Benha Vet. Med., J. Vol. 19, No1, June. 2008
Sero-Epidemiological Study Of Brucellosis And Hematological Changes
Of Infected Humans In Kalyobia Governorate
Lobna, M. A. Salem*, Shousha, S.M. ** and El-Newishy, A.A*
* Dept. of Zoonoses. Vet. Med., Benha University
** Dep. of Physiology. Vet. Med., Benha University
Abstract
This study was carried out on 300 animals (100 cows, 85 buffaloes, 65 sheep
and 50 goats)which were collected from private dairy farms, veterinary clinics,
abattoirs and sporadic cases in some villages at Kalyobia governorate and were
serologically examined using Rose Bengal Plate Agglutination Test( RBPAT) and
Tube Agglutination Test (TAT). The percentage of positive reactors was 6%, 4.7%,
16.9% & 24% by RBPAT and it was 6%, 3.5%, 15.4% & 22% by TAT respectively,
otherwise the results of Rivanol Test (Riv. T) on positive serum TAT were 100%,
100%, 90% & 90.9% respectively in the examined animals, as well as 250 persons
suffering from fever suspected to be brucellosis were collected from fever hospitals
and clinics at the same locations in Kalyobia governorate and serologically examined
using Rose Bengal Plate Agglutination Test (RBPAT), Tube Agglutination Test (TAT)
and 2-Mercapto Ethanol Test (2-MET). The results of these tests were 16%, 15.6%&
15.2% respectively. Moreover, the results revealed that the majority of positive cases
among males (18.4%) more than females (8.3%) and among humans aged between 20
to 45 years and more in those among occupational contact with animals. Most of the
cases complained from fever (60%), night sweats (52.5%), arthralgia (52.5%), back
pain (50%) and on examination, splenomegaly ranked first in 5(12.5%) cases,
hepatomegaly in 4(10%) cases and enlarged lymph nodes in 2(5%) cases. Blood
picture of serologically positive brucellosis showed anemia in 10 cases, Leukopenia in
9 cases accompanied with neutropenia and relative lymphocytosis, Thrombocytopenia
was found in 5 cases while pancytopenia was detected in one case. The public health
hazard was discussed and suggestive recommendations were recorded.
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Benha Vet. Med., J. Vol. 19, No1, June. 2008
Introduction
Brucellosis is a bacterial zoonotic disease with a broad spectrum of
clinical manifestations, transmitted directly or indirectly to humans from
infected animals. It constitutes a major health problem in many parts of the
world, particularly in the Mediterranean and Middle East (1).
It is still a serious disease problem facing the veterinary and medical
professions due to appreciable economic losses to the live stock industry in
infected areas resulting from abortions, sterility, decreased milk production
and the cost of culling and replacement animals (21). Humans are infected
through direct contact with infected animals or their discharges especially
among veterinarians, shepherds, farmers and animal attendants or through
consumption of raw milk and milk products or through the respiratory tract
especially workers in abattoirs and microbiology laboratories (12).
Brucellosis is a multi system disease with a broad spectrum of
clinical manifestations as the infection spreads hematogenously to tissues
rich in elements of the reticulo-endothelial system including liver, bone
marrow, spleen and lymph nodes and it may also localize in other tissues
including joints, central nervous system, heart and kidneys (19). This leads
primarily to the patients suffering from flu-like symptoms i.e. fever, chills,
sweating, headaches, arthralgia, fatigue and musculoskeletal involvement
(20).
As brucellosis is a disease involving the lympho proliferative
system, it may lead to changes in the hematological parameters and hypo
plastic pattern on the peripheral blood smear (18). The hematological
abnormalities including anemia, leukopenia and thrombocytopenia can be
encountered during the course of the disease. (9).
The isolation of the bacterial agent is considered the gold standard
that confirms the disease and assessment of antibody response employing
serological tests plays a major role in the routine diagnosis of brucellosis
and supported where appropriate by bacteriological examination (6). This
study was planned to investigate brucella infection among domestic animals
and humans and to determine the hematological changes on those humans
infected with brucellosis in Kalyobia Governorate.
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Benha Vet. Med., J. Vol. 19, No1, June. 2008
Materials and Methods
The present study was carried out in laboratory of zoonoses
department, Faculty of Veterinary Medicine, Benha University, while
hematological examination were done in a private medical analysis
laboratory at El-Quanter El-Khayria, Kalyobia Governorate
1- Animal samples and laboratory analysis:
Blood samples were aseptically collected from 300 animals (100
cows, 85 buffalos, 65 sheep and 50 goats) to separate serum. Animals were
randomly selected from private dairy farms, veterinary clinics, abattoirs, in
addition to sporadic animals owned to farmers in villages located in
Kalyobia governorate. All the examined animals were mature aged and were
subjected to clinical and field investigation in order to collect some
knowledge on their reproductive status. The serum samples were subjected
to the Rose Bengal Plate Antigen test (RBPA) as a sensitive screening
qualitative test using the technique described by (22) and the samples which
gave positive results were confirmed by Tube Agglutination Test then
followed by the Rivanol Test(Riv.T) in order to classify the actually positive
animals according to (6).
2- Human samples and laboratory analysis:
2-a- Human serum samples:
Blood samples were aseptically collected from 250 persons from
different fever hospitals and clinics at the same locations in Kalyobia
governorate. History was taken and physical examination considering age,
sex, occupation, fever attacks as well as any complain suggesting
brucellosis as sweating, back pain, chills or arthralgia. The obtained blood
was transferred into sterile test tubes to separate serum and then examined
serologically by RBPAT, TAT and 2 MET according to (22) & (6) as
mentioned previously. All the antigens used were supplied by Veterinary
Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt.
2-b- Anticoagulated blood samples:
Blood samples were collected from all positive sero-reactors and
transferred into sterile tubes with EDTA anticoagulant for determination of
Total Red Blood Cell count (RBCs), Total White Blood Cell count (WBCs),
Differential Leucocytic count (DLC) and Hemoglobine content (Hb). All
these tests were done as described by (14).
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Benha Vet. Med., J. Vol. 19, No1, June. 2008
Results
Table (1): Results of blood serum agglutination tests of examined animals
RBPAT
TAT
No of
animals +ve % ±ve +ve %
100
6
6
0
6
6
Cows
85
4
4.7
1
3
3.5
Buffaloes
65
11 16.9
1
10 15.4
Sheep
50
12
24
1
11
22
Goats
300
33
11
3
30
10
Total
RBPAT= Rose Bengal Plate Antigen Test.
TAT= Tube Agglutination Test.
Riv. T= Rivanol Test (Riv. T) on positive serum to TAT.
Animal
species
450
Riv-T on positive TAT
+ve
%
6
100
3
100
9
90
10
90.9
28
93.3
Benha Vet. Med., J. Vol. 19, No1, June. 2008
2-MET=2-Mercapto Ethanol Test.
451
18
13
4
38
%
5
No
1/1280
0
1/320
1
1/160
1
1/640
15.6
Total
positive
+ve
1/80
39
%
2
No
4
±ve
1/40
14
2- MET
1/20
20
1/1280
0
1/320
1
1/160
0
1/640
16
1/80
40
1/40
250
1/20
Table (2): Results of blood serum agglutination tests of examined persons
RBPAT
TAT
Total
±ve
+ve
Examined
positive
persons
+ve %
15.2
Benha Vet. Med., J. Vol. 19, No1, June. 2008
Table (3): Personal characteristics and positive sera of examined humans
No. of
No. of
Characters
examined
positive
Sex
190
35
Male
60
5
Female
250
40
Total
Age
44
3
15-20y
77
21
20-30y
57
10
30-40y
43
5
40-45y
29
1
> 45y
250
40
Total
Occupation
13
2
Veterinarians
20
4
Vet. Assistants
18
4
Animal attendants
45
5
Farmers
20
2
Butchers
53
16
Shepherds
16
1
Secretariat
40
4
House wives
10
1
Agric-engineers
15
1
Students
250
40
Total
452
%
18.4
8.3
16
6.8
27.3
17.5
11.6
3.4
16
15.4
20
22.2
11.1
10
30.2
6.3
10
10
6.7
16
Benha Vet. Med., J. Vol. 19, No1, June. 2008
Table (4): Clinical manifestations elicited in 40 persons suffering from brucellosis
Patients
Symptoms
No
%
24
60
Fever
21
52.5
Night sweats
21
52.5
Arthralgia
18
45
Headache
19
47.5
Malaise
20
50
Back pain
16
40
Loss of appetite
5
12.5
Cough
6
15
Abdominal pain
4
10
Heptaomegaly
5
12.5
Splenomegaly
2
5
Enlarged lymph nodes
More than clinical manifestation may be elicited in the person
Table(5):Hematological findings of 40 persons with brucellosis
Hematological findings
Anemia with(Hb< 11.5g/dl in ♂ and < 13 g/dl in ♀) or
(RBC count < 4.5 x 106 mm3 in ♂ & 3.8 x 106/mm3 in ♀)
Leukopenia (WBC count < 4x 103/mm3)
Thrombocytopenia (platelet count < 150 x 10 3/mm3)
Pancytopenia
The hematological values were expressed according to (14)
453
Patients
No.
%
10
25
9
5
1
22.5
12.5
2.5
Benha Vet. Med., J. Vol. 19, No1, June. 2008
Discussion
Serological tests are still the principle aid for identification of
brucella infection in animals and man. However, no single test is capable of
identifying all positive cases, so a combination of tests provides the best
basis for assessing the status of cases. Concerning results of different
serological tests among the examined animals, Table (1) indicated that
RBPAT as an initial screening test could detect more positive reactors
33(11%) than TAT 30(10%). Regarding the examined species, the
percentage of brucella positive reactors among cows, buffaloes, sheep and
goats reached 6%, 4.7%, 16.9% and 24% respectively by RBPAT, while by
using TAT, it reached 6%, 3.5%, 15.4% and 22% respectively, so the TAT
succeeded in classifying 30(10%) positive cases and 3(1%) doubtful cases
among the examined animals. Using the Riv.T in detecting true positive
cases revealed that the sensitivity of the test in classifying 100% of serum
TAT positive cases among cows and buffaloes, but 90% and 90.9% among
sheep and goats. These results were in accordance with (29 and 8) but
higher than those obtained by (7, 21 and 25) and lower than those reported
by (27 and 30).
The higher rate of brucella infection among sheep and goats may be
attributed to the fact that the majority of sheep and goats flocks are mobile
flocks and always in movement all over the year for grassing and hence can
be exposed to several routes of infection and then contaminate the pasture
and spread infection along wide distances to other animals, moreover, the
bad habit of farmers and shepherds in collecting and keeping both aborted,
pregnant and non pregnant in the same flock give a higher chance for other
sheep and goats to catch brucella infection (29). Besides, the lack of proper
sanitary condition and lack of routine serological examination. This explains
why the rate of infection was higher in small ruminants than large ones.
It is worth to mention that where brucellosis exists in animals, the
disease offers hazard for humans and serological tests appear to be the
reliable and dependable tools in diagnosis. Table (2) revealed that RBPAT
could detect more positive reactors 40 (16%) than that obtained by two
supplemental tests TAT and 2-MET 39(15.6%) &38(15.2%) for both tests.
It is evident that TAT could detect a total of 39cases (15.6%) as positive
454
Benha Vet. Med., J. Vol. 19, No1, June. 2008
reactors with titre 1/160 or higher, 1 case was doubtful with titre1/40 but 2
MET could detect 38(15.2%) as positive reactors,2 cases were doubtful and
the titres were either slightly lower or equal to those of TAT and this
coincides with the findings obtained by (31) as the immunoglobulins
responsible for agglutination in TAT are (IgM & IgG) and the agglutination
activity of IgM is destroyed by 2- Mercapto Ethanol and therefore,
agglutination is indicative only for IgG (16).
From the previously mentioned results of different serological tests
among the examined animals and man, it is cleared that RBPAT is a quick,
easy, simple screening and highly sensitive qualitative test which can detect
missed cases by TAT, while TAT had been used and still accepted in many
countries as a valuable diagnostic test as it can detect earlier brucella
infection due to the earlier appearance of IgM than other immunoglobulins
(5 and 23). Moreover, the 2MET can detect active brucella infection
explained by the presence of IgG which appears later, so 2MET is used for
accurate diagnosis and elimination of non specific agglutination titres, in
addition it is also useful in following the treatment and cure of patients (16).
Mean while Riv.T is highly specific test but it failed to detect animals in the
incubation period or at the beginning of infection, as the only IgM appeared
after beginning of infection and was discarded by Riv. solution so it detects
only IgG in the serum of infected animals (6).
Results in Table (3) revealed that brucellosis was much more
common in males (18.4%) than females (8.3%) and this could be due to
occupational exposure as farmers, shepherd veterinarians…..etc are mostly
males and this reflect an occupational exposure to the disease (11 and 1).
[
Concerning the age, the most common affected age was from 20-45
years and this may be attributed to that the majority of the workers engaged
in veterinary care, rearing, milking are of young adult. This is agree with (32
and 3) who stated that brucellosis is mostly often affect young to middle
aged adults but with a low incidence among children and the elder.
The results of this study showed that shepherds were the highest
positively infected (30.2%) followed by animal attendants (22.2%), vet.
assistants (20%), veterinarians (15.4%), farmers (11.1%), butchers,
housewives, Agric-engineers (10%) for each, students (6.7%) and
secretariats (6.3%). The highest percentage of brucella infection among
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Benha Vet. Med., J. Vol. 19, No1, June. 2008
shepherds may be due to the fact that susceptibility of caprine and ovine
species to brucella infection is high which may act as a dangerous source for
human infection (4). It is clear that a high percentage of infection among
animals seems to raise the infection in humans, which may be due to the
high risk of exposure to large numbers of brucella micro organisms via the
exposure to aborted foeti or retained placenta in infected animals or
consumption of infected raw milk or milk products, beside droplet infection.
Moreover, negligence to conduct periodical routine serological examination
and shortage in sanitary measures. This coincides with the findings obtained
by (24 and 1).
Brucellosis is usually associated with non specific clinical
manifestations, Table (4) indicated the main symptoms noted in patients
with brucellosis. Fever was the most common presenting symptoms with a
percentage of (60%) followed by sweating and arthralgia (52.5%), back pain
(50%), malaise (47.5%), headache (45%), loss of appetite (40%), abdominal
pain (15%) and cough (12.5%) and because of reticulo endothelial
involvement, splenomegaly, hepatomegaly and enlarged lymph nodes were
observed in patients with brucellosis during son graphic examination of the
abdomen with percentage of 12.5%, 10% and 5% respectively.
Regarding the hematological findings of positive sero reactors, it is
evident from Table (5) that the most hematological abnormalities including
anemia in 10(25%), leukopenia in 9(22.5%) accompanied by neutropenia
and relative lymphocytosis,while thrombocytopenia was found in 5(12.5%)
and pancytopenia found in 1(2.5%) patient. These findings substantiate what
had been reported by (17,13,2 and 26). Anemia (defined as Hemoglobin
less than 11.5 g/dl in males and 13 g/dl in females or decrease in Total Red
Blood Cell Count than 4.5 x 106/mm3 in males and 3.8 X 106/mm3 in
females) was found in 25% of patients. Several mechanisms may be
involved in the pathogenesis of anemia in patients with brucella infection as
alterations in iron metabolism which occur after infection, hypersplenism,
bone marrow suppression or auto immune hemolysis (15). Leucopenia
(Total White Blood Cell Count < 4 X 103/mm3) occurred in 9 patients
(22.5%) accompanied by neutorpenia and relative lymphocytosis. The
presence of neutropenia may be due to the aggregation of neutrophils to the
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Benha Vet. Med., J. Vol. 19, No1, June. 2008
affected tissue and their withdrawal from the peripheral circulation but
lymphocytosis is known to be a body defense mechanism and is a feature
mainly related to activation of the lympho reticular system for production
and transportation of antibodies in a trial to combat infection (10).
Thrombocytopenia (blood platelets < 150 x 103/mm3) was detected in 5
patients (12.5%) and the possible mechanisms may be hypersplenism.
Hemophagocytosis and peripheral immune destruction of thrombocytes (28
and 33) and as presence of splenomegaly and leukopenia, so the
thrombocytopenia was though to be due to hypersplenism. Moreover,
pancytopenia was occurred in 1 case of patients and may be a result of
hyper splenism with excessive destruction of formed blood elements as
brucella organisms exert a direct inhibitory effect on proliferating marrow
cells and induce parasitized macrophages and this activate lymphocytes to
release mediators that inhibit hematopoiesis which subsequently resulting in
pancytopenia (9).. The presence of hematologic alterations in brucellosis is
common but they rarely constitute a true complication and resolve promptly
with treatment (19).
Practically, every organ and system of human can be infected by
brucellosis, so the hematological consequences of brucellosis should always
be kept in mind in the differential diagnosis of anemia, leukopenia,
thrombocytopenia and pancytopenia especially in areas where brucellosis is
an endemic disease. Also, the approximate prevalence of brucellosis should
be considered as this undoubtedly points to a problem of considerably
magnitude which does not have an impact on economical loses but it is also
a disease of zoonotic importance. Thus the disease must be over looked as it
is considered internationally one of the most dangerous and notifiable
diseases. This implies the necessity of directing all efforts to determine
rapid and accurate diagnosis and elimination of infected animals in order to
reduce the risk of infection to those in regular contact with animals.
Furthermore, we should insert health education programmes and hygienic
measures among high-risk groups in order to control the spread of
brucellosis.
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Benha Vet. Med., J. Vol. 19, No1, June. 2008
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‫دراسة سيرووبائية لداء البروسيالت والتغيرات في صورة الدم لإلنسان المصاب بمحافظة‬
‫القليوبية‬
‫لبنى محمد علي سالم* ‪ -‬سعد محمد شوشة**‪-‬عادل عبد العزيز النويشى*‬
‫* قسم األمراض المشتركة – كلية الطب البيطرى‪ -‬جامعة بنها‬
‫** قسم الفسيولوجيا‪ -‬كلية الطب البيطرى‪ -‬جامعة بنها‬
‫الملخص العربي‬
‫أجريت هذه الدراسة علي عدد ‪ 033‬حيوان (‪ 033‬من األبقار و‪ 58‬من الجاامو و‪ 58‬مان‬
‫األغنام و‪ 83‬من الماعز) مان مازارخ صا اة وعيااداط بي رياة ومجااالر وحااةط يردياة ياي ب ا‬
‫قاارم مفاي ااة القليوبيااة وقااد اار يفج ا ا ساايرولوجيا باسااوزدام اصولااار الروالبنجااا ال ااري األحم ار‬
‫الفمضاا‪ ،‬ا اصولااار الولاازن األالااوب‪ ،‬الل ا لل ااار عاان داد اللروساايجط وقااد جااادط اوااا اصولااار‬
‫الروالبنجاااا ال اااري اااال ي ‪ %5‬ا ‪ %7.4‬ا ‪ %05.1‬و‪ %47‬علاااي الواااوال‪ ،‬ا بينماااا ااااات اواااا‬
‫اصولااار الولاازن األالااوب‪ ،‬الل ا هااي ‪ %5‬ا ‪ %0.8‬ا ‪ %08.7‬و‪ %44‬علااي الوااوال‪ ،‬يااي حااين اااات‬
‫اوا اصولار الريفااو علي ال يناط اإليجابية ةصولار الولازن األالاوب‪ ،‬الل ا هاي ‪ %033‬ا ‪%033‬‬
‫ا ‪ %13‬ا ‪ %13.1‬لنفس الفيواااط علي الور يب‪ .‬ماا ار أيضاا يفاد عادد ‪ 483‬مان اددمياين مان‬
‫م وااافياط الفمياااط وال ياااداط لاانفس المفاي ااة ساايرولوجيا وياااولد يااي ر ااابو ر بااداد اللروساايجط‬
‫باسااااوزدام الروالبنجااااا ال ااااري األحماااار الفمضاااا‪ ،‬واصولااااار الولاااازن األالااااوب‪ ،‬الل اااا واصولااااار‬
‫المير ابووريثااو و اات اوا هاذه اةصولااراط هاي ‪ %05‬ا‪08.5‬و‪ %08.4‬علاي الواوال‪ .،‬هاذا وقاد‬
‫لين أن ا لة اإل ابة يي الذ ور (‪ )%05.7‬أعلاي عان اإلااا (‪ )%5.0‬و اذل أعلاي ممان واراو‬
‫أعمارهر ماا باين ‪ 43‬رلاي ‪ 78‬عاماا والوا‪ ،‬اقوضات فارول عمل ار المزال اة للفيوااااط وأن ار فااخ‬
‫درجة الفرارة هي أ ثر الا اوي المو لقة بداد اللروسيجط بن لة ‪ %53‬يلي ا ال ار الليلاي والو اا‬
‫المفا ل بن لة ‪ %84.8‬ل ل من ما وادم ال ر ف ارط بن الة ‪ %83‬وقاد وجاد باالففد اإل ليني اي‬
‫للفاةط اإليجابية سيرولوجيا أن ‪ 8‬حاةط (‪ )%04.8‬لادي ا ضازر ياي ال فاا و‪ 7‬حااةط (‪)%03‬‬
‫لدي ا ضزر يي ال لد وحالوين (‪ )%8‬لدي ا ضزر يي الغدد الليمفاوية‪ .‬وقد جادط ورة يفد الادم‬
‫للفاةط اإليجابية سيرولوجيا أيضا أن ‪ 03‬حاةط لدي ر أايميا و‪ 1‬حاةط لدي ا اقاد ياي اراط الادم‬
‫الليضاد مجفوبة بنقد يي ا لة النيوروييل وار فاخ ا ل‪ ،‬يي الزجيا الليمفاوية أثناد ال د الوجنيفي‬
‫ل راط الدم الليضااد و‪ 8‬حااةط لادي ا اقاد ياي الجافا م الدموياة‪ .‬وقاد ار مناقااة األهمياة الجافية‬
‫الماور ة ل ذا المرض و ذل الوو ياط المقورحة لوجنب مزاطره والوقاية مند ‪.‬‬
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