Abstract: Background and Objective: The economic losses due to brucellosis as well as its potential public health in human worldwide encourage more researches to find novel pathways for effective control methods of the disease. The objective of this study was to investigate the most prevalent Brucella strains obtained from cattle and their virulence genes. Materials and Methods: Three hundred small-holders cows in Menoufia governorate, Egypt, were screened for brucellosis using rose bengal test (RBT) and confirmed by complement fixation test (CFT). Milk samples and supra-mammary lymph nodes of serologically positive cows were collected for bacteriological isolation and identification. The obtained isolates were genotyped using PCR and their virulence genes (omp25, omp31, manA, manB, virB and znuA) were screened. Results: The prevalence rate of bovine brucellosis was 15 (5%), 11 (3.6%) and 7 (2.33%) by RBT, CFT and bacteriological examination, respectively. The seven isolates were identified and genotyped as Brucella melitensis biotype3. Furthermore, the molecular detection of substantial virulence genes revealed that manA, manB, omp25 and omp31 genes were detected in all tested B. melitensis strains. Meanwhile, the virB genes were detected in 4 strains and the znuA genes were detected in 3 strains among the isolated B. melitensis strains. Conclusion: It was concluded that B. melitensis biotype3 was the pre-dominant Brucella spp. as well as omp25, omp31, manA and manB were the most common related-virulence genes which assumed to play a worthy function in the pathogenesis of brucellosis.
INTRODUCTION
Brucellosis is a highly contagious zoonotic disease causing significant losses in animals. Late gestation abortion was the predominant clinical sign of B. abortus infection in cows, resulting in reproductive failure and decrease in milk production1. It was estimated that more than 300 million cattle as well as more than half a million of human infections occurred2. Egypt, Middle East and Mediterranean countries were endemic areas with higher prevalence rates3. Brucella invades the host cell and interferes with the different immune system mechanism through adaption to oxygen-limited conditions inside macrophages4. Brucella has a special affinity for the reticuloendothelial system and reproductive tracts of domestic and wild animals. Stillbirth in females and infertility in males are the most common complications of the brucellosis in livestock5. Brucella spp. including B. melitensis and B. abortus have similar pathogenic effects and zoonotic concern with some differences in virulence properties and host susceptibility. These pathogens carry various furtive strategies to enter into host cells then propagate and overcome the host defense mechanism6. In dairy animals, Brucella spp. replicates in the mammary gland and supra-mammary lymph nodes and continue to be excreted into the milk along with their lives7. A recent discovery of new a typical Brucella spp., with new genetic properties was recorded8. Therefore, it was expected that a new outbreak of brucellosis may occur in the future. Brucella was already exhibiting virulence factors required to form the infection due to their activation by erythritol9. The constant researches for Brucella virulence genes such as cell envelope associated genes and other virulent genes such as omp25, omp31, manA, manB, wbkA, cβg, virB, mviN, znuA and bvfA genes are necessary to understand their role in Brucella pathogenesis, characterize the Brucella spp., genome and have efficient control measures10. As a result of the brucellosis' endemic status in Egypt and the need to develop new preventive measures against brucellosis, this study was aimed for molecular detection and genotyping of the most virulence genes of local Brucella strains affecting cattle in Egypt.
MATERIALS AND METHODS
This study was assessed and agreed by the Animal Care and Welfare Committee Ethics, University of Sadat City, Egypt with approval number (VUSC-001-4-19).
Examined animals: The present study was carried out in Menoufia governorate, which located in Nile Delta, Egypt. A total number of 300 cows of small-holders which were not vaccinated against brucellosis were examined during the period from January-April, 2018.
Serum samples: About 10 mL of blood was collected from jugular vein of cattle. Collected samples were kept in a refrigerator overnight for serum separation then were centrifuged at 3000 rpm for 5 min. Clear sera were siphoned off and stored in cryotubes at -20°C until its use for serological studies11.
Milk and tissue samples: From all serologically positive animals, milk samples were collected immediately before slaughtering from lactating cows. While the supra-mammary lymph nodes were collected after slaughtering in sterile containers and then both samples were transferred immediately to the laboratory for further bacteriological examination12.
Serological analysis: Three hundred serum samples were examined by Rose Bengal Test (RBT) as a screening field test. Positive RBT samples were confirmed by complement fixation test (CFT)13.
Phenotypical identification of Brucella species: Milk samples and supra-mammary lymph nodes of all serologically positive animals were sterile cultivated onto Petri dishes containing selective medium prepared using Brucella medium base as described (Oxiod, code CM0169) with addition of 5-10% v/v inactivated horse serum SR0035 and 1-5% w/v of a sterile solution of glucose and finally added to Brucella selective supplement as described (Oxoid, code SR0083). All plates were incubated at 37°C with and without 5-10% CO2 for 5-7 successive days11. The typical biochemical and phenotypic identification of the suspected Brucella colonies (morphology, CO2 requirements, H2S production, urease, catalase and oxidase activity, nitrate reduction, lactose fermentation, citrate utilization, growth in presence of thionine and fuchsin dyes “1:50.000 (20 μg mL–1)”, lysis by Tbilisi phage and agglutination with A and M anti-sera) were done13,14.
Molecular characterization of Brucella species and virulence determinant genes
DNA extraction: The DNA was extracted from isolates using commercial kits (Thermo Scientific Gene Jet Genomic DNA purification Kit) (Cat nu, K0721) according to manufacturer recommendations.
| Table 1: | Sequence of used primers |
PCR amplification: The PCR assay was performed to identify Brucella isolates, targeted the bcs P gene coding outer protein of Brucella, reverse the primer that targeted the Brucella insertion element IS711 and forward primer for B. melitensis and a set of duplex PCR assays that targeted six different virulence genes namely manA, manB, omp25, omp31, znuA and virB. The sequences of each primer are presented in Table 1. All reactions were performed in total volume of 25 μL reaction mix which contained 3 μL of template DNA, 20 pmol of each primer and 1X of PCR mix (Fermentas). The analysis of PCR products was carried out using 1.5% ethidium bromide stained agarose gel.
Cycling condition of PCR assays: This was performed for BCSP and B. melitensis at the following conditions: (1) About 4 min initial step at 95°C, (2) Followed by 35 cycles at 95°C for 60 sec, 55°C for 60 sec and 72°C for 90 sec and (3) A final extension step at 72°C for 10 min. While PCR amplification assays for virulence genes (omp25, omp31, manA, manB, vir B and znuA) were as following: (1) About 5 min initial step at 94°C, (2) Followed by 25 cycles at 94°C for 60 sec, 58°C for 60 sec and 72°C for 60 sec and (3) A final extension step at 72°C for 10 min.
Statistical analysis: Statistical analysis was performed using Graph Pad prism 5 software Inc., La Jolla, CA.
RESULTS
Prevalence of Brucellosis in examined cattle: Out of examined 300 cows, 15(5%) and 11(3.6%) were positive with RBT and CFT, respectively. The results of bacteriological examination revealed only 7(2.33%) isolates which were identified as B. melitensis biotype3 “4 (1.33%) were from supra-mammary lymph nodes and 3 (1%) were from milk samples” as illustrated in Table 2.
Genotyping of B. melitensis strains: The obtained seven isolates were genotyped using a common bcsP gene and a specific gene IS711. The results indicated that all 7 tested isolates were B. melitensis biotype3 as shown in Fig. 1 and 2.
The molecular detection of substantial virulence genes among the isolated B. melitensis biotype3: Successful amplifications of 228 and 465 bp specific for manB and znuA genes, respectively were obtained (Fig. 3). Also amplicons of 188 and 257 bp specific for omp25 and omp31 genes, respectively were given (Fig. 4). Regarding to 881 and 271 bp specific bands for virB and manA genes, respectively were obtained (Fig. 5).
Distribution of detected virulence genes among the isolated B. melitensis biotype3: The results of detected virulence genes revealed that manA, manB, omp25and omp31 genes were found in all seven tested B. melitensis strains. Meanwhile, the virB genes were detected in 4 strains and the znuA genes were detected in 3 strains among the screened B. melitensis isolates. All milk samples were carried manA, manB, omp25 and omp31 genes, while the virB genes were detected in 2 samples and the znuA genes were detected in 2 samples as well. Also, all samples from lymph nodes were carried manA, manB, omp25 and omp31 genes, while the virB genes were detected in 2 samples and the znuA genes were detected in only 1 sample among the screened B. melitensis strains (Table 3).
| Table 2: | Prevalence of brucellosis in examined cattle |
| RBT: Rose bengal test, CFT: Complement fixation test, *Total isolates were 7 (2.33%) strains which were identified as B. melitensis biotype3 | |
| Table 3: | Distribution of detected virulence genes among the isolated B. melitensis biotype3 |
| S: Strain number | |
| Fig. 1: | PCR product showed positive amplification of bCSP gene of B. melitensis at 223 bp (common gene) |
| Lane M: 100 bp DNA ladder, Lane 1-7: Positive B. melitensis strains | |
| Fig. 2: | PCR product showed positive amplification of IS711 gene of B. melitensis at 731 bp (specific gene) |
| Lane M: 100 bp DNA ladder, Lane 1-7: Positive B. melitensis strains | |
| Fig. 3: | PCR product; Lane M: 100 bp DNA ladder; Lanes1-7 B. melitensis biotype3 were positive for manB gene at 228 bp |
| Lanes 3, 5 and 6 were positive for znuA genes at 465 bp | |
| Fig. 4: | PCR product: Lane M: 100 bp DNA ladder, Lanes 1-7 B. melitensis biotype3 were positive for omp25 and omp31 genes at 188 and 257 bp, respectively |
| Fig. 5: | PCR product: Lane M: 100 bp DNA ladder; Lanes 1, 2, 4 and 6 B. melitensis biotype3 were positive for virB genes at 881 bp, while Lanes 1-7 were positive for manA genes at 271bp |
DISCUSSION
Brucellosis is one of the most globally distributed diseases reported by WHO as half a million new cases reported each year20. Brucellosis is a bacterial zoonotic disease transmitted via various routes such as; contact directly with abraded skin or mucosa, fluids, tissues from aborted fetus21. The high endemic pattern of the disease in many underdeveloped countries caused large economic losses in livestock animals as well as chronic infections in human22. In the current study, the prevalence rates of brucellosis were 5, 3.6 and 2.33% with RBT, CFT and bacteriological examination, respectively. These results coincided with previous results which recorded that the prevalence of brucellosis in cattle was 5.3% by RBT in Bangladesh23,24. Furthermore, other reports demonstrated that RBT and CFT were effective methods for the detection of Brucella spp. antibodies25,26. In the Gulf area, a comparative study was applied in Sultanate of Oman where higher results were obtained (11.7 and 8.3%) positives using RBT and CFT, respectively in goats’ sera27.
The difference in the prevalence might be attributed to the difference in methodology, number of samples, examined regions and management system. The current study demonstrated seven Brucella isolates which were identified and genotyped as B. melitensis biotype3. This was supported previously by studies declared that brucellosis is endemic in Egypt particularly in the Nile Delta region and B. melitensis remains the more virulent strain with public health28,29.
In the current study, results of screening the six putative virulence genes of B. melitensis from cattle samples detected omp25, omp31, manA and manB in all isolated strains. While the virB was found in 4 strains and the znuA was detected in 3 isolates only. Although, Brucellae are deficient in some of the classical factors of ferocity and strength, such as capsules, fimbria, plasmids, exotoxins, lysogenic phages and endotoxic lipopolysaccharide, they expressed some virulent genes30.
These virulence determinants were vital for Brucella spp., to live, adapt intracellularly to inappropriate conditions and resist body immune response31. From these substantial genes, Omp25/Omp31 were described as major surface proteins strongly contributed to the virulence of Brucella32. The outer membrane protein (Omp31) was a protective immunogenic component for subunit DNA vaccines of B. melitensis as well as a simulator for the cell-mediated immunity33,34. Moreover, the major function of omp25 depends on suppression of the tumor necrosis factor alpha (TNFα) produced by macrophages35. The virB type IV secretion system (T4SS) was a gene coding for an enzyme involved in the biosynthesis of LPS36-38. Concerning to the critical role of manA and manB and znuA genes among Brucella spp., many reports confirmed the contribution of manB genes in lipopolysaccharide synthesis which allowed the intracellular survival and protection against host defense39. Others pointed out the importance of znuA and manA genes in the virulence properties of B. melitensis particularly in ATP-binding mechanism40. As well as the presence of znuABC operon which is linked to Zn2+ ions as a catalyst for the Cu and Zn superoxide dismutase (sodC genes) and provided the resistance to oxidation process by phagosome41. The present findings were in contact with results of Naseri et al.42, who detected omp25, omp31, manA, manB, virB and znuA virulence genes in all isolated B. melitensis strains recovered from human patients in Iran. Nearly similar findings were obtained by another researcher who recovered that the prevalence rates of omp25, omp31, man A, manB and znuA genes were 87.5, 100, 87.5, 100 and 100%, respectively from human blood samples in Iraq43. In addition, it was detected manA, Omp25, Omp31, manB and znuA with different percentages 100, 100, 76.9, 38.5 and 19.2%, respectively44. On the other hand, many studies reported other different virulence genes as revealed that all B. melitensis and B. abortus strains were carried virB2 and virB5 genes45. Also, it was recorded that 98.1% of B. melitensis biotype3 were carried virB genes in Egypt46. Despite that, it was also confirmed by a report in which virB genes in 73.8% of B. melitensis strains isolated from goats47. This discrepancy in results might be attributed to the variation of sources, number of samples and prevalent strains in the examined geographic regions. Finally, due to the seriousness of bovine brucellosis and its impact on public health, further studies are needed to spot highlights on the role of these genes and others in the contagiousness of brucellosis and the ability to produce types of vaccines to control or minimize the disease incidence.
CONCLUSION
It could be concluded that B. melitensis biotype3 is the most predominant Brucella spp., among cows in Menoufia governorate. The most frequent virulence genes are omp25, omp31, manA and manB among isolates which are assumed to play a worthy function in the pathogenesis of brucellosis in this region. Moreover, it may be helpful for authorized affairs to develop a strategic plan for the prevention and eradication of this disease.
SIGNIFICANCE STATEMENT
This study discovered that B. melitensis biotype3 was the prevalent circulating strain among cows in Menoufia governorate, Egypt. Also, it provided a beneficial view for clarifying the substantial role of omp25, omp31, manA, manB, virB and znuA virulence determinant genes in bovine brucellosis. This study assessed the worthy values of these genes in the virulence of Brucella. Further investigations were needed to evaluate other impact roles of these genes or other genes in bovine brucellosis.
ACKNOWLEDGMENT
The researchers would like to thank the Directorate of Veterinary Medicine of Menoufia governorate for helping us to collect samples and data in this research.