الفهرس 
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Abstract
Brucellosis is a major constraint to livestock production that still enzootic in livestock in many developing countries including Egypt. The identification of Brucella spp. in farm animals of 15 Egyptian governorates highlights the role of cattle in dissemination of Brucella infection all over the country. A total of 136 Brucella isolates were recovered from cattle in different Egyptian governorates. These include, 107 isolates of Brucella melitensis biovar 3 recovered and identified on bacteriological and molecular basis from cattle at Aswan, Beheira, Beni Suef, Dakahlia, Damietta, Faiyum, Gharbia, Giza, Ismailia, Kafr El Sheikh, luxor, Monufia, Port Said, Qalyubia and Sharqia governorates. On the other hand, 29 Brucella abortus biovar 1 isolates were recovered from cattle at Beni Suef, Dakahlia, Damietta, Kafr El Sheik, Monufia, Port Said and Sharqia governorates.
The PCR assays were capable to confirm the identification of isolated Brucella species. Using primer sequences targeting IS711 gene, PCR has amplified the fragment 839 bp confirming the presence of Brucella on genus level. On the other hand, Multiplex PCR has amplified four fragments of 450bp, 587 bp, 1071 bp, and1682 bp characteristic for Brucella melitensis biovar 3, and three fragments of 450bp, 587 bp, and 1682 bp for Brucella abortus biovar 1.
Brucella was isolated from 8 retained fetal membranes out of 13 (61.5%) and from 55 milk samples out of 128 (43 %). In this study, Brucellae were isolated from 5 out 11(45.5%) stomach contents of aborted calves and 9 out of 16 (56%) vaginal discharges of aborted cows. Brucella was isolated from lymph nodes (retropharyngial, prescapular, prefemoral, internal iliac and supramammary) from carcasses of 59 out of 103 (57.3 %) serologically positive animals submitted to abattoirs by the Egyptian veterinary authorities.
The overall seroprevalence of bovine brucellosis in this study was found to be 4.42% in lactating cows, 0.0% in heifers, 0.0% in bulls and 3.04% in ewes based on RBPT and BAPAT as screening tests and cELISA as a confirmatory test.
The obtained results revealed isolation of 108 Brucella isolates. Among the 108 field Brucella isolates obtained from clinical specimens of cattle, 88 were definitely Brucella melitensis biovar 3, (81.48% %) and 20 were bacteriologically identified as Brucella abortus biovar1.
The present study involved the use of a multiplex PCR assay capable of differentiating Brucella species and vaccine strains including B. abortus S19, B. abortus RB51 and B. melitensis Rev 1. Results revealed no vaccine strains among the Brucella isolates.
Retained placenta in this study was significantly associated with brucellosis seropositivity. Among the 48 aborted and non-aborted cows that showed retained fetal membranes, 85.42% (n= 41) were serologically positive.
Sero-prevalence of brucellosis among 14 aborted cows was 92.6% (n= 13) compared with 3.22% (n= 33) sero-prevalence among 1026 non- aborted cows that indicated that abortion was significantly associated with sero-prevalence of brucellosis (p>0.05).
The sero-prevalence of brucellosis in female cattle (lactating cows and heifers) was 2.5%. (n= 46) among 1860 cows. Interestingly, none of the 12 examined bulls were serologically positive. In addition, in this study no animals were observed with clinical evidence of orchitis and/or epididymitis.
In this study, mixed farming was identified as an important risk factor associated with brucella infection in two farms where cattle were kept closely with sheep.
Isolation of viable Brucella from manure of cattle in this study combined with survival of such pathogen for significant periods in the environment as proved in other previous studies suggests and adds another mean of Brucella dissemination that complicates the infection chain of Brucella and may represent a potential risk for both animals and human health.
In this study, estimation of sensitivity, specificity, positive predictive value and negative predictive value of serological tests under evaluation using Brucella isolation as the gold standard was carried out. Brucella organisms could be isolated from 92 (46. 46 %) out of 198 lactating cows. Sensitivity, specificity, positive predictive value and negative predictive value of the sum of sero-testing were estimated as 96.73, 48.11, 61.81% and 94.44% respectively. Bacteriological examinations failed to identify 55 (38.19 %) out of 144 serologically positive cows. Interestingly, three cows out of the 198 investigated cows, whose milk yield positive culture results were serologically negative. The obtained results clarify that the specificity of a serological test is usually difficult to be evaluated on the basis of the results of culture findings as some animals yielding negative cultures may be truly infected but yielding negative bacteriological findings.
In this study, parallel testing of 960 dairy cows was carried out for detection of brucellosis seroprevalence. Relative sensitivity, relative specificity, positive predictive value and negative predictive value of BPAT were estimated as, 100 %, 99.89%%, 98.46 % and 100 % respectively. RBT revealed 100 %, 99.89 %, 98.46 % and 100% respectively. cELISA showed 95.38%, 99.89%, 98.41%, 99.67% respectively. Concerning CFT, the relative sensitivity, relative specificity, positive predictive value and negative predictive were 93.85 %, 100 %, 100 % and 99.56 %, respectively. The result of the evaluation of serological tests in this study suggests that using BAPAT. RBPT, cELISA and CFT in parallel manner produced the highest overall sensitivity and specificity when considered as their sum. It is obvious that the parallel testing increases the sensitivity of the screening.
In accordance with the results obtained in this study, cELISA shared both RBPT and BAPAT performance in term of specificity (99. 89%) but was the least sensitive compared with the other employed serological tests.
In the present work, the combined sensitivity and specificity of RBPT and CFT of 580 dairy cows using a serial interpretation at individual animal level was estimated as 30 (5.17%) and 26 (4.48%) respectively. The RBPT was conducted as screening test and positive sera were then retested using CFT. Samples were considered positive, when reacted positive for both RBPT and CFT and the total overall seroprevalence was calculated by dividing the number of RBPT and CFT positive animals by the total tested animals.
Generally, the obtained results indicate that brucellosis is still a major constraint to livestock production in Egypt. Continuous isolation and typing of Brucella isolates on both bacteriological and molecular bases represent the essential operation toward epidemiological evaluation of Brucella herd infection status and tracing back the source of infection. The actual Brucellosis status during the years 2019 and 2020 refers to that Brucella melitensis biovar 3 and Brucella abortus biovar 1 are the prevalent types circulating in different Egyptian governorates. The seroprevalence varies with important risk factors including, sexual maturity, retained fetal membranes, abortion, sex and mixed farming. Finding and possible survival of Brucella in manure of infected animals, suggest another mean of Brucella dissemination and manure could be a vehicle for indirect dissemination and could be an important potential risk factor. Due to lack of a gold standard test apart from recovery of Brucella organisms which is a difficult process, the enhancement and validation of available serological tests are required to enhance the specificity and sensitivity of the employed tests. Predictive values are more appropriate than are sensitivity and specificity during screening programs. Probably, the solution to the problems of epidemiological investigation of Brucella outbreaks with precise diagnosis will require employing of multiple tests possessing different tasks of the immune response.