Veterinary Ophthalmology (2003) 6, 1, 45 – 50 Blackwell Science, Ltd
Seasonal effects on the aerobic bacterial and fungal conjunctival flora of normal thoroughbred brood mares in Florida Stacy E. Andrew,* An Nguyen,† Galin L. Jones‡§ and Dennis E. Brooks* *Departments of Small Animal Clinical Sciences and †Clinical Microbiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610–0126, USA, ‡Department of Statistics, IFAS Statistical Consulting Unit, PO Box 110339, University of Florida, Gainesville, FL 32611, USA
Address communications to: Dennis Brooks Tel.: (352) 392–4700 Fax: (352) 392–6125 e-mail:
[email protected] §Present address: University of Minnesota 347 Ford Hall 224 Church Street SE Minneapolis, MN 55455 USA Presented in part at the 32nd Annual Meeting of the American College of Veterinary Ophthalmologists, Sarasota, FL, October 11, 2001.
Abstract Objective To evaluate seasonal effects on the presence or absence of fungal and aerobic bacterial flora of the conjunctival fornix of normal Florida Thoroughbred horses. Sample population Both eyes of 100 horses. Procedure Horses with normal anterior segment ophthalmic examinations from three farms in north central Florida were included. Each animal had the ventral conjunctival fornix of each eye swabbed with sterile culturettes. Samples were taken in October, January, April, and July (1999–2000). Aerobic and fungal cultures were plated. Bacterial cultures were reviewed at 24 and 48 h. Fungal cultures were reviewed weekly for 4 weeks. Logistic regression analysis with season as a factor and age of the horse as a covariate was performed. Statistical significance was set at P < 0.01. Results Horses ranged from 3 to 24 years of age, with a median age of 9 years. Twentyfour genera of bacteria and 35 genera of fungi were recovered. Corynebacterium sp., Staphylococcus sp., Bacillus sp. and Moraxella sp. were the bacteria most frequently isolated. Mold species, dematiaceous mold species, Chrysosporium sp., Cladosporium sp., and Aspergillus sp. were the most frequently recovered fungi. Season did not have a significant effect on the presence of microorganisms isolated for individual horses adjusted for age. Younger horses had an increased incidence of gram-negative rods and fungal isolates. The number of bacteria and fungi isolated are not uniform across seasons. Conclusion There were no significant differences between the number or type of organisms cultured during the sampling seasons in normal Florida horses. A large range of normal bacterial and fungal flora were isolated from these horses. The number of bacteria and fungi isolated are not uniform across seasons. The likelihood of detecting an organism depends on the horses’ age. Key Words: bacteria, conjunctiva, fungi, horse, microbiology
INT RO DUC T IO N
There are many descriptions of conjunctival and corneal microbial flora in the veterinary literature. The reports detail microbes of various species in different parts of the country at various times of the year. Bacterial and/or fungal flora of the normal conjunctival fornix have been reported in sheep,1 cows,2,3 pigs,4 birds,5–7 rabbits,8,9 dogs,2,10,11 cats,2,12,13 horses,2,14–16 bison,17 llamas,18,19 alpacas18,19 and guanacos.18,19 Most reports of normal ocular flora in horses show a predominance of nonpathogenic, mainly gram-positive organisms.14 However, gram-negative and fungal species are also found as part of the normal ocular flora of the horse,14 and are thought to be transitory and © 2003 American College of Veterinary Ophthalmologists
related to fungal organisms present in the surrounding environment.20 If the corneal epithelium is intact, most ocular microbial flora are not considered pathogenic. However, when a corneal abrasion occurs, resident and transient microbes may infiltrate the corneal stroma and result in an infected corneal ulcer. Once ulcers become infected, they can be extremely difficult to treat and vision loss can result. Stromal abscessation may occur if the outer epithelium covers the infected area, or the iris may prolapse as the ulcer progressively deepens.21,21 Corneal disease can be career ending for equine athletes, as these animals are more likely to be considered unsound. It is important to understand the normal conjunctival microflora so that appropriate prophylactic
46
ET AL.
antimicrobial therapy can be instituted if a corneal injury occurs. Many factors may affect the presence or absence of ocular microflora. Season, geography, bedding, habitat and husbandry have all been suggested as potential variables affecting microbial burden in normal and diseased horse eyes.2,14,16 Seasonal effects such as temperature and humidity have been determined to play a role in equine corneal disease.22 Normal conjunctival microbial flora load has been shown to be decreased in northern climates such as Wisconsin during winter months.15 Transient populations of microorganisms are established from environmental contamination, and are in flux with the resident microorganisms on the surface of the horse eye and the conjunctiva.20 Fungal flora are believed to be seeded from the environment, and thus seasonal variation could affect the equine conjunctival microflora.2,14,16 It has also been shown that the conjunctival fungal burden was significantly higher in horses that were stabled as opposed to those that were hospitalized.14 In the south-eastern United States, ulcerative fungal keratitis occurs throughout the year but more cases are detected in the fall and winter.23,24 The purpose of this investigation was to evaluate potential seasonal effects on the presence or absence of fungal and bacterial flora in the conjunctival fornices of normal horses in Florida. Information derived from this study is anticipated to be useful in determining potential causes and treatments for corneal diseases at various times of the year in order to lessen the extent of corneal infections, and minimize lesion progression and scar formation. MA TERIALS AN D M ETH ODS
One hundred Thoroughbred horses with normal anterior segment ophthalmic examinations, as determined by assessment with a transilluminator and absence of clinical signs, were used. The animals were from three farms in north central Florida. Animals were housed outdoors in field pastures and samples were obtained as the horses were brought in each morning for feeding. Animals were not sedated or nerve-blocked for sample collection. Samples were obtained in October 1999 (fall), January 2000 (winter), April 2000 (spring), and July 2000 (summer). Microbial samples from the lower conjunctival fornix of both eyes of all horses were obtained by retropulsing each eye through the closed upper eyelid and running a premoistened sterile culturette along the surface of the ventral conjunctival fornix. The fornix cultured first (right or left) was chosen randomly. Special care was taken to ensure that the culturette did not come into contact with the vibrissae, eyelids or eyelashes. Culturettes were placed in a cooler for 3–5 h until return to the microbiology laboratory. Aerobic bacterial and fungal cultures were made from these swabs at the University of Florida. Five growth media were used for each sample: Columbia sheep blood agar (aerobic bacteria), Columbia CNA agar (gram positive bacteria), MacConkey agar (gram negative bacteria),
Sabouraud dextrose agar (yeasts and fungi), and mycobiotic agar (promotes fungal and inhibits bacterial populations). Bacterial cultures were reviewed at 24 and 48 h and fungal plates were reviewed once weekly for 4 weeks. The presence or absence of bacteria and fungi were recorded, as was the amount of growth (none, scant, moderate, and heavy). Genus only was determined. Gram-negative bacteria and yeast were identified using BioMeriux® biochemical strips. Gram-positive organisms were determined using gram stains, catalase test and colony morphology. All statistical analyzes were performed with the use of SAS PROC GENMOD® with the statistical significance set at P < 0.01. Because the response (presence or absence of bacteria or fungus) was binary, a logistic regression analysis with season as a factor and age of the horse were used as a covariate. A chi-squared test for goodness-of-fit was performed to determine whether the total numbers of microorganisms were uniform across season. R E S U LT S
The horses ranged from 3 to 24 years of age, with a median age of 9 years. Ninety-nine were female and one was male. Seventy horses came from one farm, 20 were from a second farm, and 10 were from a third farm. A total of 25 genera of bacteria and 35 fungi were isolated. (Tables 1 and 2). The median number of bacterial and fungal isolates for each collection period is reported in Table 3. Season had no significant effect on the number or type of microorganisms cultured from individual horses adjusted for age. A total of 42 types of gram-positive bacteria (17 cocci, 20 rods, 1 coccobacilli, 4 bacilli) and 26 types of gram-negative bacteria (24 rods, 2 coccobacilli) were isolated. The following bacteria were the most frequently recovered during all time periods, with relative numbers changing with the seasons: Corynebacterium sp. isolated 689 times (29.2%); Staphylococcus sp. isolated 513 times (21.8%); Bacillus sp. isolated 398 times (16.9%); Moraxella sp. isolated 225 times (9.6%); and nonenteric gram-negative rods isolated 167 times (7.1%). The following fungi were the most frequently recovered during all time periods with relative numbers changing with the seasons: unidentifiable mold isolated 160 times (29.6%); dematiaceous mold isolated 84 times (15.5%); Chrysosporium sp. and Cladosporium sp. isolated 44 times (8.1% each); and Aspergillus sp. isolated 30 times (5.6%). The numbers of isolates per genera are shown in Tables 1 and 2. Younger (< 5 years) horses were more likely to have gram-negative rods (P = 0.009), Moraxella sp. (P = 0.002), and a higher incidence of fungal isolates: Aspergillus sp. (P = 0.004); Chrysosporium sp. (P = 0.006); Cladosporium sp. (P = 0.002); dematiaceous mold (P = 0.001); mold (P = 0.005); and Penicillium sp. (P = 0.001). Total numbers of bacteria and fungi isolated are not uniform across seasons based on chi-squared goodness-of-fit analysis. For fungi, the P-value is 2.2 × 10−16 and for bacteria the P-value is 3.0 × 10−7.
© 2003 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 6, 45–50
47 Table 1. Bacterial genera, number of species isolated (in parenthesis following genus) and relative frequency of isolation by month January Frequency Gram-positive cocci Streptococcus sp. (7) Staphylococcus sp. (6) Micrococcus sp. (4) Gram-positive rods Corynebacterium sp. (9) Bacillus sp. (7) Dermatophilus sp. (2) Lactobacillus sp. Unidentified Gram-positive coccobacilli Rhodocococcus equi Gram-positive bacilli Actinomyces sp. (2) Streptomyces sp. (2) Gram-negative coccobacilli Moraxella sp. (2) Gram-negative rod (GNR) Unidentified GNR (9) Unidentified nonenteric GNR (5) Unidentified enteric GNR Klebsiella sp. (2) Alcaligenses sp. Escherichia coli Enterobacter sp. Proteus sp. Sphingomonas sp. Stentophomonas sp. Pasteurella sp. Pseudomonas sp. TOTAL (68 types)
April %
July
Frequency
%
Frequency
October %
Frequency
%
37 114 23
5.36% 16.52% 3.33%
11 138 18
2.04% 25.65% 3.35%
17 121 12
3.32% 23.63% 2.34%
22 140
3.57% 22.69%
212 132
30.72% 19.13%
123 125 1
22.86% 23.23% 0.19%
179 58 1 1
34.96% 11.33% 0.20% 0.20%
175 83 1
28.36% 13.45% 0.16%
6
0.87%
2
0.29%
3
0.56%
47 7
6.81% 1.01%
42 10
7.81% 1.86%
22
4.30%
24 2
3.89% 0.32%
40
5.80%
35
6.51%
55
10.74%
95
15.40%
18 48
2.61% 6.96%
6 23 1
1.12% 4.28% 0.19%
6 31
1.17% 6.05%
6 65
0.97% 10.53%
5
0.98%
1
0.14%
1
0.16%
1 1
0.20% 0.20% 1
0.16%
1 1
0.16% 0.16%
1
1 1 1
0.14% 0.14% 0.11%
2 1
690
0.19%
100%
538
DISC USSIO N
In the majority of reports detailing conjunctival microflora, gram-positive species predominate (Table 4). Twenty-four genera of bacteria were cultured from the horses in this study. Most genera were gram-positive, including Corynebacterium, Staphylococcus and Bacillus sp. These bacteria have been commonly isolated from the conjunctiva of other domestic species, such as dogs, cats, sheep and psittacine birds. Corynebacterium sp. were the most frequently isolated bacteria in January, July and October; Staphylococcus sp. were the most frequently isolated in April. In October a somewhat surprising finding was the prevalence of Moraxella sp., a gram-negative coccobacilli, as the second most common isolate because of the usual prevalence of gram-positive microbes. Environmental conditions may have contributed to the overall similarity of aerobic bacterial isolates detected in Florida horses over the four collection periods. Previous studies have suggested that seasonal conditions should have an effect on conjunctival microflora populations, but it may hold that there are no changes in populations and these microorganisms may be commensals to the horse and not be affected by changing seasons.16 In a study of horses that were
0.39%
0.19% 100%
512
100%
617
100%
housed in a stable vs. those kept in a hospital, ocular bacteria were not significantly influenced by the horses’ immediate surroundings.14 These investigators theorized that the bacteria were more permanent residents of internal and external body surfaces compared to the ocular fungi.14 In this study, multiple fungi were cultured from clinically normal equine eyes during all collection periods. Fungi are considered part of the normal ocular microflora in many species (Table 5). Given the high numbers of fungi in the horse environment, the presence of fungal contamination of the conjunctival sac is not an unexpected finding. It has been suggested that fungi found in the conjunctival fornix probably represent transient seeding from the environment.20 Previous conjunctival fungal reports in Florida horses demonstrated 95% positive cultures at one time period, with Aspergillus sp. and Cladosporium sp. being the most common isolates.2 In this study, unidentifiable molds and dematiaceous or pigmented molds were the most commonly isolated fungi during all collection periods. Most genera isolated are considered saprophytic; however, they can become pathogenic as a result of an underlying ocular pathology which lowers the eye’s resistance to fungi.2 Given that north central Florida has a moderate climate, it would follow that although the numbers of genera varied between the horses,
© 2003 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 6, 45–50
48
ET AL.
Table 2. Fungal genera, number of species isolated (in parenthesis following genus) and relative frequency of isolation by month January Frequency Unidentified mold (5) Unidentified dematiaceous mold (4) Cladosporium sp. Chrysosporium sp. Alternaria sp. Aspergillus sp. Acremonium sp. Candida sp. Curvularia sp. Dreschleria sp. Epicoccum sp. Fonecaea sp. Fusarium sp. Geotrichum sp. Gliocladium sp. Nigrospora sp. Paecilomyces sp. Papulaspora sp. Penicillium sp. Phialophora sp. Pseudallescheria sp. Rhizopus sp. (3) Scedosporum sp. Scopularis sp. Sporothrix sp. Torulopsis sp. Trichcladium sp. Trichophyton sp. Trichosporon sp. Tripospermum sp. Verticillium sp. Wallemia sp. Wangiella sp. Unidentified yeast TOTAL (43 types)
April %
July
Frequency
%
Frequency
October %
Frequency
49 42 17 9 3 8 8
26.06% 22.34% 9.04% 4.79% 1.60% 4.26% 4.26%
40 16 7 9 6 7 7
37.74% 15.09% 6.60% 8.49% 5.66% 6.60% 6.60%
12 11 10 4 2 6 2
20.00% 18.33% 16.67% 6.67% 3.33% 10.00% 3.33%
1 3
0.53% 1.60%
4 3
3.77% 2.83%
3
5.00%
1 1
0.53% 0.53%
3
2.83%
5
2.66%
1
0.94%
10
5.32%
2
1.89%
5
8.33%
8
4.26%
1
0.94%
1
1.67%
4 2 1 1 1
2.13% 1.06% 0.53% 0.53% 0.53%
3
5.00%
1
0.53% 1
13 188
106
100%
Month
Median number bacteria (range)
Median number fungi (range)
October January April July
3 (0–12) 3 (0–12) 2 (0–15) 2 (0–10)
1 (0 –6) 1 (0 –5) 0 (0 –3) 0 (0 –3)
the fungal population themselves did not. In a study of normal camelids, it was also found that cultures taken from between seasons yielded the same fungal species.19 During the sampling seasons, north central Florida had a mild winter and drought conditions during the summer months. Because saprophytic fungi flourish in warm wet environments, it is possible that changes between seasons were not seen due to unfavorable growing seasons. A longer sampling time would help to characterize this finding. The highest number of isolates were found in October and January, corresponding to fall and winter. These are also the times in the south-eastern United States when there are more clinical cases of fungal keratitis presented to veterinary medical teaching hospitals.23,24
60
60 15 10 22
32.09% 8.02% 5.35% 11.76%
9 8 3 2
4.81% 4.28% 1.60% 0.00% 1.07%
1 4 1 1 2
0.53% 2.14% 0.53% 0.53% 1.07%
1 12 2 1 15 2 3
0.53% 6.42% 1.07% 0.53% 8.02% 1.07% 1.60%
1 1 1 2
0.53% 0.53% 0.53% 1.07%
1
0.53%
2 5
1.07% 2.67%
1.67%
6.91% 100%
%
100%
187
100%
Table 3. Median number of bacterial and fungal isolates per horse eye for the four culture times
The increased incidence of gram-negative and fungal isolates in younger animals was an additional finding of this study. Further investigation would be necessary to determine its significance. The difference may be due to variability in the ocular surface defence mechanisms, as a similar observation was made in rabbits.8 In this study, a strong gender bias was also present. Because of the necessity of collecting samples from horses over a 1-year period, the most dependable population to choose was brood mares that were residing on the farms for the entire study period. Additional study of the ocular microflora of male horses would be worthwhile. A potential gender difference was noted in the study of pig conjunctival flora where the authors found that the highest number of bacterial isolates were from females.4
© 2003 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 6, 45–50
49 Table 4. Species of animal with per cent positive for conjunctival fornix bacteria with the most common isolates Species 7
Psittacine birds Exotic birds6 New Zealand white rabbit9 Domestic rabbit8 Sheep1 Cow3 Bison17 Camelid18 Dog10 Dog11 Cat13 Cat12 Pig4 Horse15 Horse16
Table 5. Species of animal with per cent positive for conjunctival fornix fungi with the most common isolates
Per cent positive
Most frequent isolates
59% (89/151) 83% (97/117) 99% (107/108) 83% (58/70) 40% (20/50) 87% (355/408) 100% (63/63)
Staphylococcus, Streptococcus, Corynebacterium Staphylococcus, Corynebacterium Bacillus, Staphylococcus, Streptococcus Staphylococcus, Micrococcus, Bacillus Neisseria, Micrococcus, Streptococcus Unidentified gram positive cocci, Corynebacterium Bacillus, Micrococcus, Staphylococcus Staphylococcus, Pseudomonas, Bacillus Staphylococcus, Bacillus, Corynebacterium Staphylococcus, Streptococcus, Corynebacterium Streptococcus, Staphylococcus Staphylococcus Streptococcus, Staphylococcus Staphylococcus, Corynebacterium, Bacillus Corynebacterium, Staphylococcus, Bacillus
78% (78/100) 91% (137/150) 4% (2/50) 67% (33/50) 98% 30% (15/50)
Speciesreference
Per cent positive
Most frequent isolates
Cow2 Camelid19 Dog2 Cat2 Horse − stable15 Horse − hospital15 Horse2
100% (25/25) 56%(52/93) 22% (11/50) 40% (10/25 67% (12/18) 68% (15/22) 95% (41/43)
Cladosporium, Penicillium Aspergillus, Fusarium, Rhinocladiella Cladosporium, Curvularia Cladosporium, Aspergillus, Penicillium Cladosporium, Alternaria, unidentified Alternaria, Aspergillus, Saccharomyces Aspergillus, Penicillium, Alternaria, Cladosporium
This finding might also be related to age as the sows tended to be the oldest pigs tested compared to younger nursery and feeder pigs.4 A significantly higher prevalence of bacterial isolates were found in male horses whether stabled or hospitalized.14 Bacillus was isolated significantly more frequently from female than male camelids.18 The statistical analysis of this study assumed that the presence of each organism is independent of the others. However, after an examination of the data, we suspect that this is not the case. Indeed, it does appear that the presence of bacteria and fungi are positively associated. There were a very low number of animals that had negative cultures (unilateral 9 in January, 19 in April, 20 in July, 6 in October; bilateral 6 in April, 3 in July). Fungal growth without bacterial growth was even less uncommon (unilateral 6 in January, 2 in April, 1 in July, 0 in October). Bacterial growth without fungal growth occurred much more frequently (unilateral 42 in January, 53 in April, 43 in July, 33 in October; bilateral 19 in January, 17 in April, 41 in July, 21 in October). An interaction between fungi and bacteria has been suggested by the presence or absence of both types of organisms compared to the presence of bacteria or fungi alone in a previous study as well.14 Investigation of the relationship between the presence of bacteria and fungi seems a potentially fruitful area for future research. Streptococcus sp. were isolated with a high overall incidence of 2–5% in each sampling period. Corneal infection and endophthalmitis caused by Streptococcus are not uncommon in horses and can be devastating.25,26 Pseudomonas sp. are
historically the cause of rapidly progressive, suppurative corneal ulceration but was isolated only one time (0.19%) during the entire sampling period. These results might suggest a role for prophylactic antimicrobial against gram-positive organisms as a first line treatment.27 In this study, normal fungal and bacterial ocular microflora were cultured from conjunctival fornices from Thoroughbred brood mares in Florida. The fact that significant seasonal changes were not found in Florida mares suggests that relatively stable environmental conditions favor constant conjunctival microflora. Additional studies are necessary to determine how the gender and age of the horse affect the ocular microbial populations, as well as to try and determine the relationship between the presence and absence of various microorganisms on other microorganisms. A C K N O W LE D G M E N T S
The authors wish to thank Dr Corey Miller and John Davis of Franks Farm South, Ocala, FL, Suzanne Maxwell of Pyrite Farm, Ocala, FL, and Bill and Annabelle Murphy of Elangeni Farm, Ocala, FL, for their participation in this study. Funding provided by the Florida Pari-mutuel Wagering Trust Fund. R E FE R E N C E S 1. Spradbrow PB. The bacterial flora of the ovine conjunctival sac. Australian Veterinary Journal of 1968; 44: 117–118.
© 2003 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 6, 45–50
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2. Samuelson DA, Andersen TL, Gwin RM. Conjunctival fungal flora in horses, cattle, dogs, and cats. Journal of the American Veterinary Medical Association 1984; 10: 1240 –1242. 3. Wilcox GE. Bacterial flora of the bovine eye with special reference to the Moraxella and Neisseria. Australian Veterinary Journal 1970; 46: 253–257. 4. Davidson HJ, Rogers DP, Yeary TJ, Stone GG, Schoneweis DA, Chengappa MM. Conjunctival microbial flora of clinically normal pigs. American Journal of Veterinary Research 1994; 55: 949 – 951. 5. Miller PE, Langenberg JA, Hartmann FA. The normal conjunctival aerobic bacterial flora of three species of captive crane. Journal of Zoo and Wildlife Medicine 1995; 26: 545 – 549. 6. Wolf ED, Amass K, Olsen J. Survey of conjunctival flora in the eye of clinically normal, captive exotic birds. Journal of the American Veterinary Medical Association 1983; 11: 1232 –1233. 7. Zenoble RD, Griffith RW, Clubb SL. Survey of bacteriologic flora of conjunctiva and cornea in healthy psittacine birds. American Journal of Veterinary Research 1983; 44: 1966 –1967. 8. Cooper SC, McLellan GJ, Rycroft AN. Conjunctival flora observed in 70 healthy domestic rabbits. Veterinary Record 2001; 149: 232 – 235. 9. Okuda H, Campbell LH. Conjunctival bacterial flora of the clinically normal New Zealand white rabbit. Laboratory Animal Science 1974; 24: 831–833. 10. McDonald PJ, Watson ADJ. Microbial flora of normal canine conjunctivae. Journal of Small Animal Practice 1976; 17: 809 – 812. 11. Urban M, Wyman M, Rheins M, Marraro RV. Conjunctival flora of clinically normal dogs. Journal of the American Veterinary Medical Association 1972; 161: 201– 206. 12. Espinola MB, Lilenbaum W. Prevalence of bacteria in the conjunctival sac and on the eyelid margin of clinically normal cats. Journal of Small Animal Practice 1996; 37: 364 – 366. 13. Shewen PE, Povey RC, Wilson MR. A survey of the conjunctival flora of clinically normal cats and cats with conjunctivitis. Canadian Veterinary Journal of 1980; 21: 231– 233. 14. Moore CP, Heller N, Majors LJ, Whitley RD, Burgess EC, Weber J. Prevalence of ocular microorganisms in hospitalized and stabled horses. American Journal of Veterinary Research 1988; 49: 773–777. 15. Whitley RD, Burgess EC, Moore CP. Microbial isolates of the normal equine eye. Equine Veterinary Journal of Supplement 1983; 2: 138–140. 16. Whitley RD, Moore CP. Microbiology of the equine eye in health
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