CORYNEBACTERIUM RENALE CYSTITIS IN COW-CASE REPORT -

Corynebacterium renale is a common inhabitant of the the vulva, vagina and prepuce of apparently normal cattle, but also an opportunistic pathogen and the cause of cystitis and purulent pyelonephritis in cows. In this paper, we show the isolation of C. renale from the urine of cows with clinical cystitis, colonial, microscopic and biochemical characteristics of the isolates, relevant data on virulence factors, clinical manifestations of disease and basic principles of therapy.


INTRODUCTION
Bovine cystitis is an infl ammatory process of the urinary bladder. Th e causative agents are most commonly bacteria which are an integral part of the microbiota of the genital and gastrointestinal tract, such as: Escherichia coli, Corynebacterium renale group (Corynebacterium renale, cystidis and pilosum), Trueperella (formerly Arcanobacterium) pyogenes, rarely Staphylococcus spp., Streptococcus spp., Proteus spp., Klebsiella spp., (Yeruham et al., 2006; Th e Merck Manual, 2014). Infections are more common in cows, compared to male cattle (Andrews and Williams, 2004). Th e reasons are related to the anatomical features (short urethra), hormonal status (high levels of estrogen may aff ect the functional integrity of the epithelium in the urethra and urinary bladder), risks associated with pregnancy or iatrogenic procedures (Stevens et al., 2007) Corynebacterium renale belong to the Corynebacterium renale group, genus Corynebacterium, family Corynebacteriaceae (Quin et al., 2013). Th e family contains a large number of ubiquitously widespread species that are commensals on skin and mucous membranes and opportunistic pathogens to humans and animals (Stevens et al., 2007). Th e main representative of the species, is a highly contagious human pathogen Corynebacterium diphtheriae. In domestic animals, nondiphtherial Corynebacteria cause diff erent infections: Corynebacterium pseudotuberculosis causes caseous lymphadenitis in sheep and goats; C. pseudotuberculosis causes ulcerative dermatitis in cattle, while C. ulcerans and C. bovis cause mastitis. According to this new classifi cation, the Corynebacterium renale group contains three species (previously three types): C. renale (type I), C. pilosum (type II) and C. cystitidis (type III). Th e C. renale is the cause for cystitis and pyelonephritis in cows, ulcerative (enzootic) balanoposthitis in sheep and goats and osteomyelitis in goats Quin et al., 2013). C. cystitidis causes haemorrhagic infl ammation of the bladder in cows with ulceration of the mucous membrane of the bladder, urethritis and pyelonephritis. C.pilosum has lower virulence and the infection typically results in a less severe clinical picture or in an uncomplicated cystitis. In natural infections, pyelonephritis develop less frequently than C. renale (Hayashi et al., 1985).

CASE REPORT
Sample: Catheterized urine of cows (Fig. 1). Laboratory examination: An amount of 100 mL of the urine sample was inoculated on 2 plates of Columbia blood agar base supplemented with 5% sheep blood and the MacConkey agar (CM0007,Oxoid, Basingstoke, UK).
Incubation atmosphere: Th e plates are incubated aerobically (blood and i MacConkey agar) and in 5-10 per cent CO2 conditions (blood agar).

RESULTS
Colonial apperance of isolates: Aft er 24 hours of incubation under aerobic and microaerophylic conditions, a signifi cant increase of very small (up to 1mm), non-transparent (opaque), non-haemolytic colonies were noted on the blood agar. Aft er the incubation for 48 hours, colonies turned a pale yellow color (pigment production). Th ere was no growth on the MacConkey agar.
Identifi cation criteria for isolates: a pale yellow color of colony, absence of haemolysis, absence of growth on the MacConkey agar, coryneform Gram-positive rods, oxidase negative, catalase positive, strong urease production, growth in broth at pH 5.4, glucose fermentation, non acid production from xylose and negative aesculin hydrolysis.
Identifi cation of the etiological agent: Corynebacterium renale. ). Only C. renale (most isolates) possess an extracellular protein that is referred to as renalin. Renalin reacts with ceramides, the integral part of the sphingomyelin cell wall of the red blood cells of mammals. Th erefore, it is referred to as renalin "CAMP like" protein because it produces synergistic haemolysis on blood agar with sphingomyelinase of Staphylococcus aureus (beta-haemolysin). It is believed that renalin plays an important role in the lysis of the host cells . Enzyme urease is an important virulence factor in all three species from the Corynebacterium renale gruop.

Relevant data of Corynebacterium renale cystitis
Th e urease quickly (within 1 hour) and vigorously hydrolyze the urea, and the resulting products (such as ammonia) stimulate the mucosal infl ammation.
Clinical signs: Hematuria is usually the fi rst symptom of infection, and in uncomplicated cystitis, can be the only, permanent or temporary symptom. Th e ascending spread of the infection results in the development of purulent pyelonephritis (infl ammation of the renal parenchyma and the renal pelvis). Clinical signs of pyelonephritis include fever, anorexia, colic, frequent attempts to urinate, poliuria, pyuria, agitation, decreased milk production and anemia. In chronic infections, due to the infl ammatory process, the bladder wall becomes thickened, the ureters expands and fi lls with purulent exudates. Corynebacterium spp. are pyogenic bacteria, and purulent infl ammation can aff ect the kidneys and result in the development of multiple abscesses (Th e Merck Manuals, 2014). In the urinary sediment, a large number of leukocytes and bacteria has been found (Merck Manual., 2013) Although C. renale is widespread and commonly present in the mucosa of the genital tract, the aff ected individual should be isolated of the herd, in order to prevent an increase in the number of pathogens in the environment and in the prevention of the spread of infection (Merk Manual., 2013). C. renale cystitis is established across Europe and North America, but the prevalence is unknown (Andrews and Williams, 2004).

Th erapeutic recommendations:
Corynebacterium renale is sensitive to the majority of antibiotics, such as the penicillins, ampicillin, cephalosporins, quinolones, chloramphenicol, tetracyclines, cefuroxime and trimethoprim. Th e treatment of choice for pyelonephritis due to Corynebacterium spp is penicillin, because penicillin is excreted in the urine (e.g. 10 000-15 000 iu/kg daily for at least 10 days) (Andrews and Williams, 2004) or trimethoprim-sulfadoxine (16 mg combined/kg, IM, for ≥3 wk) (Merk Manual, 2014). Treatment should begin as early as possible, before the development of serious tissue damage.

CONCLUSION
Th is is also the only case of C. renale cystitis in cows which was etiologically confi rmed in the laboratory for clinical bacteriology of the Scientifi c Veterinary Institute "Novi Sad" for a period of 10 years. Cow's urine samples are usually very rarely submitted to the laboratory for bacteriological exami-nation. Th e assumption is that clinical cystitis in the fi eld is treated empirically. Th e exact prevalence of C. bovis cystitis therefore remains unknown, and research of the etiology of cystitis in cows, to our knowledge, and on our epizootic area has not been implemented.
Clinically, C. bovis cystitis is indistinguishable from bladder urinary infections caused by other, mostly Gram-negative bacteria (eg, Escherichia coli). For proper treatment, etiological diagnosis is necessary, because in this case penicillin preparations are the drug of choice. Th e treatment is successful if starts on time, hence preventing the development of purulent pyelonephritis.

ACKNOWLEDGMENT
Th is work is supported by a grant from the Ministry of Education, Science and Technological Development, Republic of Serbia, Project number TR 31071.