Eingegangen am 30. September 1972
mykosen 19 (2)63-69 0 Grosse Verlag 1976
From the Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India (Chief: Prof. Dr. L. N. MOHAPATRA)
Studies on otitis externa in dogs. 1. Survey of aetiological agents: Fungi B. K. SINHA,L. N. MOHAPATRA and R. KUMAR
Summary The bacterial and fungal flora of 200 normal and 50 infected ears of dogs were studied with the object of isolating and identifying the causative agents. Large numbers of fungi and yeasts were isolated from normal as well as infected ears. It was concluded that Pityrosporum canis may act as a primary pathogen in otitis externa in dogs and can invade the normal healthy skin of the ear canal, whereas Aspergillus niger has no capacity to establish in the normal ear canal. However, it can colonise the ears infected with bacteria and/or fungi. Zusammenfassung Die Bakterien- und Pilzflora 200 gesunder und 50 erkrankter Hundeohren wurden d u r h kulturelle Untersuchun en erfai3t und die auslosenden Ursachen bestimmt. Zahlreiche Schimmelpilze und H e en wurden sowohl von gesunden als auch von infizierten Ohren isoliert. Es wird gefolgert, dai3 Pityrosporum canis primar pathogen sein kann und durch Besiedlung der gesunden Haut des aui3eren Gehorganges bei Hunden eine Otitis externa hervorrufen kann. Aspergillus niger hingegen besitzt nicht die Eigenschaft, den gesunden Gehorgang zu befallen, sondern dieser Schimmelpilz besiedelt nur durch Bakterien undloder Pilze erkrankte Ohren.
B
Otornycosis in human beings is a well defined clinical entity (CHHANGANI et al. 1958; LAKSHMIPATI and MURTHI1960; SINHAand MOHADATRA 1961; and SOOD et al. 1966). Fifty-three different species of fungi have been recorded as aetiological agents of human otornycosis, but their role in otitis externa in dogs is not yet fullyestablished. Otitis externa in dogs is a common condition. Most of the cases have been found to be of bacterial origin (GUSTAFSON 1955; and GRONO1969), but different fungi have also been implicated in significant proporrions (AINSWORTH 1954; LAPOEVIC and CIRIC1963; and SMITH1968). The present paper deals with the survey of fungal flora found in otitis externa in dogs and in clinically normal ears, with the object of isolating and identifying the causative agents. Materials and methods Materials were collected by sterile swab sticks from the external auditory meatus of 200 normal dogs and 50 clinically suspected cases of otitis externa (Table I ) . Swabs were
brought to the laboratory and processed immediately for fungi and bacteria. The interval Key-words: otitis externa, bacterial flora, fungal flora, dogs, Pityrosporurn canis, Aspergillus niger.
B. K. SINHA, L. N. MOHAPATRA a n d R. KUMAR
64
between collection and processing was never more than one hour. All the specimens were inoculated separately on the tubes of Emmon’s modified Sabouraud’s dextrose agar a n d et al. 19631. The Sabouraud’s dextrose agar plus chloromycetin [(0.05 mg/ml) EMMONS tubes were incubated at room temperature (25-28’ C). After culturing the swabs were used to make a direct examination in 10 % potassium hydroxide (KOH) on a glass slide. The preparations thus made were examined first under low dry and then under high d r y objective of the microscope for the presence of fungal elements. The inoculated tubes were examined weekly and no tubes were discarded as negative until four weeks of incubation. Whenever the growth of filamentous fungi was accompanied by bacterial or fungal contaminants, subcultures were made on antibiotic containing slants, until a pure growth was obtained. The purification of yeasts contaminated with bacteria and/or et al. (1963). fungi was carried out according to the method described by AJELLO
Table I: The number of specimen examined from different dogs No. of dogs
No. of specimen
examined
collected
A. Infected ear 1 . O n l y one ear infectcd 2. Both ears infected
26 12
26 24
B. Normal car O n e ear normal 2. Both ears normal
16 92
16 184
Specimen
Identification of filamentom fungi: For the identification of filamentous fungi, the following techniques were used.
The colonies were examined for their various morphological characteristics, such as rate of growth, general topography, viz. fluffy, heaped, flat, round, irregular and folded, their texture (glabrous, powdery, granular, velvety and cottony). The surface pigmentation and pigmentation on the reverse side were carefully recorded. Microscopic examination of the edge of the medium and colony was done to have a preliminary idea of the type of fungus and its sporulation. A portion of the colony was removed and placed on a slide with Lactophenol cotton blue (BENEKE,1958), teased apart and covered with a coverslip. This preparation was examined under the microscope. Whenever necessary, Ridell’s slide culture method was resorted to in order to bring out the morphological details (RIDELL,1950). Identification o f yeasts: All yeasts were purified before indentification. Smears were prepared and stained with Gram’s method to study the morphology. Finally different yeasts were identified on the basis of morphology and biochemical tests. The biochemical tests employed in the differentiation of yeasts were production of urease, fermentation and assimilation of different sugars, viz. glucose, lactose, maltose and sucrose. The methods used in the biochemical tests were described by AJELLOet al. (1963). The ability of yeasts to produce mycelium or pseudomycelium were studied on corn meal agar plates (AJELLOet al. 1963). mykosen 19, Heft 2 (1976)
Studies on otitis externa in dogs. I. Survey of aetiological agent: Fungi
65
Fig. 1 : Direct 10 % KOH preparation of swab from dog suffering from otitis externa, showing spores conidiophore and mycelial fragments (ca. X 160).
Results and observations The total number of dogs investigated in this study (both normal and suffering from otitis externa) are given in Table 1. Of the 38 dogs which were suffering from otitis externa, 12 had bilateral involvement, whereas in 26 dogs only one ear was involved (Table 11).
Table 11: Frequency of involvement of ears in otitis externa No. of dogs
Dog5 w i t h otitis externa Bilateral involvement Unilateral involvement
38'' F'
26
12
No. of ears examined 50.
The findings of microscopic examination of fresh preparation (for presence of fungus) of material taken from the normal and diseased ears are shown in Table 111.
Table 111: Result of microscopic examination of fresh preparation Clinical condition
Dogs with otitis external Dogs with normal ears
mykosen 19, Heft 2 (1976)
No. of specimen positive (for fungus)
No. of sqecimen negative
50
2
48
200
-
200
No. of specimen examined
B. K. SINHA,L. N. MOHAPATRA and R. KUMAR
66
Table IV: Mycological findings of normal and infected ears Normal ears (No. of ears examined 200)
Infected ears (No. of ears examined 50)
Statistical significance
Organisms isolated Signifi-
No. of isolations
Percentage
No. of isolations
Aspergillus niger
19.0
9.5
13.0
26.0
2.7
0.01
Aspergillus f l a w s
15.0
7.5
Nil
0
7.5
0.001
Aspergillus fumigatus
30.0
15.0
1 .o
2.0
4.3
0.001
9.0
4.5
Nil
0
4.5
0.001
31.1
15.5
2.0
4.0
3.7
0.001
3.0
1.5
Nil
0
0.75
0.5 0I X 1
29.0
14.5
1 .o
2.0
4.1
0.001
Fusarium sp.
7.0
3.5
Nil
0
3.5
0.001
Hansfordia sp.
2.0
1 .o
Nil
0
1 .o
0.501X)
19.0
9.5
1.o
2.0
0.38
0.501X)
4.0
2.0
Nil
0
0.66
0.5OlXl
10.0
5.0
1 .o
2.0
1.5
O.lO(X1
Scopulariopsis sp.
4.0
2.0
Nil
0
0.66
0.50(Xl
Syncephalastrum s p .
2.0
1 .o
Nil
0
0.50
0.501X)
Pityrosporum canis
4.0
2.0
6.0
12.0
2.5
0.01
Unclassified yeasts
7.0
3.5
1 .o
2.0
0.75
0.05IX)
Aspergillus nidulans Alternaria s p . Cephalosporium sp. Curvularia sp.
Helminthosporium sp. Penicillium s p . Rhizopus sp.
N. B.:
-
Percentage
of cance level
"P"
(x) Not significant.
Of the 50 specimens examined from the otitis material, only two were positive in fresh preparation for fungal spores and mycelial fragments (Fig. I ) . The fungal isolations from the normal and infected ears are presented in T a b l e ZV. The above table shows that a large number of fungi was isolated from normal ears. The only significant difference was in the rate of isolation of Aspergillus niger and Pityrosporum canis from infected ears. The percentage isolation of these two fungi in infected ears was 26.0 and 12.0 respectively as compared to normal ears, 9.5 and 2.0 respectively. Table V presents the actual position (single or in combination) of the organisms which were isolated from the ears of dogs suffering from otitis externa. mykosen 19,Heft 2 (1976)
Studies on otitis externa in dogs. I. Survey of aetiological agent: Fungi
67
Table V: Aetiological agents isolated from infected ears Organisms
Aspergil- Pityrosporum Staphylococcus lus niger canis aureus
Proteus vulgaris
No. growth
Total
Aspergillus niger
3
0
8
2
0
13
Pityrosporum canis
0
4
2
0
0
6
No. growth
0
0
0
0
2
2
N. B.:
- Diagonal figures represent the organisms which occurred singly.
I t is evident from the above table that out of 50 infected ears fungi alone were isolated from seven ears only and in all only one type of fungus was present i. e. Aspergillus niger ( 3 ears) and Pityrosporum canis (4 ears) in the percentages of 6 and 8 respectively. Bacteria and fungi together, as aetiological agents were isolated from 12 ears. The combinations seen were Aspergillus niger plus Stupbylococcus aureus, Aspergillus niger plus Proteus mirabilis and Pityrosporum canis plus Staphylococcus aureus giving the percentages of 16,4 and 4 respectively. There was no growth in two ears and the remaining ears yielded bacterial and fungal species which were statistically insignificant in comparison to the isolations made from the normal ears (Table ZV).
Discussion In India, no diligent attempt has been made to study the role of fungi in otitis externa in dogs. Only scattered reports on examination of a few animals are available by workers, who could incidentally get the material for examination. Mycotic infections of domestic animals are important not only because of direct loss caused by the disease but because domestic animal may act as disseminating hosts and provide a source of human infection. I t ,is often seen that the several microbial species (both fungi and bacteria) are commonly found in infected as well as normal ears. However, some microbes are isolated much more frequently from the infected ears as compared to the normal ones and therefore their aetiological significance can not be ignored. Microbes isolated only from normal ears or from both normal and infected ears with the same frequency can a t best be regarded as normal resident flora. From the data in this study, it is evident that Aspergillus niger was isolated from 13 out of 50 infected and 19 out of 200 normal ears giving the percentages of 26.0 and 9.5 respectively. I t is interesting to note that Aspergillus niger was mostly found in association with the other pathogenic organisms (Table V). I t occurred with Staphylococcus aureus and Proteus mirabilis in 8 and 2 infected ears respectively. Only in 3 ears of otit.is externa Aspergillus niger was isolated alone, in two of which direct preparation of the specimen in 10 5% K O H revealed the presence of spores and mycelial elements. This confirms the aeriological relationship of Aspergillus niger as a cause of otitis externa in dogs a t least mykosen 19, Heft 2 (1976)
68
B. K. SINHA,L. N. MOHAPATRA and R. KUMAR
in these two cases. From history, it was found that one of these two dogs was having otitis externa for more than a year and was continuously on antibiotic therapy. I n the remaining 11 ears where Aspergiftus niger was isolated in culture, no mycelial elements could be demonstrated in direct 10 X KOH preparation. Therfore, it is ddficult to assign the role of Aspergillus niger in these cases. It is, however evident that Aspergillus niger thrives in ears intected with bacteria. I t is possible, if these ears are treated w(ith antibacterial agents, the fungus may keep on thriving and perpetuate the infection. This is what may have happened in one of the two dogs mentioned above. The present investigations support the view of PHILIPS (1953), who considers that otomycosis uncomplicated with pyogenic infection is rellatively rare in dogs. LAPOEVIC and CIRIC(1963) demonstrated Aspergillus niger in three cases of otitis externa and claimed it as an aetiological agent. AINSWORTH (1954) isolated Aspergillus fumigatus from 2 % of cases of onitis externa of dogs. H e considers that these saprophytic moulds may act as commensals but may become virulent and lethal pathogens in certain circumstances. The large number of fungi which occurred in normal ears in significant number, i. e. Aspergillus flaws, Aspergilllus fumigatus, Aspergillus nidulans, Alternaria sp., Curvularia sp. and Fusarium sp. giving the percentages of 7.5, 15.0, 4.5, 15.5, 14.5 and 3.5 respectively should be regarded as being fully normal specially because the skin in the external auditory meatus gave the impression of being intact and that the dog was normal without any irritation. As these fungi are ubiquitous in nature they can gain entrance in the external auditory meatus and can settle there and even multiply to a certain extent. Their significantly higher isolations from the normal ears in comparison with infected ones can be easily explained on the basis tbat they may have no capacity t o multiply in competition with other microorganisms responsible for infection. The following fungi i. e. Cephalosporium sp., Hansfordia sp., Helminthosporium sp., Penicillium sp., Rhizopus sp., Scopulariopsis sp., Syncephalastrum sp. and unclassified yeasts were isolated in equal proporDions from both normal and .infected ears (Table ZV). There is no evidence to support their aetiological relationship with the infection. Apart from filamentous fungi, yeasts play an important role in the otitis externa of human beings and dogs. Otitis externa in dog caused by Cryprococcus neoformans has et al. (1968). FRASER (1961) is of the opinion that Candida sp. been reported by WAGNER occasionally give rise to acute mycotic otitis as a result of prolonged antibiotic therapy. There appear to be different opinions regarding the incidence of Pityrosporum canis in the ears of normal dogs and dogs with otitis externa. Although FRASER(1961) considers that the part played by Pityrosporum sp. in initiating infection is not obvious as their incidence in the external ear canal of both healthy and infected dogs is very similar. (1960) consider Pityrosporum sp. to be possible GUSTAFSON (1955) and MANKTELOW aetiological factors in canine otitis externa. I n the present study Pityrosporum canis was isolated from 2 % and 12 % of normal and infected ears respectively. Of the 12 % (6 isolations) of infected ears, in 33 % (2 iso1,ations)only it occurred with Staphylococcus aureus. These flindings indicate that Pityrosporum canis can be an important pathogen of otitis externa in dogs. I n two cases, where it was found in association with Staphylococcus aureus, it is difficult to say which of them was primary. SMITH(1968) isolated Pityrosporum canis from 21 of 22 otitic ears (95 %). This high incidence of Pityrosporum canis in otitis externa of dogs has not been reported by any other workers.
Acknowledgement: The authors are grateful to Dr. J. S. VERMA, YMotibagh Veterinary Hospital, N e w Delhi, India, who very kindly helped us in collection of materials. mykosen 19, Heft 2 (1976)
Studies on otitis externa in dogs. I. Survey of aetiological agent: Fungi
69
References G. C., 1954: Fungoid Infec1. AINSWORTH, tion of Animals in Britain. Vet. Rec. 66, 844. & L. 2. AJELLO,L., L. K. GEORG,W. KAPLAN 1963: Lab. Mannual for Med. KAUFFMAN, Mycol. Pub. Hlth. Service, C. D. C.-At3.
4.
5.
6. 7. 8.
9.
lanta, Georgia. BENEKE,E. S., 1958: Medical Mycology Laboratory Manual, Burgess Pub. CO., Minnesota. CHHANGANI, D. L., S. P. GUPTA& R. N. MJSHRA,1958: Otomycosis. Ind. J. Otolaryng. 10, 1. EMMONS, C., W. BINFORDSr J. P. UTZ, 1963: Medical Mycology. Lea and Febiger, Philadelphia. FRASER,G., 1961: The Fungal Flora of the Canine ear. J. Comp. Path. 71, 1 . GRONO,L. R., 1969: Studies on the otitis externa in Dog. Aust. Vet. Jour. 45, 9. GUSTAFSON, B., 1955: Otitis externa in the Dog. A bacteriological and Experimental Study. Thesis, Royal Veterinary College. Stockholm. G. & R. B. MURTHI,1960: LAKSHMIPATI, Otomycosis. J. Indian. Med. Assoc. 34, 439.
E. & V. CIRIC,1963: Aspergil10. LAPOEVIC, lus as the cause of otitis externa in Dogs. Vet. Glas. 17, 105. 11. MANKTELOW, B. W., 1960: Yeast of the
12. 13.
14. 15. 16.
17.
Genus Pityrosporum in the mammalian external auditory canal with special reference to Dogs. N. Z. Vet. J. 8, 76. PHILIPS, S. E., 1953: "Canine Medicine", ed. Hoskin and Lacroix, Am. Vet. Pub., Illinois. RIDELL,R. H., 1950: Slide culture t e h nique. Cited by AJELLOet al. in Lab. manual for Med. Mycol. Pub. Hlth. Service, C. D. C.-Atlanta, Georgia. SINHA,A. & L. N. MOHAPATRA, 1961 : Otomycosis; A clinical and mycological study. Ind. J. Otolaryng. 13, 3. SMITH,J. N. B., 1968: The association of Yeast with chronic otitis externa in the dog. Aust. Vet. Jour. 44, 9. SOOD,V. P., A. SINHA& L. N. MOHAPATRA, 1966: Fungal flora of normal and otomycosis ears of human ear canal. Ind. J. Microbiol. 6, 4. WAGNER,J. L., J. R. PICK& M. R. KRIGMAN, 1968: Cryptococcus neoformans infection in a dog. J. Am. Vet. Med. Assoc. 153, 945.
Address of the authors: Junior Assistant Research Officer, Livestock Research Station, Patna-14, Bihar, India.
1
Referate
j
KRAKOWKA, P., E. R O W I ~ ~ Sund K AH. HALWEG: (Institut fur Tuberkulose in Warszawa) Pleuraaspergillose Grzybica kropidlakowa ophcnej Ann. Acad. Med. Lodz., XIII, Suplement 7, 21-23
(1971)
In den Jahren 1960-1968 wurden bei TBC-Kranken mit Empyema pleurae und einer bronchopleuralen Fistel 10 Falle mit Pleuraaspergillose festgestellt. Bei 9 Kranken wurde die Diagnose durch mykologische (Aspergillus fumigatus im Sputum und im Eiter aus dem Cavum pleurae) und serologische Untersuchungen, bei 1 durch histologische Untersuchungen nach der Sektion gestellt. Von 7 lokal mit Nystatin bzw. Amphotericin B behandelten Kranken wurden 6 frei von Krankheitserregern (Aspergillus fumigatus). F. SOKOLOWSKI mykosen 19, Heft 2 (1976)
mykosen 19 (2)63-69 0 Grosse Verlag 1976
From the Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India (Chief: Prof. Dr. L. N. MOHAPATRA)
Studies on otitis externa in dogs. 1. Survey of aetiological agents: Fungi B. K. SINHA,L. N. MOHAPATRA and R. KUMAR
Summary The bacterial and fungal flora of 200 normal and 50 infected ears of dogs were studied with the object of isolating and identifying the causative agents. Large numbers of fungi and yeasts were isolated from normal as well as infected ears. It was concluded that Pityrosporum canis may act as a primary pathogen in otitis externa in dogs and can invade the normal healthy skin of the ear canal, whereas Aspergillus niger has no capacity to establish in the normal ear canal. However, it can colonise the ears infected with bacteria and/or fungi. Zusammenfassung Die Bakterien- und Pilzflora 200 gesunder und 50 erkrankter Hundeohren wurden d u r h kulturelle Untersuchun en erfai3t und die auslosenden Ursachen bestimmt. Zahlreiche Schimmelpilze und H e en wurden sowohl von gesunden als auch von infizierten Ohren isoliert. Es wird gefolgert, dai3 Pityrosporum canis primar pathogen sein kann und durch Besiedlung der gesunden Haut des aui3eren Gehorganges bei Hunden eine Otitis externa hervorrufen kann. Aspergillus niger hingegen besitzt nicht die Eigenschaft, den gesunden Gehorgang zu befallen, sondern dieser Schimmelpilz besiedelt nur durch Bakterien undloder Pilze erkrankte Ohren.
B
Otornycosis in human beings is a well defined clinical entity (CHHANGANI et al. 1958; LAKSHMIPATI and MURTHI1960; SINHAand MOHADATRA 1961; and SOOD et al. 1966). Fifty-three different species of fungi have been recorded as aetiological agents of human otornycosis, but their role in otitis externa in dogs is not yet fullyestablished. Otitis externa in dogs is a common condition. Most of the cases have been found to be of bacterial origin (GUSTAFSON 1955; and GRONO1969), but different fungi have also been implicated in significant proporrions (AINSWORTH 1954; LAPOEVIC and CIRIC1963; and SMITH1968). The present paper deals with the survey of fungal flora found in otitis externa in dogs and in clinically normal ears, with the object of isolating and identifying the causative agents. Materials and methods Materials were collected by sterile swab sticks from the external auditory meatus of 200 normal dogs and 50 clinically suspected cases of otitis externa (Table I ) . Swabs were
brought to the laboratory and processed immediately for fungi and bacteria. The interval Key-words: otitis externa, bacterial flora, fungal flora, dogs, Pityrosporurn canis, Aspergillus niger.
B. K. SINHA, L. N. MOHAPATRA a n d R. KUMAR
64
between collection and processing was never more than one hour. All the specimens were inoculated separately on the tubes of Emmon’s modified Sabouraud’s dextrose agar a n d et al. 19631. The Sabouraud’s dextrose agar plus chloromycetin [(0.05 mg/ml) EMMONS tubes were incubated at room temperature (25-28’ C). After culturing the swabs were used to make a direct examination in 10 % potassium hydroxide (KOH) on a glass slide. The preparations thus made were examined first under low dry and then under high d r y objective of the microscope for the presence of fungal elements. The inoculated tubes were examined weekly and no tubes were discarded as negative until four weeks of incubation. Whenever the growth of filamentous fungi was accompanied by bacterial or fungal contaminants, subcultures were made on antibiotic containing slants, until a pure growth was obtained. The purification of yeasts contaminated with bacteria and/or et al. (1963). fungi was carried out according to the method described by AJELLO
Table I: The number of specimen examined from different dogs No. of dogs
No. of specimen
examined
collected
A. Infected ear 1 . O n l y one ear infectcd 2. Both ears infected
26 12
26 24
B. Normal car O n e ear normal 2. Both ears normal
16 92
16 184
Specimen
Identification of filamentom fungi: For the identification of filamentous fungi, the following techniques were used.
The colonies were examined for their various morphological characteristics, such as rate of growth, general topography, viz. fluffy, heaped, flat, round, irregular and folded, their texture (glabrous, powdery, granular, velvety and cottony). The surface pigmentation and pigmentation on the reverse side were carefully recorded. Microscopic examination of the edge of the medium and colony was done to have a preliminary idea of the type of fungus and its sporulation. A portion of the colony was removed and placed on a slide with Lactophenol cotton blue (BENEKE,1958), teased apart and covered with a coverslip. This preparation was examined under the microscope. Whenever necessary, Ridell’s slide culture method was resorted to in order to bring out the morphological details (RIDELL,1950). Identification o f yeasts: All yeasts were purified before indentification. Smears were prepared and stained with Gram’s method to study the morphology. Finally different yeasts were identified on the basis of morphology and biochemical tests. The biochemical tests employed in the differentiation of yeasts were production of urease, fermentation and assimilation of different sugars, viz. glucose, lactose, maltose and sucrose. The methods used in the biochemical tests were described by AJELLOet al. (1963). The ability of yeasts to produce mycelium or pseudomycelium were studied on corn meal agar plates (AJELLOet al. 1963). mykosen 19, Heft 2 (1976)
Studies on otitis externa in dogs. I. Survey of aetiological agent: Fungi
65
Fig. 1 : Direct 10 % KOH preparation of swab from dog suffering from otitis externa, showing spores conidiophore and mycelial fragments (ca. X 160).
Results and observations The total number of dogs investigated in this study (both normal and suffering from otitis externa) are given in Table 1. Of the 38 dogs which were suffering from otitis externa, 12 had bilateral involvement, whereas in 26 dogs only one ear was involved (Table 11).
Table 11: Frequency of involvement of ears in otitis externa No. of dogs
Dog5 w i t h otitis externa Bilateral involvement Unilateral involvement
38'' F'
26
12
No. of ears examined 50.
The findings of microscopic examination of fresh preparation (for presence of fungus) of material taken from the normal and diseased ears are shown in Table 111.
Table 111: Result of microscopic examination of fresh preparation Clinical condition
Dogs with otitis external Dogs with normal ears
mykosen 19, Heft 2 (1976)
No. of specimen positive (for fungus)
No. of sqecimen negative
50
2
48
200
-
200
No. of specimen examined
B. K. SINHA,L. N. MOHAPATRA and R. KUMAR
66
Table IV: Mycological findings of normal and infected ears Normal ears (No. of ears examined 200)
Infected ears (No. of ears examined 50)
Statistical significance
Organisms isolated Signifi-
No. of isolations
Percentage
No. of isolations
Aspergillus niger
19.0
9.5
13.0
26.0
2.7
0.01
Aspergillus f l a w s
15.0
7.5
Nil
0
7.5
0.001
Aspergillus fumigatus
30.0
15.0
1 .o
2.0
4.3
0.001
9.0
4.5
Nil
0
4.5
0.001
31.1
15.5
2.0
4.0
3.7
0.001
3.0
1.5
Nil
0
0.75
0.5 0I X 1
29.0
14.5
1 .o
2.0
4.1
0.001
Fusarium sp.
7.0
3.5
Nil
0
3.5
0.001
Hansfordia sp.
2.0
1 .o
Nil
0
1 .o
0.501X)
19.0
9.5
1.o
2.0
0.38
0.501X)
4.0
2.0
Nil
0
0.66
0.5OlXl
10.0
5.0
1 .o
2.0
1.5
O.lO(X1
Scopulariopsis sp.
4.0
2.0
Nil
0
0.66
0.50(Xl
Syncephalastrum s p .
2.0
1 .o
Nil
0
0.50
0.501X)
Pityrosporum canis
4.0
2.0
6.0
12.0
2.5
0.01
Unclassified yeasts
7.0
3.5
1 .o
2.0
0.75
0.05IX)
Aspergillus nidulans Alternaria s p . Cephalosporium sp. Curvularia sp.
Helminthosporium sp. Penicillium s p . Rhizopus sp.
N. B.:
-
Percentage
of cance level
"P"
(x) Not significant.
Of the 50 specimens examined from the otitis material, only two were positive in fresh preparation for fungal spores and mycelial fragments (Fig. I ) . The fungal isolations from the normal and infected ears are presented in T a b l e ZV. The above table shows that a large number of fungi was isolated from normal ears. The only significant difference was in the rate of isolation of Aspergillus niger and Pityrosporum canis from infected ears. The percentage isolation of these two fungi in infected ears was 26.0 and 12.0 respectively as compared to normal ears, 9.5 and 2.0 respectively. Table V presents the actual position (single or in combination) of the organisms which were isolated from the ears of dogs suffering from otitis externa. mykosen 19,Heft 2 (1976)
Studies on otitis externa in dogs. I. Survey of aetiological agent: Fungi
67
Table V: Aetiological agents isolated from infected ears Organisms
Aspergil- Pityrosporum Staphylococcus lus niger canis aureus
Proteus vulgaris
No. growth
Total
Aspergillus niger
3
0
8
2
0
13
Pityrosporum canis
0
4
2
0
0
6
No. growth
0
0
0
0
2
2
N. B.:
- Diagonal figures represent the organisms which occurred singly.
I t is evident from the above table that out of 50 infected ears fungi alone were isolated from seven ears only and in all only one type of fungus was present i. e. Aspergillus niger ( 3 ears) and Pityrosporum canis (4 ears) in the percentages of 6 and 8 respectively. Bacteria and fungi together, as aetiological agents were isolated from 12 ears. The combinations seen were Aspergillus niger plus Stupbylococcus aureus, Aspergillus niger plus Proteus mirabilis and Pityrosporum canis plus Staphylococcus aureus giving the percentages of 16,4 and 4 respectively. There was no growth in two ears and the remaining ears yielded bacterial and fungal species which were statistically insignificant in comparison to the isolations made from the normal ears (Table ZV).
Discussion In India, no diligent attempt has been made to study the role of fungi in otitis externa in dogs. Only scattered reports on examination of a few animals are available by workers, who could incidentally get the material for examination. Mycotic infections of domestic animals are important not only because of direct loss caused by the disease but because domestic animal may act as disseminating hosts and provide a source of human infection. I t ,is often seen that the several microbial species (both fungi and bacteria) are commonly found in infected as well as normal ears. However, some microbes are isolated much more frequently from the infected ears as compared to the normal ones and therefore their aetiological significance can not be ignored. Microbes isolated only from normal ears or from both normal and infected ears with the same frequency can a t best be regarded as normal resident flora. From the data in this study, it is evident that Aspergillus niger was isolated from 13 out of 50 infected and 19 out of 200 normal ears giving the percentages of 26.0 and 9.5 respectively. I t is interesting to note that Aspergillus niger was mostly found in association with the other pathogenic organisms (Table V). I t occurred with Staphylococcus aureus and Proteus mirabilis in 8 and 2 infected ears respectively. Only in 3 ears of otit.is externa Aspergillus niger was isolated alone, in two of which direct preparation of the specimen in 10 5% K O H revealed the presence of spores and mycelial elements. This confirms the aeriological relationship of Aspergillus niger as a cause of otitis externa in dogs a t least mykosen 19, Heft 2 (1976)
68
B. K. SINHA,L. N. MOHAPATRA and R. KUMAR
in these two cases. From history, it was found that one of these two dogs was having otitis externa for more than a year and was continuously on antibiotic therapy. I n the remaining 11 ears where Aspergiftus niger was isolated in culture, no mycelial elements could be demonstrated in direct 10 X KOH preparation. Therfore, it is ddficult to assign the role of Aspergillus niger in these cases. It is, however evident that Aspergillus niger thrives in ears intected with bacteria. I t is possible, if these ears are treated w(ith antibacterial agents, the fungus may keep on thriving and perpetuate the infection. This is what may have happened in one of the two dogs mentioned above. The present investigations support the view of PHILIPS (1953), who considers that otomycosis uncomplicated with pyogenic infection is rellatively rare in dogs. LAPOEVIC and CIRIC(1963) demonstrated Aspergillus niger in three cases of otitis externa and claimed it as an aetiological agent. AINSWORTH (1954) isolated Aspergillus fumigatus from 2 % of cases of onitis externa of dogs. H e considers that these saprophytic moulds may act as commensals but may become virulent and lethal pathogens in certain circumstances. The large number of fungi which occurred in normal ears in significant number, i. e. Aspergillus flaws, Aspergilllus fumigatus, Aspergillus nidulans, Alternaria sp., Curvularia sp. and Fusarium sp. giving the percentages of 7.5, 15.0, 4.5, 15.5, 14.5 and 3.5 respectively should be regarded as being fully normal specially because the skin in the external auditory meatus gave the impression of being intact and that the dog was normal without any irritation. As these fungi are ubiquitous in nature they can gain entrance in the external auditory meatus and can settle there and even multiply to a certain extent. Their significantly higher isolations from the normal ears in comparison with infected ones can be easily explained on the basis tbat they may have no capacity t o multiply in competition with other microorganisms responsible for infection. The following fungi i. e. Cephalosporium sp., Hansfordia sp., Helminthosporium sp., Penicillium sp., Rhizopus sp., Scopulariopsis sp., Syncephalastrum sp. and unclassified yeasts were isolated in equal proporDions from both normal and .infected ears (Table ZV). There is no evidence to support their aetiological relationship with the infection. Apart from filamentous fungi, yeasts play an important role in the otitis externa of human beings and dogs. Otitis externa in dog caused by Cryprococcus neoformans has et al. (1968). FRASER (1961) is of the opinion that Candida sp. been reported by WAGNER occasionally give rise to acute mycotic otitis as a result of prolonged antibiotic therapy. There appear to be different opinions regarding the incidence of Pityrosporum canis in the ears of normal dogs and dogs with otitis externa. Although FRASER(1961) considers that the part played by Pityrosporum sp. in initiating infection is not obvious as their incidence in the external ear canal of both healthy and infected dogs is very similar. (1960) consider Pityrosporum sp. to be possible GUSTAFSON (1955) and MANKTELOW aetiological factors in canine otitis externa. I n the present study Pityrosporum canis was isolated from 2 % and 12 % of normal and infected ears respectively. Of the 12 % (6 isolations) of infected ears, in 33 % (2 iso1,ations)only it occurred with Staphylococcus aureus. These flindings indicate that Pityrosporum canis can be an important pathogen of otitis externa in dogs. I n two cases, where it was found in association with Staphylococcus aureus, it is difficult to say which of them was primary. SMITH(1968) isolated Pityrosporum canis from 21 of 22 otitic ears (95 %). This high incidence of Pityrosporum canis in otitis externa of dogs has not been reported by any other workers.
Acknowledgement: The authors are grateful to Dr. J. S. VERMA, YMotibagh Veterinary Hospital, N e w Delhi, India, who very kindly helped us in collection of materials. mykosen 19, Heft 2 (1976)
Studies on otitis externa in dogs. I. Survey of aetiological agent: Fungi
69
References G. C., 1954: Fungoid Infec1. AINSWORTH, tion of Animals in Britain. Vet. Rec. 66, 844. & L. 2. AJELLO,L., L. K. GEORG,W. KAPLAN 1963: Lab. Mannual for Med. KAUFFMAN, Mycol. Pub. Hlth. Service, C. D. C.-At3.
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lanta, Georgia. BENEKE,E. S., 1958: Medical Mycology Laboratory Manual, Burgess Pub. CO., Minnesota. CHHANGANI, D. L., S. P. GUPTA& R. N. MJSHRA,1958: Otomycosis. Ind. J. Otolaryng. 10, 1. EMMONS, C., W. BINFORDSr J. P. UTZ, 1963: Medical Mycology. Lea and Febiger, Philadelphia. FRASER,G., 1961: The Fungal Flora of the Canine ear. J. Comp. Path. 71, 1 . GRONO,L. R., 1969: Studies on the otitis externa in Dog. Aust. Vet. Jour. 45, 9. GUSTAFSON, B., 1955: Otitis externa in the Dog. A bacteriological and Experimental Study. Thesis, Royal Veterinary College. Stockholm. G. & R. B. MURTHI,1960: LAKSHMIPATI, Otomycosis. J. Indian. Med. Assoc. 34, 439.
E. & V. CIRIC,1963: Aspergil10. LAPOEVIC, lus as the cause of otitis externa in Dogs. Vet. Glas. 17, 105. 11. MANKTELOW, B. W., 1960: Yeast of the
12. 13.
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Genus Pityrosporum in the mammalian external auditory canal with special reference to Dogs. N. Z. Vet. J. 8, 76. PHILIPS, S. E., 1953: "Canine Medicine", ed. Hoskin and Lacroix, Am. Vet. Pub., Illinois. RIDELL,R. H., 1950: Slide culture t e h nique. Cited by AJELLOet al. in Lab. manual for Med. Mycol. Pub. Hlth. Service, C. D. C.-Atlanta, Georgia. SINHA,A. & L. N. MOHAPATRA, 1961 : Otomycosis; A clinical and mycological study. Ind. J. Otolaryng. 13, 3. SMITH,J. N. B., 1968: The association of Yeast with chronic otitis externa in the dog. Aust. Vet. Jour. 44, 9. SOOD,V. P., A. SINHA& L. N. MOHAPATRA, 1966: Fungal flora of normal and otomycosis ears of human ear canal. Ind. J. Microbiol. 6, 4. WAGNER,J. L., J. R. PICK& M. R. KRIGMAN, 1968: Cryptococcus neoformans infection in a dog. J. Am. Vet. Med. Assoc. 153, 945.
Address of the authors: Junior Assistant Research Officer, Livestock Research Station, Patna-14, Bihar, India.
1
Referate
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KRAKOWKA, P., E. R O W I ~ ~ Sund K AH. HALWEG: (Institut fur Tuberkulose in Warszawa) Pleuraaspergillose Grzybica kropidlakowa ophcnej Ann. Acad. Med. Lodz., XIII, Suplement 7, 21-23
(1971)
In den Jahren 1960-1968 wurden bei TBC-Kranken mit Empyema pleurae und einer bronchopleuralen Fistel 10 Falle mit Pleuraaspergillose festgestellt. Bei 9 Kranken wurde die Diagnose durch mykologische (Aspergillus fumigatus im Sputum und im Eiter aus dem Cavum pleurae) und serologische Untersuchungen, bei 1 durch histologische Untersuchungen nach der Sektion gestellt. Von 7 lokal mit Nystatin bzw. Amphotericin B behandelten Kranken wurden 6 frei von Krankheitserregern (Aspergillus fumigatus). F. SOKOLOWSKI mykosen 19, Heft 2 (1976)
