69(2): 87-95. Blowey, R. and Edmondson, P. (1995):. Mastitis control in dairy herds. Farm- ing press books, Miller Freeman pro- feesional Ltd, Wharfedale Road,.
SCVMJ, XIII (1) 2008
27
SEASONAL PREVALENCE OF COLIFORMS IN SOME DAIRY FARMS AT ISMAILIA GOVERNORATE *Saleh, R.E., Sobeih, M.A., Hassan, A.M. and Marwa, A. Hassan *Departemnt of Animal Hygiene, Zoonoses and Animal Behaviour, Faculty of Vet.Med., Suez Canal University.
ABSTRACT The distribution of coliform organisms on three dairy farms at Ismailia Governorate was investigated seasonally during the period from November 2006 to November 2007. Seven hundred and eighty eight mastitic milk samples, five hundred and seven teat end swab and two hundred and fifty seven soil samples were collected. Bacteriological examination revealed wide distribution of Escbericbia coli and Klebsiella, and to less extent Enterobacter and Citrobacter organisms. The incidence of mastitis with coliform bacteria was higher during summer season. E. coli was the most prevalent microorganism among coliform species isolated from mastitic milk in two farms, while in the third farm Klebsiella was the most prevalent microorganism. The highest incidence of coliforms isolated from teat apex were recorded in different sea-sons at the investigated farms (spring, summer and winter seasons). Soil sam-ples examination revealed that Coliform bacteria were isolated in high inci-dence during winter season in two farms and summer season in the third farm. Key words, Coliform bacteria, Mastitis, Soil, Teat end. INTRODUCTION Prevalence of environmental mastitis relates mainly to the level of teat-end exposure to the causative agents, mainly coliform bacteria such as E. coli, Klebsiellla pneum-oniae and Enterobacter aerogenes. (Hillerton and Kliem, 2001). Economic losses develop mainly from decreased milk yield and discarded milk and the rest due to
increased labor, treatment and veterinary services, premature culling, and reduced genetic improvement. coliform bacteria are present at all times on all dairy farms (Ingalls, 2003). Causative agents of environmental mastitis occupy many habitats in the cow’s environment. Escherichia coli is the normal inhabitants of the gastrointestinal tract of warm blooded animals. Both Klebsiella
28
spp. and Enterobacter spp. populate soils, grains, water, and intestinal tracts of animals. Although the mammary gland is not considered a natural habitat for coliform bacteria, many strains are capable of surviving and multiplying in the mammary gland (Hogan and Smith, 2003). The specific aim of this study was to investigate the incidence of coliform bacteria in some dairy farms at Ismailia Gove-rnorate during different seasons. MATERIAL & METHODS Animals and facilities The study was carried out in three dairy farms, El-Salhia Agriculture Company for Investment and Development-dairy cattle breeding farm number 2 (farms A and B) and farm of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia (farm C). In farms A and B animals were kept in yards (32 animals per each yard in farm A and 36 animals for farm B) and were milked in a milking parlour, the herd size was 288 and 324 in farm A and B, respectively. The dairy cows were mechanically milked three times daily in 2 milking parlours which were localized at the center of the farm. In farm C animals were kept in separate yards, the dairy buffaloes were manually milked twice daily in the
Saleh et al.,
milking parlour. Herd size was ranged from 36 to 40 lactating animals. 1. Milk sample Seven hundred and eighty eight individual milk samples were collected from the mastitic animals. Samples were screened for mastitis using California Mastitis Test (Schalm, et al., 1971). Positive milk samples were collected according to the regulations released by the German veterinary association (DVG, 1980). 2. Teatapex swabs Five hundred and seven teat swabs were collected aseptically from all four teats in the examined dairy farms as described by Rendos et. al., (1975). 3. Soil samples A total of two hundred and fifty seven soil samples were collected from the yards of the examined farms. Collected samples were subjected to bacteriological examination according to Clegg et al., (1983). All samples were subjected to isolation of coliform bacteria according to Cherry et al., (1972). From the original samples previously prepared, a loopfulls were inoculated into MacConkey broth medium and incubated at 37ºC for 24 hours, a loopful from the broth medium was streaked onto MacConkey agar media.
SCVMJ, XIII (1) 2008 Suspected colonies on the agar media were carefully picked-up and purified by subculturing on Eosin Methylen Blue agar media; purified colonies were picked up and transferred to nutrient agar slope for further biochemical identification (Cruickshank et. al., 1980). After incubation, a representative colony of each type of organism was described, picked, and inoculated onto Triple Sugar Iron (TSI) agar (Difco), Reactions on TSI were recorded, and growth was placed in Simmon's citrate slants, Methyl Red Vogus Proskauer broth, of Christensen`s urea agar and motility test media. Collected results were recorded in Tables (1, 2 and 3). RESULTS & DISCUSSION 1. Individual milk samples Table (1) revealed that both farm A and B, showed higher incidence in coliform species in summer with a percentages of 28.68% and 24.73%, respectively. On the other hand, summer season in farm C revealed the lowest incidence of coliform Spp. (7.46 %). This could be attributed to type of dairy animal, where animals were dairy cows in farm A and B and buffaloes were in farm C. this agree with results reported by Forsman et al., (1997) who mentioned that in dairy buffaloe farm, the most isolated microorganisms causing bovine mas-
29 titis were Staphylococcus and Streptococcus spp. Seasonally comparison showed that coliform mastitis incidence in the examined farms was high in summer season; these results agree with those reported by Todhunter et al., (1991) The same table showed that the highest incidences of E. coli were recorded in mastitic milk with a percentages of 18.38, 17.92% during summer season in farms A and B, respectively. Also E. coil was the most prevalent microorganism among coliform spp isolated from mastitic milk in both farm A and B. High incidences of E. coli in summer season was in agreement with the results reported by Waage et al., (1999). On the other hand, Klebseilla spp was the most prevalent microorganism among coliform spp isolated from mastitic milk in farm C during summer season with an incidence of 5.97%, and this incidence was the highest among the three farms (Table, 1). Also the tabulated results in Table (1) showed that E. coli was isolated only in spring season in farm C with an incidence of 1.23%, this low percentage could be explained in the light of, that E. coil mainly affect young animals and those with high milk yield and with early lactation
30
stage, while in farm C most buffaloes were above 10 years and in advanced lactation stage with low milk yield. The results also indicated that the mean incidences of E. coil were 15.09, 14.36 % in farm A and B, respectively. These results were nearly similar to those obtained by Ameh et al., (1999) 16.7%, and higher than those reported by Ibtisam and El Owni (2006) 6.32%. On the other hand, the obtained results was lower than those reported by Hamed (2006) 51.1%. 2. Teat apex swab samples Results in Table (2) illustrated that spring season showed the highest incidence of coliforms isolated from teat apex in farm A (37.04%), summer season in farm B (31.3%) and winter season in farm C ( 47.83). Incidences of E. coli isolated from teat apex in farm A and B showed the same pattern during the different seasons except spring season which revealed the highest percentage of 14.81% and 11.22% with mean incidences of 11.07% and 9.75% in farm A and B, respectively. While incidence of E. coli in farm C had relatively the same pattern during spring and winter with percentages of 14.18 and 13.04, respectively. The lowest incidence was 2.06% in summer season, which was nearly similar
Saleh et al.,
to the results obtained by Chhabra et al., (1982) who isolated E. coil from teat ends of two dairy herds (28 cows and 25 cows) in percentage of 2.67% and 4%, respectively. The environment contaminated with faeces is the main source of mastitis-causing E. coli bacteria (Ne-meth et al., 1991). Incidences of Klebseilla spp. isolated from teat apex were the highest in winter season in both farm A and C with a percentages of 11.76% and 30.43%, respectively (Table, 2). While in farm B it was higher in summer season with a percentage of 14.5%. 3. Soil samples Data in Table (3) illustrated that winter season had highest incidences of coliform spp. isolated from soil in farm A and B (26.09% and 41.03%, respectively), while summer season had the lowest percentage (13.22% and 11.11%). This observation could be explained in the light of gets that soil muddy, dampy and heavily soiled with manure and with low temperature, creates favorable condition condition for the survival of microorganisms in winter season. While in summer soil gets more dried, less moisture by action of sun rays. The difference between farms could be attributed to numbers of animal per yard, as farm B had the
SCVMJ, XIII (1) 2008 largest number of animals in the yard (36 animal), meanwhile coliform especially E. coil were normal inhabitant in the feces of the animals, subsequently the degree of contamination of the soil depended on the feces voided by animals. E. coil showed the same pattern of coliform isolated from soil where the highest incidences were in winter (21.74 , 28.21 and 15.56 %) and the lowest ones were recorded in summer ( 5.97, 5.98 and 4.35 %) in farm A, B and C, respectively. On the other hand, incidence of coliform spp. isolated from soil in farm C showed that the highest percentages was recorded in summer season with a percentage of 26.09 %, and this elevation was mainly due to high incidence of Klebseilla spp. with percentage of 15.22%. Obtained results could be as a result of defect in drainage system of water trough in the yard of the lactating animals during summer season which led to increase mud and dampness of the soil and this represent good media for growth of bacteria. Humidity, damp condition facilitates the movement of faec`es onto udders and allow greater multiplication of environmental organisms (Blowey and Edmondson, 1995). Klebseilla spp. isolated from soil samples in farm A were relatively similar in winter and summer
31 and increased in spring season (9.63%). While incidences of Klebseilla spp isolated from soil samples in farm B were nearly the same (4.26, 5.13 and 4.44 %,) in winter, summer and fall, respectively, but decreased in spring season (1.8%). On the other hand, Klebseilla spp isolated from soil samples in farm C during spring and summer showed percentages of 3.92% and 15.22%, respectively (Table, 3). It could be concluded from above results that coliform bacteria are normal inhabitants of soil and the intestines of cows. They accumulate and multiply in manure, polluted water, dirt, and contaminated bedding. Infection of the dairy animals with coliform mastitis occurred mainly between milking so the prevention of coliform mastitis could be applied by: 1- Increasing space per cow to avoid manure contamination. 2- Soil should be thoroughly scraped, cleaned and soaked with suitable disinfectant. 3- Calving areas must be clean and dry and should be cleaned and sanitized regularly. 4- Establish milking orders. 5- Use milking procedures that stimulate milk ejection and result in clean and dry teats. 6- Using of pre milking teat dipping.
32
7- Keep cows on their feet for an hour after milking by providing fresh feed for them to eat.
Saleh et al.,
34
Saleh et al.,
Table (1): Incidence of coliform bacteria isolated from individual milk samples in the examined farms. Winter
Bacterial isolates
Farm A Spring Summer
Coliform spp.*
No % Mean incidence
15 16.48
33 17.28
a-E. coli
No % Mean incidence
12 13.19
29 15.18
b-Enterobacter spp.
No % Mean incidence
1 1.10
0 0.00
c-Klebseilla spp.
No % Mean incidence
2 2.20
4 2.09
No % Mean incidence
0 0.00
d-Citrobacter spp.
78 28.68
Fall
Winter
Farm B Spring Summer
36 17.48
18 16.82
45 18.99
28 13.59
15 14.02
33 13.92
10 3.68
1 0.49
1 0.93
7 2.95
13 4.78
4 1.94
2 1.87
5 2.11
19.98
15.09
45 19.31
1 4.35
4 4.94
50 17.92
27 11.59
0 0.00
1 1.23
3 1.08
6 2.58
0 0.00
0 0.00
8 2.87
9 3.86
1 4.35
3 3.70
3 1.46
0 0.00
0 0.00
5 7.46
4 7.69
0 0.00
0 0.00
0 0.00
0 0.00
4 5.97
3 5.77
1 1.49
1 1.92
0.31
0
2.68 5 1.84
Fall
6.11
1.88
2.75
0.82
69 24.73
14.36
1.32
* Represents percentages out of all isolates
Winter
19.96
50 18.38
0 0.00
Fall
Farm C Spring Summer
4.95 8 2.87
1.04
No= number of positive isolates
3 1.29
0 0.00
0 0.00 0.85
SCVMJ, XIII (1) 2008
35
Table (2): Incidence of coliform bacteria isolated from teat apex swab samples of dairy animals in the examined farms. Farm A Bacterial isolates Coliform spp.* a-E. coli b-Enterobacter spp. c-Klebseilla spp. d-Citrobacter spp.
Winter
Spring
Farm B
Summer
Fall
Winter
Spring
Farm C
Summer
Fall
Winter
Spring
Summer
Fall
No
16
60
46
27
12
53
41
24
12
53
41
24
%
31.37
37.04
28.22
24.11
23.53
27.04
31.30
25.53
23.53
27.04
31.30
25.53
No
5
24
15
10
3
22
13
8
3
22
13
8
%
9.80
14.81
9.20
8.93
5.88
11.22
9.92
8.51
5.88
11.22
9.92
8.51
No
0
28
10
1
3
11
7
3
3
11
7
3
%
0.00
17.28
6.13
0.89
5.88
5.61
5.34
3.19
5.88
5.61
5.34
3.19
No
6
8
11
9
5
8
19
12
5
8
19
12
%
11.76
4.94
6.75
8.04
9.80
4.08
14.50
12.77
9.80
4.08
14.50
12.77
No %
5 9.80
0 0.00
10 6.13
7 6.25
1 1.96
12 6.12
2 1.53
1 1.06
1 1.96
12 6.12
2 1.53
1 1.06
* Represents percentages out of all isolates
No= number of positive isolates
36
Saleh et al.,
Table (3): Incidence of coliform bacteria isolated from soil samples in the examined farms. Farm A Bacterial isolates Coliform spp.* a-E. coli b-Enterobacter spp. c-Klebseilla spp. d-Citrobacter spp.
Farm B
Winter
Spring
Summer
Fall
No
12
25
16
12
%
26.09
18.52
13.22
20.34
Winter
Farm C
Spring
Summer
16
25
13
41.03
16.13
11.11
Fall
Winter
Spring
Summer
Fall
9
10
5
12
6
20.00
22.22
9.80
26.09
15.00
No
10
9
7
6
11
14
7
6
7
3
2
3
%
21.74
6.67
5.79
10.17
28.21
9.03
5.98
13.33
15.56
5.88
4.35
7.50
No
0
3
3
2
3
6
0
1
3
0
2
1
%
0.00
2.22
2.48
3.39
7.69
3.87
0.00
2.22
6.67
0.00
4.35
2.50
No
2
13
5
1
2
3
6
2
0
2
7
0
%
4.35
9.63
4.13
1.69
5.13
1.94
5.13
4.44
0.00
3.92
15.22
0.00
No
0
0
1
3
0
2
0
0
0
0
1
2
%
0.00
0.00
0.83
5.08
0.00
1.29
0.00
0.00
0.00
0.00
2.17
5.00
* Represents percentages out of all isolates
No= number of positive isolates
SCVMJ, XIII (1) 2008
37
REFERENCES Ameh, J.A.; Edgbe-Nwiyi, T. and Zaria, L.T. (1999): Prevalance of bovine mastitis in Maiduguria Borno State, Nigeria. Vetrinarski Arhiv., 69(2): 87-95. Blowey, R. and Edmondson, P. (1995): Mastitis control in dairy herds. Farming press books, Miller Freeman profeesional Ltd, Wharfedale Road, Ipswich IPI 4 LG, United Kingdom, Page 35. Cherry, W.B.; Hanks, J.B.; Thomason, B.M.; Murlin, A.M.; Bridle, J.W. and Groon, J.M. (1972): Salmonella as index of pollution of surface water. App. Microbiol., 24:334. Chhabra, P.C.; Kapur, M.P. and Gautam, O.P. (1982): Notes on the prevalence of Echerichia coli"O" serotypes from dairy environment. Indian J. Animal Science, 52(6): 451-452. Clegg, F.G.; Chiejina, S.N.; Duncan, A.L.; Kay, R.N. and Wary, C. (1983) : Outbreak of Salmonella Newport infection in dairy herds and their relationship to management and contamination of environment. Vet. Rec., 112. Cruickshank, R.; Duguid, J.P.; Marmion, B.P. and Swain , R.H. (1980): Medical Microbiology. th E.L.B.S.12 Ed., Vo;l.11, Reprinted Chuchill Livingstone and Robert Stervenson Edinburgh , EHI, 3 AF.
Deutsche Veterinärmedizinische Gesellschaft (DVG) (1980): disubklinischeMastitis desRindes und Vorschläge für bundeseinheitliche Richtlinien zu ihrer Bekämpfung. DVG, Fachgruppe Milchhygiene des Arbeitsgebietes Lebensmittelhygiene, Kiel. Forsman, P.; Tilsala-Timisiorvi, A. and Alatossava, T. (1997): Identification of Staphylococcal and Streptococcal causes of bovine mastitis using 16S-23s r RNA spacer regions. Microbiology, 143(11): 3491-3500. Hamed, F. (2006): Bacterial and biochemical studies on mastitis of cattle in Sharkia governorate. Animal Health Research Institute (AHRI). Assiut Vet Med J., 52(109): 207-214. Hillerton, J.E. and Kliem, K.E. (2001): Aggressive therapy of clinical Streptococcus uberis mastitis. Pro-ceedings of the Second International Symposium on Mastitis and Milk Quality,: 234-237. Hogan, J. S. and Smith, K. L. (2003): Coliform mastitis. Vet. Res., 34: 507– 519. Ibtisam, E.M. El Zubeir and El Owni, O.A.O. (2006): Seasonal Variation of Incidences and Etiological Agents of Bovine Mastitis in Friesian Cattle in Sudan. Research Journal of Animal and Veterinary Sciences, 1(1): 25-29.
38
Saleh et al.,
Schalm, O.W.; Carrole, J. and Jain, N.C. (1971): Bovine mastitis . Lea and Febiger Philadelphia. Todhunter, D.A.; Smith, K.L.; Hogan, J.S. and Schoenberger, P.S. (1991): Gram-negative bacterial infections of the mammary gland in cows. Am. J. Vet. Res., 52: 184-188. ;Waage, S.; MØrk, A.; Aasland, D. Hunshamar, A. and Ødegaard, S.A. (1999): bacteria associated with clinical mastitis in dairy heifers. J. Dairy sci., 82: 712-719.
Ingalls, W. (2003): Environmental Mastitis, Source and Causes. West Agro, Inc., Kansas City, MO. available at – Service of Goat Connection. com Khimaira. Nemeth, J.; Muckle, C.A. and Lo, R.Y. (1991) Serum resistance and the traT gene in bovine mastitis causing Escherichia coli. Vet. Microbiol., 28: 343-351. Rendos, J.J.; Eberhat, R.J. and Kesler, E.M. (1975): Microbial populations of teat ends of dairy cows and bedding materials. J. Dairy Sci., 58(10): 1492-1500.
الملخص العربي االنتشار الموسمى للميكروبات القولونيه فى بعض مزارع الحليب بمحافظة االسماعيلية ربيع السيد صالح ,محمد عبد العال صبيح ,احمد محمد حسن ,مروه عبد المنعم محمد حسن
*قسم الصحه واالرماا
الرمتراهه و سووهات الحاوا ,هواة الطب الباطاي ,جترمعة قنتة السواس
رمزااع الحواب .وفاهت رم رجرماع ررم هذه الدااسه السربات رمدى رواجد رماهاوب القولو فى بع 877عانه لب رم ضاع رمورهب ؛ 708رمسحه رم الحورمت و 778عانه رابه رم رمالعب عوى رمدى عتم خالل اابعه رمواسم رم ثالث رمزااع حالب وهم رمتاوع الصتلحاه الزااعى رمحطة البت 7 (رمزاعه أ) و (رمزاعه ب) و رمزاعة هواه الطب الباطاي جترمعه قنتة السواس (رمزاعه ج).اوضح الدااسه انرتتا واسع لرماهاوب ااتاااتات القولو و رماهاوب الهوبساال وانرتتا اقل لهل رم رماهاوب الساراوبتهرا و رماهاوب االنرااوبتهرا .رم الواضح ا نسبه عزل رماهاوب القولو رم عانت البت الحاوانت الرمصتبه هتن اعالهت خالل فصل الصاف ,وهت رماهاوب ااتاااتات القولو رم اهثا الرماهاوبت الرمعزوله رم عانت االلبت الحاوانت الرمصتبه با افااد رماهاوب القولو فى الرمزاعرا أ و ب بانرمت رماهاوب الهوبساال هت اهثا الرماهاوبت الرمعزوله فى ( الرمزاعه ج ) .هرمت اوضح الدااسه ا رماهاوب القولو قد رم عزله بنسبه عتلاه رم رمسحت الحورمت خالل فصل الاباع فى الرمزاعه أ, الصاف فى الرمزاعه ب و الترتء فى الرمزاعه (ج) بانرمت رم عزل رماهاوب القولو رم عانت الرابه خالل فصل الترتء فى الرمزاعرا ( أ و ب) وفصل الصاف فى الرمزاعه (ج).
