Comparative Analysis of Indirect ELISA and Real Time PCR for the Detection of Anaplasma Marginale in Buffalo , Cattle and Sheep in District Peshawar and Lakki Marwat , Pakistan

| Anaplasmosis is disease of livestock having pandemic in nature. In the present study the specificity and sensitivity of i-ELISA and RT PCR was investigated in the samples collected from District Peshawar and Lakki Marwat. A total of 900 samples were collected from three species, Cattle, Buffalo and Sheep from two transects Peshawar and Lakki Marwat for determination of rate of Anaplasma marginale through i-ELISA and RT PCR. Overall rate for Anaplasmosis was 37.7% (339/900) for all three techniques, whereas techniques wise the rate of Anaplasma marginale was 28.7% and 34.8 % through i-ELISA and Real-time PCR, respectively. Majority of the Cattle (41.6 %) were found positive for A. marginale as compared to 23.7% and 16.2% in Sheep and Buffalo respectively. Transects were significantly different at P <0.05 in term of rate of A. marginale. Transect wise distribution of anaplasmosis was higher in Lakki Marwat (41.5%) as compared to Peshawar (30%). Gender wise rate was higher in female through i-ELISA (57.96%) and RT PCR (64.33%) as compared to male individuals having 16.27% and 23.25% rate, respectively. A significant difference was found in seasonality of tick infestation, with the high infestations in summer as compare to winter.

(Dermacentor occidentalis) are mainly responsible for Transmission of A. marginale in livestock, since this adults species (Dermacentor occidentalis) usually suck the blood from Cattle and deer, it is likely that there is inter-transmission occur deer and deer, deer and Cattle, Cattle and deer.(Oseboldet al., 1962).
In Cattle, A. marginale occurs in acute and sub-acute forms, however sub-clinical infections are common.In animals up to one year of age this diseases occur in sub-clinical form, while in animals from 12-24 months old the disease is less low scale as compare to old aged animals where it causes death on large scale (Potgieter et al., 2004).
In tropical regions Anaplasmosis causes major economic losses to the livestock owners in shape of mortality in domestic animals and also causes health related problems such as low production and poor growth rate ( Jonssonet al., 2008;Alfredo et al., 2005).It is suspected that Tickborne diseases are main cause for fifty percent Cattle mortality (Martins et al., 2008).In livestock these haemoparasitic diseases make great loss of about 250 million, and play main constraint in production and development of livestock sector in many developing countries of world (Radostits et al., 2000).
This study was conducted to investigate the rate of "A.marginale"in District Peshawar and Lakki Marwat by using Sero-detection of Anaplasma by Ind.ELISA (Indirect Enzyme-Linked Immuno-Sorbent Assay) and Molecular detection of Anaplasma by RT PCR in Cattle, Buffalo and Sheep.

MATERIALS AND METHODS
In the present study a total of 900 blood samples (300 each species) were collected from each species of Cattle, Buffalo and Sheep.These samples were collected from District Peshawar and Lakki Marwat and transported to the Molecular Laboratory of Center of Parasitology and Poultry under refrigeration.These samples were screened through Ind.ELISA and RT PCR in order to know the standard diagnostic technique in order to avoid the false positive or negative results.
A volume of 5ml blood were collected from each selected animal via jugular vein in commercially available jell added vaccutanier for Ind. ELISA and EDTA added vaccutanier for DNA extraction for RT PCR through using disposable sterilized plastic syringes.

indirecT eliSA
Recognition of A. marginale in Cattle, Buffalo and Sheep was carried out through Indirect ELISA following the manufacturer's (svanova®) protocol.

reAl-Time PolymerASe chAin reAcTion
For Simultaneous Detection and Quantification of Anaplasma Marginale in Cattle, Buffalo and Sheep Real-Time Polymerase Chain Reaction (R-T PCR) was performed.

STATiSTicAl AnAlySiS
All the data was stored in Microsoft Excel and descriptive statistics was carried out in excel and export to Statistical program.Results were presented as counts and percentages of positive samples.Significant test was calculated for the various parameters using the Fits a variance components model by residual maximum likelihood (Reml) in Genstat software.Correlations of various parameters were determined by Pearson correlations.

RESULTS overAll rATe of A. MArginAle in boTh diSTricTS
In the present study, the overall rate of A. marginale in different livestock species was summarized in the Table 1.
On real time RT PCR the rate of A. marginale in Lakki-2 (47.7%) followed Pesh-3 (40%) and Lakki-1 (30.7%).While in Pesh-3, the rate was 87% higher recorded for Real-Time RT PCR as compared to the rest of the three areas in the Peshawar transect.This table also depicted that the overall rate of Real-Time RT PCR is 21% higher in term of positive cases then Indirect ELISA.

Sex wiSe rATe of A. MArginAle in cATTle
Sex-wise rate of A marginale for Ind.ELISA was (30.2%) higher in female then the rest of the areas.In male the rate was recorded (50%) in Pesh-3 for Real-Time RT PCR, whereas in Lakki-2 the highest rate was recorded in female with (72%) and in male it was (50%) in Pesh-3 Table 6.rATe of AnAplAsMA MArginAle in buffAlo The overall rate of A. marginale in Buffalo for Ind. ELI-SA and Real-Time PCR were recorded as (16.3%) and (23.3%) respectively.Area wise the highest rate of Anaplasma marginale on Ind.ELISA, was the highest in Pesh-3 (24%) followed by Lakki-2 (21%), Similarly on Real-Time PCR, the Cattleof Pesh-3 showed high rate of rate of A. marginale which was 32% followed by Lakki-2 31% Table 7.

Sex wiSe rATe of AnAplAsMA MArginAle in buffAlo
On Ind.ELISA, the male Buffalo of Pesh-3 showed higher rate of A. marginale which was 33.4% and in female Buffalo it was (23.2%) similarly on Real-Time PCR the female Buffalo of Lakki-2 showed the higher rate of A. marginale which was (35.7%) and in female Buffalo the rate was (36%), While for male it was (33.4%) in Pesh-3 Table 8.  (32%) respectively.While Area wise, on Indirect Ind.ELI-SA, the highest rate was observed in Lakki-2 (39%) followed by Pesh-3 (28%) Similarly on Real-Time PCR, the Cattle of Lakki-2 showed high rate of rate of A. marginale which was (46%) followed by Pesh-3 (40%) (Table 9).

Sex wiSe rATe of A. MArginAle in SheeP.
Sex wise the highest rate of A. marginale for Indirect ELI-SA the highest rate was studied in female in Lakki-2 (49%) and in male the rate was observed (25%) in Pesh-4.For Real-Time RT PCR the highest rate was found in female and male in Lakki-2 with (57%), (18%) respectively.

DISCUSSION
Anaplasmosis is a tick-borne Hemorickettsial pathogen of livestock which is widespread in many parts of the world (Table 10).

Figure 3: Anaplasma marginale visibility in Microscopy
A. marginale to certain extent is particularly related to ruminants, and commonly present in Cattle and Sheep, whereas some other ruminants may act as a reservoir for disease.The transmission of A. marginale takes place naturally by ticks, whereas the biting flies that have contamination with infected blood may be the source of mechanical transmission.Area which is free from ticks, transmission occurs through mechanical way.
The specie wise analysis of the data revealed that highest rate of disease was found in Cattle and Sheep, while lowest rate of disease was recorded in Buffaloes.A similar study was conducted by Khan et al. (2004) who reported that the rate of disease of protozoandiseases in Buffaloes and Cattle were 27.69% and 16.12%, respectively.This may be justified as that the immune system of the Buffalos are stronger than Cattle.
The technique wise analysis of data in the both districts reflects that via Indirect ELISA the highest rate of disease occur in Lakki-2 (39%) while the lowest was in Pesh-1 (16%).The results obtained from Real-Time PCR showed that highest rate of disease occur in Lakki-2 (43%) while the lowest was in Pesh-1, Pesh-2 and Pesh-4 with (21.4%).
The comparative analysis of techniques showed that Real-Time PCR is more sensitive of all techniques used.Similarly a study was conducted Carelli et al. (2007) who reported that Real-Time PCR is the more sensitive technique.They further explained that Real-Time PCR is the more specific in a sense that it produces the same results in multiple runs and never confused with other haemoparasites which are antigenically related A. marginale.
Transect wise analysis of the present data explained that maximum rate of disease of A. marginale was present in district Lakki Marwat while lowest was in district Peshawar.This area wise difference is justified as Lakki Marwat as compare to Peshawar comparatively have high temperature throughout the year which directly affected the immune system of the animal which remain Immuno compromised and more susceptible for the disease.Furthermore the socioeconomic statuses of the farmers of these areas are not good and farmers do not have cemented farms and unaware of the managemental practices and control measures about the Tick Born Diseases.Area wise difference in rate of disease of A. marginale is also supported by the findings of Kocan et al. (2003).
Gender wise rate was higher in female through Ind.ELI-SA (57.96%) and RT PCR (64.33%) as compared to male individuals having 16.27% and 23.25% rate, respectively.These findings of the author are supported by khan et al, (2012) who reported the highest rate of disease in female (26%) as compare rate of disease in male (17%).However Antonio et al. (2011) reported that there is no significance relationship of anaplasmosis with sex.

CONCLUSION
Overall area-wise prevalence of A. marginale was recorded higher in samples collected from District Lakki Marwat as compare to District Peshawar, Khyber Pakhtunkhwa.An overall gender wise higher prevalence was recorded in female as compare to male for all the tested samples.Species wise prevalence was higher in Cattle (49%) followed by Sheep (32%) and Buffalo (23%).The minimum prevalence of A. marginale was recorded in the areas where proper hygienic measure like spraying, dipping and cleanliness were ensured as compare to those in which proper managemental practices were not adopted.Real-Time PCR was observed to be more sensitive technique for detection of A. marginale among all the samples collected from Cattle, Sheep and Buffalo as compare to Indirect ELISA and Microscopy.

RECOMMENDATIONS
Keeping in view the facts and findings of the present study,

Figure 1 :
Figure 1: RT PCR DNA Amplification; The positive control quantified at 19 cycles .The graph represents the quantification cycles of samples with respect to positive and negative control .The earlier the quantification cycle comes the more is the infection.

Figure 2 :
Figure 2: i-ELISA; The first four wells represent the positive control and the later represents the negative control, the intensity of the colour shows the titer of antibodies.

Table 1 :
Overall rate of A. marginale

Table 2 :
Transect-wise Rate of A. marginale

Table 3 :
Area wise Rate of A. marginale in Peshawar and Lakki Marwat.

Table 4 :
Sex wise rate of A. marginale

Table 5 :
Specie wise rate of A. marginale in Cattle.Overall rate of A. marginale in Cattle was (42%) and (49%) for Ind.ELISA and Real-Time PCR respectively.On Indirect ELISA technique, in Lakki-2 the rate was (45%) higher for Ind.ELISA as compare to the Lakki-1 and Pesh-3 in the two transects.Similarly on Real-Time RT PCR, the Cattle of Lakki-2 showed high rate of rate of A. marginale which was (66%) followed by Pesh-3 (48%) Table5.
Overall rate of A. marginale in Cattle is explained in fol-lowing table.

Table 6 :
Sex-wise rate of A. marginale in Cattle Sex was significantly different at P <0.001 in term of positivity, for both tests in female Cattle rate were more than in male.

Table 7 :
Specie-wise rate of A. marginale in Buffalo South Asian Journal of Life Sciences January 2018| Volume 6 | Issue 1 | Page 4

Table 8 :
Sex wise Rate of A. marginale in Buffalo

Table 9 :
Specie-wise rate of A.

marginale in Sheep Area No of observation Ind.ELI- SA(%) RT PCR (%) P. Value
Overall rate of A. marginale in Sheep through Ind. ELI-SA and Real-Time RT PCR were recorded as (28%) and

Table 10 :
Sex-wise Rate of A. marginale in Sheep