Abbreviations
RBT: Rose Bengal Agglutination Test
STAT: Standard Tube Agglutination Test
ELISA: Enzyme-Linked Immunosorbent Assay
FPA: the fluorescence polarization assay
CFT: Complement Fixation Test
WOAH: World Organization of Animal Health
2ME: 2-Mercapto-Ethanol
PCR: Polymerase Chain Reaction
B.: Brucella
Spp: Species
Introduction
Bovine brucellosis is most commonly caused by B. abortus, less regularly by B. melitensis, and rarely by B. suis, all zoonosis pathogens of the genus Brucella [1] . Transmission to humans occurs primarily through contact with infected reproductive secretions or consumption of infected dairy products [ 2] . In cattle, Brucella organisms tend to localize in the supra-mammary lymph nodes and mammary glands from which they may be shed into milk [ 3].
The disease must be eradicated from animals to control brucellosis in the human, and this is largely pursued through national tests and slaughter programs in the endemic areas. To identify Brucella spp. For infected cows, different methods are available, but because of the limitations of each test, the exact diagnosis of brucellosis in cows is still challenging. Antigen detection tests, such as bacterial culture and PCR, identify the presence of Brucella spp. directly. Although bacterial culture is considered the diagnostic gold standard, it is less sensitive, time-consuming, and labor-intensive, and imposes a serious biohazard on laboratory personnel [ 4, 5]. Antibody detection or serological assays including the Rose Bengal Agglutination Test (RBT), Standard Tube Agglutination Test (STAT), Enzyme-Linked Immunosorbent Assay (ELISA), fluorescence polarization assay (FPA), and Complement Fixation Test (CFT) can be used as screening tests in the control program of brucellosis [ 4]. However, these tests are hindered by cross-reactivity between Brucella species and other Gram-negative bacteria, such as Yersinia enterocolitica O9, Escherichia coli O157, Francisella tularensis, Salmonella urbana, Vibrio cholera, and stenotrophomonas maltophilia [ 6]. The structural similarity of smooth lipopolysaccharide O-chain between Brucella spp. and these bacteria, underlies this problem. For example, Muñoz and colleagues (2005) reported that up to 15% of cattle herds in brucellosis-free regions produced false-positive results in serological tests due to cross-reaction with Yersinia enterocolitica O9 [ 7]. Consequently, a single serological test is not sufficiently reliable for screening individual animals [ 4]. To improve accuracy, at least two antigen and/or antibody detection tests are required to confirm the cattle brucellosis [ 8]. According to diagnostic regime recommended by the World Organization of Animal Health (WOAH), serological tests are applied in Iran to diagnose positive reactor cows. Serum samples from semi-industrialized and industrialized dairy farms are first screened by RBT, with positive sera are tested using STAT, including Wright's test, and 2-Mercapto-Ethanol (2ME) tests. Animals identified as positive reactors are then slaughtered with biohazard precautions. Because the test results drive the slaughter decisions, the sensitivity and the specificity of these tests are critical. So, paying more attention to the test strategies used to identify Brucella spp. in farm animals is an important neglected issue.
In the present study, we tried to evaluate the agreement between serological tests and antigen detection tests for diagnosing bovine brucellosis. Specifically, we evaluated how many serologically positive reactor cows could also be confirmed by bacterial culture, conventional PCR, and real-time PCR. An additional objective was to identify the Brucella species infecting cows of Fars province, Iran. Consequently, the presence of Yersinia entrocolitica O9 strain in the lymph nodes of cows was determined to be a probable cause of false-positive results in the brucellosis serological tests.
Results
Out of 98 lymph node samples collected from positive reactor cows, conventional PCR detected Brucella genus in 15 samples (15.3%) (Figure 1).
Figure 1. The gel picture of conventional PCR for Brucella spp. detection. A 223 bp band is obvious in the positive PCR products. Lane 2 shows a 100 bp DNA ladder. Lanes 3 and 4 have different concentrations of positive controls, and lane 1 has no template controls (NTC). Other lanes show samples. An Aliquot of B. abortus IRIBA vaccine (Razi, Iran) was used as the positive control.
The Bruce-ladder multiplex PCR, designed for specie level identification of Brucella, did not produce any PCR band from DNA extracted from lymph node tissues. However, when applied to DNA extracted from cultured Brucella isolates, the Bruce-ladder PCR was successfully differentiated the species of Brucella isolates (Figure 2).
Figure 2. The gel picture of the Bruce ladder.Lane 1 was a 100 bp DNA ladder. Lanes 2 to 5 were B. abortus from cultured bacterial colonies indicated by 152, 587, and 1682 bp bands. Lane 6 was B. abortus IRIBA strain positive control, which was similar to the RB51 strain, showing 152, 587, and 2524 bp bands on the gel, and lane 7 was B. melitensis Rev1 strain positive control, confirmed by 152, 218, 587, 1071, and 1682 bp bands. As 450 and 794 bp bands of the original Bruce ladder did not apply to B. melitensis and B. abortus identification, their primers were not used in Bruce ladder PCR.
Real-time PCR analysis identified 21 Brucella spp. positive samples (21.4%) out of 98 lymph nodes tested. Melting peak analysis and sequencing of PCR products confirmed all positive samples were B. abortus (Figure 3). No statistically significant relationship was observed between real-time PCR-positive samples and the level of 2ME Brucella titer.
Figure 3. A) Melting peak analysis of B. abortus specific (A) and B. melitensis specific (B) real-time PCR. The indicator lines show the melting peaks of B. abortus IRIBA strain and B. melitensis Rev1 strain positive controls. The graphs show that all of the samples have melting peaks similar to that of B. abortus (A) and none of them were located under B. melitensis melting peak (B).
Brucella spp. was isolated from only 5 samples (5.1%) in bacterial culture, all of which were identified as B. abortus using Bruce ladder multiplex PCR.
Given that only 21.4% of positive reactor cows were confirmed positive by real-time PCR, further investigation was conducted to explore the probable reason. As Y. entrocolitica O9 strain was one of the bacteria that might cause serological cross-reactions with B. abortus (CSFPH 2018), the prevalence of this strain was evaluated in the lymph node samples. Although 20 lymph node samples tested positive for Y. entrocolitica, none were identified as an O9 serotype.
Discussion
The study demonstrated that among 98 lymph node samples collected from positive reactor cows, Brucella spp. was detected only in 15 (15.3%) and 21 (21.4%) samples by conventional PCR and real-time PCR, respectively. In a similar study, O'Leary and colleagues (2006) applied conventional and real-time PCR to different samples from serologically Brucella spp. positive cows, slaughtered under Ireland's eradication program. They reported B. abortus detected in 3 (14.2%) and 4 (19%) out of 21 supra-mammary lymph nodes samples using conventional and real-time PCR, respectively [ 9] , which these results are consistent to our findings. Also, in another study, Tiwari and colleagues (2014) reported that from 132 STAT-positive serum samples, only 14 sera (10.6%) were positive by real-time PCR with B4-B5 primers, the same primers used in our conventional PCR assay [10] . A probable reason for the low percentage of PCR-positive results in Tiwari`s study may be partly explained by the type of biological sample, as both our and O'Leary`s studies were conducted on the lymph nodes, whereas Tiwari's was based on serum.
O'Leary and colleagues (2006) also compared Brucella spp. detection rate by conventional and real-time PCR across different sample types, including milk, blood, and lymph node. They sampled from both supramammary and retropharyngeal lymph nodes and concluded that the supramammary lymph node is the most reliable tissue for PCR detection of Brucella spp. [ 9] . Their conclusion served as the basis for selecting the sample tissue, and the supramammary lymph nodes were sampled in this research. Nevertheless, according to the tropism of Brecella spp. [11] , other organs rich in phagocytes, such as the spleen, and organs of the genital system (such as the uterus), could also be suitable sample types for Brucella spp. detection.
The real-time PCR result of this study showed that 78.6% of reactor cows were Brucella spp. negative at the time of slaughtering. Given that the supramammary lymph node is considered one of the best reservoirs for Brucella detection in cows [ 9] , these results suggest that a substantial proportion of reactor cows may be free of infection and are therefore unlikely to shed Brucella spp. in milk. These cases were those that Brucella spp. bacteria do not remain as an active infection in them. However, their antibody is still detectable by serological tests or individuals never infected with Brucella spp. but exposed to other bacteria that immunologically cross-react with Brucella spp. Several organisms are known to cause serological cross-reactions with Brucella, including Yersinia enterocolitica O9 strain, Escherichia coli O157, Francisella tularensis, Salmonella urbana, Vibrio cholera, and stenotrophomonas maltophilia [ 6] . As Y. enterocolitica O9 strain was not detected in any lymph node samples, it could be concluded that immunological cross-reaction with this bacterium was not the reason for the few real-time PCR-positive results among Brucella reactor cows.
In this study, the sensitivity of conventional and real-time PCR tests was more than that of Brucella spp. culture. This observation has been inconsistently reported in the literature. In some studies, PCR sensitivity has been reported more than that of Brucella spp. culture method [12 , 13]. Hamdy and Amin (2002) compared the sensitivity of PCR and culture methods on bovine milk samples and reported that the PCR sensitivity was greater than that of Brucella spp. Culture [13]. whereas in in another study, they reported culture method to outperform PCR [14]. Also, some researchers reported similar results [ 9] . This study uses Farrell's medium, the most widely used Brucella spp. A selective medium was used for the culture prepared by adding six antibiotics to a basal medium. Because some strains of B. abortus and B. melitensis may be inhibited by nalidixic acid and bacitracin, two antibiotics in the supplement, the use of this medium may reduce the culture method sensitivity and explain the fewer positive samples of bacterial culture than those of PCR methods.
Molecular characterization in this study identified B. abortus as the predominant species infecting cattle in Fars province, Iran. This finding was by multiplex and real-time PCR, and further confirmed by sequencing. Human brucellosis caused by B. melitensis is more severe than the disease caused by B. abortus [14] , and in terms of public health, B. melitensis is considered a more important zoonosis pathogen. Similar to this study, there are many reports that only isolated B. abortus from cow samples from Turkey [15], Pakistan [16], Ireland [ 9], and Uganda [17] , but also there are some studies that isolated B. melitensis in addition to B. abortus from cows [18]. The most similar study to ours was performed by Sharifiyazdi et al. (2010), who isolated 17 Brucella spp. from 95 positive reactor cows in the same province; of which only one was B. melitensis, and the others were B. abortus [19] . By comparing these results, it could be concluded that the Brucella species infecting cows of this region have not changed from 14 years ago, and cows in Fars province are not the source of human B. melitensis infections.
Finally, it could be concluded that the current serological test combination was conducted in Iran according to WOAH to diagnose the Brucella spp. antigen detection tests do not confirm infected cows. We have to know that the lack of specificity in the test regime could waste many healthy cows, limiting the government's potential to widen the brucellosis eradication program to all of the farm animal population, including non-industrialized native cows and sheep. Although real-time PCR is not currently feasible as a routine diagnostic tool directly on serum sample, this test could provide valuable information about the Brucella species circulating in the slaughtered cows of each region.
Materials and Methods
Cattle Herds and Sampling
Serum sampling was performed on semi-industrialized and industrialized dairy farms across all regions of Fars province, Iran, under the national brucellosis control program. All cows were lactating Holstein or crossbreeds, raised in the intensive farms. They had been vaccinated against brucellosis following to the Iranian Veterinary Organization (IVO) guidelines [ 20] using a vaccine (RVSRI, Iran) containing the IRIBA strain of B. abortus. Infected cows were diagnosed using serological assays, including RBT, Wright's agglutination tests, 2-ME agglutination tests, performed in IVO laboratories according to the WOAH guidelines [ 4] . RBT was applied as the initial screening test, with RBT-Positive sera further evaluated by Wright's agglutination tests and 2-ME agglutination tests. Interpretation of results took into account cow age, vaccination history, and the prior brucellosis condition of the sampled farm. The positive RBT cows would be divided into positive reactors (≥ 4/80 Wright and 4/40 2-ME titers), doubtful, and negative (≤ 1/20 in both tests) cases. Brucellosis cases were retested 3 to 4 weeks later to confirm their status [ 20].
In this study, supramammary lymph nodes were sampled from 98 serologically Brucella spp. positive cows from 20 farms in Fars province, Iran. Lymph nodes were obtained after slaughtering under the national brucellosis control program. Samples were transferred to the laboratory in cool boxes and stored at -20 °C until use.
Bacterial culture
One of the supramammary lymph nodes was transferred to the laboratory of the Department of Brucellosis, Razi Vaccine and Serum Research Institute (RVSRI), Iran, the only nationally authorized laboratory for Brucella spp. culture from animal samples. Samples were cultured on Brucella-specific agar enhanced with 7% defibrinated sheep blood and Brucella supplement (Oxoid, UK). The supplement contained the following quantities of antibiotics for 1 liter of agar: polymyxin B sulfate (5000 IU); bacitracin (25,000 IU); natamycin (50 mg); nalidixic acid (5 mg); nystatin (100,000 IU); vancomycin (20 mg). Plates were incubated at 37°C in 10% CO2 for 21 days. Colonies were identified as Brucella spp. based on morphology, serology, and conventional biochemical assays (catalase, oxidase, and urease tests).
DNA extraction
The second supramammary lymph node was used for DNA extraction. Firstly, an emulsion was prepared using a pestle and mortar from 100 µg ground section of each lymph node. Nucleic acid was extracted using a bacterial DNA isolation kit (Denazist Asia, Iran) from emulsion samples according to the manufacturer`s instructions. Some of the extracted DNA was electrophoresed on a 1% agarose gel to check the integrity and purity and the quantity of extracted DNA were determine using a Nanodrop (Bioteck, USA) .
Conventional PCR of Brucella spp.
To detect the Brucella genus, a PCR test was conducted in all DNA samples using the following primers: B4: `5-TGGCTCGGTTGCCAATATCAA-`3 and B5: `5-CGCGCTTGCCTTTCAGGTCTG-`3 [ 21]. A total volume of 25 µl consisted of 1 µl b4 primer (10µM), 1 µl b5 primer (10µM), 12.5 µl Red master mix (Ampliqon, Denmark), 5.5 µl molecular grade water, and 5 µl template DNA. A thermal cycler (BioIntelectica, Canada) was used to run the following PCR program: 5 min at 95 °C as initial denaturation, and 35 cycles of 95 °C 1 min, 63 °C 30 sec, and 72 °C 1 min, followed by 72 °C 10 min.
Multiplex PCR of Brucella spp.
Species-level identification of Brucella was performed using the Bruce ladder multiplex PCR. This assay combines eight primer pairs in a single PCR reaction, and Brucella species are identified based on each sample's different PCR bands (ladder) [ 22] . As the bands created by two primer pairs known as BMEI0535f-BMEI0536r and BMEI1436f- BMEI1435r were similar in B. abortus and B. melitensis species (expected Brucella species in cow), they were not incorporated in a master mix of multiplex PCR, leaving six primer pairs, as shown in the Table 1.
| Primer name | Sequence (`5–`3) | Amplicon size (bp) | Target |
|---|---|---|---|
| BMEI0998f | ATC CTA TTG CCC CGA TAA GG | 1,682 | Glycosyltransferase, gene wboA |
| BMEII0844r | GCT TCG CAT TTT CAC TGT AGC | ||
| BMEII0843f | TTT ACA CAG GCA ATC CAG CA | 1,071 | Outer membrane protein, gene omp31 |
| BMEII0844r | GCG TCC AGT TGT TGT TGA TG | ||
| BMEII0428f | GCC GCT ATT ATG TGG ACT GG | 587 | Erythritol catabolism, gene eryC (Derythrulose- 1- phosphate dehydrogenase) |
| BMEII0428r | AAT GAC TTC ACG GTC GTT CG | ||
| BR0953f | GGA ACA CTA CGC CAC CTT GT | 272 | ABC transporter binding protein |
| BR0953r | GAT GGA GCA AAC GCT GAA G | ||
| BMEI0752f | CAG GCA AAC CCT CAG AAG C | 218 | Ribosomal protein S12, gene rpsL |
| BMEI0752r | GAT GTG GTA ACG CAC ACC AA | ||
| BMEII0987f | CGC AGA CAG TGA CCA TCA AA | 152 | Transcriptional regulator, CRP family |
| BMEII0987r | GTA TTC AGC CCC CGT TAC CTC | ||
| As BMEI0535f-BMEI0536r and BMEI1436f- BMEI1435r did not apply to B. melitensis and B. abortus identification, these two pairs were deleted from the original Bruce-ladder primers. | |||
The thermal program consisted of 95 °C 15 min, 35 cycles of 95 °C 35 sec, 63 °C 45 sec, 72 °C 1 min, and finally 72 °C 10 min. 0.62 µl of each forward and 0.62 µl of each reverse primer (10 µM), 12.5 µl of Tempase master mix (Ampliqon, Denmark), and 2.5 µl of DNA sample were mixed (25 µl total volume).
Real-time PCR of Brucella spp.
Two individual Real-time PCR were performed to identify two species of Brucella (B. abortus and B. melitensis) in all DNA samples using a high-resolution melting (HRM) program. Each real-time PCR differentiates one species from others by comparing the melting peak of an unknown PCR product versus that of a certified positive PCR product. These tests were designed based on a single nucleotide difference in the glk gene of B. abortus and the int-hyp gene of B. melitensis, with the nucleotide sequence of other species, which causes a slight difference in melting peaks. Real-time PCR primer pairs specific for B. abortus and those specific for B. melitensis were named Boa and Bmel, respectively. Their sequences were:
Boa For: `5-GACCTCTTCGCCACCTATCTGG-`3
Boa Rev: `5- CCTTGTGCGGGGCCTTGTCCT-`3
Bmel For:`5- GAGCGATCTTTACACCCTTGT-`3
Bmel Rev:`5- GGACGGTGTAATAAACCCATTGG-`3 [ 23].
A common thermal program was run by the Light Cycler 96® instrument (Roche, Germany) as follows: initial denaturation of 95 °C for 10 min, then 95 °C for 10 sec and 60 °C for 50 sec repeated 40 cycles followed by HRM program from 65 °C to 95 °C by 0.2 °C /step ramp rate. Some real-time PCR products were sequenced to ensure the substitution of one nucleotide in the glk gene of B. abortus.
PCR tests for the detection of Yersinia entrocolitica O9 strain
Two PCR tests were set up to evaluate the Yersinia entrocolitica strain O9 infected cows. Firstly, a PCR test for the detection of all strains of Yersinia entrocolitica was conducted, and then another PCR test was performed on the positive samples of the first PCR to detect specifically the O9 strain. In the first PCR, 227Fmod: (`5-GTCTGGGCTTTGCTGGTC-`3), and YER2: (`5-ATCTTGGTTATCGCCATTCG-`3) primer pair targeting ompF gene, and in the second PCR, perF: (`5-GACGGGGGCAAAAGTAGT-`3), and perR: (`5-CTATTGGGAACACCTCTGGA-`3) primer pair [ 24] targeting perosamine synthetase gene were used.
In both PCRs, the same master mix components (unless primers), and a common thermal program were applied. For 20 µl total volume of each Y. entrocolitica PCR test, 1 µl of each related primer (10µM) was added to 10 µl Red master mix (Ampliqon, Denmark), 5 µl PCR grade water and 3 µl extracted DNA. Following thermal program: firstly, 95 °C 5 min as initial denaturation, followed by 40 cycles of 95 °C 20 sec, 60 °C 30 sec, 72 °C 30 sec, and finally, 72 °C 7 min as the final extension was applied to PCR microtubes.
To visualize the bands, the PCR products of conventional and multiplex PCR were electrophoresed in 1.5% agarose gels stained with RedSafe (Intron Biotechnology, Korea). The gel pictures were caught by a gel documentation system.
Statistical analysis
Chi-square (χ2) tests were used to compare the amount of serological 2ME titer and the presence of B. abortus in the lymph node samples.
Figure 4 represents a graphical abstract of the materials and methods section.
Figure 4. Graphical abstract.The diagram shows the sampling and the type of experiments conducted in this study.
Authors' Contributions
All authors contributed to the study conception and design. M.Gh. and M.S.G. performed material preparation, data collection, and analysis. M.Gh. wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This study was funded by the Fars province veterinary administration (grant number: 15398-162096).
Conflict of interest
The author declares that there is no conflict of interest.
References
- Chisi SL, Marageni Y, Naidoo P, Zulu G, Akol GW, Van Heerden H. An evaluation of serological tests in the diagnosis of bovine brucellosis in naturally infected cattle in KwaZulu-Natal province in South Africa. Journal of the South African Veterinary Association. 2017 Feb 28;88.DOI
- Alamian S, Esmaelizad M, Zahraei T, Etemadi A, Mohammadi M, Afshar D, et al. A Novel PCR Assay for Detecting Brucella abortus and Brucella melitensis. Osong Public Health and Research Perspectives. 2017 Feb 28;8(1):65–70.DOI
- Poester FP, Samartino LE, Santos RL. Pathogenesis and pathobiology of brucellosis in livestock. Revue Scientifique et Technique de l'OIE. 2013 Apr 1;32(1):105–15.DOI
- WOAH (World organization for animal health). Brucellosis (Brucella abortus, B. melitensis and B. suis). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 13th edition. 2024. Chapter 3.1.4.
- Yu WL, Nielsen K. Review of Detection of Brucella sp. by Polymerase Chain Reaction. Croatian Medical Journal. 2010 Aug;51(4):306–13.DOI
- CSFPH (The center for food safety and public health) Brucellosis: Brucella abortus factsheet, 2018. Available from: http://www.cfsph.iastate.edu/DiseaseInfo/factsheets.htm
- Muñoz P, Marin C, Monreal D, González D, Garin-Bastuji B, Díaz RF, et al. Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9. Clinical and diagnostic laboratory immunology. 2005 Jan 1;12(1):141–51.DOI
- Nielsen K, Gall D, Bermudez R, Renteria T, Moreno F, Corral A, et al. Field trial of the brucellosis fluorescence polarization assay. Journal of Immunoassay and Immunochemistry. 2002 Aug 29;23(3):307–16.DOI
- O'Leary S, Sheahan M, Sweeney T. Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Research in Veterinary Science. 2006 Oct;81(2):170–6.DOI
- Tiwari A, Pal V, Afley P, Sharma DK, Bhatnagar CS, Bhardwaj B, et al. Real-time PCR carried out on DNA extracted from serum or blood sample is not a good method for surveillance of bovine brucellosis. Tropical Animal Health and Production. 2014 Sep 4;46(8):1519–22.DOI
- Carvalho TPD, Silva LAD, Castanheira TLL,Souza TDD, Paixão TAD, Lazaro-Anton L, Tsolis RM, Santos RL. Cell and Tissue Tropism of Brucella spp.. Infection and Immunity. 2023 91:e00062-23.DOI
- Leal-Klevezas DS, Martinez-Vazquez IO, Lopez-Merino A, Martinez-Soriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. Journal of Clinical Microbiology. 1995; 33(12):3087-3090.
- Hamdy MER, Amin AS. Detection of Brucella Species in the Milk of Infected Cattle, Sheep, Goats and Camels by PCR. The Veterinary Journal. 2002 May;163(3):299–305.DOI
- Amin AS, Hamdy MER, Ibrahim AK. Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Veterinary Microbiology. 2001 Oct;83(1):37–44.DOI
- Kaynak-Onurdag F, Okten S, Sen B. Screening Brucella spp. in bovine raw milk by real-time quantitative PCR and conventional methods in a pilot region of vaccination, Edirne, Turkey. Journal of Dairy Science. 2016 May;99(5):3351–7.DOI
- Ali S, Ali Q, Melzer F, Khan I, Akhter S, Neubauer H, et al. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR. Tropical Animal Health and Production. 2013 Jul 19;46(1):73–8.DOI
- Hoffman T, Rock K, Denis Rwabiita Mugizi, Shaman Muradrasoli, Lindahl-Rajala E, Erume J, et al. Molecular detection and characterization of Brucella species in raw informally marketed milk from Uganda. Infection ecology & epidemiology. 2016 Jan 1;6(1):32442–2.DOI
- Dadar M, Alamian S, Behrozikhah AM, Yazdani F, Kalantari A, Etemadi A, et al. Molecular identification of Brucella species and biovars associated with animal and human infection in Iran. Veterinary Research Forum. 2019;10(4):315–21.DOI
- Sharifiyazdi H, Haghkhah M, Behroozikhah AM, Nematgorgani E. Bacteriological and molecular investigation of B. melitensis in dairy cows in Iran. Comparative Clinical Pathology. 2010;21:269-273.
- Amiri K, Jahan peyma D, Makenali AS. et al. National control of animal brucellosis manual. Iranian Veterinary Organization Press. 2016. [in Persian].
- García-Yoldi D, Marín CM, Miguel MJ, Muñoz PM, Vizmanos JL, López-Goñi I. Multiplex PCR Assay for the Identification and Differentiation of all Brucella Species and the Vaccine Strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clinical Chemistry. 2006 Apr 1;52(4):779–81.DOI
- Winchell JM, Wolff BJ, Tiller R, Bowen MD, Hoffmaster AR. Rapid Identification and Discrimination of Brucella Isolates by Use of Real-Time PCR and High-Resolution Melt Analysis. 2010 Mar 1;48(3):697–702.DOI
- Stenkova AM, Isaeva MP, Rasskazov VA. Development of a multiplex PCR for detection of the Yersinia genus with identification of pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica). Molecular Genetics, Microbiology and Virology. 2008;3:18-23.
- Lübeck PS, Mikael Skurnik, Ahrens P, Hoorfar J. A Multiplex PCR-Detection Assay for Yersinia enterocolitica Serotype O:9 and Brucella spp. Based on the Perosamine Synthetase Gene. Advances in experimental medicine and biology. 2004 Jan 1;451–4.DOI
)
Send comment about this article