Bacteriological Analysis of a Lethal Outbreak of Pasteurella canis in Spotted Deer (Axis Axis) in a Zoological Park in Bareilly, India

Abstract

The microbiological investigation of deaths of six spotted deer (Axis axis) in three days in a zoological park in Bareilly, India revealed Pasteurella canis of resistotype B (resistant to cotrimoxazole, enrofloxacin, meropenem, cefoxitin and erythromycin) as the cause of the disease as it was isolated from heart blood of all the six dead deer. However, from the same herd of the deer four different resistotypes (A, B, C, D) of P. canis were detected, but type A, C and D were detected from 5, 2 and 1 deer, respectively. Of the 20 isolates of P. canis from deer 12 had multiple drug-resistance (MDR) and 11 were resistant to meropenem also. The six isolates from three of the ten stray dogs in the vicinity of the park harboured P. canis in their oral cavity but all isolates belonged to resistotype E (resistant to erythromycin only) indicating that the lethal strain of P. canis isolated from spotted deer had a different origin than stray dogs. Enrofloxacin therapy in a sick deer failed to protect that but the incorporation of doxycycline (2 mg/ mL) in water was successful in preventing the deaths and illness in deer of the zoological park.

Keywords: Pasteurellosis; MDR; Blackbuck; Hog-Deer; Carbapenem- Resistance; Pneumoeteritis

Introduction

Pasteurellosis is an endemic disease in India caused by Pasteurella species, primarily Pasteurella multocida and Pasteurella canis [1]. They account for significant health and economic losses, especially in the livestock sector and poultry farming [2]. Zoonotic transmission to humans generally occurs through animal bites or licking or direct contact with nasal secretions [3]. As a zoonotic infection Pasteurella canis is often associated with soft tissue, bone, joint, and wound infections in humans acquired on bites and scratches by dogs and cats [4,5]. Pasteurella canis have been reported in 2.8 to 20% of the specimen of dog oral cavity, including both healthy dogs and dogs with periodontal diseases [6,7] and also from the respiratory tract of horses [8]. Though a few P. canis isolates have been reported to secrete toxins, the complete nature of the toxin and its role in pathogenicity is not lucid [9].

Most of the animals generally harbours Pasturella canis without showing any clinical signs but it in a few it may lead to a range of pathological symptoms from asymptomatic or mild chronic upper respiratory inflammation to acute or fatal pneumonic and/or disseminated disease leading to significant morbidity [10-12]. It has also been associated with superficial infections such as pyoderma, cutaneous abscesses [1, 13, 14], penetrating infection of eye [15], external otitis, rhinitis, vertebral osteomyelitis, meningomyelitis, bronchopneumonia, tracheitis, paranasal sinus inflammation, toxicosis [16-18] and endocarditis [19]. In cattle, it causes mild to severe pneumonia and different ailments in sheep, cats, rabbits, and deer [20].

Pasteurella canis are often penicillin-sensitive and multiple drug resistance is rarely reported [21,22]. Antimicrobial therapy results reported successful recovery after amoxicillin/clavulanic acid, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, doxycycline, fluoroquinolones, third and fourth generation cephalosporins and carbapenems [21] while macrolides, firstgeneration cephalosporins, and aminoglycosides are shown as unsatisfactory options [22]. There are several reports of pasteurellosis in deer due to P. multocida [23-26], but P. canis is rarely reported to cause fatal disease in deer. We report the death of six adult deer due to P. canis infection in a small herd of 30 adults raised in an enclosed and protected area of woods and fed with cultivated green fodder.

Case History and Animals

In a small zoological park in Bareilly, India spread over five acres of a protected area bounded with a brick wall on two sides and by steel railing and mesh (9 ft high) on other two sides had 30 spotted deer (Axis axis), 14 blackbucks (Antilope cervicapra) and 11 hog deer (Hyelaphus porcinus). An adult spotted deer having barbed wire injury was found dead on 28^th Sept. 2019. Besides, one dear was sick with rectal temperature 102.5, keeping itself isolated and lagging due to malaise. It was treated with 50mg of flunixin meglumine, 100mg enrofloxacin and 8mg dexamethasone intramuscular injection, but it was found dead next morning. Rest of the herd was dewormed using triclabendazole 120mg and levamisole 75mg per 10 kg body weight. Next day (on 29 September) three more, including one treated, and on 3rd day (30 September) two more spotted deer were found dead in the morning. None of the five spotted deer died on 29^th and 30^th September 2019 had any lesion indicating injury. Heart blood samples from all the cases were submitted for culture to epidemiology laboratory for bacteriological examination and the Centre for Animal Disease Research and Diagnosis for viral agents. On 30 of September 2019 doxycycline (2 mg/ mL) was given in drinking water to the herd for three days and no more deaths were observed further. In postmortem reports, pneumoenteritis was recorded as a cause of the death without any more details and no Peste des Petits Ruminants (PPR) virus could be detected.

Follow-up Sampling

On suspecting dog bite as a source of P. canis, in the first week of October 2010 oral swabs of 10 stray dogs in the vicinity of the park were collected by sedating dogs through feeding them coconut cookies (one piece for a dog) containing 20 mg of diazepam each. All swabs were brought to laboratory within half h of collection for processing for the isolation of P. canis.

Bacteriological Examination

Each of the blood samples was streaked in triplicate on blood agar plates [blood agar base, (Difco, USA) with 5% sterile sheep blood]. One plate incubated aerobically at 37^oC for 24 h, one plate at 37^oC for 48 h in a microaerobic atmosphere with 5% C0₂, and the third plate was incubated anaerobically in jars with 90% H2 + 10% C0₂ and palladium catalyst at 37^oC for 48 h. Three to five isolated colonies were picked up from each plate for further characterization based on growth, staining and biological characteristics [27] following criteria laid down in Bergey’s Manual of Determinative Bacteriology [28]. After confirmation of the isolates, all were submitted for further confirmation to Pasteurella Reference Laboratory, ICAR-IVRI, Izatnagar. All different resistotypes of P. canis were deposited in the repository of Veterinary Type Culture Collection (VTCC), ICARIVRI, Izatnagar for future reference. All the cultures were maintained on blood agar slants until the end of the study.

All oral swabs of stray dogs were processed in the same way as the blood samples from deer but inoculated blood agar plates were incubated at 37^oC for 24 h aerobically, only. The typical nonhaemolytic, small transparent colonies showing oxidase-positive reaction using disc method [27] were further characterized for characteristic P. canis (producing oxidase, catalase and indole, fermenting glucose and sucrose but negative in methyl red and Voges Proskauer tests, not fermenting lactose, sorbitol and mannitol, not producing H²S, urease, gelatinase, gas in glucose and lysine decarboxylase negative, and not growing on MacConkey’s agar) as for those isolated from spotted deer.

Antimicrobial Sensitivity assay: All the isolates from spotted deer and dogs were tested for their sensitivity to amoxicillin (30μg), amoxicillin (30μg) + clavulanic acid (10μg), ampicillin (10μg), azithromycin (15μg), aztreonam (30μg), cefotaxime (10μg), cefoxitin (10μg), ceftazidime (30μg), ceftriaxone (10μg), chloramphenicol (25μg), colistin (10μg), cotrimoxazole (25μg), doxycycline (30μg), enrofloxacin (10μg), erythromycin (15μg), gentamicin (30μg), imipenem (10μg), meropenem (10μg), nitrofurantoin (300μg), penicillin G (10 IU), piperacillin (100μg), piperacillin (100μg) + tazobactam (10μg), tetracycline (30μg) and tigecycline (15μg) discs (Difco, USA) as per CLSI guidelines [29] on Mueller Hinton agar (MHA, Difco) supplemented with 5% bovine calf serum (Hi-Media, Mumbai) plates.

Results and Discussion

From blood samples of all the six spotted deer P. canis was isolated in pure culture. Of 20 isolates tested for their sensitivity to 24 antibiotics, they could be classified into four resistotypes, type A to type D (Table 1). Type B resistotype (resistant cotrimoxazole, enrofloxacin, meropenem, cefoxitin, erythromycin) was detected in blood samples of all the six spotted deer while other resistotypes were not detected from all the animals. The observations indicated that resistotype type B might be associated with the mortality in spotted deer. Type A detected in blood of five deer might be not important as it was sensitive to enrofloxacin but the deer treated with enrofloxacin also died i.e., antibiotic was probably not effective as the infection might be due to enrofloxacin resistant type B isolates. Similarly, type C and type D isolates may not be the cause of the mortality in spotted deer as they were detected only from two and one deer, respectively. Considering the sensitivity of all the P. canis isolates to doxycycline it was chosen for the therapy and the stoppage of death episode after inclusion of doxycycline in water indicated that P. canis might be the cause of lethality in spotted deer.

TABLECREATED

Table 1: Antimicrobial resistance pattern of Pasteurella canis isolates from spotted deer (Axis axis) and stray dogs in the vicinity of zoological park in Bareilly, India.