الفهرس 
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Abstract
This study was carried out in two dairy farms in Beni-Suef district during the period from September 2014 till April 2016 to investigate the investigate the prevalence, risk factors associated with diarrhea in cow/calf farms, assess the current patterns of antibiotic use and molecular characterization of resistance genes in enteropathogenic bacteria recovered from dairy calves and their environment.The present study was divided into two parts to achieve the above mentioned aims as following:
Part I: The Prevalence and distribution of calf diarrhea in the examined farms:
A structured questionnaire wasadministered to collect data on calf management, calf diarrhea (frequency, distribution and risk factors), pattern of antibiotic use and biosecurity measures.Samples were collected from both calves(fecal samples)and their surrounding environment(soil, water, swabs from milk buckets, milk samples, swabs from teat apices,swabs from attendant hands, flies, swabs from water devices, swabs from manager and feeding stuffs)using stratified random sampling technique throughout the study periodin the investigated farms and thenwere cultivated for the isolation of diarrhea causing agents,then the bacterial isolates were identified using biochemical and serological techniques. Based on the bacteriological findings the prevalence of calf diarrhea and frequent distribution of enteropathogenic bacteria isolated from both calves and their environment were detected.
The obtained results showed that:
1. The prevalence of calf diarrhea was significantlyhigh in farm (I) compared to farm (II) (36.0 and 26.6%, respectively)at X2= 35.9; P< 0.001,while the percentage of apparently healthy calves were (64.0 and 73.4%, respectively) in both farms (I and II).
2. The frequent distribution of bacteria isolated from diarrheic calves (fecal samples) was significantly highin both examined farms (I and II) (91.8 and 86.2 %, respectively) at X2=11.49; P< 0.009,compared tothe apparently healthy ones (31.0 and 26.3%, respectively).
3. The frequency of enteropathogenicbacteria isolated from calves was higher in farm (I) than in farm (II) (93 and 67 bacterial isolates respectively). Moreover,E. coli was the most predominantly isolated bacterial pathogen in both farms (I and II) (64.5 and 70.1%, respectively), followed by Klebsiella spp., C. perfringens, Shigellaspp. and Salmonella spp. in farm (I) (16.1, 11.8, 4.3 and 3.2%, respectively), while in farm (II) E. coli followed by Klebsiella spp., Shigellaspp.and C. perfringens (16.4, 7.5, and 6.0%).atX2= 57.57; P< 0.001.
4. Serological tests showed that the most detected serogroups from diarrheic calves in farms (I and II) were O26, O55 and O159 (29.0, 22.6 and 16.1%, respectively) followed by O111, un-typed serogroupsand O127(12.9, 9.7, 6.5%, respectively)whileO103was the least one to be detected (3.2%). Furthermore O26 in farm (I) (33.3%) and O55 in farm (II) (30.8%) were the most predominant isolated serogroups.
5. The frequent distribution of enteropathogenicbacteria isolated from calves’ environment in farm (I) indicated that E. coli was the most isolated bacterial pathogen from the different environmental samples (45.3%) as well from the calves (64.5%), followed by Klebsiella spp., Salmonella spp., Shigella spp. and C. perfringens (32.0, 10.7, 7.3and 4.7%, respectively). Whilst the highest percentage of E. coli was recovered from soil (66.0%), followed by flies (47.8%), water trough (39.1%), attendant hands (38.5%), and feed manager (30.8%) and the least percentage was recovered from milk bucket (18.8%). Klebsiella spp.were recovered in the highest percentage from milk bucket (75.0%), followed by water trough (47.8%), attendant’s hands (46.2%), feed manager (23.1%),flies (21.7%) and the least percentage was detected in soil samples (10.6%).WhilstSalmonella spp. were recovered from feed samples in the highest percentage (50.0%), followed by flies (21.7%), manager (15.4%), attendant’s hands (11.5%) and finally soil sample (10.6%). Meanwhile,Shigellaspp. were isolated from manager at a percentage of (15.4%) followed by flies, soil, milk bucket, water troughs, and attendant’s hands (8.7, 8.5, 6.3, 4.3 and 3.8%, respectively). C. perfringens was theleast microbial pathogen that was detected in the environment of farm (I) mostly in feed sample (50.0%) followed by manager (15.4%), water trough (8.7%) and soil (4.3%) at X2= 59.84; P<0.001.
6. from the frequent distribution of bacteria isolated from calves’ environment in farm (II)it has been found that E. coli was the most prevalent bacterial pathogen (50.3%) followed by Klebsiella spp., Salmonella spp. and Shigellaspp. (34.9, 8.1, and 6.7% , respectively). Furthermore E. coli was predominantly isolated from water troughs (55.6%) and teat apices (55.3%), followed by attendants hands (51.9%), manager (47.1%), and flies (38.7%). Meanwhile,Klebsiella spp. were mainly detected in manager (41.2%), followed by water trough, teat apices, attendant’s hands, and flies (40.7, 40.4, 37.0 and 16.1%, respectively). Whilst Salmonella spp. were only detected in flies and swabs from attendant’s hands at a percentage of (35.5 and 3.7%, respectively). Shigellaspp. were mostly isolated from feed manager(11.8%) followed by flies, attendant’s hands, teat apices and water trough (9.7, 7.4, 4.3, and 3.7%, respectively) at X2= 56.74; P< 0.001.
7. Serological tests showed that E. coli O55 was the most predominant serogroup to be isolated from farm (I) environment (32.0%) followed by O26, O111, O159, O127, and Un-typed (20.0, 16.0, 16.0, 8.0 and 8.0%, respectively). Moreover attendants’ hands and water trough represent a potential reservoir for O55in this farm as it was detected in the highest percentage (50.0 and 44.4%, respectively) followed by milk bucket, manager and soil (33.3% each).Water trough considers the main source of O26 (44.4%), followed by milk bucket and soil (33.3% each), then attendants’ hands and flies (25.0 and 20.0%, respectively)While,O111 was mainly isolated from soil, milk bucket and attendants’ hands (33.3, 33.3 and 25.0%, respectively), E. coliO127 was detected in water trough and flies samples (44.4 and 20.0%, respectively). On the other hand,E. coliO159was detected mainly in water trough, flies and manager (44.4, 40.0 and 33.3%, respectively), while un-typed serogroups was mainly detected in feed manager and soil samples (33.3% each).
8. E. coli O26 was the most predominant serogroup isolated in farm (II) (25.0%) followed by serogroup O103, O159 and O55 (15.0% each), then serogroups O111, O 127and un-typed (10.0% each). Furthermore O26 was mainly detected in swabs from teat apices, flies,water trough and attendant’s hands (40.0, 33.3, 25.0 and 20.0%, respectively). WhileE. coliO103 was mostly detected in water trough, teat apices and attendants’ hands (25.0, 20.0, 20.0%, respectively). Serogroup O159 was detected infeed manager, teat apices and attendant’s hands (33.3, 20.0 and 20.0%, respectively).While,E. coliO55 was mainly detected in flies,attendant’s hands andwater trough (33.3, 25.0 and 20.0%, respectively). On the other hand,E. coliO111 was detected in swabs from teat apices and attendant’s hands (20.0% each), O127 was detected in flies and water trough (33.3 and 20.0%, respectively), and finally un-typed serogroup was only detected in swabs from feed manager (66.7%).
9. Concerning the serotyping of Salmonella spp. in farm (I) the results indicated that S. enteritidis was the most frequently detected serotypes from diarrheic calves and their environment (61.1%),followed by S. kentuky, S. typhimurium and S. dublin (22.2 and 11.1, 5.6%, respectively).Moreover,S. enteritidiswas detected in feed manager and attendants’ hands (100.0% each), followed by diarrheic calves, flies and soil (66.7, 40.0 and 40.0%, respectively).WhileS. kentuckywas mainly detected in soil, diarrheic calves and flies (40.0, 33.3 and 20.0%, respectively). S. typhimurium was only detected in the environment in flies and soil (20.0% each). While,S. Dublin was just detected in flies (20.0%).
10. Serotyping of Salmonella spp. in farm (II) proved thatSalmonella isolation and identification was limited to environmental samples Furthermore S. kentuky was the most predominantly detected serotype (50.0%), followed by S. enteritidis and S. typhimurium (33.3 and 16.7%, respectively). Moreover S. enteritidisandS. typhimurium were detected only in flies’ samples (36.4 and 18.2%, respectively). was detected in flies.S. kentuky was detected in the highest percentage in swabs of attendants’ hands (100.0%) followed by flies (45.5 %).
11. Regarding the risk factors associated with calf diarrhea in both farm (I and II) there were a variation between the two farms where in farm (I) there was a calving pen but not routinely cleaned and disinfected, the management of newly born calves including umbilical care did not take place where some calves were suffering from hernia that affect their health condition and their ability to resist diseases. Calf housing was outdoor exposing them to different weather conditions, and in yard with earthy floor that was difficult to clean and disinfect with no drainage system leading to accumulation of manure under the animals contaminating their environment and aggravating of flies problem that actas a vector for disease pathogens.Mixing buffalo calves with cow calves consider a risk factor. Meanwhile, in farm (II) calves were kept with their dams for 2 weeks of life where they were naturally suckling their dams exposing them to different pathogens that contaminate their teat apices. Calves of different ages were housed together that increased the risk of diarrhea. Watering and feeding the animals from common water troughs, buckets and manager increased the probability of contamination of food and water and transmission of infection to healthy calves in both farms.
Results in part (I)revealed that miss managmental practice during raising of calves such over stocking density of calves, raising different ages together with different species, and raising of calves on earthy floor all that increased the risk of calves to theinfection withenteropathogenic bacteria causing diarrhea, together with lower standard of hygiene and absence of routine disinfection program all that have led to higher frequency of enteropathogenic bacteria isolation from both calves and their environment particularly in farm (I).
Part II: Antimicrobial sensitivity pattern of enteropathogenic bacteriarecovered from calves and their environment in-vitro
The antimicrobial sensitivity of identified enteropathogenic bacteria isolated from calves and their environment were tested in-vitro against 12 antibiotics and 4 different types of disinfectants commonly used in veterinary practice. Bacterial isolates showed resistance to 3 or more antimicrobial agents were selected for detection of antimicrobial resistance genes.
The obtained results showed that:
1. Antibiotic sensitivity testing of enteropathogenic bacteria recovered from calves and their environment in farm (I) showed thatSalmonellaspp. were significantly sensitive to enrofloxacin (63.2%), followed by florofenicol (52.6%), and erythromycin (52.6%).While theywere significantlyintermediately resistance to florofenicol (47.4%), neomycin (42.1%) and enrofloxacin (26.3%), andhighlyresistant (100.0% each)at P<0.001 to ampicillin, amoxicillin, penicillin, tetracycline, oxytertracycline, chloramphenicol, Sulfamethoxazole/trimethoprim complex and cefoxitin.Shigella spp. as well showed similar pattern to Salmonella spp. where they were significantlysensitive to enrofloxacin (68.7%), followed by florofenicol (25.0%) and significantlyintermediately resistant toflorofenicol (62.5%), followed by neomycin and erythromycin (50.0% each) thenenrofloxacin (25.0%), while they werehighly resistant (100.0% each)at P<0.001to (β-lactamases), tetracycline, oxytertracycline, chloramphenicol, Sulfamethoxazole/trimethoprim complex and cefoxitin. Referring to Klebsiella spp.were significantlysensitive to enrofloxacin and florofencicol and neomycin (100.0, 100.0 and 40.0%, respectively), and significantly intermediately resistant toneomycin (60.0%), but they weresignificantly andcompletely resistant to other antibiotics (100.0%)at (P<0.001). Meanwhile,C. perfringenswassignificantlysensitive to ampicillin and tetracycline (42.8% and 14.3%, respectively), andintermediately resistant to tetracycline (21.4%), moreover it exhibited significant resistance to the rest of the used antibiotics (100.0%) at (P<0.001).
2. The results of antibiotic sensitivity testing in farm (II) showed that Salmonella spp.were only intermediately resistance to neomycin and enrofloxacin (50% each), and completely resistant the rest of the used antibiotics.While Shigella spp. exhibited significant sensitivity to neomycin followed by florofenicol (21.4 and 14.3%, respectively) and significantintermediate resistanceto florofenicol and enrofloxacin (50.0, 42.9%, respectively)at (P<0.001) andsignificantlycompletely resistance (100.0% each)toampicillin, amoxicillin, penicillin, tetracycline, oxytetracycline, chloramphenicol, cefoxitin, Sulfamethoxazole/trimethoprim and erythromycin. Meanwhile,Klebsiella spp. showed significantsensitivity just to neomycin (40.0%), and were significantintermediately resistance toneomycin (10.0%)at (P<0.001)andexhibited significantlycomplete resistance toall of the other antibiotics.
3. Antibiotic sensitivity testing of E. coli recovered from calves and their environment revealed that E. coli obtained from calves in farm (I) showed significantsensitivity to enrofloxacin,neomycin, chloramphenicol and florofenicol (80.0, 60.0, 30.0 and 30.0%, respectively), and significantintermediate resistance to erythromycin (50.0%) while they were significantlyresistant (100.0%) to (β-lactamases), tetracycline, oxytertracycline, Sulfamethoxazole/trimethoprim complex and cefoxitinat (P<0.001). Meanwhile the environmental isolates of E. coliwassignificantly sensitive to enrofloxacin and chloramphenicol (20.0 and 10.0%, respectively), and significantlyintermediately resistant to neomycin, florofenicol, erythromycinandenrofloxacin (50.0, 50.0, 30.0and 20.0%, respectively) while they showed significantcomplete resistance to (β-lactamases), tetracycline, oxytertracycline, Sulfamethoxazole/trimethoprim complex, florofenicol and cefoxitinat (P<0.001). Whilst E. coli isolates recovered from calvesin farm (II) exhibited significantsensitivity to enrofloxacin, florofenicol and neomycin (33.3, 16.7 and 11.1%, respectively), and significantintermediate resistance to enrofloxacin, neomycin, florofenicol,oxtetracycline and chloramphenicol (66.6, 61.1, 50.0, 16.7 and 11.1%, respectively) and they weresignificantly resistant (100.0%) to the rest of tested antibioticsat (P<0.001). Moreover, environmental isolates of E. coliwere significantlymoderately sensitive to enrofloxacin and florofenicol (50% each), and weresignificantly intermediate resistant to chloramphenicol, erythromycin, neomycin,enrofloxacin,florofenicol and Sulfamethoxazole/trimethoprim (70.0, 60.0, 50.0. 50.0, 50.0 and 20.0%, respectively), whilethey significantly resistantto the rest of used antibiotics (100.0%)at (P<0.001).
4. Disinfectant efficacy testing against different bacterial isolates in farm (I) showed thatVirkon® S (1%)was significantlyefficientagainst Salmonella spp. (78.9 and 68.4%, respectively)at(P<0.05; 0.001)at 30 and 15 min exposure time.While iodine (5%) exhibited the least bactericidal effect (15.7%)at (P<0.001)after 15 min of exposure. Regarding Shigella spp. were significantly sensitive to Virkon® S (1%) followed by TH 4+ (0.5%) at exposure time 30 (75.0 and 68.8%, respectively)thenVirkon® S (1%) after 15min of exposure (50.0%)at (P<0.05), whileH2O2 (1%) exhibited the least bactericidal effect(25.0and 18.8%, respectively) at 30 minand 15min.Meanwhile Klebsiella spp. were significantlysensitive (100.0%) to Virkon® S (1%) after 30 min of exposure time,followed by TH 4+ (0.5%) at exposure time 30 min (80.0%)thenVirkon® S (1%) after15min of exposure (70.0%)at (P<0.001) and the least efficiency was exhibited by iodine (5%) (6.7%) at contact time 5 min.
5. In farm (II) disinfectant sensitivity pattern showed that Salmonella spp. were mostly sensitive (75.0 and 50.0%, respectively)to Virkon® S (1%) at contact time 30 and 15min, and they were lesssensitive (25.0 %) to H2 O2 (1%) at contact time 15 and 10 min. Meanwhile,Shigella spp. were significantlysensitive(78.6, 71.4 and 64.3%, respectively)at (P<0.05) to Virkon® S (1%) and TH 4+ (0.5%) at exposure time 30 and 15 min of exposure time toVirkon® S (1%), and the least sensitivity(21.4% each) was exhibited to H2 O2 (1%) at the same exposure time. Meanwhile, Klebsiella spp. werehighly sensitive (100.0%) toVirkon® S (1%) after 30 min of exposure while after 15 min(76.6%) and (70.0%) sensitive to TH 4+ (0.5%) afterexposure time 30minat(P<0.001; P<0.05, respectively)while H2 O2 (1%) had the lowest bactericidal effect(26.6 and 16.6%, respectively)at (P<0.001;P<0.05) at contact time 15 and 10 min.
6. Disinfectant sensitivity against E. coli isolates recovered from calves and their environment in farm (I and II) revealed E. coli isolates obtained fromthe calves in farm (I)were significantlysensitive (80.0 % each) at(P<0.001) toboth TH4+ 0.5% and Virkon® S 1% after 30 min of exposure. On the other hand, environmental strainsin farm (I)weresignificantly sensitive to Virkon®S 1% and iodine (5%) (70.0 each)after contact time 30min followed byVirkon®S 1% after 15 min (50%) at(P<0.001), while bothE. coli isolated from the calves and their environment were significantlyresistant (100.0%) to H2 O2 1% after contact time 30 minat(P<0.001). Moreover, in farm (II) Virkon® S 1% showed highly significant bactericidal effect against E. coli isolates from the calves (100.0 and 83.3%, respectively) at 30 and 15 min of exposureatP<0.001 and sensitive toTH4+ 0.5% and iodine 5% (83.3% each) after 30 min of exposure, whileH2O2 1%was significantly the lowest effective disinfectant(22.2%)at(P<0.001)after 5 min of exposure. Whilst the environmental strains were significantly sensitive to Virkon® S 1% after 30 min and 15 min of exposure (80.0, 60.0%, respectively) and 70.0% iodine (5%) after 30 min of exposure at(P<0.001), and likewise they were significantly resistant (100.0%) to H2 O2 1% after 30 min of exposure at P<0.001.
7. The distribution of antimicrobial resistance genes showed thatblaSHVand blaTEMresistance gene; responsible for resistance to β-lactams antibiotics, were detected in 8 isolates of E. colifrom the calves and their environment. Meanwhile, while blaOXA-1 resistance gene; responsible for resistance to β-lactams antibiotics, was detected in only one isolate of E. coli form the calves. On the other hand,dƒrA resistance gene of trimethoprim was detected in 4 isolates of E. coli(2 of animal origin and 2 of environmental origin). The qacED1gene; responsible for resistance to quaternary ammonium compound (QAC) disinfectants, which was detected in 5 isolates of E. coli (3 of animal origin and 2 of environmental origin). Meanwhile,ƒloR resistance gene that is responsible for resistance to phenicols was detected in only one isolate of E. coli isolated from diarrheic calves. The sul1resistance gene; responsible for resistance to sulfonamides andtetA(A) gene; responsible for resistance to tetracycline , were detected in 3 isolatesof E. coli (2 were obtained from animals and 1 was obtained from environment).
The results in part (II)indicated that most of the isolated enteropathogenic bacteria from the calves or their environment exhibited high degree of resistance to the majority of the used antibiotics and this might be a consequence of indiscriminative use of antibiotics in clinical practice, also a variable degree of resistance to most of the disinfectants used although there were no routine disinfection and this might be due to impropercleaning and presence of organic matter that inactivate the disinfectants. All of the screened resistance genes were detected in the animal isolates pointing out to their possibility to act as a reservoir to such genes in the environment and risk to human population.