The Veterinary Record, March 24, 2007 500 bp 191 bp 200 bp N M P 1 2 3 4 5 FIG 1: Agarose gel electrophoresis of peste des petits ruminants virus (PPRV) DNA amplified by M gene- reverse transcriptase- PCR. Lane N Healthy goat splenic tissue (negative control), Lane M 100 base pair DNA ladder marker, Lane P PRRV Sungri/ 96-vaccine virus- infected Vero (positive control), Lane 1 Blood sample from a goat with clinical signs characteristic of PPRV infection, Lane 2 Lung tissue, Lane 3 Caecum tissue, Lane 4 Splenic tissue, Lane 5 Nasal swab from a goat with nasal discharge FIG 2: PCR amplification of N gene in goat tissue samples suspected for peste des petits ruminants (PPR). Lane 1 Blood sample from a goat with clinical signs characteristic of PPR, Lane 2 Lung tissue, Lane 3 Caecum tissue, Lane M 100 base pair DNA ladder marker, Lane N Healthy goat splenic tissue (negative control), Lane P PRR virus Sungri/96-vaccine virus infected Vero (positive control), Lane 4 Nasal swab from a goat with nasal discharge 337 bp N M P 1 2 3 4 Mixed infection of peste des petits ruminants and orf on a goat farm in Shahjahanpur, India P. Saravanan, V. Balamurugan, A. Sen, J. Sarkar, B. Sahay, K. K. Rajak, M. Hosamani, M. P. Yadav, R. K. Singh BOTH peste des petits ruminants (PPR) and orf, also called contagious ecthyma, are important viral diseases of small ruminants causing significant economic loss. Orf is endemic in India, having existed within the country for several dec- ades, while PPR occurred more recently, being first reported in 1989 (Shaila and others 1989). PPR has been responsible for widespread outbreaks of disease since that time (Dhar and others 2002, Singh and others 2004a). This short communi- cation describes a dual infection of PPR virus (PPRV) (genus Morbillivirus) and orf virus (ORFV) (genus Parapoxvirus) in goats. The outbreak was recorded in a small herd consisting of 150 goats owned by a private entrepreneur in Shahjahanpur, India, during October 2004. The owner had recently intro- duced 24 new goats of approximately six months of age in to the herd, which had been bought from the local market in order to increase the herd strength. Nearly 10 days after introduction of these goats onto the farm, the herd started showing signs of disease. On examination, the affected goats showed signs of purulent nasal and ocular discharges, severe diarrhoea, with pyrexia up to 41·4°C. Some of the affected animals aborted. There were also small, nodular lesions, typical of orf, on the oral commissures, muzzle, lips and also on the udder and teats of some nursing does. Ulcers were found on the oral mucosa, tongue and oral commis- sures. Lesions in and around the mouth probably prevented feeding, leading to emaciation, as observed in some of the recovered animals. Palliative treatment with antibiotics and antipyretics/ analgesics provided little relief. Morbidity due to PPR and orf was 63·2 per cent (110 of 174) and 5·7 per cent (10 of 174), respectively, while that due to mixed infection was 11·5 per cent (20 of 174). Overall, a mortality of 37·35 per cent (65 of 174) was recorded, which was mainly attributed to PPRV infection and partly to the severity of the oral lesions exaggerated by ORFV infection. Approximately 20 goats had abortions. Two goats that died on the day of investigation were sub- mitted for postmortem examination, which revealed lesions characteristic of PPR, including pneumonic changes in the lungs, congestion and haemorrhages in the lining of the tra- chea. Inflammation and hyperaemia of the mucosa of the duo- denum and jejunum, together with streaks of haemorrhages in the caecum and rectum were also observed. Enlargement of the mesenteric lymph nodes, a slight splenomegaly and necrotic foci on the liver were also evident. Samples of tis- sues from different organs showing lesions and oculonasal swabs were collected from affected animals and transported on ice for examination. Blood samples with or without anti- coagulant (EDTA) were collected in vacutainers from goats with pyrexia showing clinical signs. Nasal and ocular swabs were also collected from goats with dyspnoea and ocular dis- charge. For detection of PPR antigen, the morbid tissue samples were triturated with sterile sand in 0·1M phosphate-buff- ered saline (PBS) to make a 10 per cent (w/v) suspension. Veterinary Record (2007) 160, 410-412 P. Saravanan, BVSc, PhD, V. Balamurugan, BVSc, PhD, A. Sen, J. Sarkar, B. Sahay, K. K. Rajak, BVSc, MVSc, M. Hosamani, BVSc, PhD, M. P. Yadav, BVSc, PhD, R. K. Singh, BVSc, PhD, Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus, Nainital, Uttaranchal 263 138, India Correspondence to Dr Hosamani Detection and titration of PPR antibody was performed using a competitive-ELISA, as described by Singh and others (2004c). Nine of 10 randomly collected samples of sera gave a strong positive result for PPR antibodies, with percentage inhibition values ranging from 85 per cent to 90 per cent. PPR antigen was detected using a sandwich ELISA (Singh and others 2004b) in a total of 16 of 38 clinical samples compris- ing various tissues, collected from affected animals (Table 1). Both M gene-and N gene-specific reverse transcriptase-PCR (Aleyas 2002, Saravanan and others 2004) resulted in the amplification of 191 base pair (bp) and 337 bp products, respectively (Figs 1, 2). N gene-based PCR is specific for either PPRV or rinderpest virus (RPV), while M gene PCR is specific for PPRV only. Similarly, orf antibodies and antigens were screened by employing counter immunoelectrophoresis using reference orf antigen and antisera (ORFV-Mukteswar 59/05 antigen and its antisera), respectively (available in the authors’ labora- tory). Four of the 10 samples of sera and two of the five scab samples collected from the animals gave a positive reaction. Five representative scab samples collected from affected goats were triturated in sterile PBS to obtain a 10 per cent (w/v) scab suspension, and repeatedly freeze-thawed to extrude viral particles. DNA was extracted from 0·2 ml of the scab suspension using the Auprep genomic DNA purification kit (Life Technologies). Semi-nested PCR (Inoshima and oth- ers 2000) resulted in 235 bp products as expected, from all five specimens (Fig 3). The 235 bp amplicon of ORFV was sequenced after cloning into pGEMT-Easy vector (Promega) for further confirmation. The nucleotide sequence of the isolate showed a high sequence identity (97·4 per cent) with the ORFV-SA00 strain (Accession number AY386264), a 97 per cent homology with other ORFV strains (Accession numbers AY278209 and AY386263) and a 96·6 per cent homology with a previously reported Indian ORFV strain (Accession number AY545034) (Mondal and others 2006). It also had 94·5 per cent Short Communications