SOJ Dairy and Veterinary Science

The main objective of this research was to study Bovine brucellosis in ElHawata area, ElGadarif State (2007). A total of 121 bovine sera were collected randomly from cattle of different age and breeds. Data regarding the history of the disease (frequent abortion) in the area were recorded. Samples were 1st subjected to serological investigation using Rose Bengal Plate Test (RBPT). 19 samples (15.7%) were found positive for antibodies against Brucellosis. The result was confirmed with (competitive ELISA) c-ELISA. The result was almost identical with RBPT except one sample which was found positive with c-ELISA and negative for RBPT. Only positive samples were tested with Serum Agglutination Test (SAT). the result confirmed that of c-ELISA and revealed antibody titer ranged between 17-1280. Different age groups were included in the study and the disease was detected in all groups. The disease was more prevalent in cross breeds. It was concluded that Bovine brucellosis is prevalent in ElHawata area, ElGadarif State.

Keywords: Brucella, Rose bengal test, competitive ELISA, Serum agglutination test

Brucellosis is a zoonotic disease with public health and economic implications. Losses in animal production due to brucellosis include diminution of milk and meat, abortion, infertility, longer calving intervals and higher culling rates.¹ The disease is poses great hazard to human health specially in countries like Sudan where no proper program for disease control and the microbiological quality of milk is rarely checked. The first isolation of Brucella organism from animals was made by Bang² who was the first to report contagious abortion in cattle and other animal species and he named his isolate Bacillus abortus, which was followed by other names, Corynebacterium abortus, Bacterium abortus and Alcaligenes abortus. Meyer and Shaw (1920) suggested the name Brucella for the genus.

In Sudan the first isolation of Br. abortus was made by Bennet³ in 1943 from a Friesian herd at Bulgravia dairy farm, but the first isolation of Br. abortus from local cattle was from a cow which aborted at Juba dairy farm.⁴ There after the disease was detected in many parts of the country following isolation of the causative agent or antibody detection. Detection of brucella antibodies is a useful method for diagnosis of bovine brucellosis in many countries in the final phase of an eradication program, and it is still used for monitoring when countries are certified officially free of brucellosis. Although brucellosis was extensively studied in the country, epidemiological data regarding some parts of the country are still lacking.

A total of 121 serum samples were collected from dairy cattle in AlHawata area, ElGadarif State during the period between March to April 2007. The examined animals comprised different breeds and different age groups see Table 1 and Table 2. Blood samples were taken from cattle as described by Alton.⁵ The skin over the jugular vein was rubbed with 70% alcohol and disinfected by the application of tincture of iodine. Then 7ml of blood was withdrawn using a labeled vacutainer.  Samples were put in a wire basket under shade, before taken to laboratory with minimum possible shaking. These samples were kept overnight at 4°C to separate the serum. The serum was then separated from the whole blood by centrifugation, placed in sterile bijou bottles labeled and stored frozen for months.

Rose Bengal Plate Test (RBPT)

The antigen used in the RBPT was obtained from Central Veterinary Research Laboratory (CVRL), Sudan. The sera and the antigen were brought to room temperature before testing. The test was done as described by Alton⁵ by dispensing 0.03ml of each serum to be tested to an enamel plate and equal amount of RBPT antigen was added to each serum sample and both were mixed together, rocked by hand for four minutes, after which the test was immediately read. Result was read as follows: -

1. Negative when there was no agglutination or clumping, or showing a pattern of dispersed particles without clumps.
2. Positive when there was agglutination, with moderate to large clumps.

C-ELISA

The test was carried out as described by (Veterinary laboratory agency). The conjugate solution was prepared immediately and diluted to working strength with diluting buffer according to instructions on the ampoule label. 20µl of each test serum was added per well and columns 11 and 12 were left for controls. 20µl of the negative control was added to wells A11, A12, B11, B12, C11 and C12. 20µl of the positive control was added to wells F11, F12, G11, G12, H11 and H12. The remaining wells (had no serum) were act as the conjugate controls. Immediately 100µl of the prepared conjugate solution was dispensed. This gave a final serum dilution of 1/6. The plate was then vigorously shaken (on the microtitre plate shaker) for 2 minutes in order to mix the serum and conjugate solution. The plate was covered with the lid and incubated at room temperature (21°C ± 6°C) for 30 minutes on a rotary shaker, at 160revs/min. The contents of the plate were shaken out and the plate was rinsed 5 times with washing solution and then thoroughly dried tapping on absorbent paper towel. The microplate reader was switched on and allowed unit to stabilize for 10 minutes.  the substrate and chromogen solution were prepared by dissolving one tablet of urea H2O2 in 12ml of distilled water. When dissolved the OPD tablet was added and mixed thoroughly. This took a few minutes; the use of a magnetic stirrer greatly increased the speed with which it dissolved. 100µl of this solution was added to all wells. (This solution was not stored). The plate was left at room temperature for a minimum of 10 minutes and a maximum of 15 minutes. The reaction was slowed by adding 100µl of stopping solution to all wells. Condensation from the bottom of the plate was recovered with absorbent paper towel. Read plate at 450nm in ELISA Reader.

Serum Agglutination Test (SAT)

The test was done according to Alton5 0.8ml of phenol saline was placed in the first tube and 0.5ml in each succeeding tube. 0.2ml of the serum under test was transferred to the first tube and mixed thoroughly with the phenol saline already there. 0.5ml of the mixture was carried soon over the second tube. This process is continued until the last tube, from which after mixing, 0.5ml of dilution was discarded. This process of doubling dilution results in 0.5ml of dilutions 1:5, 1:10, 1:20 and so on in each tube. To each tube 0.5ml of antigen was then added at the recommended dilution and the contents of the tube were thoroughly mixed, thus giving final serum dilution of 1:10, 1:20 etc…The tubes were then incubated at 37°C for 20 hours before the results are read.

The degree of agglutination was assessed by the amount of clearing that has taken place in the tube as compared with a standard tube. The tubes were examined, without being shaken, against a black background, with a source of light coming from above and behind the tubes. Complete agglutination and sedimentation with water-clear supernatant was recorded as ++++, nearly complete agglutination and 75% clearing as +++, marked agglutination and 50% clearly as ++, some sedimentation and 25% clearing as +, and no clearing as standards were prepared at the time the tests were done and incubated with them. The antigen was diluted by mixing 2ml of antigen, diluted as for the test, with 2ml of phenol-saline.

Serological investigation

Results are shown in Table 3-5. All serum samples were investigated using RBPT and cELISA. 19 samples (15.7%) were found positive with RBPT see table 3 and 20 samples (16.5%) were found positive with cELISA see table 4. The result of the two tests was identical except in one sample which was found positive with cELISA and negative with RBPT see table 5. Only positive samples were subjected to further investigation using SAT. Results are shown in Table 6. All samples were found positive including the sample which gave different reading with RBPT and c-ELISA they had antibody titer ranged between 17-1280.

Effect of age

Results are shown in Table 7, Table 8 and Figure 1. The two tests showed that 17.3% of animals ≥ to 8 years old in contrast to 21.4% of animals ≥12 years old were found positive. The highest antibody titer was detected in animals ≥ to 8 years old. In addition, most of animals with low antibody titer were also detected in animals ≥8 years old Figure 2.

Effect of animal’s breed

Results are shown in Tables 9, Table 10 Among the investigated animals, the cross animals were the most frequently affected group (33.3%).

Relation between brucellosis and history of abortion

Among the investigated animals Seventeen cows had a history of abortion. Only 5 of which were found positive for Brucellosis. See Figure 3. In the current study the presence of the disease in ElHawata area, ElGadarif State, Eastern Sudan was studied for the first time. Twenty samples (16.5%) out of 121 bovine sera were found positive for brucellosis. This finding was comparable to other studies, Daffalla,4 Fayza6 and Musa⁷ report that prevalence rate for brucellosis was 10.7% (in ElGazira), 15.73% (Khartoum State) and 13.7% (in Darfur States) respectively. Brucellosis may spread in ElHawata area, due to many reasons which may include mixing of different animal species together (goats, sheep & cattle), Nomads knows little about the disease, they screen cattle for brucellosis for export and retain the positive ones within their herd. Lack of control measures whether by isolation of infected animals, vaccination or stamping out of infected cattle was also thought to be responsible for the high rates of infection. The nomadic nature of the husbandry method and continuous movement of cattle from one locality to another exposing them to brucella infection.

RBPT is the only serological test used in Sudan for routine diagnosis of brucellosis. in this study its result   was confirmed with c-ELISA and further with SAT. 99.2% of results agreed with that of c-ELISA and SAT indicating that the test was sensitive. However, it may give false negative result which may lead to spread of the disease. In another study, Mukhtar8 (2006) reported that c-ELISA was more sensitive than RBPT. Antibodies against Brucellosis were detected in all animal groups. However, the higher percentage of affected animals were observed in cows ≥12 years old. This result was similar to that of Raias9 who found that the old animal is susceptible to the disease more than young one. This could be explained by the fact that once an animal is infected, it cannot be treated. In addition, infected older animals may not abort (i.e. showing no clinical signs of the disease) and accordingly remain hidden in the herd (carriers) and a source of infection to others. The highest titer was detected in animals ≥8 years old & this may probably indicate recent infection.

Four different cow breeds were included in the study and they represent breeds in ElHawata area. The highest percentage of infected animals was detected in cross breed although the number of samples from this breed was small. This may be attributed to the fact that local breeds are generally resistant to diseases in the tropics compared to foreign ones. Only 29.4% of cows with history of abortion were found positive for brucellosis. This finding indicated that the diagnosis of the disease should not be revealed only on cases of abortion and usually confirmatory tests are needed to a certain the presence of the disease. And the abortion may be due to miss use of drugs and infection with other microorganisms which cause abortion

It was concluded from this study that bovine Brucellosis was present in El Hawata area, and affects different age and breed groups.
It is recommended that:

1. The distribution of the disease in all Sudan States should be studied.
2. Proper control measure should be followed to reduce the infection rate.

Education of people like animal owners, nomads and abattoir workers is essential to increase their awareness to avoid infection and prevent their animals from the disease and environment from contamination.

Thanks, and praise the Head and Staff of the Department of Brucella, Central Veterinary Research Laboratory, Sudan, for generous help. The technical help, advice and encouragement of my colleagues Rashid EE, Hind M, Mohammed B, Huda OA, Huda AE, Amar AH, Sara A, also acknowledged. I am greatly indebted to the staff of the Veterinary Office in ElHawata area, ElGadarif State.

None.

Author declares that there is no conflict of interest.

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