ISOLATION AND MOLECULAR STUDY OF SOME BACTERIAL URINARY TRACT INFECTIONS OF SHEEP IN BASRAH PROVINCE

This study was aimed isolation and molecular detection of some causative agents of urinary tract infection (cystitis and pyelonephritis). Out of 108 tested urine samples (56 from females and 52 from males); 60 samples (55.55%) have infected with Escherichia coli and Klebsiella pneumoniae were 36 (64.2%) females and 24 (46.1%) males. The sixty infected samples contain from 56 E. coli and (4) K. pneumoniae, this samples identified by Vitek 2 (44 isolates E. coli and 2 isolates K. pneumoniae) were subjected to DNA extraction. A total of 44 E. coli isolates detected to FimH and pai genes. 44/44 (100%) were positive for presence of FimH gene, and 20/44 (45.45%) were positive for presence of pai gene. The two isolates of K. pneumoniae which detection of Ecpa gene and given positive result to this gene 100%.


INTRODUCTION
Urinary system is one of the most important system in the animal's body, this function includes removal of toxic waste from the body and regulation of the components of body fluids, as well as control the hormonal secretion which promote the bone marrow to red blood cells formation (15). Urinary tract infection (UTI) means the colonization and infections by one or more urinary tract parts (32). The sources of UTI are by emanating bacteria which come from gastrointestinal tract causing colonizing of the external genitalia, invasion of the bladder and urethra against the flow of urine (6). UTI also cause damage of vascular of urinary bladder then decrease the function of kidney competence, and disturbances the excretion of end products metabolic (9). Urinary system of sheep has been less commonly affections compared with other species of ruminant, the important bacteria infected are Corynebacterium pseudotuberculosis, C. renale, E. coli, K. pneumoniae, Actinomyces pyogens, Staphylococcus aureus, and Proteus (4,27). The urine moves to the urinary bladder through the ureter come from the kidneys. To helps urinary system from bacterial invasion, it is found the valve to prevents the come back the urine to the ureter from the bladder, if happened invasion, may be opportunistic germs caused by the normal flora. This type of infection happened from the ureter to the kidneys (35). Pyelonephritis is the inflammation of all parts of kidney. It is occasionally affected the sheep while behold firstly a bovine disease, (27). A few reports of bacterial sheep urinary system infections with E. coli and other gram-negative coccobacillus (18). Escherichia coli, as well as K. pneumoniae are from the family of enterobacteriacea, gram negative bacteria, aerobic and anaerobic growing (14). In our country, a few researches have been executed for the very important system. The significant economic losses cause by this system by quantitative and qualitative reduction of animal productivity. This study was designed for isolation and molecular detection of some causative agents of urinary tract infection (cystitis and pyelonephritis) in sheep's.

MATERIALS AND METHODS
One hundred and eight samples were collected during the period of April to December 2019 (56 from female and 52 from male suspected with UTI) were collected from Al-Basrah abattoir in Basrah province, and after collected from sheep, the case history from the sheep owner, frequent attempts to urinate, discomfort, a slight fever, anorexia, polyuria and sometimes colic. Checking the animal before slaughtering. Then collected urine and tissue samples for microbiological, molecular and histopathological analyses.

Methods of collection
Tied the urinary bladders to retain the urine, by polyethylene bag wrapped and transported to laboratory by cold recipient. Sterile syringes and needles were using, after cleaning the puncture sites with water then alcohol 70%, urine was taken from the urinary bladders (10).
To isolate E. coli and K. pneumoniae, gram stain is done (10,11). Poured the urine samples into capped sterile centrifuge tubes, then centrifuged for 10min at 2,000g, 1ml approximately of the sediment of urinary were added to 10 ml Tryptone soy broth, discarded the supernatant solution, incubated for 24hrs at 37ºC. Pre-enriched with trypton soy broth, then showing a loop-full turbidity of culture, streaked MacConkey plates and Eosin methylene blue, then incubated at 37ºC for 24 hours (9). By naked eye concerning their shape, colour and size of the colonies. Then the gram staining was done, and Vitek 2 confirmation. The antibiotic sensitivity test was carried out according to (20). In this study eight antibiotics were used, penicillin, chloramphenicol, gentamycin, streptomycin, ciprofloxacin, erythromycin, trimethoprim and tetracycline. Molecular study DNA extraction: By using bacterial DNA extraction kit (DNA extraction mini kit, Promega / USA) following the instructions of manufacturers. The concentration and purity of extracted DNA have determined using NanoDrop spectrophotometer (Optizen, Korea) at 260 nm and 280 nm and stored at -20°C (30). Polymerase chain reaction By using performing PCR technique, DNA of bacterial was amplified by used (Go Taq Green Master mix(M7122), Promega/USA). Three primers pairs were designed to identify important bacterial organisms including E. coli two primers, those are fimH gene (F: 5'-GCCAAACGAGTTATTACCCTGTT -3' and R: 5'-CCTTGATAAACAAAAGTCACGCC - 3') and Pai gene (F: 5'-TAGCTCAGACGCCAGGATTTTCCCTG -3' and R: 5'-CCTGGCGCCTGCGGGCTGACTATCAGG-3') (25) and Klebsiella Ecpa gene (F: 5'-AATGGTTCACCGGGACATCATGTCC -3' and R: 5'-AAGGATGAAATATCGCCGACATCC -3') (8). The amount used in this PCR, green master mix 25 µl, F primer 2 µl, R primer 2 µl, DNA template 10 µl and nuclease-free water 11 µl. The annealing temperature for fimH and Pai primers was 60C˚, while Ecpa primer was 62C˚. Detected the PCR product on agarose gel (1%) stained with ethidium bromide by used two ladders, 1,500 bp ladder (Promega/USA) and 1,500 bp ladder (Bioneer, Korea). Initial denaturation for PCR, 5min at 95°C. followed by 30 cycles of 95°C for 45sec, 58°C for 45sec, 72°C for 45sec. The reaction was then at 72°C for 6min, and cooled down 4°C for 5min. Detected the PCR product by agarose gel stained. PCR product then sent to Macrogene (Korea) company for sequencing. By using Parbi-Doua and NCBI BLAST programs, the sequences were edited and aligned. Macroscopically and microscopically examination of kidneys and urinary bladders of sheep's After slaughtered animals, limited the size of both kidneys and consistency, as well as urinary bladder for macroscopically examination. Then taken kidney and urinary bladder of infected slaughtered and healthy animal to histopathological examination according to (21).

RERSULTS AND DISCUSSION
The result of our study started by limited urinating as a very important sings, increased gradual with fluctuating temperature, loos of appetite, poor body condition, all these sings which represented from the case history according to the results out of the 108 tested urine samples; 60 samples 55.5% infected from suspected UTI of sheep (Table 1), all these sings which represented from the case history (28). The result of this study agreed to Petrovski in the cause of urinary tract infection are the bacteria and characterized by fever, colic and pyuria phenomenon and/or haematuria (24).  (23) who recorded that E. coli more important causative agent 21% of urine than K. pneumoniae was 5.3%. The differences of infected percentage between male and female because the female animals more susceptible to infected than male by urinary tract infection because many reasons like trauma of urethra, short urethra, effects of hormonal and reproductive system infection (26,34). The percentage of this study were disagreeing with the study of Fatihu et al (10) that reported the rate of infected female 6.3% and in male 16.7%. In our study, the Vitek 2 results for confirmation bacteria accepted with used Vitek 2 technology to identify E. coli (31).  (19). The main factor of virulence is a fimbria, which is very important in the adhesion to receptors of cells host, and protect bacteria from host response (7). The very important stage to development UTI due to the bacteria adherence to urinary epithelial cells, this allows bacteria to resistant and action flushing of the urine flow and bladder emptying, all this process stimulate the bacteria and activates to increase probability to staying in the urinary tract of the host (22). FimH gene is a gene responsible of fimbria, this give indicate the big problem because all this sample have this gene, which added the virulence of bacteria to adherence to urinary epithelial cells. The present study showed the positive result of pai gene in E. coli 20/44, the percentage rate 45.45% for presence of pai gene, the result has some deferent with the reported of Anad (1) in Iraq because he reported 57.1% of E. coli isolates by pai gene. From two sample of K. pneumoniae isolates which were identified by Vitek2, the result of PCR assay for detection Ecpa genes 2/2 (100%) were positive for presence gene. Our result agreed with Cruz-Córdova et al (8), who reported that 100% of the K. pneumoniae isolates have Ecpa gene, as well as Yassein reported 96% of clinical K. pneumoniae isolates (33). But the percentage of our result has higher than reported by Alcántar-Curiel et al (5) reach to 96%. The percentage changing of the of Ecpa gene because the K. Pneumoniae fimbriae nature played important role in the bacteria adherence to epithelial cells, it is regarded as a pathogenic an virulent agent which have related to the pathogenesis of K. pneumoniae (29).  Table (2) showed the result of forty-four isolates of E. coli identified by PCR were tested for the susceptibility to eight antibiotics by using method of disc diffusion. The result showed 100% resistance to tetracycline and erythromycin, 56.8% for penicillin but showed 100% sensitive to streptomycin and gentamycin, 90.9%; 75% sensitive to ciprofloxacin and trimethoprim respectively and 59% for chloramphenicol. Table (3) showed the result of two isolates of K. pneumoniae identified by PCR were tested for the susceptibility to eight antibiotics by using method of disc diffusion. The result showed 100% resistance to penicillin , erythromycin and tetracycline, and the result shoed 100% sensitive to streptomycin, gentamycin, ciprofloxacin and trimethoprim, while 50% sensitive to chloramphenicol. All the antibiotic sensitivity test matched to Abdullah and Mustafa (2) research because the same area of sampling and same conditions but different animals. The present study matches with Soud in E. coli resistance to erythromycin which record 97%, as well as our study agreed with Soud in sensitive to gentamycin and streptomycin (31), but disagree with Islam et al their recorded erythromycin resistant to E. coli are 73.3% and same percentage susceptible to tetracycline (16).

Histopathological result
The present study recoded high enlargement in both kidneys size, the consistency is softening and pale. While the urinary bladder was distended with cloudy urine of alkaline pH of 8. Some causes the enlargement of kidney appeared in right kidney and found patchy congestion, and haemorrhage. The result of histopathological study represented in figures 4 and 5. Figure