Assessment of Seroprevalence and Associated Risk Factors for Anaplasmosis in Camelus dromedarius

(1) Background: Anaplasmosis is an infectious disease in camels caused by an obligate intracellular bacterium that is transmitted by ticks. (2) Methods: A cross-sectional study was conducted during 2020 to study the seroprevalence of Anaplasma spp. among Camelus dromedarius in three governorates in Egypt and assess the associated risk factors. Serum samples from 365 camels were examined by a competitive enzyme-linked immunosorbent assay (cELISA) test. (3) Results: Overall, the seroprevalence of anaplasmosis among camels was 18.6%. Multivariable logistic regression was performed, and it was discovered that tick infestation, application of acaricides, grooming practice and body condition were potential risk factors for Anaplasma spp. infection (odds ratio > 1) in dromedary camels. In contrast, the locality in which the camels lived and their age were not significant effects with regard to the occurrence of anaplasmosis. (4) Conclusions: The current findings suggest that improvement of protective measures to limit the effects of the identified risk factors can help to reduce the spread of anaplasmosis among camels in Egypt.


Introduction
The camel is a multipurpose animal that lives in arid and semi-arid areas. In many countries, including Egypt, the dromedary (Camelus dromedarius), often known as the onehumped camel or Arabian camel, is a very valuable species. Camels are of socioeconomic importance in Egypt since they can be utilized as sources of meat and milk and as a mode of transportation and tourist rides. Moreover, Camel milk is a healthy food for people since it contains more vitamin C and has less cholesterol [1]. Camels outperform farmed cattle due to their unique physiological characteristics that allow them to survive lengthy periods of time without access to water [2]. Despite their vast resources, camels can get various infectious diseases that affect their health and productivity [3][4][5][6][7].
Many animals, including camels, are affected by haemoparasites. The primary vectors for the transmission of these pathogens are ticks. Many studies have been conducted on tick-borne pathogens in camels, including trypanosomiasis, theileriosis, babesiosis and anaplasmosis [8][9][10].

Ethics Statement
The ethical committee for Animal Experiment of the faculty of veterinary medicine, Benha University, approved all procedures involving the handling and collection of samples from camels used in this study. The camel's owners gave their verbal approval for the samples to be collected.

Study Area
A cross-sectional study was conducted during 2020 in three governorates of Egypt that had high camel populations. The study areas were the governorates of: Qalyubia (30  ( Figure 1). The climatic conditions of Qalyubia and Kafr ElSheikh governorates are wet winters with moderate rainfall and dry summers, while the Red Sea area has a desert climate during the whole year with virtually no rainfall. These warm climatic conditions are suitable for tick propagation. Ticks are the principal vector for transmission of Anaplasma spp. Moreover, the types of observed ticks in examined camels in the study areas were mainly Rhipicephalus annulatus, Hyalomma dromedarii and Rhipicephalus turanicus [17]. Vet. Sci. 2022, 9, x FOR PEER REVIEW 3 of 9 A cross-sectional study was conducted during 2020 in three governorates of Egypt that had high camel populations. The study areas were the governorates of: Qalyubia (30°25 N to 31°13 E), Kafr ElSheikh (31.1107° N, 30.9388° E) and the Red Sea (25°32′1″ N 33°26′18″ E) ( Figure 1). The climatic conditions of Qalyubia and Kafr ElSheikh governorates are wet winters with moderate rainfall and dry summers, while the Red Sea area has a desert climate during the whole year with virtually no rainfall. These warm climatic conditions are suitable for tick propagation. Ticks are the principal vector for transmission of Anaplasma spp. Moreover, the types of observed ticks in examined camels in the study areas were mainly Rhipicephalus annulatus, Hyalomma dromedarii and Rhipicephalus turanicus [17].

Sample Collection and Preparation
The required sample size of the study was calculated according to a formula devised by Thrusfield [36] as follows: In which n is the sample size, Pexp is the expected prevalence rate and d is precision. The expected prevalence rate that was used in this study was 34.1%, as previously reported by Parvizi et al. [4], with a 95% confidence interval and 5% precision. The majority of the study animals were chosen at random from small-scale farmers that keep camels as working animals. A total of 365 blood samples were collected from the jugular vein of camels using a vacuum tube without EDTA. The collected blood samples were

Sample Collection and Preparation
The required sample size of the study was calculated according to a formula devised by Thrusfield [36] as follows: In which n is the sample size, Pexp is the expected prevalence rate and d is precision. The expected prevalence rate that was used in this study was 34.1%, as previously reported by Parvizi et al. [4], with a 95% confidence interval and 5% precision. The majority of the study animals were chosen at random from small-scale farmers that keep camels as working animals. A total of 365 blood samples were collected from the jugular vein of camels using a vacuum tube without EDTA. The collected blood samples were transferred in iceboxes to the Veterinary Diagnostic Laboratory, Faculty of Veterinary Medicine, Benha University. The sera were collected using clean, sterile vacuum tubes and were separated Vet. Sci. 2022, 9, 57 4 of 9 by centrifugation at 3500× g for 10 min. The examined camels were categorized according to their locality (Qalyubia, Kafr Elsheikh or the Red Sea), sex (male or female) and age (≤2, >2-5 and >5 years old). Moreover, information regarding tick infestation, whether or not acaricides had been applied (trimonthly application), grooming practice (removing thick hair that has accumulated grain, grime, and mats on a regular basis) and body condition (emaciated, decrease the bodyweight than normal or healthy) was collected to investigate their association to infection.

Serological Analysis
The specific antibodies against Anaplasma spp. were investigated in the collected sera through the use of a commercial competitive ELISA (cELISA) v2 (VMRD Inc, Pullman, WA, USA), which is able to detect antibodies against the major surface protein 5 (MSP5) of A. marginale, A. centrale and A. ovis [37]. The process of the test was performed according to the guidelines of the manufacturer. This kit had previously been validated to show 100% sensitivity and specificity in the detection of Anaplasma spp. antibodies in camels [4]. The sample was considered positive if the cut-off value (Ct) was equal to 0.42.

Statistical Analysis
Data regarding the anaplasmosis surveillance were analyzed by use of the statistical program for the social sciences (SPSS) software v24 (IBM SPSS Inc., Chicago, IL, USA). The data were verified through the use of the chi-square test, and the results were considered significant if p < 0.05. The results were analyzed through the use of univariable logistic regression to evaluate the association between each variable and prevalence of Anaplasma spp. The Hosmer-Lemeshow goodness-of-fit test was applied to evaluate the fit of the multivariable logistic regression model. The variables with p ≤ 0.2 were included in the multivariable regression model, which was used to determine the risk factors, odds ratios (ORs) and confidence intervals (CIs) of each significant variable in the univariable analyses. Odds ratios of >1 suggested an increased risk of seroprevalence of anaplasma infection, whereas odds ratios of <1 suggested a decreased risk of seroprevalence of anaplasma infection.

Results
The present study demonstrated an overall 18.6% (68/365) seroprevalence of anaplasmosis among camels that lived in the three investigated areas. The highest seroprevalence for Anaplasma spp. in camels was estimated to be in the Red Sea governorate (21.3%, n = 32) ( Table 1). In order of increasing magnitude, the seroprevalence was 13% (n = 13) and 20% (n = 23) in Kafr ElSheikh and Qalyubia governorates, respectively (Table 1).
According to the univariable analysis, seropositivity to Anaplasma spp. in camels was associated significantly with female sex, tick infestation, non-application of acaricides, poor grooming practice and poor body condition (p < 0.005). The highest seroprevalence rates were observed in females (21.5%), infested camels with ticks (33%) and in cases of the absence of acaricides application (23.5.%) and grooming application (25.9%), Table 1.
In addition, a strong association was found between animals in an emaciated condition and Anaplasma spp. infection. On the other hand, there was no significant interaction between age and Anaplasma spp. infection (Table 1).
Significant variables that were obtained through univariable studies were then analyzed multivariably. The animal's age and locality were removed as factors. In this study, it was found that females were two times more likely to be infected than males (95% CI: 0.91-4.35). Furthermore, tick infestation of camels (OR = 1.12, 95% CI: 0.54-2.32), lack of acaricide application (OR = 1.02, 95% CI: 0.39-2.68), absence of grooming (OR = 1.3, 95% CI: 0.53-3.18) and an emaciated condition of the examined camels (OR = 9.36, 95% CI: 4.36-20.10) were found to be potential risk factors for Anaplasma spp. infection in camels (Table 2). The result is non-significant at p > 0.05. The result is significant at p < 0.05.

Discussion
Dromedary camels can harbor a variety of pathogens, including Anaplasma. This genus has been reported in the last few years in some studies, but the epidemiological data remains limited.
A description of the epidemiological status of anaplasmosis and evaluation of the risk factors that are potentially related to disease in camels helps to improve the understanding of the dynamics of and potential control methods for the disease [38].
In this study, the antibodies against Anaplasma spp. in camels were detected in 68 of 365 animals, and the seroprevalence rate was recorded as 18.6%. Despite the large differences in bioclimatic features between the three sites studied, the prevalence rates do Vet. Sci. 2022, 9, 57 6 of 9 not differ significantly (p > 0.05). The highest rate, 21.3%, was observed in the Red Sea, while the lowest rate (13%) was found in the Kafr ElSheikh governorate. This is likely due to the frequent movement of camels between these areas and the similarities of tick populations infesting camels in the sampling locations [39]. The high rate observed in the Red Sea governorate may be due to the nature of this area, which is a border governorate that receives camels from neighbouring countries. These camels may be carriers of haemoparasites. In addition, different humidity levels that enable the proliferation of vectors and transmission of Anaplasma can affect the prevalence rate [40].
The differences in prevalence rates may be attributable to sample numbers, the diagnostic techniques were used, demographics of the research locations and disease endemicity in each study region [46]. Furthermore, tick control programmes, farm management, husbandry practises, wildlife reservoir hosts and/or abiotic variables may all play a role in the large disparity in prevalence rates. Several studies have found that the incidence of Anaplasma species in ruminants varies depending on geographic location, as well as tick habitat and animal care [48,49]. Moreover, different humidity levels that enable the proliferation of vectors and transmission of anaplasmosis can affect the prevalence rate [40].
From the results, it is clear that the age of the camels did not affect the prevalence of anaplasmosis and animals aged >2 to 5 years were at a higher risk than those aged >5 years. This finding is in contrast to those of Farooqi et al. [50], who reported that the age of the camels was a potential risk factor for the occurrence of anaplasmosis. In addition, Kocan et al. [51] observed that animals over the age of five are found to be less affected, which can be related to the fact that low-level infections over time lead to immunity against clinical anaplasmosis.
A further finding was that the sex of the animal was a significant variable for camel anaplasmosis, and females were more susceptible to infection than males. The findings contradict those of Azmat et al. [46], who found that male animals have higher infection rates than female animals. Our result is consistent with the findings of Maurizi et al. [52] and Belkahia et al. [53], who reported a higher prevalence rate in females in comparison with males. The differences between these findings can be explained since people in the area of the Javed study kept female camels for breeding purposes, so these animals performed few draft activities and were more exposed to tick infestation. Moreover, this is may be due to female immunosuppression, which can develop during pregnancy and lactation and has the potential to last two years [54].
Other factors that were found to be significant were the tick infestation of examined camels and whether acaricides were previously applied. Camels that were infested with ticks were at high risk of anaplasma infection compared with non-infested camels. Overall, these findings are in accordance with those reported by Atif [55], who reported that tick infestation made animals more susceptible to infection. We believe that Egyptian camels can be infested by a variety of ticks, particularly hard ticks, which are the main vector for Anaplasma spp. [56].
Furthermore, in line with the results of Azmat et al. [46], the current study found that good grooming practice significantly reduced the rate of Anaplasma spp. infection, because frequent grooming led to the early observation of ticks, which could be controlled immediately. Furthermore, grooming practises have a considerable impact on disease dynamics, which could be attributed to the fact that regular grooming practice allows for early detection of vectors and prompt control. In addition, the absence of grooming allowed the existence of hiding spots for vectors that are difficult to manage through farm manipulation. Similarly, emaciated camels were found to be more susceptible to infection, as was previously concluded by Azmat et al. [46]. This may be explained by the emaciation due to infestation with ticks and concurrent infection by other parasites or bacteria, which can increase the risk of infection [57].
The limitation of this study was the use of a commercial ELISA test for the detection antibodies against A. marginale, A. centrale and A. ovis. A specific test such as PCR is needed to determine the prevalent species among camels in Egypt. In addition, the present study is a cross-sectional study able to evaluate association only; therefore, longitudinal studies are required to prove causation.

Conclusions
It has been concluded that tick infestation, tick control status, grooming practice and body condition are strongly associated with Anaplasma spp. infection. In Egypt, the link between Anaplasma spp. infection and their arthropod vectors is mostly unknown, and more research is needed. However, further epidemiological and molecular studies are required to evaluate the situation of the disease across the country and to identify the genetic features of Anaplasma spp. in camels.