Field and Molecular Epidemiology of Peste des Petits Ruminants in Pakistan

Muhammad Abubakar,1,2,* Aamer Bin Zahur,1,3 Khalid Naeem1,3, Muhammad Azeem Khan1,3 and Subhan Qureshi4 1PARC Institute of Advances in Agricultural Sciences, Islamabad, Pakistan 2National Veterinary Laboratory, Park Road, Islamabad, Pakistan 3Animal Health Program, National Agriculture Research Centre, Park Road, Islamabad, Pakistan 4Faculty of Veterinary and Animal Sciences, The University of Agriculture, Peshawar, Pakistan Article Information Received 14 September 2017 Revised 12 December 2017 Accepted 02 January 2018 Available online 20 February 2018


INTRODUCTION
P este des Petits Ruminants (PPR) is a viral disease, highly contagious and infectious in nature, and affect mainly small ruminants such as goats and sheep but can also affect wild animals infrequently.The PPRV infection is characterized by elevated temperature, purulent ocular and nasal discharges, necrosis, pneumonia, mucous membrane ulceration and gastro-intestinal tract inflammation which ultimately lead to severe diarrhea (Gibbs et al., 1979).It is a viral disease against sheep and goats herds and is considered as the strongest limiting factor to demanding sheep and goat farming in developing countries (Abubakar and Munir, 2014).
PPR has been recognized in Pakistan since 1991, earlier on the basis of clinical signs (Pervez et al., 1993;Athar et al., 1995;Amjad et al., 1996) and later by using

Field epidemiology of PPRV
A total of eighty-four PPR outbreaks were investigated during the study period (2010 to 2013).A case definition was established as a sheep or goat having a combination of respiratory and alimentary symptoms with fever.
Swabs from the conjunctival discharges, nasal discharges and buccal mucosae were collected from

A B S T R A C T
diseased animals for the detection, identification and genetic characterization of PPRV by using conventional as well as molecular tools (Diallo et al., 1988;Libeau et al., 1995;Forsyth and Barrett, 1995).
The samples were tested by Immuno-capture ELISA which was performed as the method adopted by Anderson and McKay (1994) with the help of Kit produced by BDSL Company, UK with the collaboration of Flow Laboratories and CIRAD, EMVT, France.

Genetic characterization of PPRV
A RT-PCR technique based on the amplification of the N and F genes proteins was standardized for the specific diagnosis and confirmation of PPR (Forsyth and Barrett, 1995).Representative PPRV isolates (n= 12) from different outbreaks were processed for sequencing to provide phylogenetic analysis of nucleoprotein and fusion protein gene segments to get an insight genetic picture of PPRV as well as its cross border transmission.The PCR products were purified using Purification kit (Invitrogen, Pure link PCR purification kit, Cat # k3100-01).Then the purified samples were sequenced on DNA sequencer (GeXP, BackMann Coulter, USA).

Sequence data analysis
The consensus sequences were then included in a dataset containing either all the available sequences (N gene sequences) in the Gen-Bank or representative sequences for each lineage (N and F gene sequences).All sequences datasets were aligned in the BioEdit using ClustalW algorithm and were cut to equal length.Construction of phylogenetic trees was performed with the neighbor-joining method using Kimura two-parameter model in MEGA5 version 5 (CEMI, Tempe, AZ, USA) with 2000 replications.The bootstrap values lower than 50% were not shown in the tree.Horizontal distances are proportional to sequence distances.Sequencing was carried out in National Veterinary Laboratory, Islamabad, Pakistan on GeXP, BackMann Coulter, USA (Supplementary Figs. 1 and 2).

Area-wise occurrence of PPR
During the period of 2010 to 2013, a total of eightyfour (84) PPR outbreaks were attended and investigated.A total of 471 samples were taken out of which 288 samples were found positive for PPRV antigen giving a positive percentage of 61.15% (Table I).
The disease outbreaks occurred throughout the country.The highest number of outbreaks was reported from Punjab province followed by Sindh and KPK (Khyber Pakhtunkha).More number of positive samples was from ICT (Islamabad Capital Territory) as compared to other regions (Table I).In the Punjab province, the highest number of outbreaks was confirmed from northern Punjab (65.79%) as compared to other regions.In Northern Punjab, the highest occurrence was in Attock (73.33%) followed by Chakwal and Rawalpindi.In central Punjab, the highest occurrence was in Jhang (64.29%) followed by Hafizabad and Faisalabad.In South Punjab, the highest occurrence was in Bahawalpur (77.78%) while Bahawalnagar and Layyah were at second and third place, respectively (Supplementary Table I).
In KPK, the highest occurrence was in Abbotabad (64.29%) followed by Charsadda and Peshawar.The overall occurrence of PPR positive samples was 59.32%.In Baluchistan, the percent positivity was 60%.In Sindh, the highest number of positive occurred in Mithi (75%) followed by TandoJam and Hyderabad.
In Azad-Jammu& Kashmir (AJK), the highest occurrence was in Kotli (80%) followed by Muzaffarabad and Mirpur.In Gilgit-Baltistan, it was 44.44% as compared to ICT (Islamabad Capital Territory) it was the highest as 75% (Supplementary Table I).

Seasonal occurrence of PPR
The disease outbreaks occurred throughout the study period which was highest in the year 2011 followed in 2013 while in comparison, these were less in 2010 and 2012 (Table II) which is suggestive that a cyclic pattern of disease occurred.Although the disease is not considered as seasonal but as we compared the data of four years, the disease outbreaks occurred in higher number from October to February.

Species-wise occurrence of PPR
PPR outbreaks affected both sheep and goats.In sheep, disease was less severe as compared to goats (Table III).In 48 out of 84 outbreaks, disease occurred in goats only while 18 outbreaks affected sheep only while remaining occurred in mixed herds.A total of 6221 animals were affected in these outbreaks.Sheep were less severely affected in comparison with goats as morbidity, mortality as well as case fatality rates were higher in goats.The morbidity rate was 26.79% in sheep in comparison with 34.90% in goats.The mortality rate was 10.83% in sheep as compared with 16.34% in goats.Similarly case fatality rate was 46.82 in goats which was slightly higher than sheep that was 40.41% (Table III).Statistical analysis showed p-value of 0.099 which is non-significant and depict that both specie are equally susceptible to PPRV.As the mortality rates are quite less as reported by the literature so it shows the possible endemicity of disease.Age-wise occurrence of PPR Overall, disease affected all three age groups of sheep and goats but the younger animals were more severely affected with a morbidity rate of 37.19%.The mortality and case fatality rates were also higher in young which were 46.86% and 17.39%, respectively (Table IV).
In sheep, the disease caused high morbidity and mortality rates in animals with age group less than 6 months which were 35.71 and 15.25%, respectively.The case fatality rate was almost giving a similar trend in all age groups while mortality rate was higher in younger animals as compared to aged (Table IV).In goats, the morbidity, mortality and case fatality rates were higher in first two age groups in comparison with animals with age more than 2 years (Table IV).
Statistical comparison of age groups between both species showed p-values of 0.384, 0.002 and 0.061 in age group 1, 2 and 3, respectively.This depicted that there is significant difference in second age group (6 months to 2 years) towards disease susceptibility and goats are more susceptible than sheep.

Genetic characterization of selected isolates
The sequenced samples of PPRV during this study (total 12 isolates which were selected based on geographical distinction) indicated similarity and were clustered in Lineage IV which is for Asian isolates (labeled in Phylogenetic trees as in Table V).
Detection and confirmation of PPRV by F and N gene based RT-PCR amplification with primers F1b/F2d and F1/F2, resulted an expected amplicon of 328 and 372bp, respectively.A total of 12 representative samples, were selected for sequencing.The viruses of the different locations (Islamabad, Taxilla, Khaniwal, Layyah, Attock, Faisalabad, Sargodha, Rawalpindi, TandoJam and Mithi) were analyzed for molecular epidemiology.The results of the phylogenetic tree indicated that all Pakistani PPRV strains, regardless of the gene used either F or N, were clustered in lineage IV which is the most prominent and prevalent lineage of Asia (Figs. 1, 2).

Sequence identity
Using Clustal W alignment tools within BioEdit, the sequences for the both the F and N genes were aligned and their nucleotide and amino acid similarity was graphically presented.All the sequences reported are highly similar between each other and have at least 15 substitutions in the entire stretch of N gene whereas 11 substitutions in the F genes.Majority of these substitutions are synonymous and thus do not impact the final amino acid sequence where they have shown high level of amino acid identity in all isolates except the Faisalabad strain.This strain, due to shift in the open reading frame, has unique sequence compared to the rest of Pakistani strains reported.It would be worth considering the exact biological role of these substitutions and their impact on the pathobiology of PPRV in future.

Percent identity and divergence matrix
Direct comparison of the nucleotide sequences of all reported Pakistani strains of PPRV in this study, it was evident that these share high nucleotide identity from 93% to 99% based on the N gene whereas from 94% to 100% in case of the F gene.The corresponding divergences indicated the report of unique strains reported from individual outbreaks and the internal variations between these strains of PPRV prevalent in the small ruminant population of Pakistan.

DISCUSSION
The present study provided substantial evidences on the endemicity of PPR in about variation in the incidence rate of PPR in different areas, during different season in sheep and goats of different age groups.Results of study demonstrated that highest number of outbreaks occurred in Punjab during the study period as compared to other provinces of the country.This could be due to a better disease reporting system of the Punjab as compared with other provinces.Few reports were also documented outbreaks from Punjab previously (Khan et al., 2007;Abubakar et al., 2008).Field study of Punjab also demonstrated that more number of PPR cases was reported from Southern Punjab than any other Punjab region.Zahur et al. (2006) has also reported similar findings from Punjab but their data size was less as compared to present study.
Temporal evaluation of outbreaks gives us a wave like pattern of disease outbreaks i.e., rise in one year and decline in the next year repetitively.This specific pattern can be linked to decrease in vaccination during the year when PPR cases are lower that result in more outbreaks in coming year.The present study also showed the seasonal variation of PPR outbreaks over a longer period of study.During the study period it was observed that PPR incidence is more during winter season.This can be linked to migration of susceptible, unvaccinated sheep and goat herds from mountains to plains of the country.These results are in contrast to other studies in Pakistan that reported few seasonal trends of PPR but for shorter periods of study (Zahur et al., 2009(Zahur et al., , 2011;;Abubakar et al., 2011;Ullah et al., 2015).In contrast to other studies from the world, Opasina (1983) also proved in western Nigeria that PPR incidence is increased during cold season.
In our study, all the sequenced isolates were related to PPRV lineage IV which is the Asian lineage and has been found in most of the region.The sequence and Phylogenetic analysis revealed that two groups; one of them was clustering with other previous Pakistani isolate while the others were grouped with the regional strains like India and Iran.Anees et al. (2013) conducted a similar study and specified the grouping of the sequences in lineage IV along with PPRV strains from Asian and Middle East.In contrast to our findings, Kumar et al. (2014) studied a PPRV isolate from an outbreak that occurred in sheep and goats in Nanakpur village of Mathura District in Uttar Pradesh (India) and their findings were that the PPRV/ Nkp1/2012 may not be closely related to lineage IV PPR viruses believed to be there in the Indian subcontinent.Munir et al. (2012) examined samples from two different outbreaks of PPR in goats and its phylogenetic analysis revealed that Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates, which is indicative of the true geographical pattern of PPRV.In a similar study by Munir et al. (2013) on the samples of PPR suspected outbreaks from different regions of Pakistan, fusion and nucleoprotein genes were sequenced.
The sequence identity pointed to the possibility of at least one group of PPRV from a diverse source and its passage in the small ruminants of Pakistan.In another study from the neighboring country India, Balamurugan et al. (2010) performed comparative sequence analyses of four genes (nucleocapsid (N), the matrix (M), the fusion (F) and the haemagglutinin H) of their isolates with already published sequences and found an identity of 97.7-100% and 97.7-99.8%among the Asian lineage IV and 89.6-98.7% and 89.8-98.9% with other lineages of PPRV at nucleotide and amino acid levels, respectively.

CONCLUSION
In conclusion, the rapid detection by suitable and appropriate methods of antigen and nucleic acid detection of PPRV in infected animals will assist in early judgment of infection and subsequently control of disease in Pakistan.