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المستخلص
6. SUMMARY
Advanced bacteriological and immunological studies on Campylobacters isolated from farm animals
Campylobacteriosis is known to have a world-wide distribution and to be associated with infertility and abortion of different farm animals. Once the disease is established in a herd, the syndrome of fetal death, repeat breeding and irregular estrus occur, resulting in great economic losses. The disease is generally venereal in cattle, buffaloes and camels caused by C.fetus particularly subspecies venerealis. The natural habitat of this organism in female animals is within vagina, cervix and uterus, while in males (healthy carriers) is confined to the mucosa of glans penis and prepuce.
In the present study a total of 1205 bacteriological samples were collected from cattle (607), buffaloes (135), sheep (170) and camels (293). Samples collected from cattle included 170 preputial washings, 55 preputial swabs, 27 fresh semen, 200 frozen semen, 45 vaginal swabs, 27 uterine discharges, 12 gravid uteri, 35 aborted feti and 36 fecal swabs. Samples collected from buffaloes included 17 preputial washings, 45 preputial swabs, 13 fresh semen, 36 vaginal swabs, 6 uterine discharges, 7 gravid uteri, 5 aborted feti and 6 fecal swabs. Samples collected from sheep included 7 vaginal swabs, 5 gravid uteri, 3 aborted feti, 102 bile secretions and 53 fecal swabs. Samples collected from camels included 77 preputial swabs, 104 vaginal swabs, 23 uterine discharges, 14 gravid uteri, 3 aborted feti, 27 bile secretions and 45 fecal swabs. Collected samples were subjected to different conventional bacteriological investigations (CM).
As a result of CM, out of 1205 collected samples, 59 (4.9%) Campylobacter isolates were recovered. Out of 607 cattle samples, 21 (3.46%) isolates were obtained, 8 (4.7%) isolates from preputial washes, 5(11.12%) isolates from vaginal swabs, 3(5.45%) isolates from preputial swabs, 3 (8.57%) isolates from aborted feti, 1 (8.34%) isolate from gravid uteri and also one(2.78%) isolate from fecal swabs. Out of 135 buffaloe samples, 9(6.67%) isolates were obtained, 3(8.34%) isolates from vaginal swabs, 3(6.67%) isolates from preputial swabs, 2(11.76%) isolates from preputial washes and only one(20%) isolate from aborted feti. Out of 170 sheep samples, 12 (7.05%) isolates were obtained, 7 (6.86%) isolates from bile secretions, 4 (7.54%) isolates from fecal swabs and 1 (20%) isolate from aborted feti. Out of 293 camel samples, 17 (5.8%) isolates were obtained, 5 (4.8%) isolates from vaginal swabs, 3 (3.9%) isolates from preputial swabs, 3(6.67%) isolates from fecal swabs, 2 (7.4%) isolates from bile secretions, 2(8.7%) isolates from uterine discharges, 1 (7.14%) isolate from gravid uteri and 1 (33.34%) isolate from aborted feti.
Accurate identification of the isolated strains was performed morphologically, culturally and biochemically. Different types of media, with and/or without antibiotics in addition to the use of Millipore filters (0.65um. pore size) were used through the practical part of the study. The prevalence of Campylobacter isolated from cattle was 21 (3.46%) isolates out of 607 samples (10 C.fetus subsp.. venerealis, 7 C.sputorum bv. bubulus, 2 C.fetus subsp.. fetus, 1 C.jejuni and 1 C.coli). The prevalence of Campylobacter isolated from buffaloes was 9 (6.67%) isolates out of 135 samples (5 C.sputorum bv. bubulus, 2 C.fetus. subsp. venerealis, 1 C. fetus.subsp. fetus and 1 C.jejuni). The prevalence of Campylobacter isolated from sheep was 12 (7.05 %) isolates out of 170 samples (7 C.fetus subsp.. fetus, 4 C.jejuni and 1 C.coli). The prevalence of Campylobacter isolated from camels was 17 (5.8%) out of 293 samples (10 C.fetus.subsp. venerealis, 4 C.jejuni, 1 C.coli, 1 C.fetus subsp. fetus and 1 C.sputorum bv. bubulus)
This work determined the locations and alternations of the protein components to a total number of nine recovered Campylobacter isolates, 3 out of them (No. 1,2and 3) were belonged to C.fetus subsp. venerealis, 2 out of them (No.4and5) were belonged to C.fetus. subsp. fetus, 2 out of them (No.6 and 9) were belonged to C.jejuni and 2 out of them (No.7 and 8) were belonged to C.coli using SDS-PAGE. The results revealed that Campylobacter strains had 6-11 discrete protein bands with molecular weights (MWs) ranging from 219 to 14 kDa.
• Lane-1 : C.fetus. subsp. venerealis ”isolated from aborted feti of cattle”, showed 10 protein bands with MW of 219, 175.57, 157.325, 155.91 111.55, 109.19, 107.38, 102.575, 97.633 & 54.24 kDa.
• Lane-2 : C.fetus. subsp. venerealis ”isolated from preputial swabs of buffaloes”, showed 10 protein bands with MW of 219, 175.57, 165.795, 162.92, 157.325, 155.91, 109.43, 107.38, 97.633 &75.632 kDa.
• Lane-3 : C.fetus. subsp. venerealis ”isolated from vaginal swabs of camels”, showed 9 protein bands with MW of 175.57, 157.325, 155.91, 111.19, 105.115, 75.632, 34.597, 17.882 & 15.439 kDa.
• Lane-4 : C.fetus. subsp. fetus ”isolated from aborted feti of sheep” showed 8 protein bands with MW of 213.805, 165.795, 157.325, 155.91, 107.38, 105.115, 84.703 & 34.597 kDa.
• Lane-5 : C.fetus. subsp. fetus ”isolated from preputial washes of cattle” showed 9 protein bands with MW of 213.805, 157.325, 155.91, 111.55, 109.43, 102.575, 48.703, 75.632 & 34.597 kDa.
• Lane-6 : C.jejuni ”isolated from vaginal swabs of cattle” showed 11 protein bands with MW of 111.19, 107.38, 97.633, 84.703, 75.632, 63.795, 54.24, 47.418, 41.375, 27.348 & 22.165 kDa.
• Lane-7 : C.coli ”isolated from bile secretions of sheep” showed 10 protein bands with MW of 111.19, 105.115, 97.633, 84.703, 71.671, 54.24, 47.418, 41.375, 27.348& 22.165 kDa.
• Lane-8 : C.coli ”isolated from fecal swabs of camels” showed 11 protein bands with MW of 111.55, 102.575, 97.633, 84.703, 71.671, 63.795, 47.418, 41.375, 27.348, 22.165 & 17.882 kDa.
• Lane-9 : C.jejuni ”isolated from fecal swabs of sheep” showed 9 protein bands with MW of 111.55, 97.633, 84.703, 75.632, 63.795, 54.24, 47.418, 41.375 & 27.348 kDa.
• Marker showed kDa with 205, 116, 97, 66, 29 & 14.
The dendrogram showed cluster analysis on the basis of similarity coefficient values and average linkage method of groups formed by SDS-PAGE protein patterns. The dendrogram showed that the similarity between lane 1 and 2 was 85.71%, between 4 and 5 was 76.19%, between 6 and 9 was 83.33%, between 7 and 8 was 87.50, between group of (1 & 2) and 3 was 64.71%, between group of (6 & 9) and group of (7 & 8) was 78.46%, between group of (4 &5) and group of (6, 7, 8 & 9) was 61.49% and all strains showed sharing similarity with a percentage of 54.23%.
During the current work, ELIEA was applied for detection of anti-OMP antibodies of C. fetus subsp. fetus (group-I), C. fetus subsp. venerealis (group-II) and C. jejuni (group-III) revealed that :- The optical densities (ODs) of anti-Campylobacter OMP were in the highest level when they were reacted with whole cell antigens(WCAs) or OMP antigens of the same Campylobacter species, however ODs were in the lowest levels when they were reacted with heterologous antigens.
Group-I:- The ODs of anti-C. fetus subsp. fetus OMP with whole cell antigens(WCAs) of C. fetus subsp. fetus were 0.25, 0,32 and 0.41, while with OMPs of C. fetus subsp. fetus were 0.23, 0.29 and 0.38. The ODs with C. fetus subsp. venerealis were 0.08, 0.14 and 0.16 for WCAs and were 0.08, 0.14 and 0.18 for OMPs. While the ODs with C. jejuni were 0.09, 0.12 and 0.15 for WCAs and were 0.07, 0.13 and 0.15 for OMPs , after 1st, 2nd and 3rd week postinoculation respectively.
Group-II:- The ODs of anti-C. fetus subsp. venerealis OMP with WCAs of C. fetus subsp. venerealis were 0.25, 0,32 and 0.41, while with OMPs C. fetus subsp. venerealis were 0.28, 0.36 and 0.46. However ODs with C. fetus subsp. fetus were 0.11, 0.13 and 0.16 for WCAs and were 0.11, 0.12 and 0.18 for OMPs; while ODs with C. jejuni were 0.10, 0.14 and 0.16 for WCAs and were 0.15, 0.19 and 0.21 for OMPs , after 1st, 2nd and 3rd week postinoculation respectively.
Group-III:- The ODs of anti-C. jejuni OMP with WCAs of C.jejuni were 0.28, 0,36 and 0.52, while with OMPs of C.jejuni were 0.29, 0.37 and 0.53 . However ODs with C. fetus subsp. fetus were 0.09, 0.11 and 0.14 for WCAs and were 0.07, 0.13 and 0.15 for OMPs; while ODs with C. fetus subsp. venerealis were 0.11, 0.14 and 0.18 for WCAs and were 0.15, 0.19 and 0.21 for OMPs , after 1st, 2nd and 3rd week postinoculation respectively. Antisera of controls in each group gave no results.
The cross-reactivity percentages after 1st, 2nd and 3rd week were calculated. The highest percentages were observed between anti-OMP Campylobacter antibodies and their corresponding antigens (both WCAs and OMPs). At the first week, percentages were 100%, 89.28% and 96.55% for WCAs, while were 92%, 100% and 100% for OMPs; at the second week, percentages were 100%, 88.89% and 97.3% for WCAs, while were 90.63%, 100% and 100% for OMPs and at the third week, percentages were 100%, 89.13% and 98.11% for WCAs and were 92.68%, 100% and 100% for OMPs of C.fetus subsp. fetus, C.fetus subsp. venerealis and C. jejuni respectively. However, the lowest percentages of cross-reactions occurred between anti-OMP Campylobacter antibodies and heterologous antigens (WCAs and OMPs of different serologically related Campylobacters) with different degrees (24.14% - 53.55%) at the first week, (29.73% - 53.57%) at the second week and (26.42% - 45.65%) at the third week. The cross-reactivity of controls antisera of each group gave 0%.
7. CONCLUSION
**from the results of the current study, it could be concluded that:-
• Each Campylobacter species has a distinct protein pattern which can be used to distinguish between different strains.
• SDS-PAGE can be considered as a rapid, reliable, sensitive and diagnostic tool for characterization of Campylobacter isolates.
• Construction of outer membrane protein polyvalent vaccines containing either homologous or heterologous antigens, can provide complete prophylaxis against campylobacteriosis infection, moreover, it is more economic.