Enhancement of peste des petits ruminants (ppr) diagnosis through analysis of experimental infection of sheep and goats and validation of real time polymerase chain reaction as a diagnostic tool in Kenya

Abstract

Peste des petits ruminants (PPR) is an acute viral disease of small ruminants caused by a Morbillivirus and characterized by fever, oculo-nasal discharges, stomatitis, diarrhea and pneumonia. The disease is relatively new in Kenya and is thus not well described or understood by many stakeholders. This study was aimed at enhancing the diagnosis of PPR through description of clinical course and pathological changes in sheep and goats experimentally infected with Kenyan isolates of Peste des petits virus and to validate the use of real time reverse transcriptase PCR as a diagnostic tool. A pilot study was initiated in a controlled environment using two non infected goat kids, aged three months that were tested to be free from PPRV infection by competitive ELISA to determine whether tissues from field infected goats were infective and could be used to reproduce the disease. Each kid was inoculated intranasally with 2ml of mixed tissue suspension. The animals were observed daily for development of PPR specific signs. The infected goat kids remained healthy for 4-5 days post infection. On day 6 they developed fever and cough. Nasal and ocular discharges appeared on day 9 and cleared by day 13. Severe diarrhea set in on day 15 post infection and resulted in severe dehydration and death on day 18 post infection. Competitive ELISA and real time PCR tests confirmed presence of antibodies as well as PPRV RNA in serum samples and ocular swabs respectively, indicating the infectivity of PPRV in tissue samples collected from the field. Based on this pilot study, a more elaborate experiment was designed using seven goats and seven sheep that had tested to be negative for PPR antibodies by c-ELISA. These animals were divided into two treatment groups and one control group. Each animal in the treatment groups was inoculated intranasally with 2ml of infective mixed tissue suspension while the control group was inoculated with phosphate buffered saline. Animals were then examined for development of PPR specific signs. Fever Nasal discharges were recorded from day 8.2 ±2.28 and 9 ±1.83 post infection in goats and sheep respectively. Ocular discharges were observed on day 10 ±2.24 post infection in goats and on day 9.8 ±2.17 post infection in sheep. Oral lesions were observed only in one goat and two sheep. Sheep developed diarrhea from day 13.5 ±0.58 post infection. In goats, severe diarrhea was observed in three animals from day 14 ±1 post infection. Hematology results revealed a significant increase in hemoglobin concentration, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration in goats after infection. In sheep, there was a significant increase in red blood cell count and neutrophils while there was a significant decrease in mean corpuscular volume, mean corpuscular hemoglobin and percentage of lymphocyte. In goats, there was no signicant change in neutrophil lymphocyte (N/L) ratio (p> 0.05). In sheep, there was a signifacant increase in neutrophil lymphocyte ratio (p< 0.05). Gross post mortem examination revealed lesions in the lungs, body lymph nodes, especially mesenteric lymph nodes, and the intestines. All experimentally infected animals showed detectable antibodies against PPR by day 10 post infection. Real time RT-PCR assay revealed positive amplification by the cycle 36.51. None of the PPRV negative controls was positive by the RT-PCR assay. Real time RT PCR can therefore be used as a diagnostic tool to confirm PPR infections. The results from this study indicated that whereas PPR is thought to mainly affect goats, the disease in Kenya appear to evenly affect both sheep and goats. The experimental infection model can be used to determine the efficacy of vaccines for example the thermo-stable PPR vaccine currently being developed by other researchers.