History

In a flock of 27-day-old commercial broiler chickens in southeast Alabama, there was a noticeable increase in mortality rate in 1 of the 4 houses in which the birds resided. The field technician responsible for this flock reported that several birds in the affected house were small and listless and had ruffled feathers; some birds were recumbent and unable to rise. Of 37,300 birds originally placed in the affected house at 1 day after hatching, 194 had been found dead over a 3-day period (61, 76, and 57 deaths among the 24-, 25-, and 26-day-old birds, respectively). The expected mortality rate at this age and in a flock of this size was < 15 birds/d.

Prior to the placement of birds in the house, the used litter had been removed and the house was bedded with fresh peanut hulls. The birds in the flock had been hatched at a local hatchery that participated in the National Poultry Improvement Plan. The eggs from which the birds hatched had been produced by breeder stock that were monitored according to the guidelines of the National Poultry Improvement Plan; under that certification scheme, the stock were classified as Mycoplasma gallisepticum monitored, Mycoplasma synoviae monitored, and avian influenza clean. The birds were vaccinated against Marek's disease virus in ovo at 18 days of incubation and spray vaccinated against Newcastle disease virus and the Arkansas strain of infectious bronchitis virus prior to leaving the hatchery. However, because of the recent increase in the number of deaths, 6 live birds from the affected house were submitted to the J. B. Taylor Veterinary Diagnostic Laboratory in Elba, Ala, for examination.

Clinical and Gross Findings

The 6 birds had poor uniformity of stature, and 4 birds were noticeably smaller than the remaining 2. Three of the smaller birds rested on their tibiotarsus-tarsometatarsus joints (hocks) and had difficulty remaining in an upright position. One of the smaller birds had a slight head tremor. The birds were euthanized by means of a carbon dioxide chamber. During necropsy, multiple firm, yellow to white nodules were found throughout the lungs of the 4 smaller birds (Figure 1). The thoracic air sacs had similar but fewer lesions. A small focus of white discoloration was seen in the cerebellum of one of the smaller birds. There were no gross lesions in the 2 larger birds. During necropsy, brain and lung tissue samples were collected aseptically for microbial culture.

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Photographs of the left lung (A and B) and a sagittal section of the brain (C) of a 27-day-old broiler chicken from a commercial flock that had an increase in mortality rate. Multiple pale yellow to white nodular lesions are seen throughout the lung, and a single similar lesion is visible in the cerebellum. Representative lesions are indicated (arrows). 1 = Heart. 2 = Liver. 3 = Proventriculus. 4 = Spleen. 5 = Left kidney. 6 = Left testicle. 7 = Left lung.

Citation: Journal of the American Veterinary Medical Association 241, 10; 10.2460/javma.241.10.1297

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Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →

Histopathologic and Microbiologic Findings

Samples of brain, lungs, eyelids, trachea, and the bursa of Fabricius were collected from the 6 birds and fixed in neutral-buffered 10% formalin. Tissue samples were submitted to the Thompson Bishop Sparks State Diagnostic Laboratory in Auburn, Ala, where they were routinely processed, embedded in paraffin, sectioned, stained with H&E stain, and examined by light microscopy. The lung nodules consisted of multiple coalescing granulomas characterized by foci of caseous exudate (necrotic cellular material and heterophils). The areas of necrosis were immediately surrounded by multinucleate giant cells, beyond which were dense infiltrates of macrophages, additional multinucleate giant cells, lymphocytes, fibroblasts, and heterophils. In some areas, caseous exudate appeared to be within remnants of secondary and tertiary bronchi. Unstained spaces in the shape of fungal hyphae were evident in necrotic foci and within phagocytic cells (Figure 2).

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Photomicrographs of sections of lung and cerebellum tissue of a 27-day-old broiler chicken from the same commercial flock as the bird in Figure 1. A—A nodule in the lung tissue consists of coalescing granulomas appearing as foci of caseous exudate (arrow) surrounded by dense infiltration of multinucleate giant cells, macrophages, lymphocytes, fibroblasts, and heterophils. Clear outlines of fungal hyphae can be identified within phagocytic cells (arrowheads). Unaffected lung parenchyma is visible at the lower corners of the image. H&E stain; bar = 200 μm. B—In the main image, the cerebellum has necrosis and inflammation involving white matter and folia. H&E stain; bar = 200 μm. Inset—Fragments of hyphae are present in the lumen and wall of a blood vessel (arrowhead) and in the surrounding parenchyma. Periodic acid–Schiff stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 241, 10; 10.2460/javma.241.10.1297

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In a section of grossly affected cerebellum from one of the smaller birds, extensive necrosis accompanied by dense infiltration of macrophages, lymphocytes, multinucleate giant cells, and heterophils was detected in the white matter and several adjacent folia (Figure 2). Small veins and capillaries were occluded by fibrin thrombi. Fragments of fungal hyphae (similar to those in the lung tissue) were observed in blood vessels, in areas of necrosis, and within phagocytic cells. Application of a periodic acid–Schiff (fungal) stain revealed the hyphae to be thin-walled, septate, and somewhat variable in width, with occasional 45° angled branches. These features were considered to be consistent with Aspergillus organisms.

Similar, smaller lesions were evident within other sections of cerebellum and cerebrum from the other birds. By use of periodic acid–Schiff stain, hyphae were also detected in isolated feather follicles and in small granulomas in the dermis of 1 eyelid. In 1 bird, striated muscle surrounding the trachea also contained a small granuloma with a few organisms. Examination of sections of the bursa of Fabricius from the 6 birds revealed necrosis accompanied by severe depletion of lymphoid follicles, consistent with acute infectious bursal disease.

Brain and lung tissue samples that had been collected during necropsy were placed on Sabouraud dextrose agar and incubated at 25°C. After 5 days, cultures of brain tissue samples from 5 of the 6 birds and lung tissue samples from all 6 birds yielded wooly colonies that were initially white but later became yellow-green with a white colony edge. Transparent tape was used to transfer organisms to slides for staining with lactophenol cotton blue stain; microscopic examination of these mounts revealed conidiophores with circumferentially arising biseriate phialides and smooth conidia consistent with Aspergillus flavus.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: multifocal to coalescing granulomatous pneumonia and focally extensive necrotizing granulomatous encephalitis with intralesional fungi, consistent with aspergillosis.

Case summary: A flavus mycosis in boiler chickens.

Comments

For the affected birds in the flock of commercial broiler chickens of the present report, a diagnosis of A flavus mycosis was made on the basis of gross lesions, histopathologic findings, and characteristics of the isolated pathogen. Characterization of the pathogen ruled out other known causes of avian mycosis, including infections with Aspergillus fumigatus or Ochroconis gallopavum (formerly Dactylaria gallopava). Aspergillus flavus is a ubiquitous thermotolerant mold that is commonly found in soil, grains, and decaying vegetation.¹ Aspergillus flavus is known to be a potential environmental contaminant of poultry confinement facilities.² Aspergillosis involving A flavus has been identified in many avian species, including ostriches, geese, falcons, Japanese quail, turkeys, broiler chickens, and both broiler breeder and leghorn pullets.^3–9 It has been suggested that perhaps all birds should be considered potential hosts for Aspergillus organisms.¹

Aspergillosis in mammals is uncommon, although cases involving dogs, horses, cattle, and dolphins have been reported.¹⁰ Such infections often involve A fumigatus rather than A flavus.¹⁰ Mice are susceptible to A flavus when experimentally inoculated IV with the organism.¹¹ As the number of immunocompromised humans increases, A flavus is emerging as a pathogen of importance in human medicine. It is the second most common cause of invasive Aspergillus infections in the United States and the most common cause of such infections in some arid regions of the world.¹² As a potential producer of aflatoxins and contaminant of grain stores and food products, A flavus is important to human health as a food safety issue. Because of this, much research has been devoted to establishing and understanding the relationship between peanuts and A flavus.^13–16

Peanut hulls are known to support the growth of A flavus.¹⁷ In southeast Alabama, peanut hulls are readily available for use as bedding in poultry houses. In our laboratory experience, cases of A flavus mycosis in south Alabama occur most often in the first flock to reside in poultry houses that have been bedded with fresh peanut hulls. Albeit anecdotal, this observation seems to be supported by findings of 1 study,¹⁸ in which A flavus or A parasiticus was not detected in hulls previously used as bedding, although aflatoxins were detected in the hulls prior to use. This led to the conclusion that the aflatoxins were formed by Aspergillus populations present in the hulls before use, which either were no longer present or were markedly reduced during use.¹⁸

Development of aspergillosis in poultry as well as other species is associated with exposure to large numbers of organisms and impaired immune function. Under appropriate conditions, inhalation of spores by birds may result in colonization of the lungs and air sacs. In more severe infections, the organisms may be disseminated via the bloodstream to distant sites, including the brain.¹ In the birds of the present report, hematogenous spread was most likely, although the cutaneous lesions detected in 1 eyelid may have resulted from direct environmental exposure. Susceptibility to aspergillosis in these birds was most likely increased by immune compromise associated with concurrent infectious bursal disease. Infectious bursal disease virus is a highly contagious virus of the Birnaviridae family that most severely affects the B lymphocytes of the cloacal bursa. The most important sequela of the disease is severe and prolonged immunosuppression of chickens infected at an early age.¹⁹

An effective treatment for aspergillosis in commercial poultry is not currently available. The disease is controlled by sanitation and avoidance of moldy feed and litter.¹