International Journal of Agricultural

Science and Research (IJASR)

ISSN (P): 2250-0057; ISSN (E): 2321-0087
Vol. 8, Issue 2 Apr 2018, 19-24
© TJPRC Pvt. Ltd.

ISOLATION AND IDENTIFICATION OF PATHOLOGICAL

AGENTS INDOGS WITH OTITIS EXTERNA

KASHYAP ROSHAN1, ROY MANJU2 & ROY SUSHOVAN3

1
M. V. Sc Scholar, Veterinary Clinical Medicine, College of Veterinary Science & A. H., Anjora, Durg, Chhattisgarh, India
2
Associate Professor, Veterinary Physiology & Biochemistry, College of Veterinary
Science & A. H., Bilaspur, Chhattisgarh, India
3
Professor & Head, Veterinary Medicine, College of Veterinary Science & A. H., Anjora, Durg, Chhattisgarh, India
ABSTRACT

The present study was conducted to record the suitable cytological diagnostic procedure followed by the
isolation and identification of important pathogens causing external otitis in dogs. Out of total 251 dogs presented in
Veterinary Hospitals of Drug district, ear swabs were obtained from 51 dogs of 1 to 8 years with clinically diagnosed otitis.
Samples collected were grown on special culture media and identification was based on morphological and biochemical
characters of cultured for aerobic and anaerobic bacteria, using standard microbiological technique to diagnose isolates.

Original Article
Staphylococcus Spp was found to be most common bacterial isolate (52.94%) responsible for causation of otitis externa in
dogs followed by Pseudomonas spp. (21.56%). However, other bacteria and yeast contributed for less than 10% of cases
respectively.

KEYWORDS: Dog, Otitis Externa, Bacteria, Identification, Diagnosis & Biochemical

Received: Jan 06, 2018; Accepted: Jan 27, 2018; Published: Feb 22, 2018; Paper Id.: IJASRAPR20183

INTRODUCTION

Otitis externa is defined as an acute or chronic inflammation of the epithelium of the external ear canal
which may also involve the pinna. It is one of the most common and multifactorial disorders, accounting for up to
10 to 20% of consultations in canine practice (Senthil et al., 2010). The etiological factors of otitis externa can be
parasites, foreign bodies, hypothyroidism and keratinizing diseases those responsible for initiation of the
inflammatory process in the ear and are all skin problems (Zur et al., 2011). Predisposing factors do change in the
microclimate of ear canal (Paterson, 2016).

Clinical signs of otitis externa include head shaking, ear scratching and pain on palpation of inner ear
pinna and the canal are usually erythematous and swollen Pinnal alopecia, excoriations and different colored crusts
are also noticed. In chronic cases hyperpigmentation, pinnal hyperkeratosis and as well as ear canal stenosis from
fibrosisare common (Sander, 2001)

Otitis externa occurs for the first time in middle-aged or older dogs, most frequently observed in dogs of
five to eight years of age (Paterson, 2002). Environmental factors may influence the incidence of otitis externa in
canines. It is more prevalent in the areas of high temperature and humid environment (Angus, 2004). Our study
aimed to investigate and identify through the bacterial, fungal and yeast agents present in ear swabs of patients with
otitis externa.

www.tjprc.org editor@tjprc.org
20 Kashyap Roshan, Roy Manju & Roy Sushovan

MATERIAL AND METHODS

A total of 281 dogs irrespective of age, breed and sex both healthy as well as dogs seeking therapeutic
intervention presented to the Teaching Veterinary Clinical Complex, College of Veterinary Science and Animal Husbandry
Anjora Durg (C. G.) and various Government Veterinary Hospitals in and around Durg were studied for the period of one
year from July, 2016, to June, 2017. Fifty one belonging to 24 breeds and aged between 6 months and 12 years is
represented by the pathological ear secretions. The detailed information regarding age, breed, gender, onset of symptoms
and other relevant information was collected from the owners. Ear exudates samples were obtained by inserting sterile
swabs to the junction of vertical and horizontal external ear canal. Dogs were clinically examined for their normal
appearance and diseased condition. Patient’s data, owner’s complaint and clinical signs of disease were recorded.
Restlessness and frequent ear itching were accompanied by ottorrhoea with discharge varying from yellowish brown to
white purulent in color. The most common clinical signs observed included head shaking, ear scratching, pain on palpation
of the ear, ear rubbing, and tilting of head to one side.

Otic exudates, cerumen, roll swab smears, tape impression smears were collected from the affected dogs for
laboratory examination and diagnostic study. Samples were collected, grown on special culture media for the isolation of
causative agents and identification on the basis of morphological and biochemical characters of microorganisms
(Balappanavar, &Vasanth, 2013).

Isolation and Identification of Micro Organism

Morphology of Organism

Bacterial smears were prepared by mixing 24 hour old cultures with sterile saline on a clean microscopic slide.
The smear was then air dried, heat fixed over flame and then stained with Gram’s stain and examined under oil immersion
lens. The bacteria were studied and each isolate was recorded as being gram positive cocci or gram negative, cocci, bacilli
or coccobacilli.

Yeast Identification

For isolation of yeast otic exudates were inoculated on brain heart infusion agar and Sabouraud’s dextrose
followed by incubated at 300C. The identification of yeast was done on colony and microscopic features produced on brain
heart infusion and Sabouraud’s dextrose agar.

Identification of Bacteria

The collected otic inoculum was inoculated into the nutrient broth and incubated at 370C for 24-48 hours. The
growth of enriched inoculums from all the samples was streaked on primary media like nutrient agar and blood agar, and
incubated at 370C for 24-48 hours. Colonial morphology on culture, media and microscopic morphology of gram stained
smears of the representative colonies were studied. Further, the identified colonies were streaked on selective and
differential media like Mannitol Salt agar, Pseudomonas isolation agar, MacConkey agar, Eosin-methylene blue agar, Urea
agar, Simmons citrate agar (HiMedia Laboratory Ltd., Mumbai) and incubated at 370C for 24 to 48 hours. The inoculated
plates were examined for morphological characteristics and growth of bacterial colonies after incubation period. The
isolates were then identified on the basis of colony characteristics, staining characteristics (after staining with Gram’sstain)
microscopic morphology, catalase test, slide coagulase test, and mannitol fermenting ability (Quinn, 1999).

Impact Factor (JCC): 5.9857 NAAS Rating: 4.13

Isolation and Identification of Pathological Agents in Dogs with Otitis Externa 21

RESULTS AND DISCUSIONS

A total of 51 clinically positive dogs was screened for the presence of microorganisms by cytological
examination. In the present investigation the diagnosis of canine otitis externausing tape impression smear (68.62 %) is
more as compared to other methods like roll swab and direct impression smear method. Our findings are in accordance
with Shaw, (2016) who also reported that tape impression smear was superior as compared to other tests like collecting
surface skin debris and scales for diagnosis of otitis external. Present investigation recorded that with tape impression
smear, more number of cocci (71.4%) were observed, followed by bacilli (40%) and yeast (11.42%).

The samples found positive by tape impression smears were subjected to microbial isolation and identification.
A total of 47 bacterial isolate were obtained which included maximum occurrence of Staphylocoocus followed by
Pseudomonas, Streptococcus, E. coli and Proteus spp. respectively. Malassezia organisms were also recorded in 04
samples (7.84%) during this study (Table 1).

In this study, 43.56% of all microbial isolates belonged to the group of coagulase-positive staphylococci, among
which Staphylococcus inter medius (recently called Staphylococcuspseudointermedius) and Staphylococcus aureusare
considered as common canine pathogens (Green, 2006). Petrov, (2013) revealed that the combination Staphylococcus
intermedius and Malasseziapachidermatis are the most common cause of otitis externa. The findings of the present study
corroborated by the findings of Agnihotriet al. (2014) isolated Staphylococcus aureus(59.21%), Pseudomonas
aeruginosa(24.02%), Proteus (10.61%) and Streptococci (6.15%) from clinical cases of otitis externa in dogs. Lyskovaet
al., (2007) opined Staphylococcus intermedius(58.8%), the most frequently isolated microorganism from otitic ears
followed by Streptococcus canis (29.9%), Proteus spp. (14.4%) and Escherichia coli (10.3%) respectively.

Table 1: Microbial Isolates from Otitis Externa in Dogs

Microbial species Number of isolates (n=51) Percentage
Staphylococcus spp. 27 52.94
Pseudomonas spp 11 21.26
Streptococcus spp 4 7.84
Escherichia coli 3 5.88
Proteus spp. 2 3.92
Malassezia spp. 4 7.84

Loh et al., (1998) reported that coagulase-negative Staphylococcus and Aspergillusniger were the most common
bacteria and fungi cultured respectively in otitis externa patents. They emphasized that self-cleaning of the ears was the
most common predisposing factor

It is important to note that bacteria or yeasts associated with cases of otitis externa are only opportunists and are
not primary pathogens, in fact they are normally present in low numbers in the external ear canal. When a primary disease
damages this anatomic component of the ear, the normal microflora can proliferate and exacerbate or perpetuate
inflammatory reactions (Bugden, 2013).

Table 2: Biochemical and Cultural Characteristics of Bacterial

Isolates from Otitis Extera in Dogs
Test E. coli Staph. spp... Strepto. spp Pseudo. aeruginosa Proteus spp
Indole Production - - - - -
Methyl red + + + - +
Voges Proskauer - + + - -

www.tjprc.org editor@tjprc.org
22 Kashyap Roshan, Roy Manju & Roy Sushovan

Table 2: Contd.,
Citrate utilization - + NA + +
Catalase Test + + - + +
+(6)
Coagulase Test - + NA NA
-(21)
Oxidase Test - - - + -
Urease test - - - - +

Biochemical testing is based on the metabolic activities of bacteria. Sustained appearance of bubble in catalase
test confirms the presence of Staphyloccoccus species and also positive coagulase test. Clear to blue dark colors produce in
oxidase test confirms presence of Pseudomonas species. The Development of bright red color at the interface of the reagent
and the broth indicative of E coli and proteus. The broth turns a bright pink color in urease test indicate positive for Proteus
species (Table 2).

CONCLUSIONS

Present study concluded that most common bacterial agents isolated from canine otitis externa were coagulase-
positive staphylococci. It was also observed that fungi and yeasts are also frequently involved in the etiology of ear canal
inflammations and that in many instances of clinical cases were caused by co-infections.

REFERENCES

1. Agnihotri, D., Sharma, A. &Khurana, R. (2014). XXXII Annual Convention of ISVM and International Symposium on the 21st
Centuary Road map for Veterinary Practice, Education and research in India and Developing Countries, 6,21-91.

2. Augus, J. C. (2004). Otic cytology in health and disease. Veterinary Clinics of North America. Journal of Small Animal.
Practice, 34, 411-424.

3. Bugden, D. (2013). Identification and antibiotic susceptibility of bacterial isolates from dogs with otitis externa in Australia.
Australian. Veterinary Journal, 91, 43–6.

4. Greene, C. E. (2006), Otitis Externa. In: Infectious diseases of the dog and cat. 3rd edition, Saunders, Missouri, pp., 815-823.

5. Loh, K. S., Tan, K. K., Kumarasinghe, G., Leong, H. K. &Yeoh, K. H. (1998). Otitis externa-the clinical pattern in a tertiary
institution in Singapore. Annals of the Academy of Medicine., 27(2),215-218.

6. Lyskova, P., Vydrzalova, M. &Mazurova, J. (2007). Identification and antimicrobial susceptibility of bacteria and yeasts
isolated from healthy dogs and dogs with otitis externa. Journal of Veterinary Medicine. A, Physiology, Pathology, Clinical
Medicine, 54, 559–563.

7. Paterson, S. (2002). A review of 200 cases of otitis externa in the dog. Veterinary Dermatology14:249. Balappanavar, B. R.
&Vasanth, M. S. (2013). Clinico-diagnostic and therapeutic management of canine malasseziosis. IntasPolivet, 14(2), 353-
358.

8. Shahida Azhar Ali et al., Pet Owners Motivate Dogs to Contaminate Soil, Promoting Echinococcosis, International Journal of
Zoology and Research (IJZR), Volume 3, Issue 2, May – June 2013, pp. 39-50

9. Paterson, S. (2016). Topical ear treatment–options, indications and limitations of current therapy. J. Small Ani. Prac., 7:67-
75.

10. Petrov, V., Mihaylov, G., Tsachev, I., Zhelve, G., Marutsov, P. &Koev, K. (2013). Otitis externa in dogs: microbiology and
antimicrobial susceptibility. Revue de MedecineVeterinaire, 164(1), 18-20.

Impact Factor (JCC): 5.9857 NAAS Rating: 4.13

Isolation and Identification of Pathological Agents in Dogs with Otitis Externa 23

11. Quinn, P. J., Carter, M. E., Markey, B. K. & Carter, G. R. (1999). Clinical Veterinary Microbiology, 9th Edn, London, Mosby
yearbook Limited, PP: 95-126.

12. Sander, R. (2001). Otitis Externa: A Practical Guide to Treatment and Prevention. American Family Physician Journal,
63,927-942.

13. Senthil, K. K., Selvaraj, P., Vairamuthu, S., Mala, S. and Kadiresan, D. (2010). Antibiogram patterns of microbes isolated
from otitis externa of dogs. Tamil Nadu Journal Veterinary & Animal Sciences, 6 (3),145-147.

14. Shaw, S. (2016). Pathogens in otitis externa: diagnostic techniques to identify secondary causes of ear disease. In Practice, 38,
12-16.

15. Zur, G. L., Lifshitz, B., BdolAhabram, T. (2011). The association between the signalment, common causes of canine otitis
externa and pathogens. Journal of Small Animal Practice, 52, 254-25.

www.tjprc.org editor@tjprc.org