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Measuring the level of agreement between cloacal gram's stains and bacterial cultures in Hispaniolan Amazon parrots (Amazona ventralis).

Abstract: Cloacal or fecal Gram's stains and bacterial cultures are routinely performed during avian physical examinations to assess the microbial flora of the gastrointestinal tract. Although cloacal or fecal Gram's stains and bacterial cultures are considered routine diagnostic procedures, the level of agreement between the individual tests has not been determined. To investigate the level of agreement between results from Gram's stain and bacterial culture when used to assess cloacal or fecal samples from psittacine birds, samples were taken from 21 clinically healthy Hispaniolan Amazon parrots (Amazona ventralis) and tested by Gram's stain cytology and bacterial culture. Most bacteria (97.2%) identified by Gram's stain were gram positive. However, gram-negative organisms were identified in 7 of 21 (33.3%; 95% confidence interval: 13.3%-53.3%) birds. Escherichia coli was the only gram-negative organism identified on culture. Agreement between results of Gram's stain and culture was fair (weighted k = 0.27). The results of this study suggest that Gram's stains and bacterial culture may need to be performed with a parallel testing strategy to limit the likelihood of misclassifying the microbial flora of psittacine patients.

Key words: bacteria, Gram's stain, culture, cloaca, fecal, avian, Hispaniolan Amazon parrots, Amazona ventralis

Introduction

Gram-negative bacteria have been associated with both primary and secondary infections of the avian gastrointestinal system. (1-8) In addition to causing gastrointestinal signs, such as anorexia, diarrhea, and regurgitation or vomiting, bacteria that penetrate the mucosal border of the gastrointestinal tract may lead to systemic disease, sepsis, and death. (1,6) The normal alimentary tract micro flora of psittacine birds is composed predominately of gram-positive bacteria, although some gram-negative bacteria can be isolated from apparently healthy psittacine birds. Commonly isolated gram-positive organisms from psittacine birds are Bacillus, Corynebacterium, Staphylococcus, Streptococcus, and Lactobacillus species; these bacteria are generally nonpathogenic. Pathogenic bacteria of clinical significance isolated from the avian gastrointestinal tract are primarily gram negative. (1,5) Commonly isolated gram-negative organisms from psittacine birds include Escherichia coli, Enterobacter species, Pseudomonas species, and Klebsiella species. The total amounts and ratios of autochthonous bacterial flora versus aberrant bacteria are often subjectively used in determining potential abnormal bacterial colonization of the gastrointestinal tract, so much so, in some cases, that the bird's general health is evaluated by this measure.

Fecal Gram's stains are commonly performed as a method of evaluating the gastrointestinal health of psittacine species. (3,13) In birds showing evidence of disease, Gram's stains afford timely, inexpensive data regarding morphology of potential pathogens and provides some information if culture results are unobtainable. In practice, some veterinarians use Gram's stains as part of psittacine bird wellness examinations. (13) However, one disadvantage of using Gram's stains to evaluate apparently healthy birds is that Gram's stain results and clinical health or disease are not always in good agreement. Cloacal or fecal bacterial cultures are often reserved for cases with abnormal Gram's stain results; however, organisms found on Gram's stain might not be isolated on culture media. Furthermore, we are unaware of any scientific investigations that have assessed the diagnostic value of these testing modalities in clinically healthy psittacine birds.

The purpose of this investigation was to measure the level of agreement between results of fecal and cloacal Gram's stains and fecal aerobic bacterial culture to determine the clinical value of these diagnostic tests in clinically healthy psittacine birds. The specific biological hypothesis tested in this study was that the level of agreement between results of fecal and cloacal Gram's stains and bacteriologic culture is poor.

Materials and Methods

Birds

The project was performed in accordance with the regulations established by the Institutional Animal Care and Use Committee at Louisiana State University. Twenty-one adult Hispaniolan Amazon parrots (Amazona ventralis) were used in this study. The birds were individually housed at Louisiana State University and fed a commercially available, pelleted diet (Kaytee Exact Parrot Rainbow, Kaytee Products, Chilton, WI, USA). Clean water was offered daily ad libitum. The cages used to house the birds were cleaned and disinfected daily. All birds were considered clinically normal based on results of physical examination and diagnostic blood tests (complete blood cell counts and plasma biochemical profile) performed before the initiation of the study as part of their annual examination.

Procedures

Two small, sterile swabs, lightly moistened with sterile saline were fully inserted into the cloacal opening of each bird and then rotated to allow for the collection of feces. Sample material from each swab was then transferred directly to a microscope slide for a Gram's stain or used to inoculate sheep's blood and MacConkey agar plates for culture. A random number generator (http://www.random. org) was used to determine the order that each swab was assigned to a Gram's stain or culture.

The agar plates were incubated at 37[degrees]C for 24 hours under aerobic conditions. Distinct colonies found on the original plates were further subcultured on blood agar for additional testing. Gram's stains and biochemical testing (API 20 commercial test strips, bioMerieux, Durham, NC, USA) were used to characterize each isolate. Attempts were made to differentiate each bacterial isolate to the species level, although, in some cases, the organisms could be categorized only to the generic level of Gram's stain morphologic features.

The microscope slides were heat fixed and stained by standard methodology for Gram's stains, except that only brief contact (3 seconds) with the decolorization solution was used to avoid excessive decolorizing. (14) All slides were examined by a single individual (E.E.E.). Slides were scanned at X100 to identify an appropriate evaluation site. Then, 10 fields were scanned at X1000 for each sample. The total number of bacteria per X1000 field was counted and recorded. (2,15) The number of each of the following was quantified and recorded: gram-positive cocci, gram-positive rods, gram-negative cocci, gram-negative rods, yeast, hyphae, white blood cells, and red blood cells. The relative percentages of each morphologically defined category were calculated and recorded.

Statistical analysis

The data for Gram's stains were evaluated with the Shapiro-Wilk test and were found to be not normally distributed; therefore, the median, 10-90 percentiles ([P.sub.10]-[P.sub.90]), and minimum/maximum values were collected. The 95% binomial confidence interval (CI) was calculated for each proportion. These results represent, with 95% confidence, what the proportions would be for the entire population if all animals were sampled. Agreement between the results of Gram's stains and culture results was measured by a weighted [kappa] test. SPSS 19.0 software (SPSS Inc, Chicago, IL, USA) was used to analyze the data.

Results

Most bacteria (97.2%) identified on Gram's stain were gram-positive. Of these gram-positive bacteria, 86.2% were gram-positive rods (median: 25%, [P.sub.10]-[P.sub.90]: 12.6%-76%; minimum-maximum (min-max): 7%-89%) and 13.7% were gram-positive cocci (median: 4%; P10-P90: 0%-41.6%; min-max: 0%-75%). The ratio of gram-positive rods to gram-positive cocci was 6.25:1. The remaining 2.8% of bacteria identified as gram negative were all rods. More than 70% (15 of 21) (95% CI, 51.7%-90.3%) of the birds in this study had 100% gram-positive bacteria identified on cytologic examination. However, 6 birds (28.6%) (95% CI, 9.0%-47.9%) were identified as having gram-negative bacteria on Gram's stain examination. In these 6 birds, the following percentages of gram-negative bacteria were identified: 3%, 7.5%, 10%, 11%, 12%, and 15%.

The following gram-positive bacteria were identified on bacterial culture: Brevibacterium species, Cellumonas/Microbacterium species, Corynebacterium argentoratense, Corynebacterium species, Enterococcus species, and Staphylococcus species (coagulase negative) (Table 1). An additional gram-positive rod and gram-positive cocci (oehemolytic) were identified but could not be further speciated. Escherichia coli was identified in 23.8% (5 of 21) (95% CI, 6.8%-40.7%) of cultures and was the only gram-negative organism identified on culture. In 60% of these samples (3 of 5 cultures), growth was classified as slight, and in the other 40% of these cases (2 of 5 cultures), growth was classified as moderate.

Gram-positive rods were identified by Gram's stain in all birds and by aerobic bacterial culture in 86% (18 of 21) (95% CI 72%-100%) of the birds. All birds from which gram-positive rods were recovered on culture also had gram-positive rods identified on their corresponding Gram's stain. In 2 birds (9.5%), bacterial cultures were negative for any growth despite the presence of gram-positive rods on cytologic examination.

Gram-positive cocci were identified in 81% (17 of 21) (95% CI 64.2%-97.7%) of the birds: via Gram's stain in 71.4% (15 of 21) (95% CI, 51.5%-90%) and via bacterial culture in 52% (11 of 21) (95% CI, 30.6%-73.3%) of birds. In 6 (28.6%) birds with gram-positive cocci identified on Gram's stain, no gram-positive cocci grew on culture, while in 2 (9.5%) birds from which gram-positive cocci were cultured, gram-positive cocci were not identified on Gram's stain. In 9 birds (42.9%), gram-positive cocci were identified on both Gram's stain and culture.

Gram-negative organisms were identified in 33.3% (7 of 21) (95% CI, 13.3%-53.3%) of birds. Of these 7 birds, gram-negative bacteria were identified on both aerobic bacterial culture and Gram's stain in 4 (57.1%) birds, on the Gram's stain only in 2 (28.6%) birds, and on culture only in 1 (14.3%) bird.

The overall agreement measured between culture and Gram's stain results for all bacteria was fair (weighted [kappa] = 0.27).

Discussion

Most gram-positive bacteria identified on Gram's stain of cloacal swab samples in this population of 21 Hispaniolan Amazon parrots were gram-positive rods, with a rod to cocci ratio of 6.25:1. This finding is consistent with previous research (12,16) that suggests gram-positive rods are more common than gram-positive cocci. Although most clinicians can agree that most bacteria identified on Gram's stain should be gram positive, there is a difference of opinion as to what percentage of gram-negative bacteria is acceptable. Some clinicians maintain that 100% of the bacteria identified should be gram-positive, (15) whereas others consider 5%--10% gram-negative bacteria to be acceptable. (2,13) However, both viewpoints are based on subjective information and represent common dogma in avian medicine. The findings from our study suggest that gram-negative bacteria are not uncommon on Gram's stains from clinically healthy Hispaniolan Amazon parrots and that the prevalence of gram-negative bacteria in this species identified by Gram's stain could be between 9.0%-47.9%. Although, none of the 6 birds with gram-negative bacteria identified on Gram's stain appeared to be clinically ill, 3 had between 11%-15% gram-negative bacteria, 2 had 5%-10% gram-negative bacteria, and the remaining bird had 3% gram-negative bacteria. Several exogenous (eg, diet, water source, disinfection protocols) and endogenous (eg, gastrointestinal pH, motility) factors may influence the microbial flora of psittacine birds in captivity. Additional studies are required to adequately evaluate these factors in different avian species to further elucidate the most appropriate gastrointestinal bacterial populations and gram-positive to gram-negative ratios in psittacine birds.

Gram-negative bacteria are often considered the most important pathogens associated with disease of the gastrointestinal tract of psittacine birds. In this study, a third of the clinically healthy birds sampled had gram-negative bacteria present on Gram's stain cytology, culture, or both. This finding is consistent with previous studies that have shown the presence of E coli in clinically healthy cockatoos (Cacatua species) (10) and in 12 species wild-caught parrots in Brazil. (17) The results of this study indicate that this bacterium is also found in Hispanolian Amazon parrots without causing overt disease. Multiple factors may drive the presence of different species of bacteria in the psittacine gastrointestinal system, including dietary influence, food or water contamination, (18-20) interaction with other species (including humans), and environmental sanitation. However, the presence of the organism alone should not be used to determine its relationship with the host. Instead, the culture results should be evaluated in context with the patient's history, physical examination findings, and other related diagnostic test results. For example, the presence of gram-negative bacteria, such as E coli, on a culture collected from a bird with diarrhea has a different relevance than in the case where the animal is clinically healthy. Clinicians need to recognize the limits of the diagnostic tests they pursue. For culture, one such limitation is that the presence of an organism does not necessarily relate to the presence of disease or pathologic findings.

The gram-positive bacteria identified on cultures in this study, including Corynebacterium and Enterococcus species, have previously been identified in healthy psittacine birds and are considered components of the normal intestinal microflora. (2,6,15) Staphylococcus species, a common bacteria found on the skin of psittacine birds, has been observed as both an unremarkable finding (2) and as a potential pathogen. (16) In this study, none of the birds from which Staphylococcus was cultured showed any overt clinical signs of illness, and the bacteria were considered to be either a sampling contaminant or an unremarkable finding.

To our knowledge, Brevibacterium and Cellumonas/Microbacterium species have not been reported as disease pathogens in psittacine birds. Brevibacterium species are gram-positive bacteria found in the soil, (21) on human skin, (22) and associated with several popular cheeses. (23) To date, these bacteria are known to cause disease only in people who are immune-compromised. (22-24-25) This bacterium is commonly found in the feces of poultry and is currently under investigation as a specific marker of contamination from poultry litter. (26) Poultry are the only avian group in which this bacterium has been associated with disease; affected birds develop bumble-foot lesions. (27) Cetlumonas and Microbacterium species could not be further speciated. Cellumonas species have previously been described as Oerskovia species, (28) and Cellumonas and Microbacterium species are ubiquitous in the environment, typically being found in soil and vegetation. (29-30) Both are considered opportunistic pathogens in humans and are associated with disease in immune-compromised individuals. (31-34) Cellumonas species have also been identified as a primary pathogen in equine abortions, premature births, (35) and human neonatal sepsis. (36)

Fair agreement was observed between results of Gram's stains and bacterial culture. Although it appears that Gram's stain results were more likely to identify specific groups of bacteria compared with microbiologic culture, the 95% CI suggests that there is no difference between the 2 techniques in this study. Our results do indicate a tendency for culture to underestimate the true diversity of bacterial flora. In 2 of 21 (10%) of the birds, culture results were negative for any bacterial growth despite the presence of gram-positive rods identified by Gram's stain. This result is consistent with previous findings. (10) Lack of bacterial growth on culture, despite evidence of bacteria on Gram's stain, may be caused by failure of bacteria to grow on the selected media (fastidious organisms) or because the bacteria observed are anaerobes and require special culture techniques. (6-15) Another possible explanation for this difference between the diagnostic techniques is related to sample quality or size. For most bacteria to be isolated on culture, a minimum of [10.sup.3] or [10.sup.4] organisms are needed, whereas for a Gram's stain, as few as 1 or 2 organisms are needed because they are visualized by microscopy (M.A.M., unpublished data, May 2001). We attempted to control for sample quality by the same techniques and altering the assignment of the swab to the diagnostic method (Gram's stain or culture). Interestingly, the 2 samples that were negative for bacteria on culture also had the fewest organisms observed on their respective Gram's stains.

Many of the bacteria identified by Gram's stains did not result in a corresponding identifiable isolate on culture. When a sample is being cultured, competition on the agar plate can possibly lead to inhibition of conspecifics. Because of this limitation, Gram's stains are likely to provide a more complete representation of the gastrointestinal flora in comparison to cultures. Therefore, Gram's stains may be of higher value than cultures are for use as screening tests during health assessments. However, given the wide disparities in autochthonous flora reported in a variety of species (9-11) and the reported variance seen in different species with varying levels of stress and diet, (37) determining a reference interval for the number of bacteria and the ratio of gram-positive to gram-negative organisms is recommended for any established group of birds in which one uses Gram's stains as a method of assessing the health of an avian patient.

Fecal cultures are often used either in combination with fecal Gram's stain cytology or as a second tier diagnostic test to further evaluate potential pathogens identified on cytologic examination. (13) The decision to pursue antibiotic treatment and the choice of an appropriate antibiotic are often determined or confirmed by the use of fecal culture results. Additional methods of identifying specific potential fecal pathogens at the molecular level, including polymerase chain reaction testing and enzyme-linked immunoassay, are rapidly becoming available. Some veterinary researchers have begun to use these tools as methods to assess the health of bird populations. (38) Molecular identification of potential pathogens was beyond the scope of this study, but increasing availability to practitioners may make them important future detectors of subclinical disease in psittacine birds.

The results of this study suggest that Gram's stains and bacterial culture can be used to provide insight into the gastrointestinal microflora of clinically normal Hispanolian Amazon parrots. Although these 2 diagnostic tests did not show high agreement between samples, they did show a fair level of agreement that can be used for screening patients. In cases in which characterizing the bacteria is important in managing a case, a parallel testing strategy, with both tests used simultaneously, will limit the likelihood of misclassifying the microbial flora of a psittacine patient.

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Erika E. Evans, DVM, MBA, Mark A. Mitchell, DVM, MS, PhD, Dipl ECZM (Herpetology), Julia K. Whittington, DVM, Alma Roy, MS, PhD, and Thomas N. Tully Jr, DVM, MS, Dipl ABVP (Avian), ECZM (Avian)

From the Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, 1008 W Hazelwood Dr, Urbana, IL 61802, USA (Evans, Mitchell, Whittington): the Louisiana Disease Diagnostic Laboratory (Roy), and the Department of Veterinary Clinical Science, School of Veterinary Medicine (Tully), Louisiana State University, Skip Bertman Dr, Baton Rouge, LA 70810, USA. Present address (Evans): Small Animal Clinical Sciences, Veterinary Teaching Hospital, 2407 River Dr, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
Table 1. Results of Gram's stain cytology and bacterial culture of
cloacal swab samples of healthy Hispanolian Amazon parrots (N=21).

                   Gram-positive rods

         Gram's
Bird     stain,
no.     no. (a)          Culture growth (b)

 1         27      Corynebacterium
                     argentoratense (+++)
 2         22      Corynebacterium
                     argen toratense (++)
 3         17      NG
 4         27      Cellumonas/ Microbacterium
                     species (+++)
 5         76      Cellumonas/ Microbacterium
                     species (+++)
 6         32      Corynebacterium
                     argentoratense (+++)
 7         7       Cellumonas/ Microbacterium
                     species (++)
 8         12      NG
 9         25      Brevibacterium species (++)
10         42      Brevibacterium species (++)
                   Gram-positive rod (+)
11         23      Brevibacterium species (++)
12         20      Gram-positive rod (++)
13         76      Cellumonas/Microbacterium
                     species (++)
                   Brevibacterium species (++)
14         15      NG
15         18      Corynebacterium species (+++)
16         17      Cellumonas/Microbacterium
                     species (+++)
                   Corynebacterium species (++++)
17         24      Cellumonas/Microbacterium
                     species (+++)
18         55      Corynebacterium species (+++)
19         27      Cellumonas/ Microbacterium
                     species (++)
                   Corynebacterium species (++++)
20         38      Corynebacterium species (+++)
21         89      Corynebacterium species (++++)

                  Gram-positive cocci

        Gram's                                                Gram's
Bird    stain,                                                stain,
no.       no.             Culture growth                        no.

 1         0      NG                                             0
 2         0      Gram-positive cocci, alpha                     4
                    hemolytic (++)
 3         0      NG                                             0
 4         0      Staphylococcus, coagulase                      0
                    negative (++)
 5        10      Staphylococcus, coagulase                      0
                    negative (+++)
 6         4      NG                                             0
 7        22      Staphylococcus, coagulase                      4
                    negative (+++)
 8         0      NG                                             0
 9         8      Enterococcus species (++)                      0
10         4      NG                                             0
11         9      NG                                             0
12        40      Enterococcus species (++)                      2
13        10      NG                                             0
14        22      Enterococcus species (+++)                     3
15         0      NG                                             2
16         2      NG                                             0
17         2      NG                                             0
18        42      Enterococcus species (+++)                     0
19         4      Enterococcus species (+++)                     0
20        20      Enterococcus species (+++)                     0
21        75      Enterococcus species (++++)                   21

        Gram-negative rods

Bird
no.             Culture growth

 1      NG
 2      NG
 3      NG
 4      NG
 5      NG
 6      NG
 7      Escherichia coli (++)
 8      NG
 9      NG
10      NG
11      NG
12      NG
13      NG
14      Escherichia coli (++)
15      Escherichia coli (++)
16      NG
17      NG
18      Escherichia coli (+++)
19      NG
20      NG
21      Escherichia coli (+++)

(a) Total bacteria counted over 10 fields at X1000 (oil immersion).

(b) Culture growth: NG indicates no growth; +, few colonies; ++,
slight growth; +++, moderate growth; ++++, heavy growth.
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Author:Evans, Erika E.; Mitchell, Mark A.; Whittington, Julia K.; Roy, Alma; Tully, Thomas N., Jr.
Publication:Journal of Avian Medicine and Surgery
Article Type:Clinical report
Date:Dec 1, 2014
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