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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.

Journal of Veterinary Medicine and Research

*Corresponding authorMayada Gwida, Faculty of Veterinary Medicine, Department of Hygiene and Zoonoses, Mansoura University, Mansoura 35516, Egypt, Tel: 002050 2372592; Fax: 00205023799; E-mail:

Submitted: 30 October 2015

Accepted: 10, December 2015

Published: 12 December 2015

ISSN: 2378-931X

Copyright© 2015 Gwida et al.

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Keywords•Brucellosis•Nile Delta•Egypt•Serology•Zoonoses

Research Article

Seroprevelance of Bovine Brucellosis in the Nile Delta Region, Egypt: A Preliminary StudyMayada Gwida1*, Maged El-Ashker2, Mohamed El-Diasty3, Falk Melzer4 and Heinrich Neubauer4

1Department of Hygiene and Zoonoses, Mansoura University, Egypt2Department of Internal Medicine and Infectious Diseases, Mansoura University, Egypt 3Department of Infectious Diseases, Mansoura Provincial Lab, Egypt4Friedrich-Loeffler-Institute, Institute of Bacterial Infections and Zoonoses Jena, Germany

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, Damietta 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.

INTRODUCTIONBrucellosis, caused by Brucella spp., is considered one of

the most important contagious diseases in animals causing unbearable serious impacts due to abortion, premature birth, decreased milk production, and reduced reproduction rate resulting in substantial economic losses [1,2]. It also represents a great public health problem in the Mediterranean region, western Asia, parts of Africa and Latin America and more than 500,000 humans get infected with brucellosis every year worldwide [3]. Brucellosis was first recognized as a disease affecting humans on the island of Malta in the 19th and early 20th centuries. In Egypt, it was reported in 1939 for the first time and is now considered endemic in most parts of the country [4]. The annual economic losses due to brucellosis were estimated to be about 60 million Egyptian pounds yearly in 1995 [5], but actual estimates are still missing. Despite almost 30 years of implementation of the

Egyptian control program, brucellosis remains endemic among ruminants and humans and recent reports suggest that the incidence of human infection is dramatically increased [6]. Given that infected animals are the source of human infection, the increasing incidence of human brucellosis is probably a reflection of a similar trend in domestic animals [2]. Several authors have attributed the limited success of the control program in Egypt to improper diagnosis and spreading of the disease at large animals markets where different animal species of unknown health status from different localities intermix. Additionally, small ruminant flocks present in high numbers in Egypt are highly migratory [7]. Other researchers linked the lack of Brucella control to insufficient compensation for owners and the emotional attachment of owners to their animal’s resulting in slaughtering of only 0.2% of seropositive animals [8,9]. Importantly, the failure to get access to all animals which should be tested according to the control

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program is also one reason for the lack of reliable data on animal brucellosis in the country [10].

Brucella melitensis (main hosts are small ruminants) and B. abortus (main hosts are bovids) are the species causing most human cases. Human brucellosis is well documented from all over Egypt with annual incidence ranging from 64 to 70 per 100,000 in Fayoum Governorate [6]. Three and 11% of acute febrile illness patients in Egypt were positive either by culture or serology, respectively [11]. The vast majority of brucella isolated from humans was B. melitensis [12]. The disease is spreading mainly due to close contact between farmers and their animals, occupational exposure of farmers, veterinarians and butchers to infected animals and through the food-chain via contaminated milk and dairy products.

Accurate diagnosis of the disease is considered as an elusive target. Isolation of Brucella species or detection of its DNA by using PCR is the only method that allows certainty in diagnosis [13]. Despite being the gold standard, bacterial isolation has been reported to show poor sensitivity for samples with low-level contamination and is impractical for regular screening of large populations [14]. Thus, serological tests still have a major role in the routine diagnosis of brucellosis especially in developing countries due to their ease in handling, high sensitivity and low price [15]. In this setting, Buffered Plate Agglutination Test (BPAT), Enzyme Linked Immunosorbant Assay (ELISA) and Fluorescent Polarization Assay (FPA) are still appropriate screening tests [16]. RBT, Serum Agglutination Test (SAT), Complement Fixation Test (CFT), and indirect ELISA (IELISA) have also been applied for the diagnosis of brucella infection [17]. Although these tests are well-established for the diagnosis of bovine brucellosis in Egypt, they still do not follow the scientific standards. There is also an obvious discrepancy between the officially reported seroprevalence for Brucella infection in Egypt and the data obtained from scientific reports. Therefore, the present study was planned to provide a preliminary study on the prevalence of cattle brucellosis in three large Governorates in the Eastern Nile Delta region using different serological tests.

MATERIALS AND METHODSStudy population and selection criteria

An informed consent for brucella investigation was given by the owners. All procedures were performed in accordance with the principles and specific guidelines presented in the Guidelines for the Care and Use of Agricultural Animals in Research and Teaching (3rd ed.; http://www.fass.org/), and those of Mansoura University Animal Care and approved by its Ethical Committee.

During 2012 and 2013, a total of 2.830 Holstein-Friesian dairy cattle aged between 2 to 5 years, from farms located at Dakahlia, Damietta, and Port Said Governorates were examined for Brucella infection according to the practiced routine control program (Table 1). The data retrieved from the farm medical records indicated that all cattle investigated were not previously vaccinated with Brucella vaccine. These animals were allocated into two groups. Group 1 included 1.920 cattle with various reproductive disorders; while group 2 included 910 apparently healthy cattle with no history of reproductive troubles (Table 1). All holdings were chosen because they were known to have good management practices. They were considered as closed epidemiological entities.

Sampling

Blood samples were randomly collected from cows with reproductive disorders (n = 203) and from apparently healthy ones (n = 608). As many cows as possible were sampled on a farm to detect asymptomatic and chronic cases. Briefly, ten ml of blood was collected from each animal through jugular vein puncture using plain vacutainer tubes and needles. Each blood sample was labeled with the respective animal number. The collected blood samples were kept overnight at room temperature for clotting. On the next day, sera were collected and stored in cryo-tubes at -20°C until further examination.

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.

Location Number of examined animals

Number of positive animals by all serological test

Serologicaltest

RBT ELISA FPA

+ % - % + % - % + % SUSP % - %

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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performed as described in the Manual of Standards for Diagnostic Tests and Vaccines[17] using antigen obtained from Institute Pourquier, France. Positive and negative control sera were the German national reference sera standardized according to OIE. The ELISA was performed and results were interpreted according to the instructions of the manufactures using IDEXX™ Brucella-Ab ELISA kit (Montpellier SAS, France). FPA was done and results were interpreted according to the instructions of the manufacturer (Diachemix, Whitefish Bay, and WT, USA). An animal was considered infected if it tested positive in all tests.

RESULTS The distributions of cattle investigated along with the

serological findings are summarized in tables 1-to- 5. Briefly, the individual seroprevelance among cattle exhibited reproductive disorders was 52.2%; being higher in Damietta region than in Port-Said. Collectively, ELISA gave the highest number of positive samples (n = 138; 67.9%) followed by FPA (n = 120; 59.11%) then RBT (n =109; 53.7%) (Table 2). Out of the healthy animals examined (n = 608), 532 (87.5%) tested negative; while 3.3%, 4.1% and 1.8% were serologically positive by using ELISA, RBT and FPA, respectively (Table 3). These positives were found only in Dakahlia farm; however, no positive reactors were reported among healthy cattle tested in Damietta region.

DISCUSSIONBrucellosis is an emerging threat and can be considered

one of the most common global zoonoses, especially in the developing nations including Egypt. Despite its potential impact on public health, the epidemiologic situation of Brucella infection in Egypt is still unresolved and warrant further investigation.

Likewise, most previous studies were applied in response to clinical settings such as noticing of abortion cases among the reared cattle [18] such reports do not comply with the standards for epidemiological investigations specially study design or biostatistics. We aimed at presenting a reliable preliminary data about the seroprevalence of the disease to be expected in well managed dairy farms in three large Governorates where the actual epidemiologic data are still scarce. Several authors assumed that hotspots for animal brucellosis are located in the Nile Delta region and in Upper Egypt, along the River Nile where the south of the Delta contains 32% of the Egyptian large ruminant plus 39% of the small ruminant stocks which are often kept in small mixed herds owned by single householders [19]. Interestingly, a very low rate of infection was reported in seven upper Egyptian Governorates (Benisuef, Al Minia, Assuit, Sohag, Quena, Luxor and Aswan) within the framework of the veterinary service where the prevalence was 0.79% in investigated cattle; while it was 0.2% in household cows where at least one animal was seropositive [20].The assumption of hotspots needs further clarification. We did choose well managed farms having good biosafety measures in practice for our study on purpose. We do think that the selected herds whose control programs could have the best prospect of success as introduction of new stock include serological testing beforehand. Given that these herds are of prominent importance for the supply of many (urban) consumers, the control of brucellosis here could have the biggest impact on public health. By doing so, it is obvious that our data cannot be compared with data from earlier studies including farms or holdings despite their management form on a more or less random basis. In the present study, 109 cows with 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)

44RBT ELISA FPAPos Pos Pos

40 Neg Neg Neg8 Neg Pos Pos5 Neg Pos Neg3 Neg Neg Sus

Damietta (n= 103) 62 Pos Pos Pos18 Neg Neg Neg10 Neg Pos Neg8 Neg Pos Sus1 Neg Neg Pos1 Neg Neg Sus1 Pos Pos Neg1 Pos Neg Neg1 Pos Neg Sus

Table 4: Seroprevalence of brucellosis in the study group with apparently healthy animals.

Location Number of examined animals

Number of negative animals by all serological test

Serological testRBT ELISA FPA

+ % - % + % - % + % SUSP % - %

Damietta 334 298 14 4.2 320 95.8 1 0.3 333 99.7 2 0.6 21 6.3 311 93.1Dakahlia 274 235 11 4.0 263 96.0 19 6.9 255 93.1 9 3.3 20 7.3 245 89.4Total 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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(4.1%) tested positive using RBT. Here, RBT gave lower incidence of positive reactors in the first group compared to ELISA and FPA. This could be attributed to inhibition of non-specific agglutinins by acidic pH of antigen [21]. However, the test gave a higher percentage of positive reactors among cattle of group 2. The high number of seropositive in clinically inconspicuous animals was surprising. This finding might be caused by cross reactions of brucellae LPS with that of other bacterial species including Yersinia enterocolitica O: 9 and E.coli serotype O: 157 [22]. The detection rate was higher in animals suffering from reproductive disorders when compared with those of the clinically healthy group (Table 3). Additionally, ELISA showed the highest number of positive samples among cattle suspected to be infected with brucellosis as well as the highest number of negative cases (Table 4). Our findings were in harmony with other researchers who reported that ELISA could be highly specific and of equal or greater sensitivity than CFT and RBT and could be a suitable test for large scale screening for bovine brucellosis [23,24]. Our finding is also coincided with that of Konstantinidis et al. [25] who found the same magnitude of sensitivity between ELISA, FPA and RBT. In contrast, it was assumed that FPA can be comparable to the standard tests including RBT, CFT and iELISA), thus it is now included into the European Union (EU) legislation on intra-community trade of bovines as standard test for brucellosis diagnosis [26]. It was surmised that FPA could replace the card test (CT) as a screening test for its better performance compared to CFT due to its adjustable cut-off useful in different epidemiological situations, and for its reliability, ease of performance, comparable costs, and potential application in field and high-throughput laboratories [27]. Our serological data can only show that brucellosis is endemic in dairy cow herds in the Nile delta even if biosafety is managed well. It is obvious that the different tests have to be evaluated in the epidemiological setting of the Egyptian Nile delta where Holstein Frisian cows from non endemic regions and local breeds are present. We do think that the number of false positives was reduced by the procedure to consider an animal only as positive if all three tests were positive. We assume that still a relevant number of false positives may be

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 abortions is usually considerably low again.

An earlier national serological survey which was conducted between 1994 to 1997 [4] where 40% of the total ruminant population was tested (as part of a brucellosis surveillance and control project) and have reported the seroprevalence of brucellosis among cattle, buffalo, sheep and goat at the following detection rates: 0.9%, 0.3%, 1.8% and 8.2%, respectively. Keeping in mind that the real seroprevalence was underestimated at that time and we might face a bias towards overestimation, our results 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 [28,7,29,30]. In contrast, a higher prevalence rate was also found in cattle by recent reports corroborating our idea of a steadily increasing brucellosis problem in the last years [31,32,33]. In Menufia Governorate, the individual seroprevalence for cattle and buffaloes was estimated to be 11.0% and that for household to be 15.5%, respectively [34]. B. melitensis (Bv 3) was regularly isolated from cows and buffaloes of seven Egyptian Governorates (Alexandria, Behira, Monofia, Qalioubia, Giza, Benisuef, and Assiut) despite the fact that the normal reservoir of this species is goats and sheep. The seroprevalence reported was around 5% in cattle in those Governorates [7]. In one study conducted in Sharkia Governorate, the incidence of Brucella infection was 6.72%, 5.62%, 7.61% and 10.95% in cows, buffaloes, sheep and goats, respectively; while it was 37.74% among the tested human samples [31] where B. melitensis biovar 3 was isolated from both man and animals. Hence, a different management form i.e. cohabitation of cattle and small ruminants can be assumed for those farms which is unusual on well managed farms. Thus a new control program should include only those premises where no mixed cohabitation is practiced.

CONCLUSIONTaken together, it becomes apparent that Brucella infection

could be endemic in the studied regions at North Eastern Nile Delta region of Egypt. It is very likely that this infection could pose a considerable risk to consumers and may also a potential source of infection to animal keepers, veterinarians and slaughterhouse workers etc. Great care should be taken in handling animals with reproductive disorders. Setting up a tailored program for well managed dairy herds is the first step towards a better control of the disease to supply safe dairy products for a broad range of consumers.

ACKNOWLEDGEMENTSThis work was in parts funded by the German Federal

Foreign Office program: Deutsches Partners chafts program für biologische Sicherheit und Gesundheitssicherstellung

Conflict of Interest

The authors of this paper have no financial or personal relationship with other people or organizations that could appropriately influence or bias the content of the paper.

Table 5: Detailed findings of serological tests in apparently healthy cattle. Number RBT ELI5A FPADamietta farm (n = 334) 298 Neg Neg Neg

20 Neg Neg Sus13 Pos Neg Neg1 Pos Neg Sus1 Neg Neg Pos1 Neg Pos Pos

Dakahlia (n=274) 235 Neg Neg Neg17 Neg Neg Sus3 Neg Pos Pos1 Neg Pos Sus3 Pos Neg Neg6 Pos Pos Pos7 Neg Pos Neg2 Pos Pos Sus

Abbreviations: RBT: Rose Bengal Test; ELISA: Enzyme Linked Immunosorbant; FPA: Fluorescent Polarization Assay

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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.

Cite this article

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