The prevalence of brucellosis of farm animals using serum-and milk-ELISA test in Al-Najaf province

A total of 412 milk and sera samples were collected from the same animals of 88 buffaloes, 212 cattle, 84 sheep and 28 goat were screened by indirect ELISA (i ELISA) milk confirmed with serum i-ELISA. The results of seropositive i-ELISA were 121(29.36%) from all farm animals and distributed to 20 (22.27%) buffaloes, 36 (16.98%) cattle , 56(66.6%) sheep and 9 (32.14%) goat . The positive results of milk i ELISA were 89 (21.6%) from all farm animals and distributed to 8 (9.09%) , 32 (15.09%) , 44(52.83%) and 5(17.85%) represent buffaloes , cattle , sheep and goat respectively . epidemiological informations of animals collected by questionary about ( abortion , stillbirth and previous history vaccination against brucellosis) . The prevalence was relatively higher in sheep and goat followed by buffaloes and cattle . The sera and milk i ELISA test is only the best serological tests and has a high efficiency and accuracy in the diagnosis and inexpensive and can be used to control of the disease.


Introduction
Brucellosis is a zoonotic disease which is widely spread in the world. It's importance come from its major impact on both public health and farming economy . Brucella abortus (bovines) or Brucella melitensis (small ruminants) is the most common cause of the disease which can cause abortions as well as it can be excreted by milk (1,2).
Laboratory tests used in diagnosis of brucellosis are culture and detection. Culture is the gold standard diagnostic test, this method is reliable and definitive, but it is unfavorable due to the long time required for isolation and the zoonotic nature of the organism which is a potential hazard for laboratory personnel, while detection of brucella DNA is more simple and sensitive than culture method (3). Other tests are serological tests for presence of antibodies in blood, milk, whey, vaginal mucus and seminal plasma.
The spread of this disease led the health ruling authorities to imply certain screening programs to detect and eradicate suspected cases . screening is usually done by certain serological tests (Rose Bengal Test, Wright's Serum Agglutination Test, Complement Fixation Test, Ring-Test and ELISA) , which are the only used to mass screening systems (3).
The diagnosis of brucellosis is usually done by using indirect ELISAs (iELISA), such method involve immobilization of one of the active components on a solid phase, and iELISAs the antigen bound to a solid phase is usually a polystyrene microtitre plate so that antibody.
Because of the good correlation between isolation of the causative microorganisim (brucella) and positive tests obtained from sera and milk samples, Serological test is considerd as the more reliable and economical method of diagnosis. When Brucella antibodies detection in both milk and serum is considered, the main methods for detecting infected herds are serological tests (4). The Rose Bengal plate test (RBPT)is the most famous and widely used screening test for detection of infection in humans and animals because it's easy to do and simple to read ; however, personal experience can affect the interpretations of results (5). Because of the false positive and false negative results of the RBPT and MRT, recently,several researchers reported the usage of Enzyme-linked Immuno Sorbent Assay (ELISA) for Brucella antibody in ruminant serum or milk (6,7,8,9). So the indirect ELISA is the more specific and sensitive methods that can be applied to process a lot of samples in a short time. The Aim of the study (a) a comparison between prevalences of the disease in different livestock species in Al-Najaf province. (b) provide a baseline data for further study . (c) identification of anti-Brucella antibodies in buffalos , cattle , sheep and goat serum and milk samples. (d) to find a starting point to control spread of the disease.

Materials and Methods
The study accomplished through the period between May to August on five different regions representing all over Governorate of Al-najaf . A total of 412 sera and milk samples were collected from farm animals of different species included 88 buffaloes , 212 cattle ,84 sheep and 28 goat were selected for this study. The individual animals were sampled at the same time for both serum / milk (from each one). The positive serum/milk samples were obtained from permanently infected flocks, where clinical symptoms (abortions) occurred .Samples of serum and milk were taken from buffaloes and cattle which aren't vaccinated against Brucella ,while sheep and goats with previous history of vaccination.

Collection of blood sample :
By Jugular venipuncture, samples of 10 ml blood were taken from a healthy looking animals by using needles, needle holder and plain vacationer tubes . To separation of serum ,samples were kept at 4•C overnight then centrifuged at 3000 rpm for 10 minutes. The collected sera coded and then kept at -20•C to be ready to use at the time of test.

Collection of milk samples
The collection of milk samples was done under aseptic technique. the teat cleaned well with a cotton soaked in 70% ethyl alcohol. After discard of the first three streams of milk , 10 ml of milk was taken in a sterile glass bottle then Samples were kept in a box filled with ice while transported immediatly to Al-najaf veterinary hospital's laboratory to be analysed directly. Indirect ELISA test for milk samples the test procedure and kits were applied according to ID screen brucellosis milk indirect elisa kit -ID vetgrabeis-France (BRUMILK ver 0512GB) .

ELISA test for serum
The test kits and procedure were supplied by serelisa brucella kit-synbiotic-Europe Code: ASBRU3OCB. .

Results and discussion
An indirect enzyme-linked immunosorbent assay (i-ELISA) was validated to detect Brucella antibodies in sera & milk of buffalos , cattle , sheep and goat. It is preferable to use LPS-based ELISA. The latter is more accurate & less cross-reactions in determining Brucella antibodies (10).
A total of 412 serum and milk samples were analyzed from fife regions in Alnajaf province . Among 412 farm animals (88 were buffalos , 212 were cattle , 84 were sheep and 28 were goat ) , 121 (29.36%) were found reactive positive serum ELISA The highest percentage of positive results were detected by serum ELISA (29.36%) in compared to milk ELISA (21.6%). The sensitivity of sera and milk ELISA test were 88.8% , 78.57 % and 40% of cattle , sheep and buffalo respectively table (1) , whereas specificity 100% in all farm animals .
The prevalence of was much higher in buffaloes than cattle of serum i-ELISA test 22.7% and 16.98% respectively , while contrary in milk i-ELISA test 9.09% ,15.09% in buffaloes and cattle respectively , the interpretation of these results are using artificial insemination in cattle ,in opposite buffaloes using natural mating, the risk is somewhat low that infected males transport the infection to vulnerable females . Also fatty milk of buffaloes make it less sensitive than serum ( sensitivity 40% ). In cattle colonization of the udder is frequent and excretion of brucella in the milk may be prolonged for months or years, this lead to increase production of antibrucella antibodies ( sensitivity 88.8%). Also, the contact between different herds/flocks can increase the spread of disease to susceptible animals accordance with (11). The percentage of positive serum i-ELISA test of cattle in blood samples subjected to was 16.98% , less than (12) 20.45 % in Iraq.
The sensitivity and specificity of i-ELISA were 88.8 % and100% respectively . Although the positive percentages of Brucella antibodies in milk samples tested in this study was found 15.09 % lower than (13,14 )and its higher than (15) . Its considered as high for Alnajaf state, where no previous history of vaccination against brucellosis is applied in cattle.
The reason for the differences in infection rates between this study and others is that samples may be taken randomly from the area that disease is endemic and other free of the disease , or the density of livestock , moreover cross reaction of lipopolysuccaride of other gram negative bacteria (16).
In sheep the result of indirect ELISA test(i-ELISA) total seroprevalence was (66.6% ) , in line with (17) 23.6% -100% was recorded in Mosul and (18) in Basrha (68.8%), but higher than (19) (29.34 %) , the result of milk i-ELISA test in sheep ( same flock ) were 52.83% , in reverse with (20) in Iraq. This variation in results may be due to differences in geographical region , age of animals and previous history of vaccination against Brucella in sheep and goats , subsequently cause increase titration of antibodies in serum , serological tests, can distinguish between antibodies that arise from infection and and those from vaccination, respectively, have not been developed accordance with (21). if the lactating udder is infected, less serological response will occur, and localization is confined to a few number of lymph nodes may fail to produce immunological response at all species .
The prevalence of caprine seroprevalence brucellosis was 32.14% , less than presented by (22 , 23 ) and 17.85% of milk I-ELISA test , supported by (3,24) in Iraq , the level of antibodies against brucella decline in milk after parturition , while remain high level in blood .
Although followed planned program of vaccination against brucellosis in small ruminant in Iraq , the disease still endemic , because of movement of animals through Alnajaf deserts . In sheep, goats and cattle, The Seroprevalence of disease was widely & irregularly distributed among flocks and provinces ,which refer by (25,26).
The positive reactor of serum and milk i-ELISA test was 29.36% and 21.6% respectively in all farm animals ,the reason may be due to that test is accuracy and efficiency in detecting all immunoglobulins in serum accordance to (27).
This assay does not distinguish between infected & vaccinated animals, however, it can be used for free monitoring in vaccinated areas and for diagnosis in affected areas where animals were vaccinated more than one year before.
The I-ELISA kit evaluated in our laboratory seemed to be rapid, simple, sensitive, and specific for detecting antibodies to Brucella . The I-ELISA should also be evaluated as a diagnostic tool in control programs in Iraq.

Conclusion and recommendations :
Conclusions : 1. The Bruc ELISA test is a sensitive, specific, and inexpensive method for screenin g large numbers of individual or bulk milk samples for antibody to B. abortus.
Recommendations : 1. Test and reduction of reservoir of infection and removes infected animals from the herd and reduces exposure to causative agents and transmission within the herd. 2. Use of effective vaccination program to reduce of economic loses .