A Report on the Tick Burden, Molecular Detection and Phylogenetic Analysis of Anaplasma Marginale in the Blood Samples of Cattle Collected from District Layyah in Punjab (Pakistan)

Back ground: Anaplasmosis is known as yellow bag or yellow fever and it is a tick borne disease caused by obligate intercellular gram negative bacteria, Anaplasma marginale. Aim: Present study is reporting seasonal prevalence, epidemiology and phylogeny of Anaplasma marginale in three cattle breeds from District Layyah, Southern Punjab, Pakistan. Methods and results: A total of 844 blood samples (Cross = 300, Holstein Friesian = 244, Sahiwal breed = 300) from apparently healthy cattle on seasonal basis were collected along with epidemiological data. Results: Polymerase chain reaction generated 265 base pair amplicon specic for Major surface protein–1b encoding gene of Anaplasma marginale in 8.6% (73/844) of enrolled cattle. Highest prevalence was observed during autumn (18.3%) followed by summer (9.7%) and winter season (7.1%). Holstein Friesian breed was most susceptible to A. marginale infection (13.1%) followed by Sahiwal (7.6%) and cross breed (6%). Representative amplied partial gene sequences of A. marginale were submitted to GenBank (Accession numbers MK032842 and MK032843). Its phylogenetic analysis had shown similarities with sequences submitted by other countries. 37/844 (4.3%) Giemsa stained blood smears were found positive for Anaplasma spp. Analysis of epidemiological factors revealed that female cattle and farm with water supply from pool, farms where other dairy animals and dogs were living with cattle and dogs having ticks load on them had signicant association with A. marginale prevalence. It was observed that white blood cell, lymphocytes (%), Monocytes (%) heamatocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were signicantly disturbed in A. marginale positive than negative cattle. Conclusion: We recommend that this PCR protocol should be used for the detection of A. marginale in livestock for their proper diagnosis and treatment.


Introduction
Livestock is our natural resource for the conversion of roughage into good quality-foods like milk and meat. The other livestock products like hides are raw material for industries [1]. In a country like Pakistan with rain-fed agricultural production system, livestock provides security against crop-failure [2]. According to a recent report, livestock is contributing 53.2% in agricultural sector and has about 11.4% share in Pakistan's GDP that is greater than the crop growing part [47.4% of the agriculture and 10.3% of entire GDP] [3]. Cattle constitute 43% of the total livestock population in Pakistan including exotic and crossbreed cattle in addition to 15 indigenous breeds of cattle. [1,4]. Despite good genetic potential most dairy animals in Pakistan have considerably low quantity of milk due to lack of disease control. Climatic conditions of Pakistan (being in tropics) are favorable for growth and development of tick species that are considered as major vector for several pathogens causing a variety of diseases in cattle [5].
Anaplasmosis is a tick-borne disease of cattle and is caused by Anaplasma marginale (A. marginale), an obligate intracellular Gram-negative bacteria from order Rickettsiales [6]. A. marginale are erythrocytic parasites that are transmitted by Ixodes sp., Dermacentor sp., Rhipicephalus sp. and Haemaphysailis sp. ticks, from infected to susceptible cattle by biting ies or by blood-contaminated fomites including needles, ear tagging and dehorning and castration equipment [7]. A. marginale are known to cause hemolytic anemia in host cattle and more than 70% erythrocytes are reported to be invaded by them in acute disease conditions [8]. Common clinical signs of A. marginale infection are fever, dyspnoea, lethargy, icterus, hyperexcitability, abortion due to hypoxia [9]. Recovered animals from the acute stage of anaplasmosis will develop lifelong low level of bacteraemia. These carrier animals act as a reservoir of infection for the susceptible host [6].
Present study was designed for the season based detection of Anaplasma marginale in three breed of cattle from District Layyah through conventional (smear screening) and modern tool (Polymerase chain reaction) in three different cattle breeds (cross, Holstein Friesian and Sahiwal) and to report the association of parasite prevalence with epidemiological factors and complete blood count parameters, if any.

Sample and Data Collection
Blood samples of 844 apparently cattle (Holstein Friesian, Sahiwal and cross breed) were collected from District Layyah, Southern Punjab, Pakistan on seasonal basis [summer (May till July), autumn (August till October), winter (November till January) and spring (February till April)]. After the informed consent of owners all the animals were examined by the veterinarian on the sampling sites and blood sample (approximately 10 ml) was collected from Jugular vein of each cattle and immediately preserved in a sterile tube containing 0.5M EDTA as an anticoagulant to be used for DNA extraction and for complete blood count analysis. A questionnaire was lled on the sampling site in order to collect data regarding epidemiological factors (gender, water source, tick load, other animals associated with herd, dog associated with herd and tick load on dog) associated with the prevalence of Anaplasma marginale in cattle.

Blood smear formation and screening
Geimsa stained blood smear of each animal was prepared and examined under the oil immersion lens of microscope for the microscopic detection of Anaplasma marginale following Takihi and Sandes [10].

Tick identi cation
Ticks were collected from the body of each animal during the collection of blood sample. Collected ticks were preserved in 70% ethanol and identi ed at the Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan using stereomicroscope and tick identi cation key following Madder et al. [11].

DNA Extraction from blood and tick samples
Inorganic method of DNA extraction was used following Saeed et al. [3]. The quality of the DNA extracted was assessed by measuring the optical density at 260/280 nm (O.R.I. Reinbeker, Hamburg) and by using submerged gel electrophoresis. DNA from ticks was extracted by Ammonium hydroxide method following Ammazzalorso et al. [12].

PCR ampli cation
A set of oligonucleotide primers, Fwd 5'GCT CTA GCA GGT TAT GCG TC3' and Rev 5'CTG CTT GGG AGA ATG CACCT 3' was used to amplify 265 base pairs of major surface protein-1b encoding gene of Anaplasma marginale in blood and tick sample following Asif et al. [13]. Polymerase chain reaction (PCR) was performed in a nal volume of 50 µl containing following Asif et al. [13]. PCR products were held at 4 o C until gel electrophoresis was carried out by using 2% agarose gel and visualized under a UV Trans illuminator (Biostep, Germany).

DNA sequencing and phylogenetic analysis
To con rm the PCR results of A. marginale, ve representative PCR products were randomly selected for DNA sequencing. PCR products were puri ed from agarose gel slices with NZYGelpure® (Nzytech, Portugal), and subsequently sequenced (First base Sequencing Service, Malaysia) with the same primers used for DNA ampli cation. The obtained sequences were analyzed by a BLAST search in Gene Bank for determining the accuracy of the PCR and submitted to NCBI Gene Bank.
Phylogenetic analysis was conducted using MSP1b sequences based on a 216 nucleotides aligned, and tree was created in MEGA 7.0 [14]. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3parameter model. The tree with the highest log likelihood (-549.04) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and Bio NJ algorithms to a matrix of pair wise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 20 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 155 positions in the nal dataset.

Hematological Analysis
Complete blood count parameters in all cattle blood samples were analyzed by using automated complete blood count analyzer following Saeed et al. [3].

Statistical Analysis
All the data with quantitative values is expressed as Mean ± Standard error of mean. Statistical package Minitab (version 17, USA) was used for the statistical analysis of the results. Association between the presence of Anaplasma marginale and various risk factors, i.e. gender, water source, tick load, other animals associated with herd, dog associated with herd and tick load on dog was assessed by contingency table analysis using the Fisher's exact test (for 2 × 2 tables). 2 sample t-test was calculated to compare various studied hematological parameters between Anaplasma marginale positive and negative blood samples.

Blood smear screening based prevalence of Anaplasma marginale
Following Giemsa's staining, blood smears from 844 cattle were examined microscopically and 37 (4.3%) cattle were found positive for Anaplasma spp. Upon microscopic examination, Anaplasma spp. appeared as dense, homogeneously stained blue-purple inclusions located toward the margin of the infected erythrocyte. Breed wise analysis indicated that 6.5% Holstein Friesian, 4% Sahiwal and 3% cross breed cattle blood smears were positive for Anaplasma (data not shown here). When data was analysed on season basis, it was observed that maximum Anaplasma prevalence was observed n blood smears prepared during autumn (43%) followed by autumn (29%), winter (21%) and spring (5.4%) (data not shown here).

PCR based prevalence of Anaplasma marginale
Polymerase chain reaction (PCR) ampli ed a 265 base pair amplicon speci c for Major surface protein-1b gene in 73 out of 844 (8.6%) analyzed cattle blood samples. Highest prevalence of Anaplasma marginale was observed in blood samples collected during autumn (18.3%) followed by samples collected in summer (9.7%), winter (7.1%) and spring season (1.7%) ( Table 1). Among the breeds, prevalence of Anaplasma marginale was highest in Holstein Friesian breed (13.1%) followed by Sahiwal (7.6%) and cross breed cattle (6%) ( Table.1).When prevalence data from each breed was analyzed on seasonal basis, it was observed that highest prevalence of Anaplasma marginale in Holstein Friesian breed was detected in blood samples collected during summer (24%) followed by autumn (8%), winter (8%) and spring season (2.6%). In Cross breed, the higher prevalence of Anaplasma marginale was observed in blood samples collected in autumn (14.6%) followed by winter (6.6%) and summer season (2.6%). Anaplasma marginale was not detected in blood sample of cross breed cattle collected in spring season. Similarly Higher prevalence of Anaplasma marginale in Sahiwal breed was observed in blood samples collected during autumn (18%) followed by winter (6.6%), summer (2.6%) and spring season (2.6%) ( Table 1).
Number of tick samples collected during present study was limited and Anaplasma marginale was not detected in any of them through PCR ampli cation.

DNA sequencing and Phylogenetic Analysis
Two represented amplicon of 265 bp from Major surface protein-1b gene sequence were con rmed by DNA sequencing and were submitted to the GenBank database under the Accession numbers MK032842 and MK032843. A BLAST analysis revealed nucleotide sequence identities of 97-99% with homologous sequences of Anaplasma marginale isolates registered in GenBank. The phylogenetic analysis of the obtained DNA sequence from Anaplasma marginale placed it in stable monophyletic cluster along with major surface protein 1b gene of Anaplasma marginale downloaded from public databases (Fig. 1).

Morpho-Taxonomic identi cation of ticks
In present study, Rhipicephalus microplus and Haemaphysalis punctata ticks were identi ed. The morphological structures analyzed included the capitulum, Haller's organ, adanal plates, spiracles, festoons and other aspects of general morphology (data not shown here).

Risk factor analysis
Analysis of studied epidemiological factors for Holstein Friesian breed revealed that prevalence of Anaplasma marginale was signi cantly higher during summer in farms supplied by water pools (P = 0.003) and in autumns in farms where only cattle were reared (P = 0.01) and having dogs present in farms with tick burden (Table. 2). For Sahiwal breed during autumn season, it was observed that prevalence of Anaplasma marginale was signi cantly higher in farms without dogs (data not shown here). For cross breed, none of the studied epidemiological factors were found associated with prevalence of Anaplasma marginale (data not shown here).

Complete blood count analysis
Analysis of results indicated that cattle of Holstein Friesian breed positive for Anaplasma marginale infection during summer had signi cantly reduced Mean cell hemoglobin (P = 0.017mean corpuscular hemoglobin concentration (P = 0.016) and Platelet count (P = 0.016) than Anaplasma marginale negative animals (Table 3). For Sahiwal breed, White blood cell count during summer (P = 0.01) and autumn (P = 0.02), Lymphocytes % in winter (P = 0.02) and Platelet count in winter (P = 0.05) were signi cantly reduced in Anaplasma marginale positive than negative cattle (data not shown here). For cross breed, it was observed that Anaplasma marginale positive animals had signi cantly reduced Monocytes (%) during autumn (P = 0.03), mean corpuscular hemoglobin concentration (P = 0.002) and platelet count (P < 0.001) during summer than parasite negative animals. All other studied parameters varied non-signi cantly (P > 0.05) when compared between Anaplasma marginale negative and positive cattle blood samples of all three breeds (data not shown here).

Discussion
Tick-borne rickettsial diseases are considered as one of the major constraints to livestock improvement programmes as they are responsible for serious health problems resulting in reduced animal productivity and economic losses [15]. It has been documented that ticks of Rhipicephalus sp. and Haemaphysailis sp. are responsible to transmit Anaplasma marginale to a variety of vertebrate host due to elevated temperature and favorable climate, in tropical countries [16]. Our results are in line with these established facts as in present study, ticks belonging to, Rhipicephalus and Haemaphysalis were identi ed from the cattle under investigation during morpho-taxonomic identi cation (data not shown here).
Diagnosis of A. marginale is performed by both modern and conventional methods. The most common but less reliable method is the Giemsa stained blood smear screening for the detection of these bacteria within the erythrocytes [17]. However, this technique is unable to detect pre-symptomatic, and carrier animals having low parasitemia [18]. PCR is usually considered as gold standard for the detection of tick borne blood parasites and it is more sensitive and reliable than parasite detection by blood smear screening [6]. The sensitivity of PCR for detection of Anaplasma marginale (8.6% prevalence) was higher than the blood smears screening (4.3% prevalence) during present study. Ybanez et al. [19] had also documented that PCR based detection of Anaplasma marginale is more sensitive tool than through Giemsa stained blood smear screening. infection rates of Anaplasma marginale may be due to differences in tick control programmes, habitat suitability for ticks, farm management, husbandry practices, wildlife reservoir hosts and/or abiotic factors as reported by others [20,30].
The data on genetic diversity of Anaplasma marginale is very rare from Pakistan. To investigate the genetic background of Anaplasma marginale present in Pakistan, we performed phylogenetic analysis based on a 265 base pair amplicon from major surface protein-1b gene that revealed greater genetic diversity as several diverse genetic groups can be seen in phylogenetic tree based on partial sequence homology (Fig. 2). Analysis revealed that sequences obtained during present study are clustered with the previously reported sequences from United States of America (AF348137 and CP000030) indicating that these are closely related genetic variants (Fig. 1).
Prevalence of Anaplasma marginale varied with sampling season during present study and highest parasite prevalence was observed during autumn (Table 1). High parasite prevalence in autumn during present study is probably due to abundance of vector ticks that were developed during summer months. Our result is contradictory to the ndings of Vohra et al. [31] and Roy et al. [32] who had reported high prevalence of Anaplasma marginale in summer season. These differences observed in the prevalence between these studies are due to different geographical locations, abundance of tick vector and breeding microenvironment of ticks (temperature and humidity) [33].
According to our result Holstein Friesian breed had highest infection rate of Anaplasma marginale as compared to cross and Sahiwal breed (Table 1). Our result is in line with result of Tay et al. [34] who had reported that Anaplasma spp. prevalence was higher in Holstien cattle as compared to local breeds. This result may be due to some kind of resistance of the local breed that is developed against rickettsial infection. Indeed, local breeds are known to be more resistant to tick-borne pathogens [3]. Longer and thicker hairs of Holstein Friesian breed may be the factor for their higher infestion of ticks as compared to other breed. It has been reported that there is a positive association of hair length and coat thickness with the number of ticks [35]. However, our result is contradictory to ndings of Belkahia et al. [20] who had showed Holstein cattle were signi cantly less infected by Anaplasma marginale (p < 0.001) than other breeds.
Epidemiological data analysis for gender, although remained non signi cant, revealed that female cattle were more prone to Anaplasma marginale infection than male. This is in accordance with Kamani et al. [36] who revealed prevalence of Anaplasma infection was higher in female cattle possibly due to the fact that they were kept longer for breeding and milk production purpose, supplied insu cient feed against their high demand. The immune suppression in advanced pregnancy and or lactation in high producing study animals are the possible reasons for the higher prevalence of Anaplasma marginale in female cattle [8]. The dog associated with herd of cattle is another parameter that has showed signi cant association with the Anaplasma infection indicating that dogs are acting as tick carrier and bring the infection to the herd Our results are contradictory to Ashraf et al. [6] which showed higher Anaplasma marginale prevalence in buffalo herds without dog. Signi cantly higher prevalence of Anaplasma marginale was observed in traditionally managed farms where the animals were not regularly vaccinated against tick borne diseases and water was supplied through pools with contaminated water instead of pumps providing fresh ground water. Our results is in accordance with Swai et al. [37] and Atif et al. [38] who had reported signi cantly higher prevalence of Anaplasma marginale in traditionally managed farms when compared to modern ones. Poor management, lack of tick control practices and inadequate economic sustainability of poor resource smallholders may contribute to the higher prevalence of Anaplasma marginale [38].
Analysis of the complete blood count parameters indicated signi cant decrease in hematocrait, mean cell hemoglobin and mean corpuscular hemoglobin concentration in parasite positive cattle. These observations are in line with Gannguly et al. [39] who had reported that in severely infected cases, cattle had hemoglobin level as low as 3 g/dl and packed cell volume was decreased to 1.99 × 10 6 /µl. This might be due to damage caused by Anaplasma marginale inside the RBC's during their multiplication. Our result is also in accordance with Riond et al. [40] who had reported decreased mean corpuscular hemoglobin concentration and increased mean corpuscular volume in Anaplasma marginale infected as compared to uninfected cattle. They have reported that fast destruction of RBCs by phagocytosis leads to their increased demand, therefore bone marrow cells starts to release immature RBCs. The immature RBCs are bigger in size than mature RBCs having more corpuscular volume. In present study, the mean corpuscular volume was non-signi cantly increased in infected animals. It re ects that animal's haemopiotic system was activated in response to erythrophagocytosis initiated by parasitic damage to erythrocytes and that leads to decrease in RBC, this may be also due to increased level of activated complement products and removal of destroyed cells by bovine reticuloendothelial system [41]. Analysis of hematological parameters indicated that white blood cells, platelet count and lymphocytes (%) were signi cantly decreased in Anaplasma marginale positive cattle blood samples than in blood samples where parasite was not detected (Table 3). Our result coincide with Khan et al. [42] who recorded signi cant decrease of WBCs in prepatent phase and non-signi cant decrease of WBCs in early and late stages of the disease. Our results are in agreement with the nding of Riond et al. [40] who had reported that anemic cattle showed thrombocytopenia i.e a condition in which there is a low blood platelet count. The increased platelet consumption may be attributed to an immune-mediated process or result from intravascular disseminated coagulation [43].
In conclusion, we are reporting the prevalence of Anaplasma marginale infection in three cattle breeds from Pakistan with Holstein Friesian breed being most susceptible to this infection. We have documented that PCR is more reliable and sensitive technique for Anaplasma marginale detection than blood smear screening. We are also reporting that parasite prevalence varied with the sampling season and presence of Anaplasma marginale had signi cant effects on the hematological pro le of all cattle breeds under investigation. Data generated through this study will pave the way for the control of anaplasmosis in Pakistan. We recommend this technique to the livestock owners for prophylactic detection of Anaplasma marginale in cattle blood in order to improve their economic output.

Declarations
Ethics approval and consent to participate All the animal handling procedures and lab protocols were approved by the ethical committee of Institute of Pure and Applied Biology following the instructions of Institutional Review Board (IBR) of Bahauddin Zakariya University Multan, Pakistan. Informed consent was obtained from each cattle owner before and blood, tick and data collection.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this submitted article.

Figure 1
Phylogenetic tree based on partial major surface protein-1b gene sequences from Anaplasma marginale isolates from cattle in Pakistan and cattle worldwide, available in GenBank. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter model. The three new sequences of Anaplasma marginale obtained in the present study are represented in bold.