A study on molecular surveillance of Theileria spp . infection and its impact on hematological and biochemical changes in naturally infected small ruminants at Multan , Pakistan

The current investigation was conducted to evaluate hematological and biochemical changes in naturally infected sheep and goats with Theileria spp. infection and to compare the conventional microscopic method of diagnosis with PCR amplification. A total of 200 blood samples (144 sheep and 56 goats) were collected and examined for the presence of Theileria infection. 12.5% and 39.5% blood samples were found positive with Theileria infection by microscopic examination and PCR amplification respectively. Hematological parameters were analyzed by automatic hematology analyzer (Diatron Abacus) while serum biochemistry was studied by Semi-automatic Chemistry Analyzer (Micro Lab 300). Hematological parameters revealed significant (p < 0.05) decrease in RBCs counts, Hb contents, PCV, MCH and MCHC values while non-significant (p > 0.05) correlation in MCV values of Theileria infected small ruminants. Biochemical analysis revealed significant (p < 0.05) reduction in total serum protein and albumin concentration while non-significant (p > 0.05) rise in urea and cholesterol level in infected animals compared to normal. Theileria piroplasms prevalence during early stage of disease without any clinical symptoms can only be identified through PCR amplification compared to microscopic examination.


Introduction
Theileriosis caused due to Theileria spp. (Piroplasmida, Theileridae) is a tick-borne protozoan disease found both in wild and domestic ruminants. Theileria genus belongs to Apicomplexa phylum including Babesia, Toxoplasma, Neospora, Plasmodium and others [1]. Theileriosis transmitted by ticks, caused high morbidity and mortality in infected animals resulted in higher economic losses in tropical and subtropical regions of the world [2, 3]. A total of six Theileria species caused ovine and caprine theileriosis from which Theileria lestoquardi considered highly pathogenic while T. ovis caused subclinical infection in sheep and goats [4].
The diagnosis of Theileria spp. in general is based on microscopy of stained smears and presence of clinical based symptoms in animals infected with ovine theileriosis. But the above cited methods are beneficial only in acute cases of theileriosis and insufficient for detection of piroplasms in carrier animals [9,10]. In recent years, the molecular technique Polymerase chain reaction (PCR) is frequently used for detection of ovine and caprine piroplasms. Hematobiochemical indices had been employed in attempts to get information regarding health status, performance and fitness of animals. An aberration from normal values might serve a guide for the differential diagnosis of a disease status [11]. Blood glucose and albumin level reduced significantly while serum protein and creatinine level decreased non-significantly during theileriosis [12]. Biochemical profile of Theileria infected animals showed higher level of urea, aspartate amino transferase, bilirubin (end product of hemoglobin) level and lactate dehydrogenase compared to healthy animals [13]. The significant decrease in RBCs counts, Hb contents and PCV values were identified while no changes found in MCH and MCHC values in infected goats with theileriosis [14]. Hematobiochemical changes reported in earlier studies in infected small ruminants was based on experimental animals, the current investigations was designed to delineate hematobiochemical changes in naturally infected small ruminants with theileriosis which can be utilized for diagnosis, prognosis and for better management of metabolic health status of infected animals.

Materials and methods Study area
Multan is situated in Punjab province (southern region), Pakistan between 29'-22'north latitude and 71'-4' east longitude, higher temperature 49ºC during hotter season and 1ºC during winter season. The average rainfall in district Multan is of 127 mm. The present survey was conducted on Theileria spp. infection and changes in hematological and biochemical values in Theileria infected small ruminants in Multan ( Figure 1). Blood sampling 200 blood samples (sheep = 144, goats = 56) were collected from apparently healthy small ruminants from selected herds located at different places during 2013. 10 milliliter (ml) blood was collected from sampled animals by puncturing jugular vein with sterilized syringe. 5 ml collected blood was poured in eppendorf having few drops of EDTA for extraction of DNA for PCR amplification; while other 5 ml collected blood was used for serum collection in order to determine hematobiochemical analysis. The collected blood samples were properly labeled including date, location and characteristics of animals as well as herds.

Microscopic examination
Thin blood smears were prepared in the field, then air dried and fixed in methanol (absolute) for 1 minute in the field. The blood smears were stained with Giemsa (5%) for 30 minutes in the laboratory and enquired Theileria spp. under immersion oil lens (× 1000). 250 microscopic fields were surveyed to identify the infected RBCs in order to assess the parasitemia ratio in the infected animals [15]. PCR amplification Inorganic method was used for DNA extraction [16]. The quality of extracted DNA was evaluated by spectrophotometer analysis at 260/280 nm density constant and gel electrophoresis. The extracted DNA was used for PCR amplification. The primers set 989 F; 5′-AGTTTCTGACCTATCAG-3′ and 990 R; 5′-TTGCCTTAAACTTCCTTG-3′ (Penicon) were used for amplification of 1098 bp portion of the ssu rRNA gene of genus Theileria. The final 50 µl PCR mixture contained of 5µl (1 ng µl -1) of template DNA, 5 µl of 10 X PCR buffer (100 mMTris-HCl (pH 9) 500 mMKCl, 1% Triton X-100), 5 µl of 50Mm MgCl2, 6 µl of dNTPs, 4 µl of each primer (µM) (Penicon) at a concentration of 10 pmol/µl, 2 U of Taq DNA polymerase (Vivintas) and 20.5 µl of PCR water. PCR amplification was done using a programmable thermal cycler (BIORAD). The cyclic conditions were at 94 °C for 5 min (initial denaturing step), followed by 35 cycles each at 94 °C for 1 min(denaturing step), at 60 °C for 1 min (annealing step) and 72 °C for 1 min (extension step) with a final extension step at 72 °C for 7 min. PCR amplified products were separated on 1.5% solidified agarose gel in TBE buffer and visualized by using ethidium bromide and UV-illuminator. The 100-1500 bp ladder (Vivantus) was used as DNA marker. Positive control genomic DNA of Theileria was provided by Professor Urike Seitzer (VIIRC, Borstel, Germany).

Haematological and Serum biochemical studies
The haematological values such as RBCs, WBCs, HB concentration, PCV, MCV, MCH and MCHC values were recorded of infected and healthy small ruminants. The sera of infected and healthy animals were analyzed for the measuring of total protein, albumin, globulins, cholesterol, calcitonin and urea level using commercial test kits.

Statistical analysis
Mini Tab (Version 16) was used for statistical analysis. One way Analysis of Variance (ANOVA) was used to compare and determine significant difference of hematological and biochemical parameters of laboratory obtained values between healthy and infected animals with theileriosis. All values were expressed as mean and standard error (SE). Fisher's exact test and Pearson's Chi square test was used to find association between different variables of (animals and herds) and ovine theileriosis. A p<0.05 value was considered as statistically significant

Results and discussion
Blood smear examination and clinical symptoms based diagnosis are useful in acute cases but meager in subclinical cases of theileriosis. As opponent to these methods, molecular assays are better for detection of piroplasms in carrier animals. PCR amplification method is more specific and sensitive than microscopy for diagnosis of Theileria piroplasms. A total of 12.5% and 39.5% blood samples found Theileria infected by microscopy and PCR respectively during present study. The sampling area, number of samples collected and Theileria positive samples are described in detail (Table 1). . This stress might attribute erythrocytic fragility because of membrane lysis and lower haemoglobin concentration in infected animals [23,24]. The decline of RBCs count, Hb contents and PCV values might endorsed the deficit of erythrocytes resulted by invasion of macrophages in lymph nodes, spleen and other organs of reticuloendothelial system. The oxidative stress increased in infected animals cannot be abridged by the antioxidant enzymetic activity [25]. The decline in RBCs count and HB contents was due to higher parasitaemia level in the infected small ruminants [26] and led to severe anemia in the Theileria spp. infected animals [27]. Surface membrane changes, increase the osmotic fragility and variations of glycoloipids of red blood cells membrane and oxidative damages resulted in anemia in diseased animals [28,29]. Destruction of RBCs counts during anemia in infected animals might be due to fastening of autoantibody to infected RBCs which later phagocytized [30] or due to cytokine tumor necrosis factor (TNFα1) which is effective inducer of fever and might play a role in development of anemia [31]. Removal of piroplasms from infected erythrocytes by macrophages could be the source of anemia in the Theileria infected animals [32]. The reduction in RBCs count might be due to erythropagocytosis during theileriosis and resulted in higher oxygen radicals caused anemia [29]. During present study, lower values of MCV found between healthy and Theileria spp. infected animals. Similar trend of significantly (p ˂0.05) higher MCV value was found during malignant ovine and bovine theileriosis [13]. Concerning the erythrocyte indices with parasitemia rates a significant depletion in MCV value [17].   Non-significant rise in cholesterol level found in animals infected with theileriosis during present study was in accordance to significantly higher level of cholesterol reported in cattle infected with theileriosis [39] but contradicts to [12] who reported non-significant increase of cholesterol levels in small ruminants infected with theileriosis. The higher cholesterol level endorsed to liver impairment due to parallel higher fats contents and lower sugar and protein level [40]. Conclusions This is the first preliminary study of Theileria infection in sheep and goats of Multan, southern Punjab, Pakistan which has a deleterious influence health of small ruminants. The current study epitomizes that ovine theileriosis significantly affect the hematological and biochemical parameters in the infected animals that are in the early stage of disease without any appearance of clinical symptoms. Further research is requisite to trace the variations in haematobiochemical parameters due to Theileria spp. infection.