An incidence of heavy mortality was observed among Tellicherry breed of goats in an organized farm. Mortality and morbidity rate recorded were 52% and 100% respectively. The clinical signs and necropsy findings were suggestive of PPR. Counter immunoelectrophoresis test was employed and found positive for PPR. Confirmative diagnosis was carried out with RT-PCR targeting N gene of PPR which yielded 350 bp products corresponding to PPR. Histopathological examination of lungs revealed severe serofibrinous broncho-interstitial pneumonia with multinucleated syncytia formation and intracytoplasmic inclusion bodies in bronchiolar epithelial cells. In Vero cells the CPE observed viz. rounding, syncytia and detachment of cell sheet were characteristic of PPRV. It was observed that Tellicherry breed was highly susceptible to PPR infection. Hence, the breed susceptibility may be considered while implementing control and eradication programme for PPR.
Keywords : PPR – Tellicherry – RT-PCR - Isolation
Peste des Petits Ruminants (PPR) is an acute highly contagious viral disease of wild and domestic small ruminants that mainly affects sheep and goat but the disease is more severe in goats than sheep (Dimri et al., 2002). PPR was first reported in West Africa in 1942 by Cote d’lvoire (OIE, 2014). Now, PPR is prevalent in most of the African countries, Arabian Peninsula, India, Nepal, Bangladesh, Pakistan and Afghanistan. In India first PPR outbreak was recorded in Tamil Nadu in the year1987 (Shaila et al., 1989). Since then PPR has been endemic and reported regularly from different parts of the country. PPR is caused by Morbilli virus belongs to the family Paramyxoviridae. The disease is characterized by fever, oculonasal discharge, stomatitis, diarrhoea and pneumonia (OIE, 2014). PPR virus is antigenically similar to Rinderpest virus, canine distemper and measles virus. There is only one serotype of PPR virus, but there are four lineages which can be differentiated by nucleic acid sequencing. Lineage 1and 2 occur in parts of Africa, lineage 3 occurs in Africa, Middle East and Southern India. Lineage 4 is reported from Middle East and Indian subcontinent. However, lineage IV has been now recorded in PPR outbreaks in Africa by replacing other lineages (Parida et al., 2015; Banyard et al., 2010). PPR is one of the major threats to the small ruminant industry which causes high morbidity (100%) and mortality (50-90%) (Abu – Elzein et al., 1990; Karim et al., 2016). Hence, early diagnosis is essential to prevent the disease. The present study describes an outbreak of PPR with heavy mortality among Tellicherry breed of goats in an organized farm and identification by PCR and isolation in Vero cells.
Materials and Methods
The present outbreak was recorded in an organized farm in Thiruvallur district ( 13.15˚N 80.24˚E )of Tamil Nadu during the month of October, 2013 where different breeds of goats and sheep (Goat breeds- Barbari, Beetal, Zhakrana, Jamunapari and kanni adu; Sheep breeds- Kizha Karisal, Vembur, Mecheri, Madras Red, Salem black,) were maintained. Totally 200 goats and 60 sheep were maintained in that farm. The incidence of disease was observed only in newly purchased Tellicherry goats with a flock size of 25 numbers whose vaccination status were unknown. The other breeds of goats and sheep did not show any clinical signs. Comprehensive disease investigation was carried out to identify the cause of disease. Nasal swabs, ocular swabs, buccal swabs, rectal swabs and faecal samples were collected from ten live goats. Necropsy examination two goats were carried out and representative tissue samples from lungs, spleen, mesenteric lymph node and intestine were collected on ice and in 10% buffered formalin for virological and pathological examination. Heart blood swab, heart blood smear and lung swab were collected for bacteriological examination.
Faecal samples and heart blood smears were examined for the presence of parasitic ova and for bacteria as described by Soulsby (1982) and Quinn et al. (1994).
Heart blood swab and lung swab were inoculated into nutrient agar, blood agar, Mac conkey agar, anaerobic agar and Sabouraud dextrose agar plates as per Quinn et al. (1994).
Counter Immunoelectrophoresis Test (CIE)
CIE test was carried out as per Roy et al., 2010 for initial screening of samples using hyper immune serum raised against local isolate of PPR virus.
Polymerase Chain Reaction
RNA was extracted using TRIzol (Sigma). cDNA was synthesized using first strand cDNA synthesis kit (Thermo Fisher Scientific) as per manufacturer’s instruction. PCR was carried out for N gene as per Couacy –Hymann et al., 2002. Briefly, 4 µl of cDNA was mixed with 12.5 µl of red dye master mix (Ampliqon), 1 µl of each primer (F-5’ TCT CGG AAA TCG CCT CAC AGA CTG 3’; R- 5’ CCT CCT CCT GGT CCT CCA GAA TCT 3’) and 6.5 µl of DEPC water. The reaction mixture was placed in a Eppendorf thermal cycler with following conditions: 94ºC for 5 minutes; 35 cycles of 94 ºC for 30 seconds, 55 ºC for 30 seconds, 72 ºC for 30 seconds, final extension at 72 ºC for 7 minutes. 10 µl of PCR product was electrophorosed on 1.5% agarose gel containing 2.5 µg/ ml of ethidium bromide and visualized in Bio Rad Gel doc imager XR + ®.
Virus Isolation in Cell Culture
Lung, spleen and mesenteric lymphnode suspension were pooled and filtered through 0.22 µ syringe filter. The filtrate was inoculated in to Vero cells monolayer and cytopathic effects were observed. Uninoculated Vero cell monolayer was kept as control.
All the goats in the newly purchased flock showed high fever (41°C), enteritis, mucopurulent nasal discharge, necrotic plaques and ulcers in mouth (Fig.1a & 1b) . Both the sexes of 6-8 months aged goats were affected. Out of 25 newly purchased Tellicherry goats, 13 goats were died. Morbidity and mortality rate were 100% and 52% respectively. Necropsy findings include frothy exudates in tracheal lumen, congestion and consolidation of lungs, congestion in mucosa of abomasum and intestine with patchy areas of hemorrhages (Fig. 2a, 2b & 2c); ileo caecal junction was haemorrhagic and mesenteric lymph node was enlarged and oedematous. Microscopic examination of heart blood smear and faeces did not showed any presence of bacteria and parasitic ova respectively. No bacteria of etiological importance could be isolated from bacterial culture. Histopathological examination of lungs revealed severe serofibrinous broncho-interstitial pneumonia with multinucleated syncytia formation and intracytoplasmic inclusion bodies in bronchiolar epithelial cells (Fig.3a & 3b).
In CIE test, nasal swab, ocular swab, rectal swab, buccal swabs, lungs, spleen and mesenteric lymph node were positive for PPR antigen. RT-PCR for N gene yielded 350 bp product specific for PPR virus (Fig 4). In Vero cells cytopathic effects were observed after three blind passages. CPE was completed 72h post -infection. The CPE observed were rounding of cells, syncytia and detachment of cell sheet (Fig. 5a & 5b).
|Fig 1a – Mucopurulent nasal discharge in PPR outbreak in Tellicherry Breed
|Fig 1b – Necrotic plaque and ulcers in mouth in PPR outbreak in Tellicherry Breed
|Fig 2a – Frothy exudates in trachea and terminal bronchus
|Fig 2b – Congestion and consolidation of lungs
|Fig 2c – Congestion and haemorrhage in abomasum
|Fig 3a – Lung – Severe diffuse bronco-interstitial pneumonia -100X
|Fig 3b – Lung -Intranuclear acidophilic inclusions in nuclei of syncytia -1000x
|Fig 4 – RT-PCR for N gene Lane 1, 2 –Samples Lane 3 -100bp MW marker NC – Negative control
|Fig 5a – Cytopathic effects observed in PPR infected Vero cells -rounding and syncytia observed after 72h PI.
|Fig 5b – Control Vero cells
Peste des petits Ruminants is enzootic in India and regular outbreaks have been reported from different parts of the country (Singh et al., 2004). In the present outbreak, we recorded heavy mortality (52%) and morbidity (100%) in Tellicherry breed of goats which is higher than earlier records of Selvaraju and Balasubramaniam (2013) who reported mortality and morbidity rate of 14.04% and 5.18% respectively in Tellicherry breed. Roy et al., 2010, reported higher percentage of mortality in Tellicherry kids. The current outbreak was recorded in October month when the north –east monsoon is on the peak in Tamil Nadu. Moreover, the goats were reared on slatted floor system (intensive system of rearing) which resulted in close contact between animals and enhanced the spread of disease. The close contact and seasonal conditions may be the reason for heavy mortality and morbidity. Balamurugan et al. (2012) also reported the seasonal influence on PPR outbreaks in India i.e. PPR outbreaks are most frequent during wet or cold dry months (April to February). Clinical signs, necropsy findings and histopathological observations were suggestive of PPR which correlates with earlier findings of Roy et al. (2010) and Zahur et al. (2009). In order to differentiate enteritis due to coccidiosis and helminthic infection faecal samples were examined and no parasite could be detected by microscopic examination. To rule out the involvement of bacteria which may be a cause for heavy mortality in goats as previously reported by Roy et al. (2010), bacterial culture was carried out and no bacteria could be isolated. In CIE test, all the samples were positive for PPR viral antigen. CIE test is not highly specific test for the diagnosis of PPR. Since, other Morbilliviruses also cross react with hyperimmune serum raised against PPRV. Hence, CIE test was used as an initial screening test and for further confirmation RT-PCR for N gene was carried out in which 350 bp product was obtained which is highly specific to PPR virus and confirmed that the present outbreak was due to PPR. The findings are in accordance with the findings of Couacy –Hymann et al. (2002). The PPR virus was successfully isolated in Vero cells and produced characteristic CPE for PPR virus viz. rounding and syncytia which are in accordance with the findings of Hegde et al. (2009). PPR is one of the economically important diseases of small ruminants. Venkataramanan et al. (2005) estimated the annual loss due to PPR was INR 1,800 million. Hence, earlier diagnosis is highly essential to control the outbreak. Tellicherry breeds are known for their higher multiple birth percentage and higher milk yield. But, this breed is highly susceptible to PPR as we recorded in this outbreak. Similar findings were observed by Selvaraju and Balasubrasmaniam (2013). Hence, to explore the advantages of this breed proper vaccination may be practiced to control the disease.