Seroprevalence and Molecular Epidemiology of Brucellosis in Cattle in Egypt

The study was applied on 4772 lactating and non-lactating cows distributed on different districts in Al Sharqia Governorate. Sera were collected from animals during routine diagnosis and control program. The results of screening tests Buffer acidified plate antigen test (BAPAT), Rose Bengal plate test (RBPT), Tube agglutination test (TAT), Complement fixation test (CFT) and indirect enzyme linked immunosorbent assay gave 124 and 176 seroreactive animals by incidence of 4.42% and 8.91% in private farms and individual cases respectively. 37 (29.8%) and 97 (55.1%) isolates of Brucella melitensis biovar 3 were recovered from 124 and 176 seroreactive animals respectively. In seroreactive cows, Brucella melitensis biovar 3 was isolated from 36% and PCR yielded expected products in 40%. In conclusion, more attention should be paid to the role of Brucella melitensis biovar 3 in brucellosis in cattle during the application of national program of brucella control and eradication.


Introduction
Brucellosis is a wide spread disease among animals and human and of a major economic importance due to abortions, decrease milk yield, temporarily or occasionally permanent sterility [1]. In Egypt, B. abortus was the commonly isolated species until the beginning of 1970s [2]. In the last years, B. melitensis become the most common strain prevalent in animals in Egypt [1,3,4]. Brucella is a facultative, intracellular, gram negative, bacterial pathogen and the etiologic agent of brucellosis, important zoonosis with a nearly worldwide distribution [5]. � distribution of the disease appears to be correlated with high animal densities associated with winter feeding [6]. Clinical symptoms of brucellosis are ������ and its diagnosis in sheep is currently based on serological and microbiological tests [7,8]. A variety of antimicrobial drugs have activity against Brucella, however, the results of in vitro susceptibility tests do not always correlate with clinical ����M Bacteriological isolation of B. melitensis and/or positive blood culture soon �� the infection are common laboratory procedures that are used for diagnosis. However, these procedures are not always successful as they are complicated and represent a great risk of infection for laboratory technicians [9,10]. Serological tests can also be used for diagnosis of Brucella spp. infection via detection of antibodies in serum [11].
In addition, the organism can be detected by polymerase chain reaction (PCR) in blood, semen and abomasal �� of aborted fetuses and, compare to culture method, PCR has more sensitivity and ����� [8,12]. Recently, � PCR assay has been used for detection of Brucella spp. It is a promising alternative for conventional bacteriological techniques due to its speed, safety, high sensitivity and ������ In Egypt, control of Brucellosis is yet a ����• task since it had been diagnosed by Ahmed [13], despite the exhaustive ��� and ����• concepts of approach; this ����� is mainly due to the very high cost and the wide range of maintenance factors of Brucella organisms. � aim of the present study was to determine the immune response of vaccinated animals and the presence of Brucella in blood �� vaccination using serological tests and PCR method.

Description of the study area
�� study was carried out in Al Sharqia Governorate, East of Cairo. � region was purposively selected for the study because it's have large numbers of animals and human populations. � study was carried out between September 2008 to July 2011. � region has warm and cool dry season with considerable variation of rainfall from year to year. � area has a long rainy season which extend from November to April and a dry season which extends from May to October.

Animals
A total of 4772 cattle were examined for Brucella. � animals were kept under restricted program for controlling the internal and external parasites, vaccination programs and standard level of nutrition; whereas mineral mixture and water were available add libitum.

Study design and samples size estimation
A cross-sectional study was conducted to determine the epidemiology of Brucella infection in animals in ����• districts making up the Al Sharqia region. � sample sizes for animals for serological studies, milk samples from cattle for molecular studies, were calculated by the formula of multistage random sampling described by [36]. A random sample of ����• villages of Al Sharqia Governorate was done, using a table of random numbers, from a sampling frame comprised of a list of all villages in the study area. We collected the samples from ����• private farms and ����• areas for individual animals; we select the number of animals every year.

Samples collection
Cattle were manually restrained and had blood samples taken from the jugular vein using 10 ML plain vacutainer tubes. � blood samples from cattle were allowed to clot in a slant position and serum samples were harvested �� 24 h. � harvested serum was transferred to 1.5 ML cryovials and stored in Liquid Nitrogen (LN) before being transferred to the laboratory at the Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt where they were stored in an ultradeep freezer (-80 °C) until tested. Cows from each selected households were randomly selected and sampled to obtain a total of 4772 animals. Milk samples were collected under hygienic condition from udders of cattle by hand stripping just prior to milking using sterile screw caped 50 ML falcon tubes. Each sample was composed of representative amount of milk taken from each quarter. Volumes of about 12 ML of milk sample were taken from each quarter to have a total of 50 ML of milk from cattle. First streaps of milk from each quarter were discarded. Blood was collected from lactating cattle in which milk was sampled for Brucella DNA detection using 10 ML plain vacutainer tubes for serological study. � samples were immediately stored in LN before transferred to the laboratory at the Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt to be stored in ultra-deep freezer (-80 °C) until tested.

Epidemiological investigation
Cows of ����• ages and gestation stages, Lactating and nonlactating were examined for abortion and breeding troubles including retained placenta, retained placenta with ����• birth, endometritis and repeat breeder. Data regarding beginning of these troubles were also recorded.

Bacteriological isolation
Blood samples were collected for isolation and ������� of bacteria according to Alton et al. [15] and serological tests were applied according to Hess [17] and Lambert and Amerault [18]. Bacteriological culture was carried out on specimens from retropharyngeal, supramammary lymph nodes obtained from seroreactive animals was described by Alton et al. [15]. Biochemical tests, dye sensitivity, exposure to ������ antisera, susceptibility to antibiotics and lysis by phages were performed on colonies with characteristics typical of genus Brucella.

Serological tests
� collected serum samples were examined by BAPAT and RBPT as screening tests. �� all positive serum samples were further retested by TAT, CFT and ELISA as quantitative ������ tests. � antigens of BAPAT, RBPT, and TAT were supplied by Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt and performed according to Alton et al. [15]. ELISA antigen was supplied from Synbiotics Europe 2, rue A-Fleming 69007 Lyon-France. Serum samples were performed by ELISA as mentioned by Cardoso et al. [21]. Rose Bengal Plate test (RBPT) was done according to Alton et al. [15] and Morgan et al. [19]. While, ���� ���� plate antigen test (BAPAT) was applied according to [20].In addition, tube agglutination test (TAT) was done according to the method adopted by the central veterinary laboratory (C.V.L.), Weybridge, England as described by Alton [7]. Complement Fixation test (CFT) was done according to Alton et al. [15].

PCR assay
Extraction of DNA was carried out according to [22]. PCR and oligonucleotide primers: the brucella Omp 2 gene was used as target DNA. � forward primer (p1 {5' TGGAGGTCAGAAATGAAC 3'}) and reverse primer (p2 {3' GAGTGCGAAACGAGCGC 5'}) of an Omp 2 gene segment were obtained from National Bioscience, Inc., Plymouth, Minn. PCR ������ was performed by the method of [23]. A typical reaction mixture contained 50 mM Kcl, 1.5 mM MgCl2, 0.1 % (wt/vol). Triton X-100, 0.2 mg of bovine serum albumin (fraction IV; Sigma) per ml, and mM each of the four deoxyribonucleotides, 100 ng of sample DNA and each oligonucleotide primer. For slide PCR, sample DNA was replaced with brucella that was laid on a glass slide, air dried, and �� by being heated. A sample of the dried cells was then collected with a needle, the needle was dipped in 10 ul of double-distilled water, and 2 ul from this solution was put in the PCR mixture. Otherwise, sample DNA (2 ul from a bacterial cell suspension in double distilled water boiled at 100 C for 20 min.) was used. Reactions were initiated by adding 0.5 U of Taq polymerase (Appligene, IIIkirch, France).
� reaction mixture was covered with 15 ul of mineral oil (Sigma) to prevent evaporation. Following hot start treatment at 95 C for 3 min., PCR was performed with an Eppendorf ������ (Eppendorf, Humburg, Germany) as follow 35 cycles of PCR, with 1 cycle consisting of 20 s at 95 C for DNA denaturation, 1 min at 50 C for DNA annealing, and 1 minutes at 72 C for polymerase mediated primers extension. � last cycle included incubation of the sample at 72 C for 7 min. ten micro liters of the ���� product was analyzed with electrophoresis in 1.5 % agarose gel in TEA ��� (20 mM Trisacetate, 1 mM EDTA {pH 8.0}.

PCR results
Ten (0.40) out of 25 milk samples from seropositive cattle tested positive with PCR. While Seven (0.28)out of 25 serum samples from seropositive cattle tested PCR positive. � Brucella species detected from cattle was Brucella melitensis biovar 3. All PCR positive results were shown by migration of PCR product to approximately 720-bp for Brucella melitensis fragments (Figure 1 and Table 3).

Discussion
Brucellosis is a well-documented zoonosis worldwide posing serious public health problems and extensive economic losses [22]. In these areas brucellosis represents a �����• public health issue and its incidence might reach more than 200 cases per 100000 populations [24]. � misdiagnosis and under reporting of brucellosis were make the true incidence of brucellosis remains unknown and might extend to 25 times higher than the ���� one [25]. � ultimate goals of vaccination are to control disease and reduce or eliminate transmission from reservoir species. To accomplish these goals in ruminants using brucella vaccines, the development of more ����� vaccination mechanisms are need to enhance vaccine ����M Our results revealed that the antigen reach the immune system and it delivered to the antigen presenting cells are fundamental in the induction of an optimal immune system response. � incidence of brucellosis in cows either lactating or none lactating during ����• stages of gestation, and heifer's ones in some private farms and individual animals, the percentages of serologically reactors were 4.42 % and 8.9 % for cows in private farms and individual respectively. �� results recorded by [26,16] who observed that vaccine used serves to modify the uptake and processing of antigen. Furthermore, [27] suggested that prolonged persistence of the vaccinal strain in the host needed for the development of suitable anti-brucella immunity.
Bacteriological isolation of brucella from milk samples was 8 % and 19 % and 25 % and 31.6 % from lymph nodes of seroreactive animals from private farms and individual cases respectively; this result agrees with [15] who isolate brucella species from milk samples and lymph nodes.
PCR has increasingly been used as a supplementary method in Brucella diagnosis [32]. Recently a molecular biotyping approach has been proposed on the basis of restriction endonuclease polymorphism in the genes encoding the major outer proteins of Brucella membrane [33]. � Omp2 gene exists as a locus of two nearly homologous repeated copies that ��� slightly among Brucella species and biotypes [34]. We used previous information to design ���� primers that amplify a 720 bp fragment lanes 3, 4 and 5 shows the positive samples taken from ��• farm �� vaccination with RB51 vaccine, whereas lane 7 only positive samples collected from second farms, Lanes 6 and 8 were negative for PCR against brucella species.
We assumed that the sensitivity of the test would be doubled by selecting duplicated DNA sequences of two gene, we assumed that because of the existing Pst I site polymorphism between B. melitensis and B. abortus, the test is ���� for distinguishing between 2 species [35].
In conclusion, raising goats with large dairy animals is a faulty traditional practice, whereas it may be a source of B. melitensis infection for animals in Egyptian villages. It should be focused on the problem of the disease in small ruminants as they played a role in transmission of the disease to eliminate it and reduce the prevalence of the disease among cattle.