First report of bovine anaemia associated Theileria sinensis infection and phylogenetic analyses of partial gene sequences of Theileria and Anaplasma species detected in naturally infected Malaysian cattle

Background: Serious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Trypanosoma evansi, Theileria, Anaplasma and Mycoplasma species, and studied their phylogenetic relationships, haematobiochemical abnormalities and erythrocyte osmotic fragility (EOF) in Malaysian cattle. Methods: Molecular analysis was achieved by PCR amplication and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, 16SrRNA gene of Candidatus Mycoplasma haemobos, MPSP gene of T. orientalis and RoTaT1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) Kedah-Kelantan X Brahman cattle from Pahang, Malaysia. Haemato-biochemical analyses were performed using automated analysers while EOF was determined with the aid of saline solutions. Results: PCR amplication produced the expected fragment sizes for MPSP gene of T. orientalis, msp4 gene of Anaplasma marginale, 16S rRNA gene of C. M. haemobos, RoTaT1.2VSG gene of T. evansi. Nucleotide blast demonstrated that sequences of the PCR amplicons showed a high degree of molecular similarity in comparison with reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16S rRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys as additional species detected in these cattle. Theileria species was the most detected blood pathogen in the sampled cattle. The blood picture of all cattle group revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. Erythrocyte fragility values of all the cattle groups were above the reference range. Anaemia of the macrocytic normochromic type was observed in the Trypanosoma evansi; and Anaplasma platys + Theileria sinensis double species co-infected cattle. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Signicant (p<0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin was observed in the Trypanosoma evansi infected cattle. Conclusion: We present the rst report of anaemia associated with Theileria sinensis infection in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of Anaplasma platys, there is an urgent need for appropriate preventive and control measures, as Theileria species and A. platys are of great economic and zoonotic importance respectively. marginale A signicant (p<0.05) decrease in mean PCV, RBC, Hb and high mean cell volume (MCV), icteric index and a MCHC within the reference range were recorded in the T. evansi infected groups. Plasma protein value was signicantly (p<0.05) high in all cattle groups with that of the T. evansi being highest. Cattle positive for T. sinensis had signicant (p<0.05) decreased RBC, Hb with normal PCV and icteric index. Signicantly (p<0.05) decreased RBC count and Hb value with a high MCV and normal PCV value were recorded in the A. platys + T. sinensis cattle group. Signicant (P<0.05) increase in mean plasma protein value were observed in all cattle groups. The mean relative diameter width (RDW) of C. M. haemobos and T. orientalis + A. marginale infected cattle groups signicantly (p<0.05) lower than that of other cattle groups. There no signicant (p > 0.05) difference in the mean values of MCHC and platelet count of all cattle groups. All blood pathogen infected cattle groups had signicantly (p<0.05) high myelocyte, metamyelocyte, band and segmented neutrophil values, with a normal promyelocyte, lymphocyte, basophil, white blood cell counts. AST, globulin,

predominately affects cattle in Africa, but T. annulata has a world-wide distribution [2]. T. parva and T. annulata are the most pathogenic protozoa in animals [29], and their schizonts causes incomplete neoplastic transformation, proliferation and immortalization of T cells [30]. Theileria sinensis which is a phylogenetically closely related member of T. orientalis complex is known to be the major cause of bovine Theileriosis in China [31] and has been recently reported as one of the causes of benign bovine theileriosis in Malaysia [25]. It is of low pathogenicity in affected cattle [12].
In ruminants, haematology and serum biochemical analyses are relevant for diagnosing haematological disorders and many organ and systemic diseases [32]. Results from such tests (Liver and kidney function test) provide valuable information in the diagnosis, surveillance, making a prognosis and monitoring the disease process (static, progressing or regressing) [33]. Haematological and serum biochemical parameters are usually increased or decreased in blood pathogen infections [34]. The alterations are dependent upon type of infecting species, the infectious dose, virulence, immune status of the host and environmental factors. Anaplasma and Theileria species infects and multiplies inside the red blood cells, and during this intra-erythrocytic stage, intravascular or immune-mediated haemolysis can occur leading to anaemia [35]. Anaemia is a consistent feature in vector-borne diseases, and it is usually as a result of intravascular or extravascular destruction of the red cells. Anaemia of the macrocytic normochromic to macrocytic hypochromic (trypanosomosis) or normocytic normochromic (Anaplasmosis and theileriosis) type is usually evident in affected animals [36]. Immune-mediated hemolytic anaemia has been reported in bovine anaplasmosis and theileriosis [23]. Osmotic erythrocyte fragility test is widely used as an aid to diagnosis of certain haematological disorders such as intravascular haemolytic anaemia, autoimmune diseases, spherocytosis, elliptocytosis and stomatocytosis [37]. Blood parasites cause alteration in the red cell membrane of affected animals thereby making erythrocytes highly susceptible to lysis when subjected to hypo-osmotic stress [37,38].
The ruminant industry is an integral part of the Malaysian agricultural sector and diseases caused by blood pathogens is one of such diseases with negative impact on it. Therefore, it is essential to improve detection and surveillance of these blood pathogens [39]. Use of light microscopy for identi cation in stain blood smears are usually ineffective for differentiation of the species and detection of asymptomatic, presymptomatic and carrier animals with low parasitaemia. The use of molecular (PCR) based detection method has enabled better detection of these blood pathogens with high sensitivity and speci city [25,29]. A need to determine the phylogenetic relationships and to have a better molecular taxonomic understanding of the infections caused by various species of commonly occurring blood pathogens in Malaysian cattle is required. Therefore, this present study provides the molecular occurrence of Theileria species, Trypanosoma evansi, Candidatus M. haemobos and Anaplasma species isolates in Muadzam Pahang Malaysia and ascertained the haemato-biochemical abnormalities and erythrocyte osmotic fragility in Kedah-Kelantan X Brahman crossbred cattle infected by these blood pathogens.

Ethical statement
This study was conducted according to the guidelines of the Animal Care and Use Committee, Universiti Putra Malaysia, Animal Welfare Act.

Study population and blood sampling
A total of 61 blood samples of 5 ml each were obtained by coccygeal venipuncture from randomly selected Kedah-Kelantan x Brahman cattle of varying ages (1-11 years old) and sex (male and female) at Muadzam Pahang, Malaysia. The animals were reared in an oil palm plantation, and graze forages under palm oil plantation with some mineral supplementation. Animals were physically observed, and clinical signs were noted before blood samples were obtained. The blood samples were placed in a pre-labelled K2 EDTA vacutainer tubes for haematological determinations, heparin blood vacutainers for erythrocyte osmotic fragility and plain vacutainers to harvest sera for biochemical determinations. The samples were then transported in an ice box to the haematology and clinical biochemistry laboratory, Universiti Putra Malaysia. Whole blood samples for PCR was stored at -80°C prior to DNA extraction. Extracted genomic DNA was subjected to genera and species-speci c PCR test for detection of these blood pathogens.
Microscopic examination of stained thin-blood smears Examination of Giemsa stained thin-blood smear for possible identi cation of blood pathogens were performed by light microscopy.
Determination of haemato-biochemical parameters Erythrocytic parameters, platelet, total leukocyte and differential leukocyte counts were performed using automated haemoanalysers (Advia 2120i Siemens-Healthineers, Malvern, USA). Packed cell volume was performed using the micro-haematocrit technique with the aid of haematocrit centrifuge (Haematokrit 20, Hettich Zentrifugen, Tuttlingen, Germany), and Hawksley micro-haematocrit reader (Hawksley, England). Sera were harvested using portable table centrifuge (EBA 20 Hettich Zentrifugen, Tuttlingen, Germany). Serum sodium, potassium and chloride were determined using Siemens xpand plus chemistry analyser (Siemens Healthineers, Malvern, USA). Serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT), total bilirubin (TB), total protein (TP), albumin (ALB), conjugated bilirubin (CB), inorganic phosphate (IP), urea, and creatinine were done using BiOLis 24i premium chemistry analyser (DiaSystem Scandinavia AB, Sweden). Globulin was obtained by subtracting the albumin value from total protein value. Albumin, globulin ratio was calculated by dividing the albumin value with the globulin value. Plasma protein was determined using a hand-held refractometer (Atago, USA) while icteric index was determined by comparing the colour of test plasma sample with a set of colour standards.

Erythrocyte osmotic fragility test
Erythrocyte osmotic fragility (EOF) was determined by subjecting erythrocytes to varying concentrations of buffered saline solutions with slight modi cation [40,41]. Brie y, 5 ml of different buffered saline concentrations of pH 7.4 ranging from 0.1% to 0.9% were pipetted into 5 test tubes arranged serially in a test tube rack. A set of 5 test tubes was used to analyse each sample. Whole blood (20 µl) was pipetted into each 5 test tubes and mixed thoroughly by inverting the test tubes several times to mix the content. The mixed blood was incubated at room temperature for 20 minutes. The content was mixed again before spinning at 1500 x g for 15 minutes. The supernatant was transferred into cuvette and read at an absorbance of 540 nm using a spectrophotometer (Tecan Biochem, USA). The percentage haemolysis was calculated with the following formula: Haemolysis (%): (Optical density of test/Optical density of distilled water) x 100 The EOF curve was obtained by plotting the haemolysis percentage against the different saline concentration. The result of the EOF is expressed as the saline concentration that caused 50% haemolysis, which is the mean cell fragility (MCF).

Genomic DNA extraction
Genomic DNA was extracted from whole blood stored at -80°C using the DNeasy® Blood and Tissue kit (Qiagen, Hilden Germany) according to the manufacturers' protocol with minor modi cation. Brie y, 20 µl of proteinase K and 200 µl of Buffer AL was added into 200 µl of whole blood samples in a 2 ml microcentrifuge tube. The mixture was vortexed and incubated at 56°C for 10 minutes. After incubation, 200 µl of 100 % ethanol was added into the mixture and thoroughly vortexed. The mixture was pipetted into a spin column and placed in a 2 ml collection tube. This was centrifuged at 6000 x g for 1 minute. The ow through was discarded and the spin column was placed in a fresh 2 ml collection tube. 500 µl of buffer AW1 was pipetted into the spin column and centrifuged for 1 minute at 6000 x g. Flow-through and collection tube were discarded. The spin column was placed in a new 2 ml collection tube and 500 µl of buffer AW2 was added. This was centrifuged for 3 minutes at 2000 x g to dry the DNeasy membrane. The ow-through and collection tube was discarded. The spin column was placed in a 2ml microcentrifuge tube and 200 µl of buffer AE was pipetted directly onto the DNeasy membrane. After incubating at room temperature for 1 minute, the spin column was centrifuged for 1 minute at 6000 x g to elute the DNA. The eluted DNA was stored at -20°C until use. DNA concentration and purity were measured with a Nanodrop spectrophotometer (Tecan In nite M200®, Austria). DNA samples with A 260 /A 280 ratios between 1.7 -2.2 were further analysed.

Molecular identi cation by PCR
Polymerase chain reaction test was done to amplify the partial gene fragments of Theileria, Mycoplasma and Anaplasma species using genus and species-speci c primer sets (Table 1 & 2). Detection of RoTaT 1.2 VSG gene for T. evansi was done using species-speci c primer set (Table 2). Thermocyclic conditions and primers for Anaplasma, Theileria and Mycoplasma species were as speci ed by Parola et al. [42]; Sogin et al. [43]; and 1990 and Su et al. [44] respectively. Thermocyclic conditions and primers for Anaplasma marginale, Theileria orientalis, Trypanosoma evansi and Candidatus Mycoplasma haemobos were as speci ed by Shkap et al. [45], Ota et al. [46], Urakawa et al. [47] and Su et al. [44] respectively. Each PCR run was performed in a nal volume of 25 µl reaction in 0. the PCR run consisted of a eld isolate con rmed by PCR and sequencing of the amplicon, and was obtained from the Veterinary Parasitology Laboratory, Universiti Putra Malaysia. Water for molecular biology (Millipore corporation, Billerica MA, USA) was used as a negative control. PCR ampli cation was performed using the BioER Little Genius® LED thermal cycler (Hangzhou Bioer Technology China) with the primer sequences and thermocyclic conditions for each blood pathogen presented in Tables 1-2. Ampli ed products were then analysed by electrophoresis on a 1.2-1.5% agar rose gel for 80 minutes at 80 voltage. The gel was stained with RedSafe TM (iNtRoN Biotechnology, Korea) for 10 minutes and visualized using a UV transilluminator (GeneDireX TM , USA).
All samples positive for 18S ribosomal RNA gene of Theileria species and 16S ribosomal RNA genes of Mycoplasma and Anaplasma species were further ampli ed by conventional PCR using species-speci c primer set (Table 1), targeting major piroplasm surface protein (MPSP) gene for T. orientalis, major surface protein 4 (msp4) gene for A. marginale and 16S ribosomal RNA of Candidatus M. haemobos.

Sequencing of PCR products
Amplicons from each blood pathogen species with clear bright bands were selected for sequencing. They were gel extracted and puri ed using QIAquick gel extraction kit (Qiagen, Hilden, Germany) according to the manufacturers' protocol. The amplicons were sequenced using the BigDye® Terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems, USA). Consensus sequences were obtained for all PCR products using BioEdit Sequence Alignment Editor Software (version 7.0.5.3). The resulting nucleotide sequences were analysed and compared for similarities with reference sequences from GenBank database, using the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST.cgi).
Amplicons found to be negative for T. orientalis, A. marginale and C. M. haemobos after the species-speci c primer sets were used, were sequenced using their respective genus primer sets. The obtained sequences were then compared with reference sequences deposited in the GenBank.

Phylogenetic analysis
Our sequences from the various PCR amplicons were supplemented with reference sequences from GenBank and were aligned using ClustalW algorithm. After alignment, regions with gaps were removed manually and gap-free sites were used to construct phylogenetic trees using maximum likelihood method in MEGA software version X [48]. The reliability for the internal branches of maximum likelihood was assessed using 1000 bootstrap re-samplings and molecular distances estimated by the general time reversible model [49].
The phylogenetic analysis of the msp4 gene was performed as described above using the maximum likelihood method. The ve msp4 genes of A. marginale, two 16S rRNA gene of Anaplasma species isolates from this study, msp4 and 16S rRNA reference gene sequences were used for the comparison and creation of a phylogenetic tree for Anaplasma species. The reference sequences with their accession numbers in GenBank and country of origin are presented as follows: The msp4 sequences of A. marginale isolates from Thailand The Kedah-Kelantan X Brahman cattle were later divided into 8 categories based on the number of infecting blood pathogens; single species, double species co-infection, and triple species co-infection following PCR ampli cation of species-speci c genes.

Statistical analysis
All data, expressed as mean ± standard deviation of mean, were normally distributed (P > 0.05, Shapiro-Wilk's test). One-way multivariate analysis of variance (MANOVA), followed by Duncan multiple post hoc comparison test, was applied to study the effect of varying concentrations of saline solutions on erythrocytes. One-way analysis of variance (ANOVA) followed by Duncan post hoc comparison test were applied on the data generated from the haemato-biochemical parameters and mean cell fragility of the different groups of cattle.
Mean cell fragility values were extrapolated from the erythrocyte osmotic fragility curves. Statistical analysis was performed using SPSS 25.0 (Chicago IL, USA). A Probability value < 0.05 was considered as statistically signi cant.

Microscopic examination of thin blood smears
No Theileria piroplasms or schizonts were detected in the blood cells. Anaplasma morula were absent in the erythrocytes and C. M. haemobos were not seen attached onto the surface of erythrocytes. Numerous T. evansi were seen in the thin blood smear as elongated extracellular protozoan parasites ( Figure 1). The blood picture of all cattle group revealed the presence of echinocytes, elliptocytes, macrocytes, anisocytosis, few acanthocytes, rouleaux formation and numerous immature neutrophils (Figures 1-5). Clinical signs such as anorexia, profuse ocular discharge, dyspnoea and recumbency in few animals were observed.
Identi cation, percentage occurrence and similarity analysis of the blood pathogens detected in the KK x Brahman cattle Molecular analysis by PCR ampli cation using genus speci c primers produced expected fragment sizes of 1750 bp for 18S rRNA gene of Theileria species (Figure 6 Percentage occurrence of single, double and triple species co-infection in Kedah-Kelantan X Brahman cattle A total of six species of blood pathogens were detected by PCR and sequencing of amplicons and these include Theileria orientalis, Theileria sinensis, Anaplasma marginale, Anaplasma platys, Candidatus M. haemobos and Trypanosoma evansi. A high percentage (89%) of the sampled cattle were co-infected with more than one blood pathogen. Analysis of blood pathogen single species infection among the sampled cattle revealed that 15% of sampled cattle were infected by T. sinensis while sole infections with C. M. haemobos infected cattle and T. evansi were 8% and 3% respectively ( Figure 14). Theileria sinensis+ C. M. haemobos had the highest double species co-infection (31%), followed by T. orientalis + A. marginale (17%), T. orientalis + T. sinensis (15%), and the lowest being A. platys+ T. sinensis (3%) (Figure 14). Candidatus M. haemobos + T. sinensis + A. marginale triple species co-infection was detected in 8% of the sampled cattle ( Figure 14). T. sinensis + C. M. haemobos species co-infection had the highest occurrence while C.M. haemobos+ T. sinensis+ A. marginale triple species co-infection was the least (Figure 14).

Phylogenetic analysis
Phylogenetic analyses were done to determine whether the vector-borne blood pathogens detected in this study are genetically diverse within different geographical regions of the world. Phylogenetic analysis based on the MPSP gene of T. orientalis grouped Malaysian isolates, TO 34 and TO 41 together with reference isolates from Myanmar (AB871365.1) and China (KX375396.1). Sri Lanka, Thailand and Mongolia reference isolates grouped together and formed a separate branch from our Malaysian isolates TO 14 and TO 61 ( Figure   15). Malaysian isolate TO 17 grouped together with China (KU356867.1) and Thailand (AB562570) reference sequences from GenBank. Analysis based on the 18S rRNA gene grouped all our Theileria sinensis isolates in the same branch as the T. sinensis reference isolates from China (KF559355.1; EU274472.1; HM538203.1) and T. sergenti (AB000271) (Figure 15).
Phylogenetic analysis based on msp4 gene grouped our 5 isolates together with reference sequences from Cuba, Mozambique, Brazil, India, Thailand and Colombia (Figure 16). Five A. marginale msp4 gene sequences from this study formed separate branches with our two A. platys sequence (Figure 16). A 95% bootstrap support of reference sequences from India (MG050139.1) and Brazil (JX392984.1) were formed with our two A. platys sequences (Figure 16).

Haemato-biochemical and electrolyte abnormalities in the different cattle groups
The haemato-biochemical parameters and electrolyte levels in the KK X Brahman crossbred cattle were presented in Tables (Table 3). Signi cant increases in serum ALP, AST, GGT activities, total protein, globulin, total and conjugated bilirubin and decreased mean serum albumin value was observed in the T. evansi cattle group when compared to the other infected cattle groups ( Table 4). The mean serum alkaline phosphatase (ALP) value of the T. sinensis cattle was lower than that of other cattle groups and the reference range of values. Mean serum total protein and globulin values were increased in the T. evansi, C. M. haemobos and T. orientalis +A. marginale infected cattle group. Low albumin with low albumin: globulin ratio was recorded in the T. evansi group, while albumin:globulin ratio was decreased across all cattle groups. A signi cant (p<0.05) increase in mean serum total and unconjugated bilirubin values of the T. evansi cattle when compared to the other cattle groups (Table 4).

Mean erythrocyte osmotic fragility
The erythrocyte osmotic fragility curves showed an evident shift to the right in all cattle groups, with the T. evansi osmotic fragility curve being farthest, followed by the T. sinensis fragility curve ( Figure 17). Duncan's post hoc comparison test showed an increase of erythrocyte osmotic fragility at 0%, 0.1%, 0.3%, 0.5%, 0.7% and 0.9% saline concentration in all cattle groups ( Figure 17). The mean cell fragility (MCF) values of all the cattle groups were above the reference range (0.40 -0.45%) and at all sodium chloride (NaCl) concentrations, no signi cant (p>0.05) difference was recorded in the EOF values of all cattle groups (Figure 18).

Discussion
Theileria species were the highest occurring blood pathogen in the sampled cattle, with T. sinensis being the most detected Theileria species. Theileria sinensis is the major causative agent of benign bovine theileriosis in China [31]. Therefore, it's detection in Malaysian cattle suggests possible introduction or importation or close contact of asymptomatic carrier cattle to naïve cattle in Malaysia. However, it would be worthy to investigate whether T. sinensis is now indigenous to Malaysia or whether it was introduced from China. The sequence similarity among the Malaysian isolates and those in other parts of the world might have been caused by the movement/importation of cattle into South-East Asia. This study also reports the presence of Anaplasma platys which is a rickettsial bacteria that infects a wide range of host such as man, horses, small ruminants and dogs [20,21]. Our discovery of this species in cattle agrees with Chien et al. [22], who reported same for Vietnamese cattle. Detection of A. platys raises an urgent concern about the zoonotic implication of this blood pathogen in Malaysia.
The blood pathogen infections caused signi cant changes in haemato-biochemical parameters of the T. evansi infected cattle; T. sinensis infected cattle; and A. platys + T. sinensis double species co-infected cattle groups. Haemato-biochemical abnormalities in T. evansi infected cattle include a mild macrocytic normochromic anaemia, high icteric index, degenerative left shift, eosinopenia, hypernatraemia, hyperkalaemia, hyperchloridaemia, azotaemia (high creatinine and urea level), increased serum liver enzyme (ALP, AST, GGT) activities, hyperproteinaemia with hypoalbuminaaemia, and hyperglobulinaemia, low albumin:globulin ratio, hyperbilirubinaemia due to increased unconjugated bilirubin level. Anaemia in trypanosomosis include mechanical injury to erythrocytes and subsequent lysis caused by the lashing action of trypanosoma agella and attachment of the trypanosomes onto red cell surface via sialic acid receptors [50]. Other causes of anaemia in T. evansi include fever, toxins and metabolites from trypanosomes (proteases, neuraminidase, phospholipases, free fatty acids), dyserythropoiesis, lipid peroxidation and malnutrition [4,51]. Elevation in serum total and unconjugated bilirubin in the T. evansi group was due to massive intravascular haemolysis with increased release and breakdown of haemoglobin and conversion to bilirubin by macrophages in the spleen and other mononuclear phagocyte system. Free bilirubin is transported to liver when bound to albumin for conjugation with glucuronic acid. If the rate of bilirubin production exceeds the rate at which the conjugation takes place, there will be increase in the concentration of unconjugated bilirubin in plasma, and hence high icteric index [52]. Elevation of liver enzyme activities in the T. evansi group was attributed to cellular hypoxia and/or centrilobular necrosis due to low liver blood volume that leads to hepatocyte injury [52]. This hepatocellular injury could have contributed to slow rate of bilirubin conjugation. Albumin is a negative acute phase protein which decreases in acute in ammatory conditions, therefore Induction of in ammatory condition by the trypanosomes could have led to hypoalbuminaemia and hyperproteinaemia due to hyperglobulinaemia. Albumin:globulin ratio decreases with increase in in ammation. Increased urea and creatinine levels in T. evansi infected cattle could be attributed to possible damage to the kidneys or renal insu ciency caused by the toxic metabolites of the protozoa [53].
The haemato-biochemical abnormalities associated with T. sinensis infection in cattle include mild normocytic normochromic anaemia, degenerative left shift, hyperproteinaemia with a low albumin:globulin ratio, hyperkalaemia, hypernatraemia, hyperchloridaemia and low serum alkaline phosphatase activity. Mild anaemia recorded in T. sinensis infected cattle group could be attributed to extravascular haemolysis of abnormal shaped red cells by the mononuclear phagocyte system [54]. The nding of normocytic normochromic anaemia in bovine theileriosis agrees with [34].
Haematology and serum biochemistry ndings in the A. platys + T. sinensis cattle group include decreased RBC count and Hb concentration with a high MCV and normal PCV value. Normal PCV value with a low red blood count and haemoglobin concentration is highly suggestive of a decrease in plasma volume due to dehydration. Therefore, a macrocytic normochromic anaemia reported in this cattle group, is responsive because anisocytosis and macrocytic indices supports regenerative status in ruminants [55]. Degenerative left shift, hypernatraemia, hyperkalaemia, hyperchloridaemia, hyperproteinaemia with low albumin:globulin ratio were other haematobiochemical features reported in the A. platys + T. sinensis double species co-infection.
Haemato-biochemical abnormalities in other blood pathogen infected cattle groups without anaemia such as the C. M. haemobos infected cattle group, had erythrocytes of low relative diameter width (RDW), degenerative left shift, hyperproteinaemia with a low albumin:globulin ratio, hypernatraemia, hyperkalaemia and hyperchloridaemia. The erythrocytes of Theileria orientalis + A. marginale infected cattle group had a low RDW, hyperproteinaemia with a low albumin:globulin ratio, degenerative left shift, hypernatraemia, hyperkalaemia and hyperchloridaemia. T. sinensis + C. M. haemobos infected cattle group had degenerative left shift, hyperproteinaemia with low albumin:globulin ratio, hypernatraemia, hyperkalaemia and hyperchloridaemia. Theileria orientalis + T. sinensis double species co-infection showed degenerative left shift, hyperproteinaemia with a low albumin:globulin ratio, hypernatraemia, hyperkalaemia and hyperchloridaemia in their haemato-biochemical pro le. The cattle group infected with all 3 blood pathogen genera (C. M. haemobos+T. sinensis+A. marginale) had a low-normal red blood cell count, degenerative left shift, hypernatraemia, hyperkalaemia and hyperchloridaemia, high serum gamma glutamyl transferase activity and hyperproteinaemia with a low albumin:globulin ratio.
Relative diameter width (RDW) is a measure of anisocytosis [56], and a low or narrow RDW observed in the C. M. haemobos and T. orientalis+A.marginale cattle groups signi es the presence of larger than normal sized red cells (macrocytes) in these cattle [32]. Increased globulin concentration in the T. evansi, C. M. haemobos and T. orienatlis + A. marginale infected cattle groups indicates an ongoing in ammatory condition consequent upon rickettsial bacteria or protozoal infections. Hyperglobulinaemia leads to increase in circulating immunoglobulins in response to the invading pathogens by the host immune system [57].
Degenerative left shift was the commonest feature observed in all the blood pathogen infected cattle groups was highly suggestive of a severe in ammatory condition. This signi es that the demand for neutrophils from an in ammatory foci is overwhelming and granulopoietic capacity has been exceeded [58,59]. The functional storage compartment of mature neutrophils in the bone marrow of cattle is low when compared to other mammalian species [60], and in severe in ammatory conditions, increased demand for neutrophils rapidly exhausts the marrow storage pool and immature neutrophils such as the band, metamyelocyte and myelocyte are released into the peripheral circulation. Rickettsial bacteria and protozoal infections tend to cause an immunosuppression that makes the host susceptible to secondary infections. Natural infection of cattle by these blood pathogens induced an increase of erythrocyte osmotic fragility. This suggest that even when the blood pathogens have been cleared or reduced from peripheral circulation, they 'left behind' red blood cells with altered cell membrane that are susceptible to hypo-osmotic stress. Also, abnormal red blood cell shapes (echinocytes, elliptocytes, acanthocytes, macrocytes) due to an altered cell wall might have attributed to cell membrane instability and thus, increased fragility [7]. It is physiologically expected that fewer RBCs should hemolyse at higher saline concentration (0.9%), but in this case, more RBCs were still haemolysed at this concentration suggesting that RBC membrane has been compromised by the effect of the blood pathogens. Also, the erythrocyte osmotic fragility curves exhibited a rightward shift and this further con rmed the increased instability of red blood cell membrane induced by the blood pathogens [61]. In general, blood pathogens induce structural, biochemical and functional damage to the erythrocyte membrane (degradation of the membrane polyunsaturated fatty acids) and makes them vulnerable to reactive oxygen species and susceptible to haemolysis [7,37].
Our nding of hypernatraemia in all cattle groups was not in agreement with [62], who reported a hyponatraemia in theileria infected cattle. Therefore, we suggest that there might have been a decrease in serum sodium level but was probably masked by dehydration. Hyperkalaemia and hyperchloridaemia observed in all cattle groups were attributed to pre-renal dehydration [63]. The Theileria orientalis, Candidatus M. haemobos, and Anaplasma marginale infection con rmed in the sampled cattle by PCR test were considered to be subclinical/asymptomatic based on the absence of anaemia, blood pathogens on thin blood smears, presence of blood pathogen DNA and presence of non-speci c clinical symptoms.

Conclusion
Trypanosomosis, theileriosis due to T. sinensis and A. platys + T. sinensis double species co-infection were observed to be the main causes of anaemia in the affected herd. Therefore, we present the rst report of anaemia associated with T. sinensis; and Anaplasma platys + T. sinensis double species co-infection in Malaysian beef cattle. Anisocytosis, poikilocytosis, degenerative left shift, increased plasma protein, hyperkalaemia, hypernatraemia, hyperchloridaemia and normal lymphocyte, leukocyte and platelet counts were consistent ndings in all groups of cattle. Both T. evansi and T. sinensis infected cattle with low erythrocytic values and other blood pathogen infected cattle groups with normal erythrocytic values possess circulating erythrocytes that are very susceptible to hypoosmotic stress. Discovery of A. platys in cattle raises a considerable concern about zoonotic transmission in Malaysia, as this species can infect man. It is pertinent to know a current status of the commonly occurring blood pathogens that affect the ruminant industry and design natural control measures for mitigating the issues facing this aspect of the agricultural sector in Malaysia.

Availability of data and materials
All data generated and analysed during the study are included in this published work.

Ethics approval and consent to participate
This study was approved by the Universiti Putra Malaysia Animal Care and Use Committee and all guidelines for the use of animals were followed.

Consent for publication
Not applicable. Table 1 Genus-speci c primers, thermocyclic conditions and amplicon size of the various genes used for the detection and ampli cation of cattle blood pathogens. Thermocyclic conditions include initial denaturing (ID), denaturing (D), annealing (A), extension (E), and nal extension (FE).

Blood Pathogens Gene
Genus-speci c Primer Sequences (5'-3') Forward (F) and Reverse (R)        Black and blue arrows showing numerous Trypanosoma evansi in Giemsa stained Kedah-Kelantan X Brahman cattle thin blood smear. Red arrow shows a lymphocyte and background red blood cells were crenated. Magni cation x1000 Figure 2 A wright-stained thin blood smear from Anaplasma platys + Theileria sinensis (con rmed by PCR and sequencing) double species coinfected Kedah-Kelantan X Brahman cattle group showing an echinocyte (orange arrow) and a macrocyte (blue arrow). Poikilocytosis is evident. Presence of macrocytes in signi es bone marrow response. Magni cation x1000 Wright stained thin blood smear from Anaplasma marginale x T. orientalis (con rmed by PCR) double species co-infected Kedah-Kelantan X Brahman cattle group showing metamyelocyte (Black arrow), lymphocyte (blue arrow) with background erythrocytes of different sizes, and rouleaux formation. Magni cation x1000.

Figure 4
Wright stained thin blood smears from Theileria orientalis + T. sinensis (con rmed by PCR and sequencing) double species co-infected Kedah-Kelantan X Brahman cattle group showing numerous background erythrocytes of different shapes (double blue arrow) elliptocytes, (Blue double arrows) echinocytes (black arrowhead) and red cells of varying sizes. Black arrow shows a monocyte. Magni cation x600 Figure 5 Wright stained thin blood smear of C. M. haemobos + T. sinensis (con rmed by PCR and sequencing) double species co-infected Kedah-Kelantan X Brahman cattle group from Muadzam Pahang showing marked rouleaux formation with normal sized red blood cells.
Rouleaux formation occurs in cases of hyperglobulinemia in mammals. Magni cation x1000    Percentage occurrence of Theileria, Mycoplasma, Anaplasma and Trypanosoma species detected and con rmed by PCR ampli cationand sequencing, in the Kedah-Kelantan x Brahman cattle sampled in Muadzam, Pahang Malaysia.        The erythrocyte osmotic fragility curves of the different blood pathogen infected Kedah-Kelantan X Brahman cattle groups from Muadzam Pahang Malaysia. Blood pathogen infection was con rmed by PCR ampli cation and sequencing. The erythrocyte osmotic fragility values were obtained by subjecting blood samples to different sodium chloride (NaCl) concentrations (0 -0.9%). All curves showed a rightward trend which signi es high fragility of red blood cells. TE: Trypanosoma evansi infected cattle; AP + TS: Anaplasma platys + Theileria sinensis ;CMH: Candidatus Mycoplasma haemobos infected cattle; TS: Theileria sinensis infected cattle; TO + AM: Theileria orientalis + A. marginale infected cattle, TS+CMH: Theileria sinensis +Candidatus Mycoplasma haemobos infected cattle, TO+TS: Theileria orientalis + Theileria sinensis infected cattle; CMH + TS + AM: Candidatus Mycoplasma haemobos + Theileria sinensis + Anaplasma marginale infected cattle.