Using and Interpreting the Fecal Gram's Stain

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WHAT YOU CAN SEE IN A PSITTACINE FECAL GRAM’S STAIN

n psittacines, the fecal Gram’s stain is a significant TECHNIQUE FOR part of a complete patient PERFORMING A FECAL GRAM’S STAIN Ievaluation. Although not defini- 1. With the wooden end of a cotton-tipped applica- • Rinse immediately, as above. tive in making a diagnosis, it tor, pick up a small amount of feces and apply it • Add 5 drops saffron to stain gram-negative provides a visual screen of the to a pre-cleaned glass slide. Use the applicator bacteria red. stick to spread the sample into a uniform, thin, • Rinse immediately, as above. percentages of bacteria present even film, using a single swath. • Blot dry with lens paper or tissue. in the gastrointestinal tract at 2. Heat-fix the sample for 5-6 seconds by applying 4. Scan the slide under low microscopic power the time the sample was the flame from a cigarette lighter to the for an ideal evaluation site and apply 1 drop collected and smeared. With underside of the slide. immersion oil to the site. the help of this information, • Using the oil immersion lens, scan several 3. Prepare to stain by placing the slide on a the veterinarian can determine fields for a further idea of uniformity. staining tray. • Choose a uniform field and begin to estimate the next diagnostic step — • Apply 3 drops gentian violet to the sample the total number of bacteria. One way to do whether to proceed to a culture and allow to stand for 30 seconds. This stains that is to count 10 bacteria, assess the propor- all bacteria blue. tion of the entire field occupied by those 10 and aggressive therapy, or to • Rinse with water (elevate one end of the treat conservatively with bacteria, and then estimate the total bacterial slide to drain excess water). population per 1000x field. husbandry changes. • Apply 3 drops Gram’s iodine and allow to Normal intestinal flora of stand for 30 seconds. 5. Record the results (see Table 1). • Rinse as above. parrots, seen as gram-positive • Apply 5 drops 95% ethyl alcohol to decolorize The entire process takes an experienced person less (blue) bacteria on a fecal blue stain from gram-negative bacteria. than 2-3 minutes to complete. Gram’s stain, represents both aerobic and anaerobic bacteria aceae on culture; therefore, such as Bacillus, Corynebacterium, NORMAL GRAM’S STAIN cultures are not warranted in Streptomyces, Lactobacillus, Strepto- evaluating asymptomatic birds. coccus and Enterococcus spp.1 The anaerobic portion is difficult to grow using standard laboratory OBTAINING techniques, so the Gram’s stain A FECAL SAMPLE is the only practical means to The most reliable results from assess the entire group. fecal Gram’s stains in the clini- Enterobacteriaceae are gram- cal setting are obtained with a negative (red) bacteria that consistent technique performed include pathogenic (e.g., Salmo- on fresh feces by a single person. Fecal samples obtained nella, E. coli, Acinetobacter) and FIG 1 Budgerigar, 4-year-old male: Hx = Apparently healthy bird, fed nonpathogenic species/strains. in the examination room are Harrison’s Bird Foods.™ CS = none. GS = Normal distribution of organ- Enterobacteriaceae are not nor- not optimal because of the ner- isms: 157 total bacteria per field, 70% gram-positive rods, 30% gram-pos- mal components of unstressed vousness of the patient. Ideally, itive cocci, 0 gram-negative bacteria, 0 yeast. Digestion of food is complete. parrots’ microflora.1 the owner should be instructed A Gram’s stain is not intended to collect a sample at home and COMMON CLINICAL to replace a culture for the eval- keep it cool until arrival at the GRAM’S STAIN RESULTS uation of pathogenic bacteria in office. This prevents prolifera- The following images represent psittacine fecal Gram’s stains com- a sick bird. On the other hand, tion of saprophytic gram-nega- monly seen in clinical practice (1000x oil field). Most birds were fed many healthy birds may show tive bacteria that may be primarily a seed-based diet plus some supplements. Distribution of some transient Enterobacteri- interpreted as pathologic. fecal components and possible causes are described. FIG 2 Cockatiel, 14-year-old male: Hx = Bird presented for boarding, FIG 3 African grey parrot, 4 years old, sex unknown: Hx = Intermittent seed diet. CS = Dull feather color, retained pin feathers. GS = 55 bacteria vomiting or loose stool, not as playful. GS = 400 bacteria per oil field, 95% per field; 90% gram-positive rods, 10% gram-positive cocci. Hyperkeratotic gram-positive short rods, 5% gram-positive rods, 0 yeast. Overgrowth of cell with characteristic straight sides suggests intestinal microflora imbal- intestinal bacteria, enterotoxemia, malnutrition. Rx = Aggressive. ance, probably due to malnutrition, early liver disease. Rx = Conservative.

FIG 4 Psittacine: Iatrogenic gram-negative rods due to staining error. An FIG 5 Amazon parrot, 8-year-old, female: Hx = Finicky eater, occasion- error is suspected when the demarcation of gram-positive and -negative is ally grumpy. CS = Failure to molt correctly, balding of feet, obvious layer- linear and the groups are similar in shape and size, differing only in color. ing of beak, overgrowth of nails, minor feather-picking. GS = 40 bacteria Note the presence of a normal intestinal epithelial cell, which is rounded per field, 90% gram-positive rods, 0% gram-positive cocci, 10% gram-neg- and takes on a blue color. Compare this to the straight, pointed edges of ative rods. (The normal binding of urates by protein is occasionally seen in the hyperkeratotic cell in Figure 2. Rx = None. fecal Gram’s stains.) Rx = Conservative.

FIG 6 Severe macaw, 7 years old, sex unknown: Hx = Depressed, not eat- FIG 7 Meyer’s parrot, 6 years old, sex unknown: Hx = Diet of seeds and ing, weak. CS = Underweight, scant feces, dark yellow urine and urates, supplements, treated previously for bacteria. CS = Depressed, fluffed, poor malcolored feathers. GS = 200 bacteria per field, 1% gram-positive rods, appetite. GS = Scant bacteria, two budding yeast organisms, suggesting 0% gram-positive cocci, 98% gram-negative rods. Rx = Aggressive. early malnutrition. Rx = Aggressive. FIG 8 Ring-necked parakeet, 9-year-old male: GS = Scant gram-positive FIG 9 Cockatiel, 8-year-old female: GS = 80 bacteria per field, 80% bacteria, occasional gram-negative, many apparent bacterial forms and gram-positive rods, 20% gram-positive cocci; 20 non-budding, yeast-like colors; invasive filament of yeast budding bi-directionally. Rx = Aggressive. structures (possibly from bakery products in diet, not clinically significant). Rx = None.

FIG 10 Umbrella cockatoo, 6-year-old female: Hx = Exposure to carniv- FIG 11 Budgerigar, 4-year-old male: CS = Digestive upset. GS = 200 orous pets, seed only diet. CS = Fetid stool, weight loss, passing undi- bacteria per field, 5% gram-positive cocci, 95% gram-positive rods, of gested food. GS = 200 bacteria per field, 10% gram-positive rods, 90% which half are large filamentous rods. Rx = Aggressive. gram-negative rods, of which 50% are Clostridium sp. Rx = Aggressive.

FIG 12 Moluccan cockatoo, 7-year-old male: CS = smelly stool. GS = 50 FIG 13 Budgerigar, 3-year-old male: Hx = Frequent masturbation. GS = bacteria per field, 90% gram-positive rods, 10% gram-positive cocci, 30 Presence of sperm. Rx =None. Clostridium sp. organisms. Rx = Aggressive.

KEY Hx = History CS = Clinical Signs GS = Gram’s stain results Rx = Therapy FIG 14 Psittacine: Various forms of gastrointesti- FIG 15 Psittacine: GS = Large amounts undi- FIG 16 Psittacine: GS = 20 bacteria per field, nal diseases can be suspected if digestion of fiber gested fiber (low microscopic power). 100% gram-positive rods, lots of undigested food or dietary ingredients is improper. Top = Normal particles cluttering field, suggesting some form of fiber content of feces. Bottom = Undigested fiber. gastrointestinal disturbance.

INTERPRETATION OF FECAL GRAM’S STAIN RESULTS According to Cheville,2 “All combination with cholesterol, distribution of bacteria on the body surfaces and orifices have produce more bacteria. This fecal Gram’s stain. In the early TABLE 1 Recording Results characteristic normal resident bacterial proliferation and fer- stages, the change is reflected by: of Fecal Gram’s Stain microflora, the secretions of mentation produces acids and •decrease in total bacteria Date ______which prevent overgrowth of other protective by-products. •decrease in percentage of Species ______pathogenic bacteria — for Such acids inhibit the presence gram-positive cocci. example, intestine. These of gram-negative rods and yeast. • increase in gram-positive Case ID ______normal microbial populations An imbalance in the home- rods. Results: reduce growth rates of some ostasis of the bird would result In the later stages of mal- _____ total bacteria/1000x field pathogenic bacteria through in upset of the normal func- nutrition and liver disease, the _____ % G+ rods/field competition for nutrients and tions of the microflora, and Gram’s stain generally shows: _____ % G+ cocci/field _____ % G- rods/field adhesion sites.” thus the distribution of bacteria •increase in presence of _____ number yeast/field Normal intestinal secretions in the gastrointestinal tract. gram-negative rods (the _____ % budding yeast and the by-products from A healthy psittacine should more gram-negative rods, _____ high fiber in feces digestion are the most have predominately gram- the more pathologic the _____ undigested food significant factors maintaining positive rods and cocci and less situation) _____ parasites normal bacterial populations. than 1% gram-negative rods. •presence of yeast (the _____ clostridia organisms Microflora in parrots degrade Malnutrition and liver disease, more budding yeast per _____ hyperkeratotic cells various fibrous carbohydrates which are very common in pet field, the greater the like- _____ normal intestinal cells (for energy), digest protein, birds, are characterized by lihood that the immune assimilate nitrogen, and, in changes in the number and system is breaking down).

REFERENCES 1. Ritchie BW, Harrison GJ, Harrison LR (eds): Avian Medicine: Principles and Application. Lake Worth, Wingers Publishing, 1994, pp 951, 968, 978. 2. Cheville NF: Introduction to Veterinary Pathology 2nd ed. Ames, Iowa State University Press, 1999, p 155. 3. Ritchie BW: Case report presentation. Proc No Am Vet Conf, 2000.

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