PROGRESS IN THE CHICK EDEMA PROBLEM

By Dr. Leo Friedman, Food and Drug Administration

It has been almost four years since I first became aware of the problem that we know today as "chick edema disease." Because of our activity on this problem, my colleagues and I have had the opportunity to become acquainted with many scientific groups and individuals working in the same area with whom we have enjoyed a fruitful cooperation and pleasurable association. We hope that they feel as kindly toward us as we do to them, but sometimes, I known, they wish as we do that they had never heard of chick edema disease. This problem has been most difficult and progress frustratingly slow. Despite the meager amount of new information that can be added at this time, especially since the most recent advances were reported by Dr. Artman recently, it is nevertheless worthwhile to review the several aspects of this problem and see its present status in full perspective.

As you recall, during 1957 an epidemic disease caused millions of dollars in losses among broiler flocks throughout a large part of the U.S. After elimination in succession of all other possibilities, attention was focused on the fat ingredient of the feed as the etiologic agent. A series of reports in 1958 from several laboratories described the manifestations of the disease and definitely implicated a toxic fat or a toxic substance in fat as the cause. The characteristic symptoms were droopiness, ruffled feathers, labored breathing and high morbidity and mortality. Autopsy findings revealed hydropericardium, abdominal ascites (water belly), subcutaneous edema, swollen liver, swollen and pale kidneys, etc. In laying hens the toxic fat caused a rapid drop in egg production. Pullets receiving toxic fat during the full growing period did not come into production, and mortality was very high. Hydropericardium, the most common lesion found in young birds, was not found in birds of laying age.

SYMPTOMS DIFFERENT

These differences in susceptibility and symptoms in different age groups of the same species should be noted. The feeding of toxic fat to other species has not produced such striking results as with young chicks, with the exception possibly of monkeys. However, every species that has been tested has shown evidence of deleterious effects. Very little work has been done with rats. Our very limited experience indicates that they are much more resistant than chicks in short-term feedings, but that when fed in sufficient dosage, extracts of the toxic fat produce definite deleterious effects as shown by growth depression, enlarged and fatty livers, and marked involution of the thymus.

I recall few reports of the effects of toxic fat on swine, but again, in our own limited experience we have seen depressed growth . . . and have demonstrated the presence of toxic factors in the meat of hogs that had been fed toxic fat.

I am indebted to Dr. Wilcke of Ralston Purina for reports of studies on guinea pigs and dogs. Guinea pigs fed 22% toxic fat stopped growing after six weeks, and death losses occurred after eight weeks. At a level of 42% toxic fat weight losses occurred after three weeks and deaths after four weeks. Control groups receiving non-toxic fats did not show weight loss or deaths. The only observed pathology at the conclusion of the experiment was congestion of the lungs and mottled livers.

In experiments with three different breeds of dogs, using Purina Dog Chow in which 10% of toxic fat was substituted for the usual normal fat, there was poor reproduction and lactation performance. The females on the toxic fat ration whelped pups that were dead or weak, and, furthermore, the mothers seemed to have an insufficient milk supply. When the pups were removed before weaning and fed a normal ration the increase in growth was immediate and dramatic. Also, the females on the toxic fat ration tended to lose hair on their backs and shoulders. With the ration containing toxic fat, post-weaning growth tests (6-18 weeks) with five litters of pups demonstrated inferior growth performance using either weight gain or increases in body length as the criterion.

WORK WITH OTHER SPECIES

In these experiments with other species, fat that had first been proved to be toxic to chicks had been used. In the case of monkeys, a sample of triolein

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that had produced irreversible toxic symptoms for no apparent reason later was proved toxic to chicks and was the source from which we isolated a highly purified crystalline "chick edema" factor. At the present time, then, "toxic fat" that produces "chick edema disease" has been demonstrated also to produce deleterious effects in rats, guinea pigs, swine, dogs and monkeys. It should be emphasized that the toxic fat undoubtedly contains many other substances that may have effects in these other species. Definitive information as to the effect of "chick edema factor" (CEF) in other species must await tests with purified CEF. Triolein-fed monkeys probably received the "purest" source of CEF. However, purified CEF should be administered to monkeys to verify the implication that the severe toxic symptoms observed with triolein were due to CEF.

Relatively little has been done to throw light on the mechanism of the toxic effect. Flick and Gallo in our laboratories have reported that in young chicks showing symptoms of the disease, the intra-cellular water was not changed. Neither were the total blood and plasma volumes altered so that the observed edema was primarily interstitial. Hemoglobin and hematocrit levels were low and blood glucose levels were decreased in advanced stages of the disease. Plasma sodium, potassium and chloride were not affected. In the liver, neutral fat was decreased and phospholipid increased. Preliminary experiments by Flick give some indication of increased membrane permeability, but these must be repeated under much more rigorous conditions.

DISSERTATION ABSTRACT

A most interesting study in chicks has been reported by J. R. Allen, Jr. Part of the results were presented at the federation meetings in March, 1961, and the complete study is available as the dissertation of J. R. Allen, Jr., University of Wisconsin, 1961. I will quote portions from the dissertation abstract [Dissertation Abstracts 22. [2], 545 (Aug., 1961)]:

"Experiments conducted to determine the effects of toxic fat' on mice, pigeons and turkeys demonstrated a reduction in growth without hydropericardium or ascites."

In chicks, "Microscopic examination of the tissues of the test animals revealed lymphocytic foci in the epicardium and myocardium. Edematous fluid separated the myocardial fibers. Edema of the lungs was a frequent observation in the experimental birds.

"... Blood pressure indicated the test birds had an elevated average mean pressure in the right ventricle of 6 cm. water and 2 cm. water in the vena cava. Electron micrographs of the myocardium revealed shrunken, vacuolated mitochondria in the test animals.

""Toxic fat' produces a reduction in growth rate of experimental animals. This reduction depends on the age of the animal and the level of 'toxic fat' added to the diet. Hydropericardium and ascites are a frequent lesion in the animals receiving from 1.0 to 5.0% toxic fat. When this level was reduced to 0.25% in the diet, reduced testicular development was a more sensitive criterion than hydropericardium, ascites or weight gain for evaluating chronic toxicity.

"The mechanism by which 'toxic fat' induces hydropericardium and ascites appears to be associated with degeneration and edema of the myocardial fibers. These data would tend to eliminate the kidneys, liver and endocrines as the primary cause of edema. The early development of hydropericardium, increased venous pressure, enlarged hearts, mitochondrial changes in the myocardium and generalized edema suggest that the myocardium may be directly inhibited; however, altered capillary permeability has not been excluded. It is believed that cardiac decompensation and increased capillary permeability act together in producing the excessive extravascular fluid collection and the demise of the animal."

The wide range of susceptibility within and among species and the variety of toxic effects that have already been noted would make it appear logical that some primary unit of structure and function such as the mitochondrion, may be the target of the toxic factor and that the observed differences may be explained by factors such as absorption, specific binding, transport, detoxication, etc., that determine the local concentration of any substance in a specific site.

Another possible mechanism that had suggested itself quite early is that the toxic factor interferes with the normal regulation of electrolyte and water balance. Selye and Stone, back in 1943, described the production of edema symptoms in chicks by certain steroids and the accentuation of these symptoms by increasing the salt intake of the chicks. Alexander has shown that it is possible to produce hydropericardium in chicks by increasing the NaCl in the ration and to prevent its occurrence even with CEF by eliminating NaCl from the diet. Work in our own and other laboratories has illustrated beautifully the interaction of nutritional factors on the susceptibility of the chicks to a toxic agent. For example, the present AOAC bioassay diet for "chick edema factor" is probably four times more sensitive than the assay diet we used originally, although both have approximately the same NaCl content.

A point to remember is that hydropericardium as a symptom of toxicity in chicks is not new. In addition to NaCl and certain steroid hormones, chapter 40 in Biester and Schwarte on Poisons and Toxins indicates that:

(1) Zinc phosphide, used as a rodenticide produces "various degrees of congestion with the accumulation of some serous fluid in the pericardial sac as well as in the abdominal cavity in some cases."

(2) Alpha naphthyl thiourea, the rodenticide ANTU, shows in poisoned chicks evidence of lung edema and excessive quantity of fluid in the pericardial

sac.

(3) Sodium monofluoroacetate, compound 1080, another very effective rodenticide, produces in chicks distention of the pericardial sac with clear strawcolored fluid, in addition to other marked pathological changes on the heart and lungs.

(4) Chlordane. "The primary lesions found in all fatal cases were in the heart. Excessive quantities of fluid were found in the pericardial sacs. ...” The weed "corn cockle" and several species of Crotalaria produce seeds which are toxic, and in chicks the toxic symptoms include hydropericardium.

However, in each case other characteristic pathology is usually present, and in no case is the purified toxic principle of the same high order of activity as the toxic substances that have been isolated from toxic fats.

It is well known now that chicks can efficiently utilize large amounts of fat in properly balanced rations. The use of fat as a standard ingredient of poultry feeds grew as the price of fat calories dropped and became competitive with calories derived from corn. The chick edema disease epidemic of 1957 was a totally unexpected consequence of this growing practice. Many of you are familiar with the story of how the toxicity was associated with a fatty byproduct of stearic and oleic acid manufacture that had been blended with feed grade fats.

Very large quantities of fatty acids are used industrially in the manufacture of lubricants, rubber, paints, asphalts, roofing, chemicals, and to a much smaller extent in foods. Relatively low grades of fat are split into fatty acids and glycerol at high temperatures and pressures, sometimes with the aid of catalysts. The glycerol is recovered and the fatty acids are distilled under vacuum. The first distillate may be used directly as the highest grade of mixed fatty acids or it may be separated by a low temperature crystallization process into stearic (saturated) and oleic (unsaturated) fractions. The residue from this distillation is resplit and redistilled. The second distillate yields a lower grade of fatty acids. The residue from the second distillation is usually suitable for use on highways or in rubber manufacturing, but occasionally it is again recycled to obtain a third distillate and another residue. It was this third residue that had been blended with feed grade fat for use in feeds.

Every sample of residue of this type from several manufacturers of fatty acids proved to be rich in chick edema toxicity. Our first impression, therefore, was that the toxic factor was produced during the splitting and distillation steps and that it was concentrated in the residue along with other non-volatile unsaponifiable substances. Closer study of the various stages of fatty acid production soon revealed that the toxic factor was distillable and was present to some extent in the first distillates which were used for the production of the best grades of fatty acids, and those intended for food purposes. This discovery was made independently and reported by Ames, et al., who had found several samples of oleic acid and a monoglyceride made from such an oleic acid to be contaminated.

All this happened just after the passage of the Food Additives Amendment. The fatty acid manufacturing firms were very cooperative in providing us