Gram's stain results should not be relied upon solely to make a diagnosis of any disease or condition in an avian patient. While the Gram's stain can often be a valuable diagnostic screening test in both well and ill birds, the Gram's stain results must be interpreted in light of the history, age and condition of the bird, the bird's diet, culture results (if applicable), CBC results (especially the WBC and differential), and other diagnostic tests.
If a bird is showing upper GI signs, such as regurgitation or crop stasis, it may be valuable to perform a Gram's stain of the choanal slit and crop, as well as a fecal or cloacal Gram's stain. Fresh regurgitated material is also acceptable for Gram's staining and cytology. If a bird is showing upper respiratory signs, or if there are lesions present in the choanal slit, infundibular cleft or oropharynx, a Gram's stain of the choanal slit or lesions should be performed. Cytology of the area may also be diagnostic. If there is discharge present or excessive mucus in the choanal slit or oropharynx, consider performing a Gram's stain as well as cytology of the area. If lesions in the oropharynx are suspicious for Trichomonas, it is best to remove a lesion and swab the tissue underneath, or swab epithelium to cause a small amount of excoriation and bleeding, to have the best chance of diagnosing this disease.
Greater than 10% Gram-negative bacteria may be abnormal. Normal flora should consist of predominantly Gram-positive rods and cocci (approx. 90%.) Gram-negative spirochetes are abnormal and may cause pathology. Budding yeast or pseudohyphae may indicate an infection with Candida albicans. Non-budding yeast or a small percentage of budding yeast may be considered normal flora and may occur from additives to hand-feeding formulas. Rarely, very large Gram-positive bacteria (megabacteria) may be found in a choanal Gram's stain. Gram-negative rods may not always indicate disease, and may result from contaminated water, unwashed produce or bacterial that are just "passing through."
In addition to the observation of blunted choanal papillae as a clinical sign, sheets of epithelial cells on cytology or Gram's stain may indicate hypovitaminosis A, as will squamous metaplasia. Budding yeast and/or pseudohyphae from the choana, oropharynx or crop may also indicate hypovitaminosis A and secondary candidiasis. Inflammatory cells may indicate infection or trauma (from feeding syringes, hot formula, etc.) Trichomonads may be found intracellularly in psittacines, which may make diagnosis difficult.
With regurgitating birds or those with crop stasis, it may be valuable to gently swab the crop for Gram's staining and/or cytology. Normal flora should consist primarily of Gram-positive rods and cocci (approx. 90%.) Greater than 10% Gram-negative rods may indicate infection. Very large, Gram-positive rods may be megabacteria. Candida or trichomonads may be identified in crop swabs or regurgitated material. If a large number of similar Gram negative rods are seen, these are indicative of bacterial ingluvitis. Cytology from the crop may show inflammatory cells, bacteria within cells, epithelial cells, trichomonads, pseudohyphae, Candida or squamous metaplasia.
Less than 20 bacteria per 1000-x field are considered a reduced number of bacteria in adult psittacine birds. Gram-positive rods and cocci should make up at least 90% of the total bacterial population. Generally, Gram-negative rods should account for less than 10%. However, some Gram-negative rods can be found as normal flora in some species of psittacines (for example, some strains of E. coli can be considered normal in cockatoos). If the Gram's stain shows an increased amount of abnormal bacteria, it is recommended that bacterial culture and sensitivity be performed to identify potential pathogens. The presence of budding yeast may be abnormal (check to see if the diet contains active brewer's yeast, for example). Neonates may normally have a higher percentage of Gram-negative rods. Passerine birds normally have low numbers of bacteria.
Gram-positive rods with spores present may indicate infection with Clostridium sp. and related toxins. An anaerobic culture may help in making a diagnosis in these cases. If a choanal Gram's stain has been performed in conjunction with a cloacal Gram's stain, it is possible to compare the relative amounts of bacteria and yeast between the two locations. Increased levels of Gram-negative rods in the choana compared to the cloaca may indicate an upper respiratory or upper GI bacterial infection. Increased levels of Gram-negative rods in the cloaca, when compared to the choana, may indicate bacterial enteritis. C+S is helpful in these cases, as is evaluation of the WBC. Isolation of a bacterial organism in an almost pure culture (approximately 80% of the colonies present) may indicate that the bacteria are a component of a disease process.
Black stools are often mistaken for melena, but usually indicate a bird that has not eaten for at least 24 hours, and is passing biliverdin. This stool might show decreased levels of bacteria. A bird passing black droppings should be gavage fed for a period of time and it is suggested that a Gram's stain of the cloaca should be performed again once the bird is passing more normal droppings.
Mycobacterium cannot be diagnosed on a Gram's stain. An acid-fast stain of the stool may test positive when lesions are associated with the GI tract. However, false-negatives can occur when lesions are found in the lungs or are walled-off as granulomas. Avian TB, due to Mycobacterium sp., is most commonly found in Grey-cheeked parakeets, Red-hooded siskins, geriatric macaws or Amazons, and in birds with access to the ground.
A Gram's stain in a bird with true diarrhea may be helpful in diagnosing the cause. Warm saline wet-mounts may help by demonstrating motile bacteria or Giardia. Bacterial C+S may be helpful, as may the WBC and differential.
Copyright © 2006 Margaret A. Wissman, D.V.M., D.A.B.V.P.
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