dr. A. Rajan
Former Dean, College of Veterinary and Animal Sciences, Mannuthy, Trissur - 680 651, India

Dr. M. Krishnan Nair
Former Director of Veterinary Education and Research, College of Veterinary and Animal Sciences, 

Mannuthy - 680651, Trissur, India

Dr. T. Gopal
Former Director, Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore - 560 024, India

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).