The recent episode of multi-state mortality in dogs due to toxic levels of aflatoxin highlights the problem of mycotoxicosis which is both seasonal and regional in the USA.

Aflatoxin is the most common of the economically significant mycotoxins and in the USA is most frequently associated with contaminated corn. Drought exposes the developing ears to infection with Aspergillus flavus and A. parasiticus. These fungi elaborate at least four related toxins of which aflatoxin B1 is most frequently associated with adverse effects centering on the liver and vascular system of susceptible livestock.

Concurrent with the outbreak of aflatoxicosis in dogs, egg producers noted an increase in blood spots ranging from pinpoint accumulations of blood in the albumen adjacent to the vitelline membrane to blood-filled eggs. Small blood spots should be differentiated from protein inclusions (“meat spots”) which occur normally in up to 4% of brown-shelled eggs. These particles are formed from the protein matrix of the eggshell and have a characteristic tan to red color caused by incorporation of porphyrin pigment which colors the shell. In fact, the albumen of white-shelled eggs also contains protein particles, but these non-pigmented inclusions are translucent and are virtually invisible.

Blood spots occur as a result of hemorrhage at the time of ovulation from ruptured small vessels adjacent to the stigma of the ovarian follicle. This results in release of blood into the infundibulum of the oviduct with subsequent appearance in the albumen, usually adjacent to the vitelline membrane surrounding the yolk.

Causes of Blood Spots

Any factor which decreases the rate of clotting or impacts the integrity of blood vessels will predispose to the presence of blood spots. Aflatoxin binds to mitochondrial DNA, resulting in adducts which impair oxidative phosphorylation, critical for synthesis of proteins and the metabolism of lipids. Hepatic dysfunction as a result of aflatoxicosis is indicated by elevation and disturbance in the ratio of specific serum enzymes which can be assayed. Aflatoxicosis results in lowered serum protein, lipoproteins and carotenoids. Due to reduced synthesis of prothrombin, coagulation is adversely affected, leading to a greater potential for hemorrhage. The permeability of capillaries may also be increased due to degenerative changes induced by toxins.

Aflatoxicosis is characterized in broilers by bruising, and in highly susceptible immature species, such as ducklings, subcutaneous hemorrhages are observed. It is evident that high-producing hens will be susceptible to aflatoxicosis since the liver is responsible for synthesis of the precursors of both yolk lipids and albumen incorporated into eggs. Over short periods, hen-week production may be maintained despite the presence of low to moderate levels (100 ppb) of aflatoxin in feed although egg weight is frequently reduced.

Chronic aflatoxicosis may affect shell strength as the rate of conversion of dietary vitamin D3 (cholecalciferol) to the active metabolic form is diminished. This decreases the efficiency of calcium absorption since the activity of calcium-binding protein in the intestine is reduced. Absorption of carbohydrates and lipid nutrients is also impaired due to reduced output of pancreatic amylase and lipase. Clinical problems associated with mycotoxicoses in laying flocks also include the indirect effects of immunosuppression manifested as septicemia and peritonitis, deterioration in internal egg quality, reduced yolk pigmentation and defective shells.

The differential diagnosis of blood spots includes vitamin A deficiency and anticoagulant rodenticide toxicity, both of which are extremely rare. Deletion of vitamin K from premixes fed to caged hens will also predispose to blood spots. It is possible that hyperexcitablity (“flightyness” or “hysteria”) among hens may result in hemorrhage at the time of ovulation. Data obtained during the mid-1990s in Pennsylvania showed that flocks infected with Salmonella enteritidis had a 3 to 4-fold increase in the prevalence of blood spots compared to non-infected flocks.

Occurrence of Blood Spots

The prevalence of blood spots in white-shelled eggs was determined in North Carolina random sample tests conducted between 1980 and 1984. “Small blood spots” were present in an average of 0.93% of eggs examined with an additional 0.88% classified as “large blood spots” over 5 mm in diameter. In contrast, brown-shelled eggs showed prevalence rates for small and large blood spots of 3.2% and 2.1%, respectively.

Consumer complaints relating to the presence of blood spots and free blood in eggs can be regarded as an indication of the severity of a problem. Since approximately 1.5% of all white-shelled eggs have detectable blood spots, any marked increase in complaints might suggest a problem over and above the accepted normal prevalence.

A review of records maintained by a large integrator showed considerable variation in the prevalence of blood spots among various complexes in the group. Complaints ranged from zero to 7 per 10,000 cases marketed, with an average of 2.4 complaints per 10,000 cases. If random sample data are representative of US production,10,000 cases would theoretically contain 3,000 dozen cartons with at least one egg with a detectable blood spot, assuming a 1% prevalence rate. The discrepancy between the average of 2.4 complaints recorded and the potential for 3,000 observations suggests a high tolerance for blood spots among consumers.

Detecting Blood Spots

It is impossible to detect blood spots using conventional candling as the opacity of even white-shelled eggs limits visual detection by a candler observing 12 rows of eggs at a throttle setting of 300 cases per hour. Electronic blood detectors function by passing light beams at two different frequencies through eggs to detect the haem molecule of blood. Validation of the sensitivity and specificity of a blood spot detector by breakout examination in two large plants confirmed that among samples of rejected eggs, there were three normal eggs for every egg with a visible blood spot.

A plant operating at 300 cases per hour over an 8-hour shift would process 864,000 eggs. With a 0.5% rejection due to blood spots, the plant would normally reject 360 dozen eggs due to apparent blood spot defects. Of this quantity, approximately 240 dozen eggs would be unjustifiably rejected as “false positives” each shift. Since the manufacturers of commercial blood spot detectors cannot provide data on sensitivity (the ability to accurately detect eggs with blood spots) or specificity (the ability to accurately differentiate between blood spots and other defects), it must be assumed that not only is there false rejection but that a proportion of eggs with blood spots are released to the market.

Prevention of Aflatoxicosis

Feed mills should be equipped to screen corn and other cereal ingredients for the presence of aflatoxin. Any consignment of corn with moisture content in excess of 12% should be regarded as suspect and should be assayed using a commercially available rapid immuno-based test kit. Levels in excess of 50 ppb aflatoxin B1 are regarded as potentially toxic. It is noted that the uneven distribution of mycotoxins within a consignment of an ingredient may lead to considerable sampling bias and result in either false negative assays or non-representative high values in a car or truck load as delivered.

Commercial and state diagnostic laboratories are equipped with gas-liquid chromatography installations to provide quantitative results for a wide range of mycotoxins in feed samples. Although the cost of assays is generally low, delay in receiving results creates logistic problems of segregated storage of suspect batches of ingredients.

A number of feed additive compounds have been shown to inhibit adsorption of aflatoxins by selective binding in the intestinal tract. Although bentonite clays may inhibit uptake, their efficacy both in vitro and in vivo is extremely variable. Natural and synthetic hydrated calcium aluminum silicates are more efficient adsorbers of aflatoxins than clays. Esterified glucomannans derived from the cell wall of yeast (Saccharomyces cerevisiae) also are used as aflatoxin binders, intended for action against aflatoxins as well as fusariotoxins which are frequently co-contaminants.

Current FDA regulations prevent manufacturers of zeolites and esterified glucomannans from promoting their products as mycotoxin adsorbents or binders, which would constitute a health claim. Compounds can however be purchased and incorporated in diets as anti-caking agents at an additive cost of less than $2 per ton. Since aflatoxin is usually accompanied by other toxic fungal metabolites including fusariotoxins and ochratoxin, a broad spectrum mycotoxin binder is recommended when aflatoxin is present in cereal ingredients and as reports of blood spots are received from consumers.