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-specific PCR test for detection of these blood pathogens.
Microscopic examination of stained thin-blood smears
Examination of Giemsa stained thin-blood smear for possible identification 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 modification [40,41]. Briefly, 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 modification. Briefly, 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 flow 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 flow-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 Infinite M200®, Austria). DNA samples with A260/A280 ratios between 1.7 – 2.2 were further analysed.
Molecular identification by PCR
Polymerase chain reaction test was done to amplify the partial gene fragments of Theileria, Mycoplasma and Anaplasma species using genus and species-specific primer sets (Table 1 & 2). Detection of RoTaT 1.2 VSG gene for T. evansi was done using species-specific primer set (Table 2). Thermocyclic conditions and primers for Anaplasma, Theileria and Mycoplasma species were as specified by Parola et al. ; Sogin et al. ; and 1990 and Su et al.  respectively. Thermocyclic conditions and primers for Anaplasma marginale, Theileria orientalis, Trypanosoma evansi and Candidatus Mycoplasma haemobos were as specified by Shkap et al. , Ota et al. , Urakawa et al.  and Su et al.  respectively. Each PCR run was performed in a final volume of 25 µl reaction in 0.2 ml PCR reaction tubes, comprising of 0.3 µl of 10mM dNTP mix (dATP, dCTP, dGTP, dTTP), 1 µl of 10 µM of each primer, 5 µl of 5X Green GoTaq® flexi buffer (Promega Madison WI, USA), 5 µl of 25mM MgCl2, 0.3 µl of 5 U GoTaq®G2 flexi DNA polymerase (Promega Madison WI, USA), 8.4 µl of water for molecular biology ((Millipore corporation, Billerica MA, USA) and 4 µl of template DNA. The positive control DNA used in the PCR run consisted of a field isolate confirmed 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 amplification 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. Amplified 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 RedSafeTM (iNtRoN Biotechnology, Korea) for 10 minutes and visualized using a UV transilluminator (GeneDireXTM, USA).
All samples positive for 18S ribosomal RNA gene of Theileria species and 16S ribosomal RNA genes of Mycoplasma and Anaplasma species were further amplified by conventional PCR using species-specific 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 purified 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 18.104.22.168). 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-specific 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.
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 . 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 .
Five (5) and fourteen (14) sequences of the corresponding PCR products obtained with the MPSP and 18SrRNA primer sets for Theileria orientalis and Theileria sp. respectively were analysed using BLASTn to confirm their identities. They were then compared with MPSP sequences of T. orientalis from Sri Lanka (LC438477.1; AB701444), Thailand (KU886285.1; KU886290.1; AB562570), Mongolia (AB602388.1), China (KU356867.1), Vietnam (LC125432.1), Myanmar (AB871365.1), China (KX375396.1) and 18SrRNA sequences of T. sinensis isolates from China (KF559355.1; EU274472.1; HM538203.1). In addition to T. orientalis and T. sinensis, a closely related species T. sergenti (AB000271) isolate from Japan was included in the phylogenetic tree for comparison. Plasmodium malariae (MF796946.1) isolate from Thailand was used as an outgroup.
The phylogenetic analysis of the msp4 gene was performed as described above using the maximum likelihood method. The five 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 (MK140740.1; MH939156.1), Mozambique (MH172467.1; MH026093.1), India (MG676459.1; KX989517.1), Brazil (MK570463.1), Cuba (MK809386.1), Colombia (MF771080.1) and 16Sr RNA sequences of A. platys isolates from India (MG050139.1), Brazil (JX392984.1), China (KU585997.1). Plasmodium falciparum isolate (EY977394.1) from UK was used as an outgroup.
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 amplification of species-specific genes.
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 significant.