SOME EPIDEMIOLOGICAL, HAEMATOLOGICAL AND BIOCHEMICAL STUDIES ON BRUCELLOSIS IN CATTLE AND HUMAN CONTACTS IN SOME EGYPT GOVERNORATES

SOME EPIDEMIOLOGICAL, HAEMATOLOGICAL AND BIOCHEMICAL STUDIES ON BRUCELLOSIS IN CATTLE AND HUMAN CONTACTS IN SOME EGYPT GOVERNORATES

H.A. SHALABY^*; M.M. EL-DIASTY^*; R.M. DHRYG^** and M.S. EL-SHRBYNY^***

^*Animal Health Research Institute, Al. Mansoura Provincial Lab.,

^** Animal Reproduction Research Institute, Al-Harm,

^*** Faculty of Medicine, Mansoura University, Egypt.

Email: hamed_shalaby56@yahoo.com

INTRODUCTION

Brucella is a highly contagious zoonotic infectious organism. Infection is generally manifestated clinically in animals by abortion; decrease of milk yield, breeding troubles and infertility while in human being a brucella infection cause undulant fever with its all complications, (Mehanna and Soliman, 1998). The estimated annual economic losses due to brucellosis were about 60 million Egyptian pounds yearly (AOAD, 1995). Because of being a serious zoonotic disease, brucellosis has been a subject of great concern of Official Veterinary Authorities and researchers since the disease was firstly reported by Bruce (1887). More than 70% of Egypt’s total livestock population is owned by small holders, who keep a few cattle and buffalo in their household as a source of milk and dairy products for home consumption or to sell, often unpasteurized, in local markets (El-Rafey, 2005 and Aidaros, 2005). The WHO considers brucellosis to be a neglected as a zoonotic disease, despite its widespread distribution and its effect on multiple species. It is not prioritized by national and international health systems, (WHO, 2005). Brucellosis was first reported in Egypt in 1939 and is now considered endemic in most parts of the country, (Refai, 2002). Despite its economic and public health importance, in recent years, the Official Egyptian Brucellosis Control Program does not appear to have been fully implemented, (Refai, 2002 and Hegazy     et al., 2009). Aggad (2003) recorded the disease in Egypt in Damietta (10.4%) and while in Dakahlia (7.18%) Governorates (El-Diasty, 2009). In 2007 a population-based survey of acute febrile illness patients was conducted in Fayoum, a rural Governorate of Egypt, from the results of the survey human incidence of brucellosis was estimated to be 64 and 70 per 100 000 people in 2002 and 2003, respectively. If incidence estimates in the study had been based purely on hospital admissions, they would have been 3.8 per 100 000, (Jennings et al., 2007). Brucellosis is considered an occupational hazard; risk factors identified for human brucellosis in the study were close contact with animals, exposure to aborted materials and consumption of dairy products. As close contact with animals is common in rural areas of Egypt and healthcare is not easily accessible for rural populations, rural residents are likely to have a high risk of brucellosis. The most effective way of reducing incidence in humans, is by controlling brucellosis in livestock, (Jelastopulus et al., 2008 and Nicoletti, 1992).

Regarding the biochemical changes with brucellosis the available literature is little. The liver is the most important organ of the reticuloendothelial system (RES), that occupies a central position in the metabolism of the animal and the disturbance in hepatic function which may be accompanied with brucellosis in cattle have serious consequences for the productivity and reproductively of the diseased animals, (Deghedy et al., 1990). Serological screening tests have been proved to be the most satisfactory monitor for brucella infection and during eradication programs, (Dokoo et al., 1986). Successful control of the disease depends largely on successful diagnosis which in turn is based upon the use of efficient serological tests for picking up positive cases, (Hosein et al., 2002). The object of this work was to study the sero and biochemical changes as a result of brucella infection in the diseased dairy cattle and human at high risk when compared with healthy ones.

MATERIALS and METHODS

A)      Prevalence of brucellosis:-

Animals and samples:

A total of 5188 lactating cows; 1210 from Dakahlia Governorate, 2378 from Damietta Governorate and 1600 from Alexandria Governorate were examined in this study to determine the prevalence of brucellosis and studying biochemical changes as a result of brucella infection. Blood samples were collected from animals, human and sera separated.

B) Antigens and tests used:

Were obtained from Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt and used in the corresponding serological test for each.

a- Buffered Acidified Plate Antigen Test (BAPAT):          

The test was carried out according to Alton et al. (1988).

b- Rose-Bengal Plate Test (RBPT):

The test was carried out according to Morgan et al. (1978).

c- Tube Agglutination test (TAT):

The test was carried out according to Alton et al. (1975)

Signs on human cases:

These cases suffer from undulant fever 39-41⁰c, sever back pain, heavy sweating with bad odour, influ-like symptoms, only one complicated case suffer from sever orchitis.

Suggested treatment for human cases.

The infected persons have been divided into two groups:

-      First group take Rifamycin + Doxycyclin.

-      Second group take Rifamycin + Doxycyclin + Amikacin.

C) Biochemical and haematological examination:

Blood samples of (7) positive cattle for brucellosis and (7) human cases of brucella infection were collected (part an EDTA as anticoaguled for haematological examination and the other part left for serum separation without anticoagulant for biochemical studies.

Blood analysis:

Hematological examination was performed according to Jain (2000). Serum total proteins according to Doumas, et al. (1981), albumin according to Frank (1950), AST, ALT activity levels according to Retman and Frankel (1957), while total globulin calculated mathematically by subtracting albumin from total protein. The calcium level was determined according to Gindler and King (1972), inorganic phosphorus El-Merzobani et al. (1977), magnesium Bauer (1982), iron Khan et al. (1995), sodium Guder et al. (1982), Potassium Henry (1974).

Statistical analysis:

All data were subjected to statistical analysis according to Snedecor and Cochran (1982).

RESULTS

1- Prevalence of cattle brucellosis among individually reared animals in Dakahlia, Damietta and Alexandria Governorates.

Results are shown in Table (1) and Figure (1).

Table 1: Prevalence of brucellosis among individually reared animals in Dakahlia, Damietta and Alexandria Governorates.

Figure (1) Prevalence of brucellosis among individually reared animals in Dakahlia, Damietta and Alexandria Governorates.

2 - Biochemical studies on the serum of (7) serologically positive cattle and human contacts:

Seven positive and (7) control cattle, (7) positive and (7) control human were used to determine mean values of some haematological parameters, mean values of liver function test and mean values of some macro and micro elements in examined dairy cows and human contacts. Results of haematological parameters are shown in Table (2), while biochemical parameters in Tables (3) and (4).

Table 2: Mean values of some hematological parameters in the examined dairy cows and human contacts.

     *Significant at P ≤ 0.05 ** Highly significant at P ≤ 0.01 *** Very highly significant at P ≤ 0.001

Table 3: Mean values of liver function test in the examined dairy cows and human contacts.

*Significant at P ≤ 0.05 ** Highly significant at P ≤ 0.01 *** Very highly significant at P ≤ 0.001

N.S. Non significant.

Table (4): Mean values of some macro and micro elements in the examined dairy cows and human contacts.

* Significant at P ≤ 0.05  ** Highly significant at P ≤ 0.01     N.S. Non significant

2- Symptoms in human and treatment:

Twenty cases of human brucellosis were detected in present study. All the cases were serologically positive to BAPAT, RBPT and TAT. These cases have misdiagnosis from doctors as human typhoid but we suggest the infection with brucella due to the observed clinical signs positive serological tests, the relation between these cases and the veterinary field and the long term of wrong treatment.

These cases suffer from undulant fever 39-41^°C, sever back pain, heavy sweating with bad odour, influ-like symptoms. Only one complicated case suffer from sever orchitis. Doctors have diagnosed this case as abscessiation of one tests and decide the castration, but after the right diagnosis as brucellosis, the case respond to the treatment after 4 days from the beginning of treatment.

Treatment:

Co- treatment of these cases in co-operation with their doctors to coordinate the program of treatment. The twenty persons were five persons from different infected farms and 15 sporadic cases have been contacted with infected animals (cattle, buffaloes, sheep and goat) during parturition, abortion, retention of placenta and only one case during castration of infected buck suffer from orchitis.

The infected persons have been divided into two groups:

-  First group treated by Rifamycin + Doxycyclin.

-  Second group treated by Rifamycin + Doxycyclin + Amikacin.

Dose:

- Rifamycin 10 mg/ kg body weight per day every morning.

-  Doxycyclin: 100mg / kg body weight twice a day.

-  Amikacin :7.5 mg / kg body weight twice a day I.M. for seven days

These two regimes of treatment and their doses were applied as recommended by the jointWHO (2005).

Result of treatment:

1 - The second group had a higher efficacy and more rapid action in terms of relief of symptoms compared to the first group, so adding Amikacin to first group in the treatment seems to be beneficial.

2- No adverse effect seen in the two groups.

DISCUSSION

Blood serum samples of 1210 dairy cattle from Dakahlia Governorate, 2378 dairy cattle from Damietta Governorate and 1600 dairy cattle from Alexandria Governorate, Table (1), were tested for the prevalence of brucellosis. Different serological tests; namely Buffered Acidified Plate Antigen Test (BAPAT), Rose Bengal Plate Test (RBPT) and Tube Agglutination Test (TAT) were employed in this study for detection of brucella infected animals. An animal was classified as positive when two or more tests were positive.

The results summarized in Table (1) showed that 216 (17.8) of cattle in Dakahlia Governorate were positive reactors to brucella, while in Damietta Governorate, positive reactors were 212 (8.9%), but in Alexandria Governorate positive reactors were 189 (11.8%). The obtained results indicate the high prevalence of brucellosis among investigated cattle in these Governorates and the importance of the disease due to the expected losses as well as its public health significance.

These results also indicate that brucellosis is one of the important persistent and endemic diseases that have been reported at variable prevalences in Egypt. Prevalence of brucellosis has been determined by different authors at different localities as, Shalaby (1986) (in cattle 21.96%, 0.99%, 54.61%, 28.09%, 7.68% and 15.7% at El-Sharkia, El-Gharbia, El-Fayoum, El-Dakahlia, Alexandria and Giza Governorates), Hamouda (1989) (6.13% in cattle, 0.54% buffaloes and 8.5% sheep at El-Behaira Governorates), Mahmoud (1989) (2.27% in cattle and 1.88% in buffaloes at Kafr El-Sheikh Governorate), Gadalla (1991) (1.75% cows and 1.88% buffaloes in Assiut Governorate), Abdel-Hafeez (1996) (0.89% cows in Assiut Governorate), Girgis (1998) (2.72% cows and 1.31% buffaloes in Beni-Suef Governorate), Youssif (1998) (1.36 cows, 0.39 buffaloes and 2.04% sheep in El-Menia Governorate), El-Diasty (2004) (4.95-5.8% cows in Dakahlia and Damietta Governorates), Nashwa and Afaf (2004) (8.57% cows in El-Kalubia Governorate), Bassiony  et al. (2007) (4.29%-12.33% cow and 0.00% buffaloes in Assiut Governorate), Khoudair and Sarfenaze (2007) (2.10% buffaloes in El-Behaira, El-Menofia, El-Sharkia, Beni-Suef and Assiut Governorates) and (7.18% in Dakahlia Governorate and in Damietta Governorate 10.49%), El-Diasty and El-Beskawy (2012).

Such variation in the prevalence of brucellosis by different authors were attributed to variation in the sensitivity and specificity of the employed tests in each study, variation in localities and the rate of exposure in certain years.

Haematological and Biochemical studies in examined dairy cattle and human contact:

In present study (7) positive and (7) control cattle, (7) positive and (7) control human were analyzed for some haematological parameters, mean values of liver function tests and mean values of some macro and micro elements in examined dairy cows and human contacts. Regarding the serum biochemical changes, in the diseased dairy cattle and man with brucellosis, Table (2), revealed that both of total erythocytic count (TRBCs), Hemoglobin concentration (Hb), and packed cell volume were highly significant decreased (p<0.01) when compared with the control group. The marked reduction could be attributed to the reduction of red cell formation as a result of inadequate production of erythropoietin hormone (Schalm et al., 1986). These results completely agreed with those El-Olemy et al. (1984) and Ghazi et al. (2001). On contrary, the total leukocytic count (TLC) showed an opposite trend where the mean value of parameter very highly significant (p<0.001) increased over the level of the control group. The observed leukocytosis could be attributed to the activation of the lymphoreticular system for production and transportation of the antibodies in trial to combat the infection (Ahmed and Nada 1992). Recorded results were supported by those findings of Ghazi et al. (2001) and Ahmed       et al. (2004) in camel who’s recorded that leukocytosis might be due to stimulation of cell mediated immunity.

Concerning the biochemical profile results, (Table, 3) revealed significant and highly significant (p<0.05 and p<0.01) decrease in total proteins and albumin levels respectively and non significant increase in serum globulins in diseased men were estimated by comparing with the healthy one. Such results are similar to those recorded by Thabet et al. (1993) and Omima et al. (2003) who stated that brucella affected the concentration of the total protein and albumin due to hepatocellular failure to produce protein and albumin as a result of damaged liver cells. Hyperglobulinemia was attributed to humeral antibody response and increased proportion of the total titer representing. The activities of serum Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) enzymes as shown in (table, 3) showed a highly significant and very highly significant (p<0.01 and p<0.001) increase in diseased cattle and human contact respectively in comparison with the control group. The elevation in the enzymatic activities of the liver may be related to hepatic malfunction (Wattak and John 1971) as a result of increased breakdown of hepatocytes as in case of granulomates hepatitis (Benkirane 1997 and Young 1995). These results agreed with those findings of Ahmed et al. (2004) in camel and Khoudair and Sarfenaze (2007) in buffaloes. Concerning analysis of some minerals, Table (4) shows a highly significant (p<0.01) decrease in calcium and inorganic phosphorus levels below the control group level. The obtained results agreed with those of Coles (1986) and Tietz (1987) who attributed the hypocalcaemia and hypophosphatemia to non-diffusible albumin bound fraction caused by decreased protein associated with low albumin. It may be a result of decreased mobilization of calcium ions due to decreased metabolic activity during the disease condition, while Ahmed et al. (2004) reported non significant changes in camel brucellosis. It is evident from Table (4) that, there was significant (p<0.05) decrease in serum magnesium, and sodium levels, and non significant changes in potassium levels when compared with the control groups. This reduction may be due to anorexia and fluid lost during abortion and premature birth in animal (Hafez, 1980). This finding coincided with those of Nashwa and Afaf (2004). Significant (p<0.05) drop in the level of iron below the control groups was observed in positive cattle and human contact, as shown in Table (4). These results were completely agreed with those of El-Sawalhi et al. (1996) in camel and Ghazi, et al. (2001). The decreased level of serum iron may be attributed to the involvement of spleen in those animals in combating the infection and consequently disturbance of its function in storage and metabolism of iron, (Schalm et al., 1986). Also decreased absorption of calcium and other minerals from the intestine were, due to diarrhea, vomition, resulting from severe colitis caused by brucellosis in man (Stermer et al., 1991) is claimed.

Treatment of human patients:

Brucellosis remains the most common zoonotic infection worldwide (Georgios et al., 2005).The purpose of chemotherapy for brucellosis was to decrease symptoms, reduce complications, and prevent relapses (Aygen et al., 2002). The World Health Organization (WHO) guidelines from (1986)are still considered the gold standard for the treatment of brucellosis, suggesting either the combination of Doxycycline and rifampicin for 6 weeks, or the combination of Doxycycline for 6 weeks with streptomycin for 2 or 3 weeks (Georgios et al., 2005).

In present study two regimens in treatment were tried, the first one was Rifampicin and Doxycycline and the second one was Doxycycline, Rifampicin and Amikacin. Better response was recorded in patients received the second regimen which included Amikacin. In spite of aminoglycosides have multiple side-effects such as nephrotoxicity or ototoxicity; no any of these side effects was recorded in the patients.  This finding copes with Mitra et al. (2007) that had the same results in their studies. The authors concluded that, with short-term use of Amikacin for seven days, they did not have any aminoglycoside-specific side-effects (Mitra et al., 2007).

The response of treatment with Doxycycline and Rifampicin was good, the recovery rate with the Doxycycline and Rifampicin regimen was 88% (Mitra et al., 2007). In another study in Spain in 1995, the failure rate with the Doxycycline and Rifampicin regimen was 8% (Mitra et al., 2007).

It is thus concluded that the use of Doxycycline and Rifampicin in treatment of patients with brucellosis had a good response. Adding amikacin for seven days in the treatment regimen will improve the results but the patients will need close follow up to avoid occurring of amikacin side effects.

It could be concluded that brucellosis plays a role in the remarkable disturbance of the biochemical parameters in the blood of the diseased cows and man. This reflects a disturbance in the general health condition of the host. More attention should be paid for careful, early diagnosis of this zoonosis followed by eradication of the sero-reactor dairy cattle, vaccination of non reactors and a therapeutic plan for the diseased farm workers. Veterinary autheroties must encourage the cooperation between the Veterinary Services and Public Health Authorities to put a plan for the control of brucellosis in Egypt.

REFERENCES

Abdel-Hafeez, M.M. (1996): A serological study on brucella infection among cattle in Assiut Governorate. Assiut Vet. Med. J. 36 (71): 1-8.

Aggad, H. (2003): Serological studies of animal brucellosis in Alegria.Assiut Vet. Med. J. Vol. 49 No. 98: 121-130 July 2003.

Ahmed, T.M.; Samira, A. Emara; Manal, G. Fadlallah and Mohamed, S.S.M. (2004): Camel brucellosis, serological, hematological and biochemical   profile. J. Egypt Vet. Med. Ass. 64, No. 4: 195-206.

Ahmed, W.M. and Nada, A.R. (1992): Hematological picture of camels with testicular and epididymal affections. Egypt J. Comp. Pathol. 5(2): 195-202.

Aidaros, H. (2005): Global perspectives - the Middle East: Egypt. Rev. sci. tech Off int Epiz 2005, 24(2):589-596.

Alton, G.G.; Jones, L.M.; Angus, R.D. and Verger, J.M. (1988): Techniques for the Brucellosis Laboratory. INRA, Puplication, Paris, ISEN, France.

Alton, G.G.; Maw, J.; Rogerson, B.A. and McPherson, G.G. (1975): The serological diagnosis of bovine brucellosis: an evaluation of the complement fixation, serum agglutination and Rose Bengal tests. Aus. Vet. J. 51 (2): 57-63.

AOAD (1995): Arab organization for agriculture development report. December, 1995, Khartoum, Sudan: 414 -474.

Aygen, B.; Dog˘anay, M.; Sümerkan, Yildiz O.; Kayabas, U. (2002):  Clinical manifestations, complications and treatment of brucellosis: a retrospective evaluation of 480 patients. Médecine et maladies infectieuses32,         485–493.

Bassiony, T.; Enas El-Prince; Sohair Zein El-Abdeen and Sadek, O.A. (2007): Diagnosis of brucella infection in dairy cattle with serological tests in Assiut Governorate. Assiut Vet. Med. J. Vol.53 No. 114 July 2007.

Bauer, V.P. (1982): Clinical laboratory methods. 9^th Ed. G. V. Co., 11-1830.

Benkirane, A. (1997): Analysis of the international animals health code for brucellosis (with special focus on North Africa and the Middle East).  J. Egy. Vet. Med. Ass. 57(1): 5-6.

Bruce, D. (1887): Note on the discovery of a MicroOrganism in Malta fever. Practitioner 39:161-170. Cited after stableforth, A. and Gallowy, I. (1959): Infectious Diseases of animals. Vol. I, P. 53. Brtterworth, London.

Coles, E.H. (1986): Veterinary Clinical Pathology. 4^th Ed. W. B. Saunders Company, Philadelphia.

Deghedy, N.S.; Amin, A.A. and Ghazt, A.A. (1990): Effect of fascioliases on line function before and after treatment in sheep. Egypt. J. of Comp. Clin. Pathology 3(1): 65-74.

Dokoo, I.R.; Wright, P.F. and Forbest, B. (1986): Serological test for bovine brucellosis. Canadian, J. Vet. Res. 50: 485-493.

Doumas, B.T.; Cartor, R. J.; Peers, t. and Schaffier, R. (1981): A candidate reference method for determination of total protein in serum. Clin. Chem. 27, 1642.

El-Diasty, M.M. and El-Beskawy, M.A.A. (2012): 7^th Int. Sci. Conf., Fac. Vet. Med., Mansoura University, Egypt.

El-Diasty, M.M. (2004): Some epidemiological and immunological studies on cattle brucellosis. M.V.Sc. Thesis (Inf. Dis.), Faculty of Vet. Med., Ismailia, Suez CanalUniversity

El-Diasty, M.M. (2009): Studies on causes of maintenance of brucella infection among animals in Egypt. Phd. Thesis (Inf. Dis.), Dep. Ani. Med. Faculty of Vet. Med., Beni-SuefUniversity.

El-Merzobani, M.M.; El-Aser, A.A. and Zakhary, N.I. (1977): J. Clin. Chem. Clin. Biochem. 15: 715-718.

El-Olemy, G.M.; Atta, A.A.; Mahmoud, M.H. and Hamza, E.G. (1984): Brucellosis in man, isolation of the causative organisms with special references to blood picture and urine constituents. Dev. Biol. Stand. 56: 573-8.

 El-Rafey, MS. (2005): Milk Hygiene Practice in Egypt. WHO; 2005 [http://whqlibdoc. who.int /monograph/WHO_MONO_48_  (p635). pdf].

El-Sawalhy, A.A.; Montaser, A.M. and Rizk, L.G. (1996): Diagnostic and biochemical evaluation of camel brucellosis. Vet. Med. J., Giza, 44: 323-329.

FAOSTAT (2008): Food and Agricultural commodities production Egypt. 2008 [http://faostat.fao.org/site/339/default.aspx].

Frank, H.M. (1950): Photometric analysis of albumin- globulin in serum. Dtsch. Arch. Klin. Med., 197-181.

Gadalla, Z.B. (1991): Epidemiological studies of brucellosis in Upper Egypt with special referance to its prevalences among different species of animals. M.V.Sc. Thesis, Faculty of Vet. Med., AssiutUniversity.

Ghazi, Y.A.; El-Deed, E.D.E. and Hala, A.A. Abou-Ziena (2001): Some metabolic profile of brucella infected buffaloes with special emphases to endometritis. J. Egy. Vet. Med. Ass. 61 No.1: 157-171.

Gindler, M. and King, J.D. (1972): Am. J. Clinc. Path. 58, 376.

Girgis, S.I. (1998): Evalution of different serological tests for diagnosis of bovine brucellosis. M.V.Sc. Thesis (Micro)Fac.Vet.Med.Beni-Suef, Cairo, Univ.

Georgios, P.; Javier, S.; Nikolaos, A.; Epameinondas, T. (2005):  New approaches to the antibiotic treatment of brucellosis. International Journal of Antimicrobial Agents 26, 101–105.

Guder, W.; Hoffman, G. and Oppitz, K. (1982): A normal bere ichklinischer. Chemischer befunde in den Staditischer Krankenhausen Munchens. Cited in Biochemical Kit.

Hafez, E.S.E. (1980): Reproduction in farm animals. 4^th Ed. Lea. and Febiger, Philadelphia, USA.

Hamouda, H.A. (1989): Epidimiological studies on brucellosis in El-Behera Governorment. M. V.Sc. Thesis, (Department of Vet. Med. and Forensic Med.), Fac. of Vet. Med., AlexandriaUniversity.

Hegazy, YM.; Ridler, AL. and Guitian, FJ. (2009): Assessment and simulation of the implementation of brucellosis control programme in an endemic area of the Middle East. Epidemiol Infect, 137(10):1436-1448.

Henry, R.J. (1974): Harper row clinical chemistry, 2^nd Ed., PP. 644 New York.

Hosein, H.I.; Dawood, F.Z. and El-Sheery, M.N. (2002): Evaluation of the policy of test and slaughter for control of brucellosis in Egypt. 10^th Sci. Con. 2002, Fac. Vet. Med. Assiut Univ., Egypt.111-116.

Jain, N.C. (2000): Schalm’s Veterinary Haematology. 8^th Ed. Lea & Febiger, Philadelphia, USA.

Jelastopulu, E.; Bikas, C.; Petropoulos, C. and Leotsinidis, M. (2008): Incidence of human brucellosis in a rural area in Western Greece after the implementation of a vaccination programme against animal brucellosis. BMC Publ Health, 8(1):241.

Jennings, GJ.; Hajjeh, RA.; Girgis, FY.; Fadeel, MA.; Maksoud, MA.; Wasfy, MO.; Sayed, NE.; Srikantiah, P.; Luby, SP. and Earhart, K. (2007): Brucellosis as a cause of acute febrile illness in Egypt. Transactions of the Royal Society of Tropical Medicine and Hygiene, 101(7): 707-713.

Khan, A.T.; Diffy, B.C.; Dattivi, B.E. and Forester, D.M. (1995): Evaluation of heavy metals in air, water, soil and blood of farm animals in Albania. Bull. Environ. Contam. Toxicol., 55: 568-573.

Khoudair, R.M. and Sarfenaz, S.A. (2007): Bacteriological, serological and pathological studies in buffaloes naturally infected with brucellosis. Egy. J. Comp. Path. & Clinic. Path. Vol. 20 No. 1 (March) 2007; 309-332.

Mehanna, A.M.A. and Soliman, T.M.A. (1998): 8^th Sci. Con. 1998, Fac. Vet. Med., Assiut University, Egypt.

Mahmoud, A.A. (1989): Prevalence of brucellosis among farm animals in Kafr El-Sheikh Governorate. Assuit Vet. Med. J. Vol. 23, No.46.

Mitra, R.; Fariba, K.; Mojgan, M.; Alireza, R.K.; Fatemeh, K.; Seyed, H.H. and Marzieh, N. (2007): Comparison between doxycycline-rifampin-amikacin and doxycycline-rifampin regimens in the treatment of brucellosis: International Journal of Infectious Diseases 11, 152-156.

Morgan, W.J.B.; Mackinnon, D.J.; Gill, K.P.W.; Gower, S.G.M. and Norris, P.I.W. (1978): Standard laboratory techniqes for the diagnosis of brucellosis. Report Series No. I, Weybridge Cent. Vet.Lab., England.

Nashwa, O. and Afaf, D. (2004): Some serological and biochemical studies on brucellosis in dairy cattle and human contacts. Zag. Vet. J. (ISSN. 1110-1458) Vol. 32, No.2 (2004) PP.157-162.

Nicoletti, P. (1992): The control of brucellosis -a veterinary responsibility. Saudi Med J, 13:  10-13.

Omima, M. Mohamed; Kamla El-Said; Abbas, H.E. and Soliman, T.M. (2003): Some biochemical studies on clinically healthy and brucella infected camels. Egy. J. Basic and Appl. Physiol., 2(1): 121-127.

Refai, M. (2002): Incidence and control of brucellosis in the Near East region. jvetmic, 90(1-4):    81-110.

Retman, S. and Frankel, S. (1957): Colorimetric determination of GOT and GPT activity. Am. J. Clin. Path. 28: 56.

Schalm, O.W.; Jain, N.C. and Carroll, E.J. (1986): Veterinary haematology. 4^th Ed. Lea. and Febiger, Philadelphia USA.

Shalaby, M.N.H. (1986): A survey on brucellosis as a cause of reproductive disorders in farm animals in Egypt. Ph.D. Thesis, (Dep. Obest., Gyna. And A.I.), Fac. Vet. Med., CairoUniversity.

Snedecor, W.C. and Cochran, W.G. (1982): Statistical methods. 7^th Ed. The IowaUniv. Press, Ames. Iowa USA.

Stermer, E.; Levy, N. and Potasam, L. (1991): Brucellosis as a cause of severe colitis. Am. J. Gastroenterol. 86, 917-919.

Thabet, A-El-R.; Abd El-Fattah, A.M.; Manaa, A.M.; Sayed, A.M. and Ibtisam M.H. El-Miligy (1993): Prevalence of brucellosis among camels in New Valley Governorate and its effect on serum iron, copper, glucose and   total protein. 2^nd Sci Cong. Egyptian Society for Cattle Diseases, Assiut, Egypt.

Tietz, N.W. (1987): Fundamentals of clinical chemistry. W. b. Saunders, Philadelphia.

Wattake, K. and John, J. (1971): Investigation and treatment of sterility in dairy herd. Deusche, Tierarztliche. Wochenschrift 78, 437.

WHO (World Health Organisation) (2005): The Control of Neglected Zoonotic Diseases. Programme (DFID-AHP), with the participation of FAO and OIE 20-21September 2005. Edited by: WHO/SDE/FOS. WHO Headquarters, Geneva; 2006:

Young, E J. (1995): Brucella species in Principles and Practice of Infectious Diseases. By Mandell, Bennett and Dolin. 4^th Ed. Churchill Livingstone, NY.

Youssif, A.M. (1998): Epizootiological Studies on brucellosis in El-Menia Governorate. Ph.D. Thesis (Inf. Dis.), Fac .Vet .Med. Beni-Suef, Cairo, University.