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Occurrence of anaplasmosis among sheep (Ovis aries) and goats (Capra hircus) in Madina and Tabuk, Saudi Arabia
⁎Corresponding author at: Department of Zoology, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia.
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Anaplasma spp. is a group of intra-erythrocytic bacteria that various species of ticks can transmit. Information regarding the prevalence of infections in sheep and goats caused by this group of organisms is scarce in Saudi Arabia. The present study was carried out during 2020–2021 to examine the prevalence of anaplasmosis among sheep and goats in two cities in western Saudi Arabia. The study included samples from 177 sheep (77 from Madina, 100 from Tabuk) and 226 goats (123 from Madina, 103 from Tabuk). The samples were investigated using direct microscopy method as well as a competitive Enzyme-Linked Immuno-Assay (cELISA) for the detection of anti-Anaplasma spp. antibodies. A total of 93 (23.1%) of the samples were positive on direct microscopy, whereas 84 (20.7%) were positive on cELISA. Of those samples positive on direct microscopy, 44 (19.5%) were from goats while 49 (27.7%) were from sheep. Of the positive samples on cELISA; 38 (17.0%) were from goats, and 46 (26.0%) were from sheep. A significant difference in the prevalence of anaplasmosis was reported (p < 0.05) using microscopic and cELISA in goats and sheep in both regions studied, having a higher prevalence in Tabuk. There was no significant difference in the prevalence of anaplasmosis in males and females from Madina and Tabuk using both methods (p > 0.05). However, there was a significant difference in the prevalence of anaplasmosis in older goats (>2 years) and in the summer compared to the winter in samples collected from Tabuk (p < 0.05). Seroprevalence of anaplasmosis was detected for the first time In the Tabuk region from sheep and goats. The difference in the prevalence in the two locations studied was probably due to the variation in climatic conditions and the availability of the vector responsible for the transmission of anaplasmosis.
Keywords
Anaplasmosis
Serology
cELISA
Saudi Arabia
Sheep
Goats
1 Introduction
Members of the genus Anaplasma are obligate, intraerythrocytic, gram-negative, tick-borne rickettsial organisms that infect a wide range of domestic and wild mammals, including humans. There are nine species, as well as seven candidate species, included in the genus Anaplasma (Dumler et al., 2001; Vanstreels et al., 2018). Of these, A. ovis, A. capra, and A. phagoytophilum infect sheep and goats, with the latter infecting multiple hosts, including man. A. marginale primarily infects cattle. However, it was also found to infect sheep and goats (Yousefi et al., 2017; Barbosa et al., 2021). Ticks of various genera can transmit the infection from one individual animal to another. Infection may result in anemia when transmitted to hosts outside of their natural cycle (Parola et al., 2005; Nicholson et al., 2010).
The zoonotic potential of infection with A. ovis has been proven, and some variants of A. ovis were detected in human patients from Iran and Cyprus in recent studies (Chochlakis et al., 2010; Hosseini-Vasoukolaei et al., 2014). A. ovis has a cosmopolitan distribution and is responsible for considerable losses in sheep and goats as a result (Renneker et al., 2013). In Saudi Arabia, limited studies on the disease and its epidemiological burden have been conducted. Hemoparasite investigation resulted in the detection of several blood protozoan parasites in camels, sheep, goats, and cattle in different regions of Saudi Arabia (El-Azazy et al., 2001; Diab et al., 2006). Al-Khalifa et al. (2009) conducted a microscopic examination of blood from several animals in the Riyadh region, Southern region, and the Eastern and Northern frontiers of Saudi Arabia. They found evidence of A. ovis in 2% of the sheep samples collected from the Eastern and Northern regions of the Kingdom of Saudi Arabia. Furthermore, they found A. marginale in 3.4% of cattle from the Eastern region. The overall prevalence of anaplasmosis in some areas like Pakistan was 29.63% and 1.66 % in sheep and goats, respectively (Muhammad et al., 1999). Prevalence in Mosul, Iraq, was found to be (62.6%) in sheep (Sulaiman et al., 2010).
The most common method for diagnosing hemoparasites such as Theileria spp. or intraerythrocytic bacteria such as Anaplasma spp. depends on the demonstration of the parasite or the bacteria in Giemsa-stained thick or thin blood smears using light microscopy (Ali et al., 1996; Nagore et al., 2004). In addition, indirect methods such as different serologic tests or DNA-based molecular methods of the agent are used to detect the specific antibodies or pathogens (Goff et al., 1990; Sumbria and Singla, 2015).
Enzyme Linked Immunosorbent Assay (ELISA) is routinely used for the detection of specific parasitic antibodies, antigens, and immune complexes. It is commonly used as the basis for epidemiological surveys (Passos et al., 1998; Sekiya et al., 2013). Nowadays, various studies have been performed in small ruminants and other domestic livestock for sensitive detection of hemoparasites and intraerythrocytic bacteria. Serological evidence has been provided for anaplasmosis in dogs, horses, and camels from Algeria, Morocco, and Tunisia (M’ghirbi et al., 2009; Ben Said et al., 2014; Azzag et al., 2015; Elhamiani Khatat et al., 2017). A. marginale has been reported in cattle from Sudan, Morocco, and Egypt (Salih et al., 2008; Hamou et al., 2012; Fereig et al., 2017).
The present study aimed to investigate the occurrence of anaplasmosis among sheep and goats in Medina and Tabuk, KSA using direct microscopic bacterial examination and competitive ELISA. In addition, the study also aimed to investigate the role of some risk factors that might be associated with direct infection or previous exposure to the species members of Anaplasma.
2 Materials and methods
2.1 Study sites and samples collection
Blood samples were collected from sheep and goats in Madina (24.8404° N, 39.3206° E) and Tabuk (28.2453° N, 37.6387° E) regions in the north and northwestern areas of the Kingdom of Saudi Arabia.
A total of 403 blood samples were collected from apparently healthy sheep (n = 177; 77 from Madina and 100 from Tabuk) and goats (n = 226; 123 from Madina and 103 from Tabuk). Samples were collected from animals kept for breeding and production. Some were obtained from Veterinary clinics affiliated with the Ministry of Environment, Water, and Agriculture at Madina and Tabuk. The samples were collected during the summer season (n = 219) and the winter season (n = 184). Sheep and goats’ age, gender, location, system of rearing, and season during which samples were collected are regarded as risk factors for infection with anaplasmosis.
Blood samples were collected from the jugular vein using a 20 g × ½ inch needle into 5 ml syringes. Each sample was then transferred into two clean vacutainers, one of which was coated with ethylene diamine tetra-acetic acid (EDTA) to be used in preparing direct blood thin smears for microscopic examination. The second vacutainers (non-anticoagulant tubes) were used to obtain serum.
Serum samples were obtained after obtaining the blood samples which were collected in plain vacutainers being clotted overnight at room temperature and then centrifuged for 10 min at 2500 g using a tabletop centrifuge (Gemmy Industrial Corp. Taiwan associated with Cannic, Inc. USA). Serum samples were then transferred into clean Eppendorf tubes and stored at – 20 °C until used.
2.2 Microscopic examination
A drop of blood (up to 5 µl) was placed on a clean glass slide and spread along the slide, then air-dried, stained with 5% Giemsa stain, and examined microscopically using X100 objective lens for the detection of intraerythrocytic bodies typical of Anaplasma spp.
2.3 Serological methods
Anti-Anaplasma spp. antibodies in serum samples from sheep and goats were detected using a competitive enzyme-linked immunosorbent assay (cELISA) for Anaplasma spp. antigen using (VMRD, ANAPLASMA ANTIBODY TEST KIT, cELISA v2 Pullman, WA 99163 USA) according to the manufacturer’s protocol. A volume of 50 µl of negative and positive controls and undiluted samples were transferred to the Anaplasma spp. antigen-coated plates. In the positive serum samples, the antibodies block the binding of the secondary antibody (the horseradish peroxidase labeled monoclonal antibody conjugate). Hence, when adding the substrate, there will be no color development on the positive wells and a dark color in the negative wells. The plate was read in SpectraMax M series multimode microplate reader (Molecular Devices, LLC. 3860 N First Street, San Jose, CA 95134) with a wavelength of 630 nm. The % of inhibition (I) was calculated according to the following formula: Samples with an inhibition of ≥30% were considered positive, while samples with inhibition of <30% were considered negative.
2.4 Sensitivity and specificity of tests calculations
The microscopic examination is considered the gold standard method. The sensitivity and specificity were calculated as the proportion of all the positive samples on ELISA out of all the samples positive in microscopic examination.
2.5 Statistical analysis
Results were analyzed using the Chi-square test in the statistical software SPSS 20.0 (IBM, White Plain NYS, USA). Significant differences were indicated when the p value was less than 0.05. The likelihood of detection or exposure as an indicator for the risk to the parasite was quantified using the odds ratio.
3 Results
Intraerythrocytic organisms suggestive of Anaplasma spp. (Fig. 1) were microscopically demonstrated in 23.1% of the total examined animals (44 goats and 49 sheep) (Table 1). Serological investigations revealed that antibodies to anaplasmosis were detected in 20.7% (84/403) of the studied populations, with 46 and 38 positives from sheep and goats respectively. There was a significant difference in the prevalence of anaplasmosis using microscopic and cELISA tests in goats and sheep in both regions studied, having higher prevalence in Tabuk (p < 0.05).
Intraerythrocytic Anaplasma spp. on blood smears stained with Giemsa from sheep (A) and goat (B). Scale bar 5 μm for both photographs.
Animals Examined
Results of Microscopy
Results of Ab ELISA
Positive (%)
p value
Odds Ratio (95% CI)
Positive (%)
p value
Goats
Madina (124)
14 (11.3)
<0.05
0.3 (0.2, 0.6)
12 (9.7)
<0.05
Tabuk (102)
30 (29.1)
26 (25.5)
Sheep
Madina (77)
8 (10.4)
<0.05
0.2 (0.1, 0.3)
6 (7.8)
<0.05
Tabuk (100)
41 (41)
40 (40)
The prevalence of anaplasmosis in goats and sheep is shown in Tables 2 and 3. There was no significant difference in the prevalence of anaplasmosis using both microscopic and ELISA methods in male and female goats in the Madina and Tabuk regions (p > 0.05). Likewise, there was no significant difference (p > 0.05) in the prevalence in sheep with different ages in both regions studied. However, there was a significant difference in the prevalence of anaplasmosis in older goats (>2 years old) using the cELISA method (p < 0.05) in the Tabuk region. The prevalence of anaplasmosis in goats in Tabuk was significant (p < 0.05) in the summer compared to the winter (Table 2) using the cELISA method (Table 2). There was no significant difference in the prevalence of anaplasmosis in different seasons in goats from Madina using both methods. Goats and sheep kept under intensive conditions had a higher prevalence compared to animals kept under open systems using both methods in Madina and Tabuk. However, the difference was significant (p < 0.05) in goats and sheep from Tabuk using both microscopic as well as ELISA methods (Tables 2 and 3).
Variable
No Examined
Results of Microscopy
Results of Ab ELISA
Madina
Tabuk
Positive (%)
p value
Positive (%)
p value
Medina
Tabuk
Medina
Tabuk
Medina
Tabuk
Medina
Tabuk
Sex
Male
64
39
8 (12.5)
9(23.1)
>0.05
>0.05
8 (12.5)
8 (20.5)
>0.05
>0.05
Female
60
63
6 (10)
21(33.3)
4 (6.7)
18 (28.6)
Age (Months)
<2 years
34
60
2 (5.9)
18 (30)
>0.05
>0.05
3(8.8)
11 (18.3)
>0.05
<0.05
≥2 years
90
42
12 (13.3)
12 (28,6)
9(10)
15 (35.7)
Season
Summer
49
58
3 (6.1)
25 (43.1)
>0.05
<0.05
4 (8.2)
24 (41.4)
>0.05
<0.05
Winter
75
44
11(14.7)
5 (11.4)
8 (10.7)
2 (9.1)
Rearing System
Open
50
50
6(12)
5(10)
>0.05
<0.05
3(6)
2(4)
>0.05
<0.05
Intensive
74
52
8(10.8)
25(48)
9(12)
24(46)
Variable
No Examined
Results of Microscopy
Results of Ab ELISA
Madina
Tabuk
Positive (%)
p value
Positive (%)
p value
Medina
Tabuk
Medina
Tabuk
Medina
Tabuk
Medina
Tabuk
Sex
Male
36
48
6 (16.7)
15(31.3)
>0.05
>0 0.05
1(2.8)
15(31.3)
>0.05
>0.05
Female
41
52
2(4.9)
26(50)
5(12.2)
25(48)
Age (Months)
<2 years
16
43
4(25)
15(34.9)
> 0.05
>0.05
2(12.5)
15(34.9)
>0.05
>0.05
≥2 years
61
57
4(6.5)
26(45.6)
4(6.6)
25(43.9)
Season
Summer
57
55
2(3.5)
19(34.5)
< 0.05
>0.05
0(0)
18(32.7)
–
>0.05
Winter
20
45
6(30)
22(48.9)
6(30)
22(48.9)
Rearing System
Open
42
51
2(4.8)
31(60.8)
>0.05
< 0.05
0(0)
27(52.9)
–
<0.05
Intensive
35
49
6(17.1)
10(20.4)
6(17.1)
13(26.5)
The majority of animals showed positive antibodies (34.5%) against anaplasmosis with inhibition between 30 and 40%. While 13.1% showed inhibition between 90 and 100%.
4 Discussion
In the present study, we investigated the occurrence and the associate risk factors of a tick-borne disease pathogen anaplasmosis in sheep and goats using direct microscopy and the cELISA method in two regions of the Kingdom of Saudi Arabia. To our knowledge, this is the first report of anaplasmosis from the Tabuk region in Saudi Arabia. Using direct microscopic examination of Giemsa-stained blood smears, we detected a prevalence of 23.1%, whereas using the cELISA method, antibodies against anaplasmosis were detected in 20.7% of goats and sheep screened from both regions studied. Ghafar and Amer (2019) detected a prevalence of A. ovis as low as 9% of the goats investigated from Taif using molecular techniques. On the other hand, Shabana et al. (2018) detected a much higher prevalence of antibodies against anaplasmosis in 43.6% of the sheep and goats investigated in the western part of Saudi Arabia. In Pakistan, a prevalence of 22.2% of the sheep and goats studied have shown antibodies against anaplasmosis (Khan et al., 2019). Obaidat and Salman (2019) reported as high as 90% seroprevalence in sheep and goats in Jordan. Lower rates, but higher than what has been reported in the present study, were detected from sheep in Portugal (82%), Sudan (42%), Iraq (67%), and Turkey (31%) (Renneker et al., 2013) using molecular techniques. Detection of anaplasmosis in the blood of sheep and goats depends mainly on the method used for the detection, i.e., if it was direct or indirect. Direct methods such as demonstrating the organisms in blood smears is useful; however, it requires expertise and high levels of bacteremia. Low bacteremia can simply result in false-negative results. Indirect methods such as cELISA and molecular techniques detect circulating antibodies and the organism’s DNA, respectively. Only two previous reports have dealt with direct microscopy in estimating the prevalence of anaplasmosis infection in small ruminants in the Kingdom of Saudi Arabia (Al-Khalifa et al., 2009; Shabana et al., 2018). Al-Khalifa et al. (2009) reported a prevalence of 2% in sheep from eastern and northern provinces, whereas Shabana et al. (2018) reported a much higher prevalence of 40.7% and 45.5% in goats and sheep, respectively from the western part of Saudi Arabia.
It is interesting, in the present study, to note that 13.1% of the samples investigated showed high levels of antibodies with optical densities greater than 90%, whereas most of other samples revealed optical densities between 30 and 40.
Previous studies related to the prevalence of anaplasmosis in small ruminants have shown contradicting results between males and females (Rajput et al., 2005; Shabana et al., 2018; Khan et al., 2019). Our study observed similar results with no significant difference between the prevalence in males and females in both regions studied. However, the prevalence was higher in females in Tabuk and in males in Madina. In Pakistan, Nasreen et al. (2016) attributed the higher prevalence of anaplasmosis in sheep to some stress factors such as malnutrition and pregnancy in females which may be the case in our study.
We observed that there was a high prevalence of anaplasmosis in older sheep and goats (>2 years) compared to younger animals (<2 years) in both regions. However, the difference was not statistically significant. Similar findings were reported from sheep by Khan et al. (2019) in Pakistan as well as in bovine anaplasmosis in the USA (Aubry and Geale, 2011; Okafor et al., 2019). One explanation for this finding was due to the fact that younger animals were less exposed to tick infestation as they were handled differently compared to adults. Furthermore, as animals and humans get older, they develop fewer T-lymphocytes, and the MHC class II response is lower over time (Graham et al., 2006).
The prevalence of anaplasmosis in different animal species is affected by the season. We found that goats in Tabuk, unlike sheep, showed significantly high prevalence in the summer compared to the values obtained from the winter. Whereas in Madina, sheep showed higher prevalence in the winter. Mohammed et al. (2021) reported a high prevalence of a tick-borne piroplasm in hedgehogs in Saudi Arabia during the summer compared to the winter. El-Bahy et al. (2008) reported a higher prevalence of Theileria ovis in central Saudi Arabia. There may be different susceptibilities to sheep and goats for tick infestation, as in the case of the hedgehog and sheep in studies conducted by El-Bahy et al. (2008) and Mohammed et al. (2021).
The system of rearing is important in determining the prevalence of anaplasmosis infection, where in the present study, animals reared under intensive rearing system showed higher prevalence compared to free-range animals. An intensive rearing system or what is called zero-grazing would enable keeping the animals in a pen or a paddock where food is provided without allowing the animals to mix with others. It is likely that there will be an accumulation of tick vectors among hosts in an intensive system if the management practices are poor, such as not following preventive measures regarding external parasites. Khan et al. (2019) reported that animals that are at zero grazing are not affected by anaplasmosis and they attributed that to the control of ticks.
More animals were positive by direct microscopy (23.1%) compared to the cELISA method (20.7%). Hence, the sensitivity of the cELISA was found to be 84.5%, while the specificity was found to be 93.1%. The cELISA method used in the present study employs using the Main Surface Protein 5 (MSP5), which is highly sensitive, specific, and conserved for Anaplasma spp. (Visser et al., 1992; Knowles et al., 1996).
5 Conclusion
The occurrence of anaplasmosis was investigated in sheep and goats both in the Madina and Tabuk regions of the Kingdom of Saudi Arabia using direct microscopy and cELISA. Evidence of anaplasmosis was revealed for the first time in the Tabuk region from both sheep and goats using a serological method with se seroprevalence of 40 % and 25.5 % in sheep and goats, respectively. Occurrence of anaplasmosis in the Madina region was significantly higher than in the Tabuk region (p < 0.05) (7.8 % and 9.7 % from sheep and goats orderly). Risk factors, such as sender, age, system of rearing and season were found to be contributory risk factors for the occurrence of the diseases in both regions studied. Further work is recommended in order to determine which species of Anaplasma is causing anaplasmosis in both regions.
Acknowledgements
This work was financially supported by Researchers Supporting Project number (RSP–2021/94), King Saud University, Riyadh, Saudi Arabia.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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