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Evaluation of etiology and antibiogram in otitis--a clinical study of 40 dogs.

Introduction

Otitis is a common medical terminology for inflammation of ear. Primary causes of otitis include parasites, hypersensitivity, keratinization disorder, foreign bodies, ear gland disorder and autoimmune diseases. These conditions are responsible for altering the ear canal allowing abnormal colonization of microorganisms. Treatment of these underlying conditions cannot be overlooked for eventual resolution of otitis.

Perpetuating factors include bacteria (Staphylococcus spp. and Pseudomonas spp.), yeast (Malassezia spp.) and pathologic changes such as glandular hyperplasia, epithelial folds, neoplasia, edema, mineralization and fibrosis. Secondary otitis media often perpetuates otitis externa. These conditions are frequently seen as etiology of ear infection, when in actuality they are sequelae to an underlying primary cause (Ettinger and Feldman 2000). The present study was planned to confirm the etiology and evaluate efficacy of different therapeutic regimen.

Material and Methods

In the present investigation, dogs suffering from ear infection and presented with the complaint of head shaking with itching in the ear were considered for the study. Thorough clinical examination of ear was carried out with cytological, mycological and bacteriological culture examination of otitic exudates for isolation of fungi, yeasts and bacteria. Antibiotic sensitivity test was carried out in case of bacterial otitis to appropriate proper line of treatment.

Otitic discharges were collected from affected ears using sterile cotton swabs for isolation of bacteria. Swab material collected was inoculated into nutrient broth and incubated at 370C for 6 hrs. Later on loopful of broth was streaked on Nutrient agar and MacConkey agar plate and incubated at 37[degrees]C for 24 hrs.

The characteristics of distinctly looking colonies were studied and isolated colonies were picked up and subjected to Grams staining to study morphological characters and staining reaction of organisms. Colonies of different types of bacteria were picked up and sub-cultured for further identification.

For isolation of yeast and fungi, the otitic exudate was inoculated on Sabouraud's dextrose agar (SDA) with Chloramphenicol slants, incubated at 25[degrees]C and examined every 4-6 days. The slants were held for at least 2 weeks before considering as negative.

Identification of isolates was carried out on basis of various biochemical tests as described by Markey et al. (2013).

Antibiogram of Bacterial Isolates

In-vitro antibiotic sensitivity of isolates was studied by disc diffusion method (Bauer et al., 1965). Briefly a small quantity of 6 hrs culture grown in nutrient broth at 37[degrees]C was taken with the help of Pasteur pipette and transferred on to Muller Hinton Agar plate. Using a sterile spreader a homogenous lawn was made and plates were allowed to dry for a brief period. The antibiotic disc were then placed 20 mm apart from each other and gently pressed on surface of agar. The plates were incubated at 37[degrees]C for 18-24 hrs.

The diameter of zone of inhibition including disc diameter was measured and results were interpreted as sensitive, intermediate and resistant based on recommendations of the manufacturers.

A total of 10 antibiotic discs--Ampicillin (A-10 mcg,) Amoxycillin (Am-30mcg), Cefalexin (Cn 30mcg), Norfloxacin (Nx-10mcg), Chloramphenicol (C-30 mcg), Ceftriaxone (Ctr--30mcg), Azithromycin (Azm- 30 mcg), Ciprofloxacin (Cip- 1 mcg), Ampicilin-Cloxacilin (Ax- 10 mcg) and Erythromycin (E-30 mcg) supplied by Hi-Media laboratories were used in the present study.

Exfoliative Cytological Examination

Otitic exudate swab was used for preparing smears and microscopic examination for presence of bacteria, yeast/fungi. The smears were examined after staining with Gram's stain/ Giemsa stain/ Lactophenol cotton blue stain.

Skin scraping examinations

Skin was scraped with a scalpel to collect skin tissue scraps. Skin scraps were gently mixed with potassium hydroxide 10% solution and processed for microscopic examination to detect mites or dermatophytes.

Results and Discussion

The microbial flora of ear canal of canines suffering from otitis has been studied by several workers and results differ widely. Similarly the present investigation revealed different etiological agents, Details of recovered isolates are presented in (Table 1).

In all 20 bacterial isolates were recovered, of these Staphylococci spp. were 8 (40%) followed by E. coli 5 (25%), Pseudomonas spp. 4 (20%), (Fig. 1 and 2) Proteus spp. 2 (10 %) and Bacillus spp. 1 (5%) respectively. Dakshinkar et al. (1992) recovered 25.93% of Staphylococcus aureus, 14.8% Staphylococcus epidermidis, 14.8% Streptococci spp., Proteus spp., 5.7% and 5.7% E. coli and Klebsiella spp.

In the present study, mycological investigation was carried out in which 12 cases of fungal otitis were observed on exfoliative cytology. Of these Malessezia pachydermatis was recovered in 9 dogs and Aspergillus spp. 3 dogs (Table 1) were confirmed on SDA agar with chloramphenicol slant. Similarly Blanco et al. (2014) used Sabouraud's dextrose agar medium for isolation of yeasts especially M. pachydermatis in which they observed growth after 24 hours of incubation.

Exfoliative Cytology

Exfoliative cytology is the inexpensive method and less time consuming to assess presence of etiology in number and type of etiology whether bacterial, fungal or parasitic. Cytology revealed presence of gram positive cocci, gram negative rods and Malessezia spp. on grams staining (Fig. 3 and 4). It helped in diagnosis prior to start effective treatment. Fernandez et al. (2006) found cytology as the most efficient method for identification of Malassezia pachydermatis. Also Murphy (2001) expressed that cytological examinations of otitic exudate were the most appropriate method for determining type and number of microorganisms present.

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Skin scraping

Similarly, the parasitological study was carried out to explore parasitic etiology. Total eight dogs with otitis were found to be positive for parasitic otitis. Of these, Sarcoptic mites (Fig. 5) were recorded in 4 cases, Demodex mites in 2 dogs (Fig.6) and Rhipicephalus ticks in 2 dogs were recovered.

Antibiotic sensitivity test

Antibiotic sensitivity tests were conducted to evaluate the sensitivity of causative organisms for antibiotics was evaluated. The antibiotic sensitivity tests were carried out by collecting swab of otitic discharge and fallowing standard laboratory procedure The antibiogram of bacterial isolates is presented in Table 2.

In-vitro antibiogram of Staphylococcus spp. indicated highest 62.5% (8) sensitivity to Ceftriaxone and least sensitivity to Chloramphenicol 12.5% (1). Next in order of effectiveness were Ciprofloxacin 25%, Norfloxacin 25% and Azithromycin 25%. In-vitro antibiogram of E. coli indicated highest 100% (5) sensitivity to

Ceftriaxone and least sensitivity to Norfloxacin (20%). Next in order of sensitivity was Ciprofloxacin 80% (4) followed by Chloramphenicol (40%).

In-vitro antibiogram of Pseudomonas spp. indicated highest 100% (4) sensitive to Ceftriaxone and next to Ciprofloxacin 80% sensitivity (Fig. 7). The in-vitro antibiogram of Proteus spp. indicates highest 2 (100%) sensitive to Ceftriaxone and Bacillus spp. were sensitivity to Norfloxacin, Chloramphenicol, Cephalexin, Azithromycin and Ciprofoxacin equally. These findings are in agreement with Chaudhary et al. (2003) who observed similar sensitivity pattern of antibacterial agents to different bacterial isolates as Ciprofloxacin 86.67%, Norfloxacin 56.67% and Chloramphenicol 38%. Baksi et al. (2004) also observed that out of 8 cases of Staphylococci spp. 2 were sensitive to Ciprofloxacin and 3 were sensitive to Chloramphenicol.

In-vitro antibiotic sensitivity pattern of different bacterial isolates was evaluated and presented in Table 3.

Ceftriaxone is found to be effective against almost all bacterial isolates viz. Staphylococcus spp., E. coli, Pseudomonas spp. Proteus organism followed by ciprofloxacin which found effective against Staphylococci, E. coli, Pseudomonas and Bacillus organism. Other antibiotics under study showed sensitivity to one or two bacterial isolates only. However, Ampicillin-Cloxacilin and Ampicillin has not shown sensitivity to any bacterial isolates justifying the rationality of Ceftriaxone and Ciprofloxacin in the treatment of canine otitis. This finding is in akin with the finding of Kumar and Rao (1997) Chaudhary et al. (2003) and Senthil Kumar (2010) who also recorded nearly same degree of sensitivity to Ceftriaxone and Ciprofloxacin to isolates from otitic dogs.

The present study indicates that there may be different etiological agents causing otitis in dogs. Confirmation of etiology causing otitis is necessary for correct selection of antibiotic and related therapy, instead of going for haphazard treatment, which may lead to complications and frustration for clinician also.

References

Baksi, S., Jana, P.S. and Chakraborti, A. (2004). Bacterial otitis externa in dogs and its treatment. Ind. Vet. J. 81: 1402-03.

Bauer, A.W., Kirby, W.M., Sherris, J.C. and Turck, M. (1965). Antibiotic susceptibility testing by a standerdized single disc method. The Amer. J. Clin. Pathol. 45: 493-94.

Blanco, B., Grico, V., Giancamillo, M.D., Greci, V., Travetti, O., Martino, P., Mortellaro, C.M. and Giudice, C. (2014). Canine aural cholesteatoma: a histological and immune-histochemical study. The Vet. J. 200: 440-45.

Chaudhary, M., Mirakhur, K.K. and Jand, S.K. (2003). Antibiogram and microbiological patterns of external ear canal of dogs with reference tootitis. Indian Vet. J. 80: 951-52.

Dakshinkar, N.P., Kalorey, D.R., Ganorkar, A.G. and Harne, S.D. (1992). In-vitro sensitivity pattern of bacterial isolates from cases of canine otitis. Indian Vet. J. 69:1075-76.

Ettinger, S.J. and Feldmen, E.C. (2000). Disease of the ear. In: Textbook of Veterinary Internal Medicine. Vol. II 5th ed. W. B. Saunders Philadelphia, p. 993.

Fernandez, G., Barboza, G., Villalobos, A., Parra, O., Finnol, G. and Ramirez, R.A. (2006). Isolation and identification of microorganisms present in 53 dogs suffering otitis externa. Rev.Cientffica.16 :23-30.

Kumar, A. and Rao, P. (1997). Bacterial and fungal flora in otitis externa of dogs. Indian Vet. J. 79 : 727-29.

Markey, B., F. Leonard, M. Archambault, A. Culinare and D. Maguire (2013). Clinical Vet. Microbiology 2nd Ed., Elsevier : pp 11-901.

Murphy, K. M. (2001). A review of techniques for the investigation of otitis externa and otitis media. Clin. Tech. Small Anim. Pract. 16: 236-41.

Senthilkumar, K. P., Selveraj, S. Vairamuthu, M. Shammi and D. Kathiresan (2010). Antibiogram pattern of microbes isolated from otitis externa of dogs. Tamil Nadu J. Vet. and Animal Sci. 6: 145-47.

(1.) Corresponding author E-mail: avinashrode@ymail.com

M.J. Umale, A.M. Rode (1), N.P. Dakshinkar, C.G Panchbhai and A.A. Sanghai

Department of Veterinary Clinical Medicine, Ethics and Jurisprudence

Nagpur Veterinary College

Maharashtra Animal and Fishery Science University (MAFSU)

Nagpur--440001 (Maharashtra)
Table 1 : Etiological agents isolated from
otitic dogs

                       No.of      No.of
Etiology              samples    isolates   (%)
                      screened

Malessezia spp.          92         09      22.5
Staphylococci spp.                  08      20.0
E. coli                             05      12.5
Pseudomonas spp.                    04      10.0
Sarcoptic mites                     04      10.0
Aspergillus spp.                    03      7.5
Proteus spp.                        02      5.0
Demodex mites                       02      5.0
Rhipicephalus ticks                 02      5.0
Bacillus spp.                       01      2.5
Total                               40      100

Table 2: Antibiogram of bacterial isolates in canine otitis

Bacteria             Isolates      Antibiotic Sensitivity (%)

Staphylococci spp.      08      Ciprofloxacin     Norfloxacin
                                   2 (25%)          2 (25%)
E. coli                 05       Norfloxacin      Ceftriaxone
                                   1 (20%)         5 (100%)
Pseudomonas spp.        04       Ceftriaxone     Ciprofloxacin
                                  4 (100%)          3 (75%)
Proteus spp.            02       Ceftriaxone          --
                                  2 (100%)
Bacillus spp.           01       Norfloxacin    Chloramphenicol
                                  1 (100%)         1 (100%)

Bacteria                Antibiotic Sensitivity (%)

Staphylococci spp.    Ceftriaxone      Azithromycin
                      6 (62.50%)         3 (25%)
E. coli              Ciprofloxacin       Chloram-
                        4 (80%)      phenicol 2 (40%)
Pseudomonas spp.          --                --

Proteus spp.              --                --

Bacillus spp.         Cephalexin       Azithromycin
                       1 (100%)          1 (100%)

Bacteria                Antibiotic
                     Sensitivity (%)

Staphylococci spp.   Chloramphenicol
                       1 (12.50%)
E. coli                    --

Pseudomonas spp.           --

Proteus spp.               --

Bacillus spp.         Ciprofloxacin
                        1 (100%)

Table 3: In-vitro antibiotic sensitivity patterns
of different bacterial isolates in canine otitis

Antibiotic        Staphy    E-coli   Pseudo-   Proteus   Bacillus
                  lococci             monas     spp.       spp.
                   spp.               spp.

Ciprofloxacin        2        4         3         0         1
Cephalexin           1        0         0         0         1
Chloramphenicol      1        2         0         0         1
Norfloxacin          2        1         0         0         1
Ampicilin+
Cloxacilin           0        0         0         0         0
Amoxycilin           1        2         0         0         0
Ampicilin            0        0         0         0         0
Ceftriaxone          6        5         4         2         0
Azithromycin         3        1         0         0         1
Erythromycin         0        2         0         0         0
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Title Annotation:Clinical Article
Author:Umale, M.J.; Rode, A.M.; Dakshinkar, N.P.; Panchbhai, C.G; Sanghai, A.A.
Publication:Intas Polivet
Article Type:Report
Date:Jul 1, 2015
Words:1939
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