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, Br. abortus was the commonly isolated species until the beginning of 1970s [2]. In the last years, Br. 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]. The distribution of the disease appears to be correlated with high animal densities associated with winter feeding [6]. Clinical symptoms of brucellosis are non-specific and its diagnosis in sheep is currently based on serological and microbiological tests [7,8]. While there is no treatment of choice for animal brucellosis. Bacteriological isolation of Br. melitensis and/or positive blood culture soon after 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 fluid of aborted fetuses and in compare to culture method, PCR has more sensitivity and specificity [8,12]. Recently, The PCR assay has been used for detection of Brucella spp. It is a promising alternative for conventional bacteriological techniques due to its speedy, safety, high sensitivity and specificity. In Egypt, control of brucellosis is yet a difficult task since it had been diagnosed by Ahmed [13], despite the exhaustive efforts and difficult concepts of approach; this difficulty is mainly due to the very high coast and the wide range of maintenance factors of Brucella organisms. The aim of the present study was to determine the immune response of vaccinated animals and the presence of Brucella in blood after vaccination using serological tests and PCR method [14].

Animals

A total of 710 lactating and non-lactating cows from were examined for Brucella during the period from 2008 to 2011 in Sharqyia Province, Egypt. Case history and/or owner complain were recorded.

Samples collection

Blood samples were collected from jugular veins of all animals. Serum samples were kept frozen (-20°C) till analysis. Tissue specimens were taken from supra-mammary lymph nodes, spleen, uterus and mammary glands of obligatory slaughtered animals.

Epidemiological investigation

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

Bacteriological isolation

Blood samples were collected for isolation and identification of bacteria according to Alton et al. [15] and serological tests were applied according to Hess and Lambert [16-18]. Bacteriological culture was carried out on specimen from retropharengeal, supramamary, lymph nodes obtained from seroreactive animals were described by Alton et al. [15]. Biochemical tests, dye sensitivity, exposure to monospecific antisera, suscesptability to antibiotics and lysis by phages were performed on colonies with charateristics typical of genus Brucella.

Serological examination

Rose Bengal Plate test (RBPT) was done according to Alton et al. and Morgan et al. [15,19]. While, buffered acidified plate antigen test (BAPAT) was applied according to Angus et al. [20]. In addition, tube agglutination test (TAT) was done according to the method adopted by the central veterinary laboratory (C.V.L.), Weybride, England as described by Alton et al. [7]. Complement Fixation test (CFT) was done according to Alton et al. [15]. While Enzyme Linked Immunosorbent Assay (ELISA) was done according to Cardoso et al. [21].

PCR

Extraction of DNA was carried out according to Donis- Keller et al. [22]. PCR and oligonucleotide primers: the Brucella omp 2 gene was used as target DNA. The 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 amplification was performed by the method of Mullis et al. [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 fixed by being heated. A sample of the dried cells was then collected with a needle, the needle was dipped in 10 μl of double-distilled water, and 2 μl from this solution was put in the PCR mixture. Otherwise, sample DNA (2 μl 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).

The reaction mixture was covered with 15 μl of mineral oil (Sigma) to prevent evaporation. Following hot start treatment at 95°C for 3 min., PCR was performed with an Eppendorf Thermocycler (Eppendorf, Humburg, Germany) as follow 35 cycles of PCR, with 1 cycle consisting of 20 sec 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. The last cycle included incubation of the sample at 72°C for 7 min. ten micro liters of the amplified product was analyzed with electrophoresis in 1.5% agarose gel in TEA buffer (20 mM Trisacetate, 1 mM EDTA {pH 8.0}.

According to case history and/or owner complains, cows in this experiment suffered from reproductive disorders such as repeat breeding, infertility and abortion.

Serological investigation

Table 1 show the highest incidence of positive reactors was given by BAPAT, RBPT, TAT, CFT, RIVT and ELISA (4.42%).

Table 1: Incidence of brucellosis in lactating, non-lactating and heifers cows examined using different serological tests.

Bacterial isolation

Brucella melitensis biovar 3 was isolated in 17 out of the 50 positive reactors (Table 2).

Table 2: Bacteriological isolation of seroreactive animals.

PCR

PCR was indicative of brucellosis in 40% of the positive reactors as shown by the typical PCR product specific for Brucella (720 base pair) (Figure 1 and Table 3).

Figure 1: Electrophoretic pattern of PCR product 720-bp bp in 1.5% agarose gel stained with Ethidium bromide. Lane M standard DNA marker, lane 1 positive control, lane 2 negative control, Lanes 3, 4 and 5 positive blood sample DNA PCR; lane 6 and 8 negative Blood sample, lane 7 positive vaccine DNA PCR.

Table 3: Evaluation of different diagnostic tests in Brucella seroreactive animals.

Evaluation of different Brucella diagnostic test

Evaluation of the different tests used for diagnosis of brucellosis was recoded in Table 3. It is evident that the most comparable serological tests with bacterial isolation and PCR were CFT and ELISA.

Abortion and breeding troubles

The percentages of abortion in pregnant cows suffering from brucellosis in some private farms were 1.55%, 2.61%, 3.16% and 3.06% for yeas 2008, 2009, 2010 and 2011 respectively (Tables 4 and 5). While, the percentages of abortion in pregnant cows suffering from brucellosis collected from individual animals were 2.29%, 1.41%, 2.59% and 2.83% for yeas 2008, 2009, 2010 and 2011 respectively (Table 6).

Table 4: Percentage of abortion in private farms.

Table 5: Percentage of abortion in individual investigated animals.

Table 6: Percentage of cows suffering from brucellosis associated with breeding troubles.

Brucellosis is a well-documented zoonosis worldwide posing serious public health problems and extensive economic losses [22]. In these areas brucellosis represents a significant public health issue and its incidence might reach more than 200 cases per 100000 populations [24]. Due to misdiagnosis and underreporting though its true incidence remains unknown and might extend to 25 times higher than the official one [25]. The 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 efficacious vaccination mechanisms are need to enhance vaccine efficacy.

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. The incidence of brucellosis in cows either lactating or none lactating during different 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. These results recorded by Sun et al. [16] and Donis-Keller et al. [26] 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 [28-31].

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]. The Omp2 gene exists as a locus of two nearly homologous repeated copies that differ slightly among Brucella species and biotypes [34]. We used previous information to design specific primers that amplify a 720 bp fragment lanes 3, 4 and 5 shows the positive samples taken from first farm after 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 specific for distinguishing between 2 species [35].

The percentages of abortion in pregnant cows suffering from brucellosis in some private farms were 1.55%, 2.61%, 3.16% and 3.06% for yeas 2008, 2009, 2010 and 2011 respectively. While, the percentages of abortion in pregnant cows suffering from brucellosis collected from individual animals were 2.29%, 1.41%, 2.59% and 2.83% for yeas 2008, 2009, 2010 and 2011 respectively. These results were lower than those reported by Kahl-McDonagh, et al [28], Sayour [29], Hamdy [30] whose recorded 16.1%, 37.4%, and 26%, respectively.

The breeding troubles of investigated animals were retained placenta, difficult birth, retained placenta and difficult birth, endometritis and repeat breeder in random investigated animals were 2.7%, 4%, 1.3%, 2% and 0.7% respectively. From this data we concluded that breeding troubles increase the susceptibility to infection with Brucella, this agrees with [15,21,31] who proved that breeding troubles and poor feeding increasing the infection with Brucella.

In conclusion, raising goats with large dairy animals is a faulty traditional practice, whereas it may be a source of Br. 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.