297 1Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh 13736, Egypt. 2Animal Health Research Institute, P.O. Box 264-Giza, Cairo 12618, Egypt. 3Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany. *Corresponding author at: Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany. e-mail: gamalwareth@hotmail.com. Keywords Brucella spp., MALDI, AMOS-PCR, Cattle and buffaloes, Egypt. Summary Brucellosis is a widespread disease in Egypt which cause huge economic losses in the dairy industry. The present study aims at isolating and identifying Brucella (B.) spp. circulating in bovine and buffalo dairy herds kept at farmers houses in four districts of the Delta region of Egypt. One hundred and five  tissue specimens were collected from seropositive cattle and buffaloes. The samples included 10 vaginal swabs, 3 placentas, 3 uteri and 86 supra-mammary lymph nodes from dams, as well as 3 stomach contents from aborted fetuses. Matrix-assisted laser desorption ionization (MALDI) and the conventional biotyping techniques were used for preliminary identification of isolates into the genus level. AMOS-PCR was applied to differentiate Brucella isolates into species level. Nineteen Brucella strains have been identified, four B. abortus strains were recovered from cattle and 15 B. melitensis strains were isolated from both cattle (n  =  8) and buffaloes (n  =  7). The predominant occurrence of B. melitensis in bovines raises the fact that B. melitensis clone can cross species barriers and can establish a permanent reservoir in cattle and buffaloes. Presence of culture-positive animals at householders represent a high-risk factor for human infection. This knowledge is of significant importance in the control of brucellosis in bovines. Ashraf Abd Eltawab1, Fatma El-Hofy1, Mahmoud Hamdy2, Shawky Moustafa1, Enas Soliman1, Wedad Ahmed1, Mohamed Ramadan1 and Gamal Wareth1,3* Isolation and molecular identification of Brucella spp. in bovine herds kept at householders in the Delta region of Egypt by MALDI-TOF and AMOS-PCR Veterinaria Italiana 2020, 56 (4), 297-300. doi: 10.12834/VetIt.1980.10596.3 Accepted: 23.01.2020 | Available on line: 31.12.2020 of the others are not proved yet. Regarding their capability of causing disease in humans, B. melitensis is considered the most virulence species followed by B. suis, and B.  abortus which, amongst the three species, is considered the mildest one (Galinska and Zagorski 2013). In Egypt, Brucella was reported for the first time in 1939 (Refai 2002). In the 1980ies, attention was directed to the animal diseases when an open-door policy was applied and Friesian cows were imported to establish governmental farms. In that period, the incidence of brucellosis in some farms reached 38% (Refai 2002). Recent National serological investigations gave indirect proof of the presence of Brucella in cattle, buffalo, sheep, and goat herds nationwide (Wareth et  al. 2014a). In Egypt only few laboratories are capable of isolating Brucella. Despite the implementation of the control program, Brucellosis is an infectious zoonotic disease worldwide caused by Brucella (B.) species. The disease might have serious public and animal health impact as infections are often associated to chronic debilitation in humans and reproduction failure in animals (Godfroid et  al. 2011). In the sexually mature female animals, the disease provokes metritis, abortion, stillbirth, retention of the placenta and drop in milk production, in males the disease can cause orchitis and epididymitis. Infertility may be seen in both sexes (Ducrotoy et al. 2014). Up-to-date, the genus Brucella comprises of 12 accepted phenotypically recognized species; B.  abortus, B. suis, B. melitensis, B.  neotomae, B. ovis, B. canis, B. inopinata, B. microti, B. pinnepedails, B.  ceti, B. papponi, and B.  vulpex. Only B. melitensis, B. suis except bv 2, B.  abortus and to some extent B. canis are well-known pathogens for humans (Chiliveru et  al. 2015), while the zoonotic potential SHORT COMMUNICATION 298 Brucellosis in Egyptian dairy herds Eltawab et al. Veterinaria Italiana 2020, 56 (4), 297-300. doi: 10.12834/VetIt.1980.10596.3 performed in 25 µl of a reaction mixture containing final concentrations of 10 pmol/µl for each primer with 0.2 µl of 5  U/µl of Taq DNA polymerase (Promega), and 10  mM/1µl of deoxynucleotide triphosphates (dNTP) (Eppendorf ). One μl DNA template was added to each reaction mixture. The PCR mixtures were overlaid with 2.5 µl of PCR-Puffer (Genaxxon) and complete with HPLC until 25  µL. The amplification was performed at a denaturation temperature of 93  °C for 5 min. This was followed by 35 cycles at 90  °C for 60 sec, 60  °C for 60 sec, 72  °C for 60 sec and 1 final extension at 72  °C for 5 minutes. Positive control (B. abortus 544 DNA) and negative control (HPLC water) were included in each reaction. After amplification, all reaction mixtures were analyzed by electrophoresis in a 1.5% agarose gel, stained with ethidium bromide, and photographed. Visible bands of appropriate sizes of (223 bp) for Brucella spp. were considered positive reactions. To confirm results of MALDI-TOF and secure identification of Brucella species, the AMOS-PCR (B.  abortus, B. melitensis, B. ovis and B. suis-PCR) was done according to Bricker and Halling and Matope and colleagues (Bricker and Halling 1994, Matope et  al. 2009) with few modifications. Briefly, the PCR was performed in a final volume of 25 µl consisting of a reaction mixture containing 10 × PCR buffer (Genaxxon) without Mgcl. 0.2 μM each of B. abortus, B. melitensis, B. ovis, B. suis, and IS711-specific primer added to the reaction (Table I). 10  mM of dNTPs (Eppendorf ), 0.2 µl of 5 U/µl of Taq DNA polymerase (Eppendorf ) were added and complete with HPLC water until 25  µL. 1 μl DNA template was added to the 24 μl reaction mixture. The cycling conditions utilized were initial denaturation of 95 °C for 5 min; followed by 30 cycles of amplification using the following parameters: at 95 °C for 60 sec, annealing for 120 sec at 58  °C, extension for 120  sec at 72  °C, and a final elongation step of 72  °C for 5  min. The PCR products were separated by electrophoresis the disease is still endemic among livestock and humans. Buffaloes and cattle are a valuable and economic component of Egyptian rural household and number of buffaloes is higher than in any other neighboring country in the Near East region. Control programs have been instituted by the general organization of veterinary services (GOVS) to prevent the spread of brucellosis in the country particularly in large dairy herds (Wareth et al. 2014a). More than 70% of total livestock populations are owned by smallholders. Few cattle and buffaloes are kept in the household to produce milk, meat, and dairy products for home consumption or to sell often unpasteurized products in the local markets. The current study was aimed to identify the Brucella species circulating in small buffalo and cattle dairy herds in different localities of Delta region in Egypt using Matrix-assisted laser desorption ionization (MALDI), conventional and AMOS PCRs. A total of 105 seropositive animals (90 cattle, 15  buffaloes) were used in the study. All animals were serologically positive for Brucella by Rose Bengal Test (RBT) and ELISA within the surveillance and eradication program instituted by the GOVS, Egypt. The animals originated from Qalyubia, Gharbia, Dakahliya, and Menofia. From each animal one sample was collected at abattoirs. Different tissue specimens have been taken: 86 lymph nodes, 3 uteri, 3 placenta and 10 vaginal swabs from dams, as well as 3 stomach contents from aborted fetuses. The specimens were collected aseptically and were kept in an icebox and transferred directly to the laboratory for isolation. The organ surface was burned and the internal parts were smeared by the swab. The swabs were plated on Brucella agar for primary isolation and subsequent identification of Brucella species was done according to colony morphology, Gram stain, motility, CO 2 requirement, H 2 S production, phage lysis, and the biochemical tests e.g. catalase, oxidase and urease tests (Alton et  al. 1988). A single colony was selected from the Brucella selective agar media and sub-cultured again on blood agar media to obtain uncontaminated colonies. After 48 hours of incubation, one colony was picked and submitted to Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF) for genus Brucella identification as described previously (Murugaiyan et  al. 2014). The score values between 2.0 and 2.29 considered ‘secure genus identification and probable species identification’. Genomic DNA was extracted with the High Pure template preparation kit (DNA HP kit, Roche Applied Sciences, Mannheim, Germany) according to the manufacturer’s instructions. Two pairs of primers amplifying different regions of the Brucella genome were used, the primer B4 and B5 that amplify a 223  bp fragment of the 31-kDa outer membrane protein. PCR assay conditions were Table I. Primers used in conventional and AMOS-PCR for identification of Brucella in samples collected from seropositive cattle and buffaloes in the Delta region of Egypt. Conventional PCR primer Nucleotide sequence 5'-3 ' Amplicon size B4 primer TGG CTC GGT TGC CAA TAT CAA 223 bp B5 primer CGC GCT TGC CTT TCA GGT CTG AMOS-PCR - B. abortus GAC-GAA-CGG-AAT-TTT-TCC-AAT-CCC 498 bp B. melitensis AAA-TCG-CGT-CCT-TGC-TGG-TCT-GA 731 bp B. ovis CGG-GTT-CTG-GCA-CCA-TCG-TCG 976 bp B. suis GCG-CGG-TTT-TCT-GAA-GGT-TCA-GG 285 bp IS711 TGC-CGA-TCA-CTT-AAG-GGC-CTT-CAT - 299 Eltawab et al. Brucellosis in Egyptian dairy herds Veterinaria Italiana 2020, 56 (4), 297-300. doi: 10.12834/VetIt.1980.10596.3 19  isolates, twelve strains were isolated from cattle and seven from buffaloes. Failure of isolation from the other cases may be due to a low number of viable Brucella organisms in specimens and/or due to massive contamination with other bacteria (Wareth et  al. 2014b). B. abortus was isolated from cattle, while B. melitensis was isolated from both cattle and buffaloes. Previously it was assumed that brucellosis in cattle was mainly caused by B. abortus, less frequently by B. melitensis and rarely by B. suis (Radostits et  al. 2000). In recent years, B. melitensis has been described as cause of great outbreaks in cattle and now is becoming a worldwide emerging problem and difficult to be controlled. This is likely due to the ineffective vaccination programs and lack of knowledge regarding the causative agents in different hosts (Alvarez et  al. 2011). The major limitation of the control program in Egypt is the difficulty to detect all infected animals, especially carriers hosts i.e. dogs, cats, rats (Wareth et al. 2017). Seronegative/culture-positive animals within the herds also play a significant role in re-emerging and dissemination of the infection (El-Diasty et  al. 2018). In Egypt, B. melitensis bv3 and B. abortus bv1 are the predominant source of brucellosis. B. melitensis have been isolated from sheep, goat, cattle, buffaloes, rats and Nile catfish (Wareth et  al. 2014a), while B.  abortus has been isolated from cattle, buffaloes and once from dog and cat (Wareth et al. 2017). Isolation of B. melitensis from cattle and buffaloes, the non-original hosts, can be attributed to raise sheep and goats together with cattle and buffaloes. The current results are in accordance with previous studies that highlighted the fact that B. melitensis clone can cross species barriers and establish a reservoir in cattle and buffaloes (Abd-El Halim et  al. 2017, Hosein et  al. 2018, Wareth et  al. 2014a). Different molecular typing methods such as PCR techniques can differentiate Brucella at the species and the biovars level (Al  Dahouk et  al. 2005, Bricker and Halling 1994). These methods require little expertise, easy to perform and provide valuable alternatives to biochemical typing (Fretin et  al. 2008). Application of MALDI-TOF MS was used successfully for microbial identification into the subspecies level, demonstrating that this technology is also a potentially effective tool for detection of microorganisms (Croxatto et  al. 2012). Combination of AMOS-PCR and MALDI-TOF were able to identify Brucella spp. from a single colony in a short time and are promising techniques for fast and accurate identification. The results obtained in the current study should be considered during control and eradication program of brucellosis. All strains were recovered from small dairy farms which should be included in the control programs. using 1.5% agarose gel (w/v). Visible bands were considered positive reactions at appropriate sizes of 498 bp for B. abortus, 731 bp for B. melitensis, 976 bp for B. ovis and 285 bp for B. suis. As shown in Table II, the bacterial examination of the collected samples from seropositive cattle and buffaloes revealed 19 Brucella strains, 12/90 strains were recovered from cattle and 7/15 were recovered from buffaloes. All recovered isolates showed the characteristic colonial morphology of Brucella on Brucella agar, Brucella selective media, and Blood agar. The colonies were convex, round, 1-2 mm. in diameter, with smooth margins, round edges, translucent and of golden color (pale honey-colored). All isolates were Gram-negative, non-motile, catalase and oxidase-positive. They required CO 2 for growth and produced H 2 S. Conventional PCR and MALDI-TOF confirmed the classical bacteriology results on the 19 strains as Brucella with clear bands and score values >  2.3, respectively. The score >  2 is considered positive for genus Brucella, while the score values between 2.3 and 3.0 indicate ‘highly probable species identification’. MADI-TOF identified four strains as B. abortus and 15 strains as B. melitensis. AMOS-PCR differentiated four B.  abortus strains at appropriate band size of 498 bp, while a total of 15 strains were identified as B. melitensis at appropriate band size of 731 bp. All B. abortus strains identified in the current study were recovered from cattle samples, while B. melitensis strains were isolated from cattle (n = 8) and buffaloes (n = 7). Brucellosis is endemic and prevalent in the Middle East, in the Mediterranean basin, in some countries of Africa and Asia, and in some areas of Latin America. However, still the epidemiological situation is not clear in some of those regions. The present work was planned to isolate Brucella spp. from cattle and buffaloes in different governorates of the Delta region in Egypt. Bacteriological examination of 105 collected samples produced Table II. Number of collected samples and number of Brucella isolates obtained from seropositive cattle and buffaloes in the Delta region of Egypt. Source of samples Cattle Buffaloes No. of samples No. of isolates % No. of samples No. of isolates % Lymph node 74 8 10.8% 12 5 41.6% Uterus 3 1 33.3% - - 0 Placenta 3 2 66.6% - - 0 Stomach content 3 1 33.3% - - 0 Vaginal swabs 7 - 0 3 2 66.4% Total 90 12 13.3% 15 7 46.6% 300 Brucellosis in Egyptian dairy herds Eltawab et al. Veterinaria Italiana 2020, 56 (4), 297-300. doi: 10.12834/VetIt.1980.10596.3 Acknowledgments We would like to thank the Academy of Scientific Research and Technology (ASRT) Egypt for financial support. The work belonging to BrucMedNet project (ID: 698) funded by the ARIMNet2. Abd-El Halim M., Mohamed A. & Shalaby N. 2017. Prevalence of brucellosis in buffaloes and its control measures. Journal of Veterinary Medical Research, 24, 161-170. Al Dahouk S., Tomaso H., Prenger-Berninghoff E., Splettstoesser W.D., Scholz H.C. & Neubauer H. 2005. Identification of Brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Crit Rev Microbiol, 31, 191-196. Alton G.G., Jones L.M., Angus R. & Verger J. 1988. Techniques for the brucellosis laboratory. Institut National de la recherche Agronomique (INRA). Alvarez J., Saez J.L., Garcia N., Serrat C., Perez-Sancho M., Gonzalez S., Ortega M.J., Gou J., Carbajo L., Garrido F., Goyache J., & Dominguez L. 2011. Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Research in veterinary science, 90, 208-211. Bricker B. & Halling S. 1994. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. Journal of clinical microbiology, 32, 2660-2666. Chiliveru S., Appari M. & Suravajhala P. 2015. On Brucella pathogenesis: looking for the unified challenge in systems and synthetic biology. Systems and synthetic biology, 9, 73-75. Croxatto A., Prod'hom G. & Greub G. 2012. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS microbiology reviews, 36, 380-407. Ducrotoy M., Bertu W., Ocholi R., Gusi A., Bryssinckx W., Welburn S. & Moriyón I. 2014. Brucellosis as an emerging threat in developing economies: lessons from Nigeria. PLoS neglected tropical diseases, 8, e3008. DOI:3010.1371/journal.pntd.0003008. El-Diasty M., Wareth G., Melzer F., Mustafa S., Sprague L.D. & Neubauer H. 2018. Isolation of Brucella abortus and Brucella melitensis from seronegative cows is a serious impediment in brucellosis control. Vet Sci, 5 (1), 28. doi: 10.3390/vetsci5010028. Fretin D., Whatmore A.M, Al Dahouk S., Neubauer H., Garin-Bastuji B., Albert D., Van Hessche M., Ménart M., Godfroid J., Walravens K. & Wattiau P. 2008. Brucella suis identification and biovar typing by real-time PCR. Veterinary microbiology, 131, 376-385. Galinska E.M. & Zagorski J. 2013. Brucellosis in humans - etiology, diagnostics, clinical forms. Annals of agricultural and environmental medicine: AAEM, 20, 233-238. References Godfroid J., Scholz H., Barbier T., Nicolas C., Wattiau P., Fretin D., Whatmore A., Cloeckaert A., Blasco J. & Moriyon I. 2011. Brucellosis at the animal/ecosystem/ human interface at the beginning of the 21st century. Preventive veterinary medicine, 102, 118-131. Hassanain N.A. & Ahmed W.M. 2012. Sero-prevalence of brucellosis in Egypt with emphasis on potential risk factors. World Journal of Medical Sciences, 7, 81-86. Hosein H.I., Zaki H.M., Safwat N.M., Menshawy A.M.S., Rouby S., Mahrous A. & Madkour B.E. 2018. Evaluation of the General Organization of Veterinary Services control program of animal brucellosis in Egypt: an outbreak investigation of brucellosis in buffalo. Veterinary world, 11, 748-757. Matope G., Bhebhe E., Muma J.B., Skjerve E. & Djønne B. 2009. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR. BMC research notes, 2, 261. Murugaiyan J., Walther B., Stamm I., Abou‐Elnaga Y., Brueggemann‐Schwarze S., Vincze S., Wieler L., Lübke‐ Becker A., Semmler T. & Roesler U. 2014. Species differentiation within the Staphylococcus intermedius group using a refined MALDI‐TOF MS database. Clinical Microbiology and Infection, 20, 1007-1015. Pappas G., Papadimitriou P., Akritidis N., Christou L. & Tsianos E.V. 2006. The new global map of human brucellosis. Lancet Infect Dis, 6, 91-99. Radostits O., Gay C., Blood D. & Hinchcliff K. 2000. A textbook of the diseases of cattle, sheep, pigs, goats, and horses. WB Saunders, London, 1329-1337. Refai M. 2002. Incidence and control of brucellosis in the Near East region. Veterinary microbiology, 90, 81-110. Wareth G., Hikal A., Refai M., Melzer F., Roesler U. & Neubauer H. 2014a. Animal brucellosis in Egypt. J Infect Dev Ctries, 8, 1365-1373. Wareth G., Melzer F., El-Diasty M., Schmoock G., Elbauomy E., Abdel-Hamid N., Sayour A. & Neubauer H. 2017. Isolation of Brucella abortus from a dog and a cat confirms their biological role in re-emergence and dissemination of bovine brucellosis on dairy farms. Transbound Emerg Dis, 64, e27-e30. Wareth G., Melzer F., Elschner M.C., Neubauer H. & Roesler U. 2014b. Detection of Brucella melitensis in bovine milk and milk products from apparently healthy animals in Egypt by real-time PCR. J Infect Dev Ctries, 8, 1339-1343.