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Research Article *Corresponding author Mayada Gwida, Faculty of Veterinary Medicine, Department of Hygiene and Zoonoses, Mansoura Seroprevelance of Bovine University, Mansoura 35516, , Tel: 002050 2372592; Fax: 00205023799; E-mail: Submitted: 30 October 2015 Brucellosis in the Nile Delta Accepted: 10, December 2015 Published: 12 December 2015 Region, Egypt: A Preliminary ISSN: 2378-931X Copyright Study © 2015 Gwida et al. OPEN ACCESS Mayada Gwida1*, Maged El-Ashker2, Mohamed El-Diasty3, Falk Melzer4 and Heinrich Neubauer4 Keywords 1Department of Hygiene and Zoonoses, Mansoura University, Egypt • Brucellosis 2Department of Internal Medicine and Infectious Diseases, Mansoura University, Egypt • Nile Delta 3Department of Infectious Diseases, Mansoura Provincial Lab, Egypt • Egypt 4Friedrich-Loeffler-Institute, Institute of Bacterial Infections and Zoonoses Jena, Germany • Serology • Zoonoses

Abstract There is currently an obvious discrepancy between the officially reported data for seroprevalence of human and animal Brucellosis in Egypt and the data obtained from scientific reports. The present study gives a preliminary data about the seroprevalence of Brucella infection in dairy cattle herds from three large Egyptian Governorates located in the Nile Delta region. The study population consisted of 2.830 dairy cattle from private farms in Dakahlia, and Port-Said Governorates. Serum samples were randomly collected from 811 cows (203 animals showed reproductive disorders group 1); while 608 cows were apparently healthy (group 2). The collected sera were tested by Rose Bengal Test (RBT), Enzyme Linked Immunosorbant Assay (ELISA) and Fluorescent Polarization Assay (FPA). The overall seroprevelance among the tested cows in the first group was 52.2% (106/203). ELISA showed the highest number of positive reactors (n = 138; 67.9%) followed by FPA (n = 120; 59.11%) and RBT (n = 109; 53.7%); while in group 2, the number of positive animals were 25 (4.2%), 20 (3.3%) and 11 (1.8%) by using RBT, ELISA and FPA, respectively. It is very likely that brucellosis Could poses a great risk to consumers in the study region and Could be a potential source of infection to animal keepers, veterinarians and slaughterhouse workers. Further studies are warranted to unravel the epidemiological situation of human, animal and environmental brucellosis in other regions of Egypt.

INTRODUCTION Egyptian control program, brucellosis remains endemic among ruminants and humans and recent reports suggest that the Brucellosis, caused by Brucella spp., is considered one of incidence of human infection is dramatically increased [6]. Given the most important contagious diseases in animals causing that infected animals are the source of human infection, the unbearable serious impacts due to abortion, premature birth, decreased milk production, and reduced reproduction rate of a similar trend in domestic animals [2]. Several authors have resulting in substantial economic losses [1,2]. It also represents a increasing incidence of human brucellosis is probably a reflection great public health problem in the Mediterranean region, western attributed the limited success of the control program in Egypt to Asia, parts of Africa and Latin America and more than 500,000 improper diagnosis and spreading of the disease at large animals humans get infected with brucellosis every year worldwide [3]. markets where different animal species of unknown health status from different localities intermix. Additionally, small ruminant the island of Malta in the 19th and early 20th centuries. In Egypt, 7]. Brucellosis was first recognized as a disease affecting humans on Other researchers linked the lack of Brucella flocks present in high numbers in Egypt are highly migratory [ endemic in most parts of the country [4]. The annual economic compensation for owners and the emotional attachment of control to insufficient lossesit was reporteddue to brucellosis in 1939 for were the estimated first time andto be is about now considered 60 million owners to their animal’s resulting in slaughtering of only 0.2% of Egyptian pounds yearly in 1995 [5], but actual estimates are seropositive animals [8,9]. Importantly, the failure to get access still missing. Despite almost 30 years of implementation of the to all animals which should be tested according to the control

Cite this article: Gwida M, El-Ashker M, El-Diasty M, Melzer F, Neubauer H (2015) Seroprevelance of Bovine Brucellosis in the Nile Delta Region, Egypt: A Preliminary Study. J Vet Med Res 2(5): 1037. Gwida et al. (2015) Email:

Central Bringing Excellence in Open Access program is also one reason for the lack of reliable data on animal MATERIALS AND METHODS brucellosis in the country [10]. Study population and selection criteria Brucella melitensis (main hosts are small ruminants) and B. An informed consent for investigation was given by abortus (main hosts are bovids) are the species causing most brucella human cases. Human brucellosis is well documented from all the owners. All procedures were performed in accordance with over Egypt with annual incidence ranging from 64 to 70 per Guidelines 100,000 in Fayoum Governorate [6]. Three and 11% of acute for the Care and Use of Agricultural Animals in Research and the principles and specific guidelines presented in the febrile illness patients in Egypt were positive either by culture or Teaching (3rd ed.; http://www.fass.org/), and those of Mansoura serology, respectively [11]. The vast majority of brucella isolated University Animal Care and approved by its Ethical Committee. from humans was B. melitensis [12]. The disease is spreading During 2012 and 2013, a total of 2.830 Holstein-Friesian mainly due to close contact between farmers and their animals, dairy cattle aged between 2 to 5 years, from farms located at occupational exposure of farmers, veterinarians and butchers to Dakahlia, Damietta, and Governorates were examined infected animals and through the food-chain via contaminated for Brucella infection according to the practiced routine control milk and dairy products. program (Table 1). The data retrieved from the farm medical Accurate diagnosis of the disease is considered as an elusive records indicated that all cattle investigated were not previously target. Isolation of Brucella species or detection of its DNA by vaccinated with Brucella vaccine. These animals were allocated using PCR is the only method that allows certainty in diagnosis into two groups. Group 1 included 1.920 cattle with various [13]. Despite being the gold standard, bacterial isolation has been reproductive disorders; while group 2 included 910 apparently reported to show poor sensitivity for samples with low-level healthy cattle with no history of reproductive troubles (Table contamination and is impractical for regular screening of large 1). All holdings were chosen because they were known to have populations [14]. Thus, serological tests still have a major role good management practices. They were considered as closed in the routine diagnosis of brucellosis especially in developing epidemiological entities. countries due to their ease in handling, high sensitivity and Sampling low price [15]. In this setting, Buffered Plate Agglutination Blood samples were randomly collected from cows with reproductive disorders (n = 203) and from apparently healthy screeningTest (BPAT), tests Enzyme [16]. RBT, Linked Serum Immunosorbant Agglutination Assay Test (ELISA) (SAT), ones (n = 608). As many cows as possible were sampled on a farm Complementand Fluorescent Fixation Polarization Test (CFT), Assay and (FPA) indirect are still ELISA appropriate (IELISA) have also been applied for the diagnosis of brucella infection [17]. was collected from each animal through jugular vein puncture Although these tests are well-established for the diagnosis of usingto detect plain asymptomatic vacutainer tubes and chronic and needles. cases. Each Briefly, blood ten sample ml of blood was labeled with the respective animal number. The collected blood standards. There is also an obvious discrepancy between the samples were kept overnight at room temperature for clotting. bovine brucellosis in Egypt, they still doBrucella not follow infection the scientific in Egypt On the next day, sera were collected and stored in cryo-tubes at -20°C until further examination. presentofficially study reported was seroprevalenceplanned to provide for a preliminary study on the Serological tests prevalenceand the data of cattle obtained brucellosis from scientificin three large reports. Governorates Therefore, in the Eastern Nile Delta region using different serological tests. All sera were tested by RBT, ELISA and FPA. RBT was

Table 1: The numbers and distribution of the cattle investigated (n = 2.830) in the selected regions. Location Number of animals on the farm Number of samples being tested Health status Port said 520 100 Reproductive disorders Damietta 1.400 103 Reproductive disorders Damietta 600 334 Apparently healthy Dakahlia 310 274 Apparently healthy Total 2.830 811

Table 2: Seroprevalence of brucellosis among cattle showed reproductive disorders. Number Serologicaltest of positive Number of animals RBT ELISA FPA Location examined by all animals serological + % - % + % - % + % SUSP % - % test Port said 100 44 44 44 56 56 57 57 43 43 52 52 4 4 44 44 Damietta 103 62 65 63.1 38 36.9 81 78.6 22 21.4 68 66 10 9.7 25 24.3 Total 203 106 109 53.7 94 46.3 138 67.98 65 32.02 120 59.113 14 6.896 69 33.99

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Central Bringing Excellence in Open Access performed as described in the Manual of Standards for Likewise, most previous studies were applied in response to Diagnostic Tests and Vaccines[17] using antigen obtained from clinical settings such as noticing of abortion cases among the Institute Pourquier, France. Positive and negative control sera reared cattle [18] such reports do not comply with the standards for epidemiological investigations specially study design or to OIE. The ELISA was performed and results were interpreted biostatistics. We aimed at presenting a reliable preliminary accordingwere the German to the instructions national reference of the manufacturessera standardized using according IDEXX™ data about the seroprevalence of the disease to be expected in Brucella-Ab ELISA kit (Montpellier SAS, France). FPA was done well managed dairy farms in three large Governorates where and results were interpreted according to the instructions of the actual epidemiologic data are still scarce. Several authors assumed that hotspots for animal brucellosis are located in animal was considered infected if it tested positive in all tests. the Nile Delta region and in Upper Egypt, along the River Nile the manufacturer (Diachemix, Whitefish Bay, and WT, USA). An where the south of the Delta contains 32% of the Egyptian large RESULTS ruminant plus 39% of the small ruminant stocks which are often The distributions of cattle investigated along with the kept in small mixed herds owned by single householders [19]. Interestingly, a very low rate of infection was reported in seven individual seroprevelance among cattle exhibited reproductive upper Egyptian Governorates (Benisuef, Al Minia, Assuit, , disordersserological was findings 52.2%; are beingsummarized higher in tables Damietta 1-to- region 5. Briefly, than the in Quena, and ) within the framework of the veterinary Port-Said. Collectively, ELISA gave the highest number of positive service where the prevalence was 0.79% in investigated cattle; samples (n = 138; 67.9%) followed by FPA (n = 120; 59.11%) while it was 0.2% in household cows where at least one animal then RBT (n =109; 53.7%) (Table 2). Out of the healthy animals was seropositive [20].The assumption of hotspots needs further examined (n = 608), 532 (87.5%) tested negative; while 3.3%, 4.1% and 1.8% were serologically positive by using ELISA, RBT biosafety measures in practice for our study on purpose. We and FPA, respectively (Table 3). These positives were found only doclarification. think that Wethe didselected choose herds well whose managed control farms programs having could good in Dakahlia farm; however, no positive reactors were reported have the best prospect of success as introduction of new stock among healthy cattle tested in Damietta region. include serological testing beforehand. Given that these herds are of prominent importance for the supply of many (urban) DISCUSSION consumers, the control of brucellosis here could have the biggest Brucellosis is an emerging threat and can be considered impact on public health. By doing so, it is obvious that our data cannot be compared with data from earlier studies including developing nations including Egypt. Despite its potential impact farms or holdings despite their management form on a more onone public of the health, most the common epidemiologic global situation zoonoses, of especiallyBrucella infection in the or less random basis. In the present study, 109 cows with in Egypt is still unresolved and warrant further investigation. reproductive disorders (53.7%) plus 25 apparently healthy cows

Table 3: Detailed serological investigations of cattle with reproductive disorders in the region of Port Said (n = 100) and Damietta (n = 103). Port said (n= 100) RBT ELISA FPA 44 Pos Pos Pos 40 Neg Neg Neg 8 Neg Pos Pos 5 Neg Pos Neg 3 Neg Neg Sus Damietta (n= 103) 62 Pos Pos Pos 18 Neg Neg Neg 10 Neg Pos Neg 8 Neg Pos Sus 1 Neg Neg Pos 1 Neg Neg Sus 1 Pos Pos Neg 1 Pos Neg Neg 1 Pos Neg Sus

Table 4: Seroprevalence of brucellosis in the study group with apparently healthy animals. Number of Serological test Number of negative RBT ELISA FPA Location examined animals by all animals serological + % - % + % - % + % SUSP % - % test Damietta 334 298 14 4.2 320 95.8 1 0.3 333 99.7 2 0.6 21 6.3 311 93.1 Dakahlia 274 235 11 4.0 263 96.0 19 6.9 255 93.1 9 3.3 20 7.3 245 89.4 Total 608 532 25 4.1 583 95.9 20 3.3 588 96.7 11 1.8 41 6.7 556 91.4

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Central Bringing Excellence in Open Access (4.1%) tested positive using RBT. Here, RBT gave lower incidence seen. Hence, it is not astonishing that we found a high number of positives in group 2 as brucellosis are an important abortifacient agent in chronically affected herds when the total number of byof positive acidic pHreactors of antigen in the [21]first. group However, compared the test to ELISA gave and a higher FPA. abortions is usually considerably low again. Thispercentage could be of attributed positive reactors to inhibition among of non-specific cattle of group agglutinins 2. The An earlier national serological survey which was conducted high number of seropositive in clinically inconspicuous animals between 1994 to 1997 [4] where 40% of the total ruminant of brucellae LPS with that of other bacterial species including population was tested (as part of a brucellosis surveillance Yersiniawas surprising. enterocolitica This finding O: 9 and might E.coli be serotypecaused by O: cross 157 [reactions22]. The and control project) and have reported the seroprevalence of detection rate was higher in animals suffering from reproductive brucellosis among cattle, buffalo, sheep and goat at the following disorders when compared with those of the clinically healthy group (Table 3). Additionally, ELISA showed the highest number in mind that the real seroprevalence was underestimated at that detection rates: 0.9%, 0.3%, 1.8% and 8.2%, respectively. Keeping of positive samples among cattle suspected to be infected with time and we might face a bias towards overestimation, our results brucellosis as well as the highest number of negative cases indicate that ruminant brucellosis in the study area has increased in the last 15 years. In general, the prevalence that found in this study (4%) was higher than those reported by other researchers or(Table greater 4). Oursensitivity findings than were CFT in andharmony RBT andwith could other be researchers a suitable [28,7,29,30]. In contrast, a higher prevalence rate was also found testwho for reported large scale that screening ELISA could for bebovine highly brucellosis specific and[23,24]. of equal Our in cattle by recent reports corroborating our idea of a steadily increasing brucellosis problem in the last years [31,32,33]. In who found the same magnitude of sensitivity between ELISA, FPA andfinding RBT. is In also contrast, coincided it was with assumed that of that Konstantinidis FPA can be comparable et al. [25] and buffaloes was estimated to be 11.0% and that for household to the standard tests including RBT, CFT and iELISA), thus it is toMenufia be 15.5%, Governorate, respectively the [34]. individual B. melitensis seroprevalence (Bv 3) was forregularly cattle now included into the European Union (EU) legislation on intra- isolated from cows and buffaloes of seven Egyptian Governorates community trade of bovines as standard test for brucellosis diagnosis [26]. It was surmised that FPA could replace the Assiut) despite the fact that the normal reservoir of this species card test (CT) as a screening test for its better performance is(, goats and Behira,sheep. The Monofia, seroprevalence Qalioubia, reported , Benisuef, was around and compared to CFT due to its adjustable cut-off useful in different 5% in cattle in those Governorates [7]. In one study conducted epidemiological situations, and for its reliability, ease of in Sharkia Governorate, the incidence of Brucella infection was 6.72%, 5.62%, 7.61% and 10.95% in cows, buffaloes, sheep and and high-throughput laboratories [27]. Our serological data can goats, respectively; while it was 37.74% among the tested human performance, comparable costs, and potential application in field only show that brucellosis is endemic in dairy cow herds in the samples [31] where B. melitensis biovar 3 was isolated from Nile delta even if biosafety is managed well. It is obvious that the both man and animals. Hence, a different management form i.e. different tests have to be evaluated in the epidemiological setting cohabitation of cattle and small ruminants can be assumed for of the Egyptian Nile delta where Holstein Frisian cows from non those farms which is unusual on well managed farms. Thus a new endemic regions and local breeds are present. We do think that control program should include only those premises where no the number of false positives was reduced by the procedure to mixed cohabitation is practiced. consider an animal only as positive if all three tests were positive. We assume that still a relevant number of false positives may be CONCLUSION Taken together, it becomes apparent that Brucella infection Table 5: could be endemic in the studied regions at North Eastern Nile cattle. Delta region of Egypt. It is very likely that this infection could pose Detailed findings of serological tests in apparently healthy Number RBT ELI5A FPA a considerable risk to consumers and may also a potential source Damietta farm (n = 334) 298 Neg Neg Neg of infection to animal keepers, veterinarians and slaughterhouse 20 Neg Neg Sus workers etc. Great care should be taken in handling animals with 13 Pos Neg Neg reproductive disorders. Setting up a tailored program for well 1 Pos Neg Sus 1 Neg Neg Pos of the disease to supply safe dairy products for a broad range of 1 Neg Pos Pos consumers.managed dairy herds is the first step towards a better control Dakahlia (n=274) 235 Neg Neg Neg ACKNOWLEDGEMENTS 17 Neg Neg Sus 3 Neg Pos Pos This work was in parts funded by the German Federal 1 Neg Pos Sus program: Deutsches Partners chafts program für 3 Pos Neg Neg biologische Sicherheit und Gesundheitssicherstellung Foreign Office 6 Pos Pos Pos Conflict of Interest 7 Neg Pos Neg 2 Pos Pos Sus Abbreviations: The authors of this paper haventent no financialof the paper. or personal RBT: Rose Bengal Test; ELISA: Enzyme Linked relationship with other people or organizations that could Immunosorbant; FPA: Fluorescent Polarization Assay appropriately influence or bias the co J Vet Med Res 2(5): 1037 (2015) 4/5 Gwida et al. (2015) Email:

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