Epidemiology and molecular detection of Anaplasma spp. in goats from Chattogram district, Bangladesh

Abstract Objectives Anaplasmosis is an economically important disease affecting cattle, buffalo, sheep, goat etc. The study was conducted to determine the prevalence, potential risk factors and molecular identification of circulating Anaplasma spp. in goats in Chattogram district, Bangladesh. Material and methods Four hundred blood samples were collected from goats of different ages, breeds, sex, coat color and body condition. These goats were selected based on some inclusion criteria through the period of July 2017 to June 2018. Samples were examined microscopically (Giemsa staining method) followed by polymerase chain reaction (PCR) and sequencing to identify of Anaplasma spp. Results The overall prevalences were estimated 5.75% (23/400) and 15.75% (63/400) by microscopy and PCR, respectively. Anaplasma ovis (A. ovis) and Anaplasma marginale (A. marginale) were identified with the prevalence of 14.75% (59/400) and 1.0% (4/400), respectively through PCR. Among different risk factors, jamnapari breed (p = 0.027), no use of acaricide (p = 0.025) and presence of tick (p < 0.01) were found to be significantly associated with anaplasmosis. Sequence analysis of msp4 gene revealed that, Anaplasma spp. detected in the present study were highly similar with those of China, Venezuela, Mongolia, Spain, Tunisia, Cyprus, Italy, Brazil, Argentina, Australia, Japan and Columbia. Conclusions In conclusion, strategic use of acaricide can control tick that ultimately will control the anaplasmosis in goats. Besides, rearing local goats in compare to cross and exotic breed are also recommended for the farmer to prevent the disease.


INTRODUCTION
Goats are one of the important livestock species in rural economy and nutrition with great potentiality to alleviate the poverty of Bangladesh. It is also known as 'poor man's' cow as it is generally reared by ultra poor women having little capital investment (Nath et al., 2014). It helps the landless and marginal farmers through women empowerment, youth employment and foreign exchange earnings (Rana, 2015). However, various diseases of goats may cause huge economic losses to the farmer and ultimate the economy of the country through mortality, production losses and addition of extra cost of treatment for sick animals (Singh & Prasad, 2008). Anaplasmosis is among them considering one of the top 10 economically important diseases affecting endemically in tropical, subtropical, and sporadically in temperate region (Rajasokkappan & Selvaraju, 2016). It is a vector borne (tick) disease of ruminants caused by obligate intra-erythrocytic rickettsial organism of the genus Anaplasma and characterised clinically by fever, inappetance, decrease milk yield, progressive anaemia, icterus, brownish urine, pale mucous membrane, labored breathing and constipation (Razmi et al., 2006). Currently, there are six recognised species that have been identified under this genus: Anaplasma ovis, Anaplasma marginale, Anaplasma centrale, Anaplasma platys, Anaplasma bovis and Anaplasma phagocytophilum. Among them, Anaplasma ovis (A. ovis) found as most pathogenic and Anaplasma marginale (A. marginale) as subclinical form in small ruminants (Razmi et al., 2006).
The climate of Bangladesh is usually hot and humid in nature and the geographical location of Chattogram (study area) is very conducive to a wide variety of ticks that can transmit Anaplasma spp. in goats (Ananda et al., 2009;Belal et al., 2014;Kakarsulemankhel, 2011). However, little information is known on caprine anaplasmosis though the bovine anaplasmosis has been extensively studied in this region. In cattle, the prevalence of anaplasmosis has been reported as 70% in Sirajganj,8.21% in Chattogram, 33% in Baghabari milk shed areas (Belal et al., 2014;Mannan, 2017;Talukdar & Karim, 2001); and in goats 2.48% in Chattogram (Nath et al., 2014). In these studies, diagnosis was done on the basis of clinical signs and Giemsa staining method though these methods of diagnosis have several limitations; for example, the carrier animal may goes unnoticed and differentiation between Anaplasma spp. is quite difficult as they are morphologically very similar (Ahmadi-Hamedani et al., 2009). Thus, polymerace chain reaction (PCR) has become preferred method for the diagnosis of anaplasmosis due to its high sensitivity and specificity than other conventional methods (Lew et al., 2002). Moreover, the msp4 genes involve in interactions with both vertebrate and invertebrate hosts, and evolve more rapidly than other genes are used to identify Anaplasma sp. (Ahmadi-Hamedani et al., 2009). Most importantly, as far our concern, reports on molecular detection, prevalences and associated risk factors of anaplasmosis in goat have not yet been documented in Bangladesh. Therefore, the study was designed to detect the prevalence through the amplification of 16SrRNA and msp4 genes, along with potential risk factors which may help to establish the preventive and control measure of anaplasmosis in goats.

Description of study area
The study goats were selected from Teaching Veterinary Hospital

Study population and sample collection
The study was conducted from July 2017 to June 2018. The study population was goats that were visited to the Teaching Veterinary Hospital from different area of Chattogram district for the purpose of health check up, vaccination, deworming and treatment of illness.
Immediately after registration, clinical history was taken and physical examination was performed for each goat. Physical examination included observation of general appearance, examination of backbone and ribs, observation of visible mucous membrane, palpation of superficial lymhnode, recording of body temperature etc. Goats more than 6 months old that had at least one or more following criteria like presence of vector ticks on the body, fever, anorexia, anaemia, swollen lymphnode and jaundice were considered for sample collection. Based on these criteria, the required numbers of study goats were selected. Sample size (numbers of goats) was determined by the following formula (Thrusfield, 2005).
where N is required sample size, Pexp is expected prevalence = 50% = 0.5 (we assumed prevalence was uknown), d is desired absolute precision = 5% = 0.05, 1.96 is the value of z at 95% confidence interval Accordingly, the required numbers of goats were 384. However, to increase precision the sample size was increased and total of 400 goats were included in the study.
All relevant data such as breeds (bengal goat, jamnapari, and crossbreed), age category (young, adult and old), sex (male and female), body color (black, brown, gray, white and mixed), body condition score (very thin, thin and good), different management system like rearing system (backyard, intensive and extensive), grazing (group, individual and zero grazing) and flock size (large, medium and small), acaricide using history (yes or no), presence of tick on the body surface (yes or no) and date of registration were recorded using a close ended questionnaire by face to face interview of owner and close examination of goat. After that, blood sample (5 ml) was collected from jugular vein of each goat using sterile disposable needle. Before collection, the puncture area was cleaned and disinfected with 70% alcohol. Thin smear was prepared using a drop of blood on a slide. After labelling, slide was air-dried and fixed in methyl alcohol. Remaining blood was transferred into vacutainer containing anticoagulant (EDTA) and transported to the laboratory for further analysis (Kessell, 2015).

Microscopic examination of blood smear
The fixed thin smears were stained with Giemsa stain at a dilution of 10% in buffer solution and allowed to stay for 20 min followed by washing with tap water to remove extra stain. Then the slides were air dried and examined for the presence of Anaplasma inclusion bodies under light microscope.

DNA extraction
Genomic DNA Extraction Kit (Addbio ® , Korea) was used to extract DNA according to the manufacturer's instructions. Briefly, 20 μl of proteinase K solution was added to 1.5 ml micro-centrifuge tube containing of whole blood sample (200 μl). Then binding solution (200 μl) was added to the same tube and thoroughly mixed (vortexing for 15 s) followed by incubation at 56 • C for 10 min. After incubation, absolute ethanol (200 μl) was added and mixed similarly for 15 s. The lysate was carefully transferred into the upper reservoir of the spin column and centrifuged at 13,000 rpm for 1 min. After discarding the flow through, column was washed two times with 500 μl of washing solution 1 and washing solution 2 by centrifugation at 13,000 rpm for 1 min each time.
After washing, column was dried by additional centrifugation at 13,000 rpm for 1 min to remove the residual ethanol of spin column. Finally, spin column was transferred to the new 1.5 ml Eppendorf tube and genomic DNA was eluted with 100 μl of elution buffer by centrifugation at 13,000 rpm for 1 min and stored at -20 • C for further analysis.

DNA amplification by PCR
PCR amplifications were carried out using a 2720 thermal cycler (Applied Biosystems, USA). It was performed in a total volume of 20 μl for each reaction using mastermix (10 μl), 10 pmol primer (1 μl for each primer), DNA template (4 μl) and nuclease free water (4 μl Table 1.

Purification of PCR products and DNA sequencing
Four PCR products (two from A. ovis and two from A. marginale) were purified using commercial PCR purification Kit (Addbio ® , Korea) following the procedures described by the manufacturer. Briefly, 40 μl of PCR product was mixed thoroughly with 200 μl of FADF buffer by vortexing. The mixture was then transferred to a FADF column and centrifuged for 1 min and the flow through was discarded. Wash buffer (750 μl) was added to the column and centrifuged for 1 min. After discarding the flow through the column was centrifuged again for 3 min to dry and placed on a new microcentrifuge tube. Elution buffer (40 μl) containing 10 mM Tris-HCl (pH 8.5) was added to the column and incubated at room temperature for 2 min. Column was then centrifuged for 2 min to collect the eluted DNA. Sequencing of purified PCR products were done by commercial sequencing company by conventional Sanger sequencing (Addbio ® , Korea).

F I G U R E 1
Anaplasma inclusion body (magnified circle) within the Red Blood Cell (RBC) (a). Amplification of 16SrRNA gene (345 bp) of Anaplasma sp. using genomic DNA extracted from blood of goat. Lane L is for 100 bp plus DNA ladder; Lane P is for positive control and N is for negative control; Lanes 1-8 is suspected samples; Lanes 1-3 having amplicons of 345 bp indicated presence of Anaplasma sp. (b). Amplification of msp4 gene specific to A. ovis using genomic DNA extracted from blood of goat. Lane L is for 100 bp plus DNA ladder; N is for negative control; Lanes 1-5 is suspected samples; Lanes 1-4 having amplicons of 347 bp indicated presence of A. ovis (c). Amplification of msp4 gene specific to A. marginale using genomic DNA extracted from blood of goat . Lane L is for 100 bp plus DNA ladder; N is for negative control; Lanes 1-8 is suspected samples; Lanes 1, 2, 5, 6 having amplicons of 344 bp indicated presence of A. marginale (d)

Phylogenetic analysis
Once sequences were available, checked initially using BLAST through  (Saitou & Nei, 1987). The evolutionary distances were computed using the p-distance method by Mega 10 software (Nei & Kumar, 2000). The tree stability was estimated by a boot strap analysis for 1000 replications (Felsenstein, 1985).

Statistical analysis
The obtained information was imported and stored using Microsoft

Risk factor associated with anaplasmosis in goats
Different risk factors like season, age, sex, breed, body condition score, coat color, tick infestation, acaricide practice and other management systems (rearing system, grazing, flock size) were considered in univariable logistic regression analysis. Among factors, breed, acaricide uses TA B L E 2 Overall prevalence of anaplasmosis in goat (N = 400)   (Tables 3 and 4). The chance of getting anaplasmosis is eleven times higher in tick infested goats than non infested goats.

Phylogenetic analysis
The multiple alignment and phylogenetic analysis were performed for nucleotide sequences corresponding to A. ovis and A. marginale. The

DISCUSSIONS
The study was conducted to detect anaplasmosis in goats and estimate their prevalences and associated risk factors of anaplasmosis in goats brought to the Teaching Veterinary Hospital from different areas of Chattogram district. To the best of our knowledge, this study for the first time identified the caprine anaplasmosis with circulating species in Bangladesh. Microscopic examination of thin blood smear is the commonly used tool to detect anaplasmosis in developing countries like Bangladesh. However, with the advancement of modern DNA-based diagnostic tools, it is now high time to apply state-of-the-art procedures like PCR for comprehensive molecular investigation. Therefore, classical microscopy was complemented by PCR amplification of two different genes of Anaplasma sp.
A total of 400 samples were examined by both classical microscopic and molecular technique and estimated prevalences were 5.75% and 15.75%, respectively in goats. The variation in the rate of prevalence in both techniques could be due to high sensitivity of PCR than classical microscopic examination on persistent subclinical infected individuals (Lew et al., 2002). The overall prevalence of anaplasmosis based on classical microscopy (5.75%) found higher than the findings of previous study (2.48%) in goats in Chattogram (Nath et al., 2014) possibly because of differences in diagnostic methods where they only considered clinical signs as diagnostic tool. Again, prevalence rate in this study was lower than that obtained from goats of southern part of India

F I G U R E 2
The phylogenetic tree of A. ovis obtained from the goat in this study and known A. ovis in GenBank using A. marginale as related species and outgroups F I G U R E 3 The phylogenetic tree of A. marginale obtained from the goat in this study and known A. marginale in GenBank using A. ovis as related species and outgroups (5.93%) was reported earlier in Bangladesh though the species was cattle (Samad et al., 1989).
For detection of caprine anaplasmosis, we used molecular technique PCR and overall prevalence was estimated as 15.75% which was lower than the prevalence reported in Iran by Yousefi et al. (2017) and Ahmadi-Hamedani et al. (2009) where they recorded 34.6% and 63.7% prevalences through PCR, respectively. These differences might be due to different geographical area and climate differences and variation in mechanical and biological vectors. In our study, the prevalence of A.
ovis (14.75%) found higher than the A. marginale (1.00%) which is in agreement with the findings of Yousefi et al. (2017) in Iran where they reported the prevalence of A. ovis (34.6%) was higher than A. marginale (0.00%). Differences between two species may be due to variation of susceptibility of goats to A. ovis and A. marginale. However, the prevalence of A. ovis (14.75%) of this study was inconsistent with the findings of Yousefi et al. (2017)  This variation may have been resulted from the differences of geography, climate and absence of potential vectors in this region.
The prevalence of anaplasmosis was found to be higher in winter (17.32%) and in summer (17.29%) than the rainy (12.85%) season though they were not statistically significant. Similar seasonal variation was also observed by previous investigators both in goats and cattle, reported higher incidence in winter season (Mannan, 2017;Nath & Bhuiyan, 2013;Rajasokkapan & Selvaraju, 2016). However, Velusamy et al. (2014 reported that there is no seasonal influence on anaplasmosis. All these variations are thought to be due to changes in macroclimate that is essential for breeding of ticks (Vairamuthu et al., 2012).
Moreover, contaminated fomites and some biting insects (flies) are also capable to transmit the disease and probably flies found higher in late rainy to winter seasons.
In univariate and multivariate logistic regression analysis, higher risk of Anaplasma infection found in extensive (17.64%) than intensive (16.17%) and backyard farming (15.30%). This finding concurs with previous findings of Angwech et al. (2011) and Wesonga et al. (2017) where they reported that, higher prevalence of infection of tickborne diseases found in extensively managed cattle. Supporting this findings, this study also revealed that Anaplasma infection was more in goats generally used to graze (19.10%) than non-grazed (16.21%) animal and which is supported by the findings of Rajasokkapan and Selvaraju (2016) who found that tick-borne diseases found higher in animal grazed in pasture than non grazed. The extensive system of rearing animal used to graze in pasture has direct effect on tickborne diseases as possibility of vectors exposure from nature become higher.
In this study, goats from large flock (30.0%) showed more susceptibe than medium (20.0%) and small flocked goats (14.29%). Though these finding found statistically nonsignificant but supported by Kispotta et al. (2016) and Shahnawaz et al. (2011) where they reported that tickborne infection increases with the increases of flock size. This could be due to increase chances of tick infestations through contact and instruments of medication and feeding like syringe and feeder.
Our data showed significant effect of breeds on prevalence of anaplasmosis in goats (p = 0.027). Higher infection was recorded in jamnapari (23.52%) followed by crossbreed ( Belal et al. (2014) in cattle. They reported that females were more susceptible to anaplasmosis because of stress and insufficient supply of feed during high demand in pregnancy (Kamani et al., 2010). Besides, hormonal imbalances during milk production and breeding time may causes impaired immunity (Kabir et al., 2011;Kamani et al., 2010;Sajid et al., 2009 Body condition score disclosed nonsignificant association of occurrence of anaplasmosis in goats in this study. As expected, poor (19.89%) body-conditioned goats are more likely to get anaplasmosis than good (11.69%) body-conditioned goats which is supported by previous studies (Hamsho et al., 2015;Sitotaw et al., 2014;Wodajnew et al., 2015).
This variation might be due to the fact that poor body condition has lower immunity and higher chance to get infection with different organisms like anaplasma.
No significant relation found between coat color and the prevalence of anaplasmosis of goat. However, goats with mixed coat color (25.00%) found more susceptible to anaplasmosis followed by black (15.43%), white (14.92%), brown (13.91%) and gray (12.50%) which was supported by Kispotta et al. (2016) who mentioned that breeds with red or black coat and combination of two or more color have higher risk of infection than those with white coat in regions where biting flies were the insect vector.
This study showed a significant association of anaplasmosis with the use of acaricide in goats. Goats not using acaricides were more likely to be positive for anaplasmosis than acaricide using goats. A similar observation has been reported by Kispotta et al. (2016) in cattle. Another study also supports our statement where stated that cattle and goats receiving no routine veterinary care like acaricides, anthelmintics, antiprotozoals were more prone to haemoparasitic infection than those receiving routine veterinary care (Weny et al., 2017). This difference could be due to the fact that animal with routine acaricide uses have better ability to protect tick infestations.
Availability of vectors is one of the potential risk factors for anaplasma infections (Constable et al., 2017). The final model showed that risk of anaplasmosis was significantly higher in goats having tick than non-tick infested goat (p < 0.05). A similar observation was reported by Costa et al. (2013) and agreement with the observation of Yousefi et al. (2017) and Azmat et al. (2018) where they reported the increased pattern of disease incidence with abundance of tick population. This could be because the most of the haemoparasites are harbored and transmitted by the different species of tick. In the absence of vector ticks, the possible way of anaplasma infection is blood-sucking arthropods and fomites as mechanical transmission vector (Constable et al., 2017;Dantas-Torres & Otranto, 2017). This form of mechanical transmission is considered to be the major route of dissemination of bovine anaplasmosis in areas of Central and South America and Africa where tick vectors are merely absent (Abdela et al., 2018).
A phylogenetic tree was constructed by the Neighbour Joining test using the Mega 10 software. The minimum similarity was seen for A. ovis with the isolate from Iran (KY091899.1) (Yousefi, 2018) and maximum similarity with the isolate from China (JN572931.1) (Liu et al., 2012). The A. marginale phylogenetic tree shows the highest relationships with the isolate from Venezuela (AY737009.1) (Franco et al., 2014) and lowest relationship with the isolate from Nigeria (EU106082.1) (Zivkovic et al., 2007). In topology of A. ovis we found that our isolates showed higher tree scale value (long branch length) in their respective clade. This high value indicates that there is some genetic divergence among the isolates.

CONCLUSION
Anaplasmosis was moderately prevalent in goats of Chattogram region where A. ovis and A. marginale were exist as both pathogenic and subclinical form. Different breeds of goats, no use of acaricide and presence of ticks on host were found as significant risk factors for anaplasmosis in goats. Sequence analysis revealed that isolates of the study were identical to the isolates reported from countries like Iran, Venezuela, China, Hungary, Japan, Italy, Brazil, Mexico, Australia and the United States. In context of our study, it is highly suggestive for the farmers to rear local breed in compare to exotic and crossbred to prevent the disease. Besides, regular acaricide practices are also recommended to control the tick as well as to control the disease.