Pyoderma caused by Proteus mirabilis in sheep

Abstract Pyoderma is a purulent skin infection usually caused by bacteria and can be divided into primary and secondary categories based on histology. In the present study, an 18‐month‐old female mixed breed sheep was examined for pyoderma at the injection site of the enterotoxemia vaccine. After routine bacteriology and histopathology procedures, secondary pyoderma caused by Proteus mirabilis was diagnosed. The bacterium analysed using genome sequencing and new strain called AJJ 2021 was diagnosed. This is the first report of pyoderma caused by Proteus mirabilis in sheep.


INTRODUCTION
Bacterial infections of the skin are usually purulent and are therefore commonly referred to as pyoderma. Pyoderma is classified into superficial and deep, depending on the level of tissue involvement (Denerolle et al., 1998). Pyoderma is further categorised as either primary or secondary. Primary pyoderma has no underlying cause in contrast to secondary pyoderma, in which the skin's health or integrity is compromised for other reasons, including an underlying disease process such as an endocrine or immunological disorder (Scott et al., 2012). Local disturbances of the skin's barrier integrity can also form a suitable ground for the occurrence of secondary pyoderma. These can include external parasite bites, rashes, fissures, other skin injuries or the entry of foreign objects such as plant thorns (Scott et al., 2011). This study describes the first report of secondary pyoderma caused by Proteus mirabilis in a sheep.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.

CASE HISTORY
In February 2020, an 18-month-old female crossbred sheep was examined at Alian farm in Sorkheh city (Iran) for a skin lesion. This closed farm was in a desert area and had 170 breeding sheep. All animals were fed with hay (50%) and grain materials (50%) twice a day. Daily DM was 3%. The affected sheep was anorexic, but other animals had a normal appetite. There was no history of external parasites, itching and poor quality of the sheep external coating. In affected sheep, the skin lesion was located behind the scapula and was necropurulent in nature ( Figure 1a and b). There was no previous treatment for this lesion. The lesion was painful to the touch. The history revealed that an enterotoxemia polyvalent vaccine (Razi Institute, Alborz, Iran) containing bacterial toxoids for Clostridium perfringens types b, c and d and Clostridium septicum was injected at the lesion site ten days before the examination. The vaccine was injected subcutaneously using

Bacterial culture
Without any prior preparation, bacteriological specimens consisted of two pre-sterile pre-labelled cotton swabs were rolled over the centre of the lesion and placed into Cary-Blair transport medium and sent to the laboratory in a cool box containing frozen ice packs. The collected samples were inoculated on blood agar (Merck, Germany) and purified bacterial isolates streaked on differential (MacConkey agar,

Extraction and PCR
Isolated bacteria were cultured in TSB broth (Merck, Germany) and incubated at 37 • C for 24 h. After that, 3 ml of TSB broth was cen-

Histopathology
Three samples (0/5×0/5 cm) were taken from the lesion margin with disposable biopsy punches and placed in 10% neutral buffered formalin and sent to the histopathological laboratory. The samples routinely processed, dehydrated, embedded in paraffin wax, sectioned at 5 µm in thickness using a rotary microtome (RM2 145; Leica, Wetzlar, Germany) and stained with haematoxylin and eosin (H&E) for histopathological evaluation.

Treatment
The distance between the animal and the veterinary diagnostic laboratory was distance (>300 km), for this reason empirical treatment was initiated and prescribed while diagnostic results were pending.
The wool around the lesion was sheared and cleaned using sterile gauze impregnated with 0.9% sodium chloride sterile solution. The lesion was debrided of necrotic debris using tissue forceps.

Diagnostic results
Histopathologically, moderate epidermal hyperplasia, orthokeratotic and parakeratotic hyperkeratosis were observed. There was severe ulceration, fluid debris, cellular exudate, degenerate neutrophils, necrotic keratinocytes, and basophilic bacterial colonies were also present (Figure 3a-c). In the dermal layer, interstitial neutrophilic to mixed mononuclear inflammation and destruction of adnexal units (follicles and sebaceous glands) were seen (Figure 3d). These findings were consistent with a diagnosis of deep pyoderma.
The underlying bacterial cause of the pyoderma was determined to be Proteus mirabilis. Proteus mirabilis was identified through differential and selective media, Gram-staining and biochemical tests, as shown in

Outcome and follow-up
The results from the culture and antibiotic susceptibility testing indicated that the AJJ 2021 strain of Proteus mirabilis was sensitive to dihydroestreptomycin sulphate and silver sulphadiazine. A positive response to treatment was observed and 2 weeks after the initial visit the lesion had the wound contracting (Figure 1d). During the 1-year follow-up via the phone, the wound had completely healed, leaving a persistent scar. The sheep was apparently health and gone through a successful gestation period.

DISCUSSION
The present report found that the referred sheep suffered from pyo-  (Jeong & Oh, 2011).
In this report, the observed pyoderma was classified as secondary and deep pyoderma. Subcutaneous injection of an enterotoxemia vaccine can cause a local swelling due to inflammation secondary to a foreign substance (Armbruster et al., 2018). Indeed, minor reactions to inactivated vaccines may result from the irritant nature of some adjuvants, such as aluminium (Chung, 2014) which is the adjuvant in the vaccine administered to this sheep before developing the lesion.
Other causes for localised vaccine reactions include the immunising antigens, conjugating agents, preservatives, stabilisers, antimicrobial
agents, and culture media used to prepare the vaccine (Chung, 2014).
Further, according to the manufacturer of the specific vaccine used to inoculate this sheep, the vaccine may cause local allergic reactions.
Changes in the skin's barrier integrity due to a local response to the vaccine at the injection site could have predisposed this sheep to bacterial pyoderma. According to the history of this patient, a non-sterile needle was used to administer the vaccine, which could have caused contamination leading to pyoderma is also a possibility. In the case of this sheep, it is difficult to determine if the underlying cause was the non-sterile needle, the vaccine or a combination of both.
Based on the results of the antibiotic sensitivity test of the isolate, among the antibiotics used in this study, two (dihydroestreptomycin sulphate and silver sulphadiazine) were effective antibiotics and one (procaine penicillin) was ineffective antibiotics against isolated Proteus mirabilis.

ACKNOWLEDGEMENT
Mr Abbas Alian, the owner of Alian Sheep Breeding Company, is appreciated for her cooperation in this research.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

FUNDING INFORMATION
There is no source of fund to declare.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available within the manuscript and also are available from the corresponding author upon reasonable request.

ETHICAL CONSIDERATION
After sampling, the code of ethics in the research was taken from research committee of Alian Sheep Breeding Company (ethics code: 0061891274).

ETHICAL APPROVAL
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.