Serological investigations of Peste des Petits Ruminants in cattle of Nepal

Abstract Peste des Petits Ruminant (PPR) is an infectious viral disease of small ruminants caused by PPR virus. Although goat and sheep are the primary hosts of PPR, studies have continuously reported the prevalence of circulating antibodies in large ruminants, which could bring a potential challenge to effectively control and eradicate PPR. In Nepal, seroprevalence of PPRV antibodies in cattle have not been monitored yet. To address this, a total of 255 cattle sera were collected from Rupandehi, Banke, Bara and Chitwan districts of Nepal where outbreak of PPR in small ruminants was reported previously. The sera samples were tested by competitive ELISA and the result indicated the prevalence of 5.88% PPRV antibodies in cattle which indicates the exposure of cattle to PPR virus. To make the disease control program effective, intensive monitoring of both domestic and wild animals is very important.

In Nepal, the disease was first reported in 1995 from Dhanusha, Mahottari, Bara, Sarlahi, Rautahat and Gorkha districts of Nepal (Banyard et al., 2010;Dhar et al., 2002). Since then, the disease is widespread in Nepal and every year outbreak of disease occurs.
Moreover, there is a great possibility that many outbreaks of disease might go underreported due to the absence of PPR specific surveillance programs in Nepal and therefore the real incidence of PPR might be higher. Out of 77 districts, the disease has spread to 71 districts in Nepal (Veterinary Epidemiology Section, 2018).
Outbreaks of PPR in sheep and goats from 2008 to 2017 have been detailed in Table 1. Likewise, district-wise distribution of PPR outbreak is shown in Figure 1. According to the data from 2008 to 2017, out of 77 districts, outbreaks were reported from 60 districts, among which the higher number of outbreaks were reported from Dhanusha (214) and Bara (83) Balamurugan et al., 2012Balamurugan et al., , 2014Lembo et al., 2013). The role of these animals in virus transmission and maintenance remains unclear. Lembo et al. (2013) and Sen et al. (2014) have reported the isolation of PPRV from subclinically infected cattle. The role of cattle in the epidemiology of the disease is vague and needs to be investigated in detailed. In Nepal, seroprevalence of PPRV antibodies in cattle have not been monitored yet. To address it, this study was conducted.

| ME THODS
Blood samples of cattle were collected from four districts of Nepal where outbreak of PPR was reported previously using 10-ml sterile syringe. The syringe was kept undisturbed with the needle-holding end positioned until the blood clots and the clear serum was separated. A total of 255 cattle sera were purposively collected from Rupandehi (92), Banke (45), Bardiya (47) and Chitwan (71) districts of Nepal ( Figure 2) with the approval of the farmers and the concerned veterinary officer. The samples were then stored at −40°C for future test regardless of their age, sex and breed.
Then, a competitive enzyme-linked immunosorbent assay (C-ELISA) was performed for detection of PPRV antibodies in the collected sera samples using a competitive screening ELISA kit ID Screen® PPR Competition kit (IDVet Innovative Diagnostics) following the manufacturer's instructions. The prevalence rate was calculated as: where OD sample is the OD value of sample and OD NC is OD value of negative control.
Samples presenting an S/N%: less than or equal to 50% are considered positive, greater than 50% and less than or equal to 60% are considered doubtful and greater than 60% are considered negative as per the protocol. The average diagnostic specificity and relative sensitivity of the assay is estimated to be 97.98% and 93.95%, respectively (Acharya, Poudel, & Acharya, 2018). The statistical analysis was carried out using Fisher's exact test. p value less than 0.5 was considered for statistical significance.

| RE SULTS
In Rupandehi, Chitwan, Banke and Bardiya, the seroprevalence rate was found to be 8.69%, 4.22%, 4.44% and 4.25%, respectively, as   Table 2. The overall seroprevalence of PPR in cattle was found to be 5.88% (Table 2). Statistical analysis using Fisher's exact test showed no any significant difference in seroprevalence of PPR antibodies according to location (p-value > 0.05; Table 3).

| D ISCUSS I ON
Cattle of Nepal has never been vaccinated against PPR and rinderpest. Rinderpest has been eradicated from Nepal and worldwide, therefore the seroprevalences could have resulted from field infection with PPR virus. Even though the sample size is very small, detection of PPRV antibodies in cattle indicated that these animals were exposed to PPRV via contact with infected small ruminants which might lead to subclinical infection. buffalos, pigs and wild ruminant animals of Nepal. These animals should be included in the sero-monitoring program of PPR to give a better indication of the national herd immunity and to assess in the ongoing eradication program.

E TH I C S S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as no animal was treated in this study.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.354.

DATA AVA I L A B I L I T Y S TAT E M E N T
Available upon request to the corresponding author. Note: p-value < 0.05 is considered significant.

O RCI D
Abbreviation: OR: odds ratio.