CC BY
02018, Scienceline Publication l's
Worlds Veterinary Journal
World Vet J, 8(4): 65-72, December 25, 2018 issn 2322-4568
Seroprevalence and Associated Risk Factors of Brucellosis in Sheep and Human in Four Regions in Matrouh Governorate, Egypt
Mohamed Said Diab1, Yasser Fathey Elnaker2, Nermin Awade Ibrahim3, Eman Khalifa Sedeek4 and Sherif Abd Allah Zidan5
1 Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, New Valley University, Egypt
2 Department of Animal Medicine (Infectious Diseases), Faculty of Veterinary Medicine, New Valley University, Egypt
3 Department of Bacteriology, Faculty of Veterinary Medicine, Mansoura University, Egypt 4Department of Microbiology, Faculty of Veterinary Medicine Matrouh. University, Egypt 5 Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, University of Sadat city, Egypt
^Corresponding author's Email: yasserelnaker@yahoo.com
ABSTRACT
Brucellosis is a worldwide zoonosis that has major public health concern in Egypt. The present work was conducted to investigate the seroprevalence of brucellosis in sheep and human in four localities in North Western region of Egypt, on basis of the Rose Bengal plate test (RBPT) and further confirmation by complement fixation test (CFT). A total of2471 sheep serum samples and 371 human samples were collected. The prevalence of brucellosis in sheep and human by using RBPT were 11% (272/2471) and 24.3% (90/371), respectively while by CFT were 10.56% (261/2471) and 22.91% (85/371). There was significant relationship between age and in infection rate in sheep (P<0.01), with higher percentage of infection was indicated in age group over than 24 months by 14.19% (264/1860) followed by age group less than 24 month and over 12 months by 2.39% (8/335). On studying the relation between locality and infection rate there was no significance in human samples while in sheep it was significant (P<0.01) with higher percentage of infection found in Siwa region by 20.30% (94/463) in sheep and in human by 27.6% (27/98). Concerning season there is highly significant relationship between season and percent of infection with Brucella, the high percent of infection found in human and sheep by 43.1% (62/144) and 16.51% (123/745) respectively and lower percent found in spring months by 8% in sheep. From our result, it is concluded that RBPT and CFT used as screening tests for detection the prevalence of species in serum samples, Brucella infection is found with high percent in north, west region of Egypt, which need further examination and studying another risk factor associated with infection and isolation of Brucella in this area.
Key words: Brucellosis, Complement fixation test, Human brucellosis, Rose Bengal plate test, Sheep INTRODUCTION
Brucellosis is a highly contagious zoonosis caused by genus Brucella affecting both humans and animals (Schelling et al., 2003). Sheep brucellosis divided into typical zoonotic brucellosis that caused by Brucella melitensis and non-zoonotic ram epididymitis that caused by agent B. ovis (Acha and Szyfres 2003). Sheep and goats are primary hosts for Brucella melitensis (B. melitensis) which is common Brucella species in humans (Godfroid et al., 2011). Direct contact with fluids from infected animals in birthing products and other bodily fluids such as urine is the major rout of infections between animals and humans. Symptoms of B. melitensis in animals include abortions, stillbirths, infertility and decreased production (Corbel, 2006). While Major symptoms of human brucellosis are undulant fever, headache, muscle pain, lumbar pain and arthritis (Acha and Szyfres 2003; Pal et al., 2017).
Despite of continuous effort for zoonotic brucellosis control, that represent a major public health threats, it remains endemic in the vast majority of middle eastern countries, accused of tens of thousands of new cases yearly (Pappas and Memish 2007; Patel et al., 2017). There are about half a million new human cases of brucellosis occur every year worldwide making it the most common zoonosis (Seleem et al., 2010). Transmission of brucellosis to human occurs through ingestion of the infected product, direct contact with infected animals and its materials and through inhalation of the infected particles (Dieckhaus and Kyebambe 2017). The causative agent has a very low infectious dose, only 10 organisms of Brucella melitensis initiate infection (Lopes et al., 2010). Rose Bengal plate test (RBPT) is simple, good, rapid and easy to implement and can be used as herd screening test at remote places (Gul and Khan 2007; Teng et al., 2017). Moreover, Complement Fixation Test (CFT) used as a confirmatory test for diagnosis of brucellosis (Ashraf et al., 2014).
Therefore, this study was intended to study the seroprevalence of brucellosis in sheep and humans in four localities in Matrouh Governorate, Egypt by using Rose Bengal Plate Test and confirmed by complement fixation test.
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MATERIALS AND METHODS
Study area
This study completed in four regions (Matrouh, Elhamam, El dabaa, Siwa) in Matrouh Governorate, Egypt, to study seroprevalence of brucellosis, with history of non-vaccination.
Animals
This study was performed during the period between April 2016 to Feb ruary 2017 , a total of 2471 serum samples from B arki and Rahmani free grazing sheep in addition to 271 serum samples from humans at fever hospital in the same area of sheep rearing, the distribution of sheep and human illustrated in table 1 the data of sex and age and previous illness were recorded and there is no previous vaccination against brucellosis.
Sampling
Serum samples. In human blood samples were collected aseptically by vein puncture from each patient at the initiation of therapy and serum separated in two sterile Eppendorf that frozen for serological examination. In sheep, blood was collected (5 ml) from each sheep using plain vacutainer tube, the blood was allowed to clot at room temperature for 1-2 h, stored horizontally overnight at 4° C, then the serum was separated from the clot by centrifugation at 2000-3000 rpm for 10-15 minutes, the serum was labeled and stored at -20° C till tested.
Table 1. Number of serum samples examined from Barki and Rahmani sheep and human in four areas of Matrouh governorate between April 2016 to February 2017.
Locality Humans sheep
Matrouh 89 526
El Hamam 90 881
El dabaa 94 601
Siwa 98 463
Total 371 2471
Serological test
Rose Bengal test on human serum samples. The RBPT is a valuable screening test for diagnosis of Brucella (Agasthya et al., 2007) spectrum diagnostics Brucella Rose Bengal reagent obtained from (MDSS GmbH Schiffgraben 41 30175 Hannover, Germany) it contains ready to use standardized, killed, stained, smooth specific antigen suspensions of Brucella having specific reactivity towards antibodies to Brucella antigens which planned for quick recognition of Brucella (Melitensis, suis, and abortus) specific agglutinins (Alton et al., 1988).
Principle of the test. The smooth, killed stained Brucella antigen suspensions are mixed with the patient's serum. Specific antibodies to Brucella antigens if present in the patient serum will react with the antigen suspension to produce an agglutination reaction. No agglutination indicates the absence of specific antibodies to Brucella antigens.
Rose Bengal plate-agglutination test on sheep serum samples. Prepared from heat-killed Brucella abortus cells (strain 99) stained with rose Bengal stain and kept in a stable acidic suspension, the antigen is produced by veterinary serum and vaccine research institute Abbasia, Cairo, Egypt, used for rapid detection of brucellosis in animals (as cattle, buffalo sheep and goat) by rapid agglutination test. The test procedure recommended by Alton et al. (1988) was followed. B riefly , 25 ^l of RBPT antigen and 25 ^L of the test serum were placed alongside on the plate, and then mixed thoroughly by a tooth pick or glass rod, the plate was shaken for four minutes by electric rocker and the degree of agglutination reactions was recorded. The sample was classified positive if any agglutination was observed and negative if no agglutination was noted.
Complement fixation test. The positive serum samples by RBT were retested using CFT. B. abortus S99 antigen for CFT was used to detect the presence of anti-brucella antibodies in the sera. The test antigen obtained from veterinary serum and vaccine research institute Abbasia, Egypt, and the CFT was done at Brucella unit in central laboratory evaluation for veterinary biologics, Abbasia, Cairo, Egypt according to Alton et al. (1988). The technique is usually performed using standard 96-well microtitre plates with round (U) bottoms firstly volumes of 25 ^l of diluted (1/5) inactivated test serum placed in the well of the first, second and third rows. The first row is an anti-complementary control for each serum. Volumes of 25 ^l of CFT buffer added to the wells of the first row to compensate for lack of antigen. Then 25 ^l of CFT buffer added to third row. Serial dilutions are made by transferring 25 ^l of serum from the third row onwards , 25^l of the resulting mixture in the last row are discarded. Volumes of 25 ^l of antigen, added to each well except in the first row. Finally , 25 ^l of complement, are added to each well. The plates are incub ated at 37°C for 30 minutes , and a volume of 25 ^l of sensitized sheep Red B lood Cells is added to each well. The plates are re -incubated at 37°C for 30 minutes. The results are read after the plates have been left to stand at 4°C for 2-3 hours to allow unlysed cells to settle. The degree of haemolysis is compared with standards corresponding to 0, 25, 50, 75 and 100% lysis.
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Statistical analysis
Chi square test was used in statistical studies. The P value is the probability of the event occurring by chance if the null hypothesis is true, P-values 0.0001 (<0.01)
Ethical approval
All procedures performed in this study including collection of human serum samples and animals were in accordance with the Egyptian ethical standards of the national research committee. All human subjects gave their consent for the collection of the serum samples, with the agreement that any identifying details of the individuals should not be published.
RESULTS
In table 2 and figure 1, the overall seroprevalence of brucellosis were 11% (272/2471) and 10.56% (261/2471) in sheep serum samples using RBPT and CFT respectively while in human samples were 24.3% (90/371) and 22.91% (85/371). In table 3 and graph 1, studying age as risk factors in infection with brucellosis theirs is highly significant between age of sheep and infection with Brucella P<0.01, the high percent of infection found in age group over than 24 months by 14.19% (264/1860) followed by age group less than 24 months and over 12 months by 2.39% (8/335). In table 4 and graph 2, studying locality as other risk factors, theirs is highly significance between locality and percent of infection with brucellosis in sheep P< 0.01 while in human samples there is no significance. The high percent of infection found in Siwa region by 20.30% (94/463) in sheep and in human by 27.6% (27/98).
In table 5 obtained the high percent of infection found in human females by 13.6% (21/154) and in sheep by 11.35% (259/2282). In table 6 in studying human the high percent of infection found in contact animals with sheep by 31.5% 68 (216) compared with non-contact human in which the percent of infection were 14.2% (22/155) while table 7, graph 3, studying a season as a risk factors explain that theirs highly significance between season and percent of infection with Brucella, the highest percent of infection found in human and sheep in winter by 43.1% (62/144) and 16.51% (123/745) respectively and lower percent found in spring months by 8% in sheep and no examination to human in spring months.
Table 2. Overall prevalence of Brucella melitensis by rose Bengal plate test and complement fixation test in Barki and Rahmani sheep at four areas of Matrouh governorate between April 2016 to February 2017
Test RBPT CFT
Species Total No Positive No % Positive No %
Sheep 2471 272 11 261 10.56
Human 371 90 24.3 85 22.91
RBPT; Rose Bengal plate test, CFT; Complement fixation test
Figure 1. Map for Matrouh governorate, Egypt showing four areas of study between April 2016 to February 2017
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Table 3. Prevalence of Brucella melitensis by rose Bengal plate test in relation to age in Barki and Rahmani sheep at four areas of Matrouh governorate, Egypt between April 2016 to February 2017
Age (months) Total Positive %
1-12 276 0 0
>12 to 24 335 8 2.39
>24 1860 264 14.19
Total 2471 272 11
Chi- square 78.82**
P value 0.0001 (<0.01)
Highly significant
Age
16 14 12 10 8 6 4 2 О
%
14JUL
1-12 month
2.2
>12 to 24 M
>24 M
Graph 1. Prevalence of Brucella melitensis by rose Bengal plate test in relation to age in Barki and Rahmani sheep at four areas of Matrouh governorate, Egypt between April 2016 to February 2017
Table 4. Prevalence of Brucella melitensis by rose Bengal plate test in relation to age in serum samples of Barki and Rahmani sheep and human at four areas of Matrouh governorate, Egypt between April 2016 to February 2017
Samples Human serum samples Sheep serum samples
Locality Total Positive % Total Positive %
Matrouh 89 20 22.5 526 57 10.84
El Hamam 90 19 21.1 881 52 5.90
El dabaa 94 24 25.5 601 69 11.48
Siwa 98 27 27.6 463 94 20.30
Total 371 90 24.3 2471 272 11
Chi- square 1.3NS 64.43**
P-value 0.73 0.0001 (<0.01 )
NS: Non-Significant; **: Highly significant
%
30 25 20 15 10 5 0
I Human ■ Sheep
MATROUH
25.5
EL HAMAM
EL DABAA
Locality
27.6
SIWA
Graph 2. Prevalence of Brucella melitensis by rose Bengal plate test in relation to age in serum samples of Barki and Rahmani sheep and human at four areas of Matrouh governorate, Egypt between April 2016 to February 2017
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Diab MS, Elnaker YF, Ibrahim NA, Sedeek EKh and Zidan Sh-A-A (2018). Seroprevalence and Associated Risk Factors of Brucellosis in Sheep and
Human in Four Regions in Matrouh Governorate, Egypt. World Vet. J. 8(4): 65-72. www.wvj.science-line.com
Table 5. Prevalence of Brucella melitensis in serum samples of Barki and Rahmani sheep and human at four areas of Matrouh governorate, Egypt between April 2016 to February 2017 by rose Bengal plate test in relation to sex
Sex Samples Human serum samples Sheep serum samples
Total Positive % Total Positive %
Male 217 69 31.8 189 13 6.88
Female 154 21 1З.6 2282 259 11.35
Total З71 90 24.3 2471 272 11
Chi- square 16.17** 3.56ns
P-value 0.0001 (<0.01) 0.06
NS: Non-Significant; **: Highly significant
Table 6. Prevalence of Brucella melitensis by rose Bengal plate test in relation to history of human contact with sheep in serum samples of Barki and Rahmani sheep and human at four areas of Matrouh governorate, Egypt between April 2016 to February 2017
Item Total Positive Brucella melitensis %
Contact 216 68 31.5
Non -contact 155 22 14.2
Chi- square 14.68**
P value 0.0001 (<0.01)
** Highly significant
Table 7. Prevalence of Brucella melitensis by rose Bengal plate test according to season in serum samples of Barki and Rahmani sheep and human from four areas of Matrouh governorate, Egypt between April 2016 to February 2017
Season Species Sheep Human
Total Positive % Total Positive %
Spring 400 32 8 - - -
Summer 645 65 10.08 107 16 15
Autumn 681 52 7.64 120 12 10
Winter 745 123 16.51 144 62 43.1
Total 2471 272 11 371 90 24.3
Chi- square 35.19** 46.01**
P-value 0.0001 (<0.01) 0.0001 (<0.01)
** Highly significant
Spring Summer Autumn Winter -Sheep -Human
Graph 3. Prevalence of Brucella melitensis by rose Bengal plate test in serum samples of Barki and Rahmani sheep and human at four areas of Matrouh governorate, Egypt between April 2016 to February 2017
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DISCUSSION
Brucellosis is a zoonosis of veterinary, major public health and economic importance in most unindustrialized countries including Egypt (Afifi et al., 2005; Asiimwe et al., 2015). The diagnosis is based mainly on the serologic testing, as it is fast and simple in addition to the fact that culture techniques are not available in laboratories in endemic countries (Young, 1995). RBPT and CFT have been used for many decades, confirming to be successful for eradicating brucellosis in some countries (Garin-Bastuji et al., 1998). Therefore, in the present work RBPT were used for determination of seroprevalence of brucellosis in both sheep and human in North West region of Egypt. Results in table 2 indicated that the overall prevalence of brucellosis by RBPT in sheep were 11% (272/2471). Our results were nearly similar to that found by Hegazy et al. (2011) who confirmed that seroprevalence among sheep were 12.2%, Hussain et al. (2014) and who concluded that the overall seroprevalence of ovine brucellosis was 10.0%. The differences in prevalence of brucellosis may be attributed to time and place of sampling in addition to people habits in reporting cases.
On the contrary, higher prevalence were recorded by Al-Majali et al. (2007) 33.1% Ahmed et al. (2010), Kaoud et al. (2010), Mahboub et al. (2013) and Nagati and Hassan (2016) by rates of 24%, 26.6%, 18.09% and 16.4% respectively. On the other hand, lower results were documented by Ferede et al. (2011), Rahman et al. (2011), Horton et al. (2014), Tsehay et al. (2014) and Patel et al. (2017) by rates of 0.74%, 3.08%, 4% , 7% and 8.70 respectively.
RBPT is the ideal screening test for human brucellosis. This is for the reason that it is highly sensitive, simple and rapid technique (Smits and Kadri 2005; Teng et al., 2017). In our study, the overall prevalence of human brucellosis by RBPT as illustrated in table 2 was 24.3% (90/ 371). Nearly similar results were obtained by Fouad et al. (1996) (26%), Kumar et al. (1997) (28.57%) and De Massis et al. (2005) who concluded that Human brucellosis is more widespread in areas where Brucella is prevalent in sheep. Hussain et al. (2014) confirmed that nomadic life is characterized by some factors that favor brucellosis infection such as regular migration, inadequate health services, close animal contact, poor hygienic procedures and ingestion of raw animal products.
Lower results were obtained by Afifi et al. (2007) (11%), Hassanain and Ahmed (2012) (6.26%), Hussain et al. (2014) (6%), Ali et al. (2015) (8.6%.) , Nagati and Hassan (2016) who declared that the seroprevalence of brucellosis among human was 15.2% and Tsegay et al. (2017) (4.7%). However, higher results were recorded by Ahmed et al. (2010) who founded that the overall seroprevalence of human brucellosis in Libya were 40%.
Data presented in table 3 showed that there is a highly significant difference between age groups, with higher percent of infection in age group more than 24 Month (14.19%) than other groups (2.39%) the obtained data were agreed with Abdallah et al. (2015) and Alhamada et al. (2017) who confirmed that seropositivity were significantly higher older animal ages. On the other hand, the infection rate in human were highly significant in male than female. Our results were agreed with results obtained by Afifi et al. (2005), Khan et al. (2009), Shahid et al. (2014).
A total of 2471and 371 sheep and human samples were collected from four different localities. Data presented in table 4 showed that however, there a difference in the infection rates in four localities, statistical analysis showed that significant differences found only in sheep samples. Ecological dissimilarity has been reported to influence the seroprevalence of brucellosis (Rahman et al., 2011). Prevalence of brucellosis in relation to sex were illustrated in table 5 and showed that the infection rate in sheep were higher in female (11.35%) than male (6.88%). However statistical analysis showed no significant difference. These results were agreed with that obtained by Ferede et al. (2011), Hussain et al. (2014), Tsehay et al. (2014) and Ali et al. (2015). Rams could be lower than ewes as they may be culled or sold faster. In addition to erythritol and sex hormone in ewes (Rahman et al., 2011).
On the other hand, the infection rate in human were highly significant in male than female. Our results were agreed with results obtained by Afifi et al. (2005), Khan et al. (2009), Shahid et al. (2014). Analysis of the seasonal variation of brucellosis illustrated in table 7 and showed a highly significant differences with the highest infection rate during winter season in both human and sheep. Our results disagreed with Al-Ballaa et al. (1994) who founded that the predominance of cases occurring during spring, summer and early fall and De Massis et al. (2005) who confirmed that the seasonal occurrence of human cases of brucellosis showed a peak in summer.
CONCLUSION
High prevalence of brucellosis in sheep and human in North West region of Egypt, representing major public health threats. We need further investigation including isolation and molecular identification and further analysis of other possible risk factors associated with Brucella infection.
DECLARATIONS
Competing interests
All authors have no conflict of interest.
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Author's contributions
Mohamed S. Diab, Yasser Elnaker, Nermin Awade, Eman Khalifa and Sherif Zidan conceived and designed the
experiments. Yasser Elnaker, Nermin Awade, Eman Khalifa performed the experiments. Sherif Zidan and Yasser
Elnaker analyzed the data. Mohamed S. Diab, Sherif Zidan, Nermin Awade and Eman Khalifa contributed reagents/
materials/ analysis tools. Yasser Elnaker, Mohamed S. Diab and Nermin Awade wrote the paper.
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