BACKGROUND
peste des petits ruminants disease , caused by ppr virus , is a highly contagious disease of sheep and goats that has been widely reported in sub saharan africa  <cit> .
the disease has been characterized by fever, erosive-ulcerative stomatitis, fibrino-necrotic tracheitis, brochointestitial pneumonia and diarrhea  <cit> .
morbidity and mortality can be as high as 100% and 90%, respectively, depending on the endemic status of the disease in an area  <cit> .
this disease was first described in west africa in 1940s  <cit>  and the first outbreak was describe in nigeria in  <dig> by taylor and abegunde  <cit> .
the virus has since spread to other countries.
ppr has been reported in southern asia, near east, arabian peninsula and in recent years outbreaks have been reported in turkey , tibet china , morocco , tanzania  and uganda .
since the earlier reports on nigeria in  <dig>  the disease has become endemic giving rise to economic loses to the rural poor who rely on these animals as a source of livelihood.
earlier studies have suggested that ppr might have been around for quite some time in different countries but was wrongly diagnosed  <cit> .
it has also been confused with rinderpest because of the clinical similarity  <cit> .
laboratory techniques that were used in diagnosis were virus neutralization test , agar gel immunodiffusion test , counter immuno-electrophoresis  and virus isolation which has been time consuming and laborious  <cit> .
recently, molecular biology tools have made it possible to diagnose this disease rapidly and with great sensitivity compared to earlier test  <cit> .
etiological agent of ppr is a member of the family paramyxoviridae and genus morbillivirus  <cit> .
the viral genome is  <dig>  nucleotides long  <cit>  and contains six genes encoding six major polypeptides: nucleocapsid protein , phosphoprotein , matrix protein , fusion  protein, hemagglutinin , and large rna-dependent polymerase protein.
it is a linear, single stranded, non segmented, negative sense rna virus  <cit> .
although pprv has been known to occur as one strain or serotype  <cit> , partial sequence analysis of the fusion protein gene, indicates occurrence of four lineages , of which three have been reported in africa  <cit> .
the fourth  lineage is the only one that exists in the indian sub continent but east african lineage  <dig> has been reported to coexist with the lineage  <dig>  this phylogenetic analysis is helping in the epidemiological understanding of the spread of the disease among animal populations.
in nigeria there is continuing pockets of outbreaks of ppr in small ruminants necessitating studies to delineate the molecular details of the circulating field virus .
the aim of the present study was to determine the phylogenetic relatedness of ppr viruses from outbreaks in the north central states of nigeria.
RESULTS
detection of pprv f gene
of the  <dig> clinical samples collected from sheep  and goats ,  <dig> were positive for pprv by f1/f <dig> primer amplification .
the remaining  <dig> samples were negative for pprv.
all the  <dig> samples collected from kaduna were positive while out of the  <dig> samples from plateau only  <dig> gave the target  <dig> bp amplicon .
the reference vaccine strain  also yielded the  <dig> bp amplicon.
sequencing
the  <dig> bp region of the f gene of  <dig> pprv positive samples were obtained.
of the ten  positive pcr products, nine  sequences were generated .
one sample  did not yield any sequence.
the remaining  <dig> positive samples were not sequenced.
all the  <dig> sequences were submitted to the genbank
sequence similarity analysis
a sequence comparison of the nine   <dig> bp f gene sequence fragments showed 98-100% nucleotide homology.
the two sequences from kaduna  showed 99% nucleotide homology while the seven sequences from plateau showed 98-100% similarities.
two sequences  from plateau  were entirely identical in their nucleotide.
inter- state, one  sequence sample from kaduna  and one  from plateau  were also entirely identical among them.
all the nine  field sequences also showed 93-95% nucleotide similarity with the vaccine.
identity levels of deduced amino acid sequences of the  <dig> samples ranged from  <dig> - <dig> % .
phylogenetic analysis
the results of phylogenetic analysis of the sequences  obtained from this study based on the  <dig> bp lineage specific sequences of f gene are shown in figure  <dig>  they were aligned with other available sequences from the genbank, including sequences from turkey, india, pakistan and china.
all the nigerian sequences clustered into lineage  <dig> together with the vaccine strain nig 75/ <dig> and nig 76/ <dig> separate from the turkey and indian strain which belong to the lineage  <dig>  none of the sequences clustered with cote d'ivoire which belongs to lineage  <dig>  there were no sequences of lineage  <dig> in the genbank to be used in generating the tree.
from plateau state, hq <dig>  and hq <dig>  showed close relationship .
these findings suggested that the nigerian ppr viruses vary in the level of similarity but are homologous and similar to the vaccine strain isolated in  <dig>  used in the country.
discussion
ppr is a very serious economic disease that has persisted in nigeria for decades.
one of the most important outbreaks occurred in  <dig> and  <dig>  <cit> .
since then the disease has continued with outbreaks occurring sporadically  <cit> .
due to the endemic situation in nigeria, control has been mainly hinged on vaccination  <cit> .
outbreaks are detected clinically and confirmed either serologically or using molecular techniques.
the f gene based rt-pcr developed by forsyth and barrett  <cit>  has gained much popularity, though pcr tools based on other gene targets have become available  <cit> .
the ppr specific primers f <dig> and f <dig> developed by aforementioned workers give an amplification of  <dig> bp between positions  <dig> to  <dig> nucleotides of pprv f gene.
in the present study, pprv was detected by f gene based rt-pcr in  <dig>  of the  <dig> clinical samples tested, which confirmed pprv in the two north central states.
previous survey by obi and ojeh  <cit>  in nigeria reported a positivity rate of  <dig> % and  <dig> % from tissue homogenate using dot elisa and standard indirect elisa, respectively, from the southern states of nigeria.
our findings suggest that ppr is less prevalent in the northern states  as opposed to the southern.
arguably, the differences in positivity rate may also be related to the detection method, sample size and the presence of other exacerbating diseases such as pasteurellosis, caprine contagions pleuropneumonia , blue tongue , contagious ecthyma and foot and mouth disease   <cit>  which can occur concurrently.
nonetheless the ppr positivity rate we found was similar to that reported recently in an endemic region in india  using rt-pcr  <cit> .
although ppr has been a problem in nigeria for a long time, studies did not go as far as sequencing of the isolates apart from the full genomes sequence of nig 75/ <dig>  and nig 76/ <dig>  which were done in pirbright  <cit> .
our study is the first to carry out sequencing and sequence analysis of f gene from ppr viruses involved in the recent disease outbreaks .
a sequence comparison showed high level of homology  of the circulating viruses suggesting that these viruses do not undergo rapid genetic changes in the f gene.
our data appear to be in agreement with other studies  <cit>  who reported that the ppr virus is more prone to mutations on the n gene compared to the f gene.
between states, the ppr viruses were also highly homologous.
this suggests criss-cross movement of infected small ruminants between states.
geographically, the two states share boundaries but also human related activities such as persons buying and transporting animals which may account for long distance spread of ppr.
according to ezeibe et al.,  <cit>  vaccinated animals can shed the virus from faeces for up to  <dig> weeks post vaccination.
arguably, naturally infected animals if moved from place to place or state to state can form a big source of infection to naive populations.
approximately  <dig> years ago, ppr was reported in nigeria from sheep and goats purchased from open markets at vom and potiskum  <cit> .
since then the tissue culture rinderpest vaccine  was used to confer protection against ppr until recently when the homologous ppr vaccine was developed by diallo  <cit>  from the attenuation of nig 75/ <dig> pprv strain.
interestingly, all the nigerian strains from this study demonstrated 93-95% homology to the latter vaccine strain currently used for the control of the ppr in nigeria.
our study revealed that the nig 75/ <dig> vaccine is indeed the suitable one for use in nigeria.
based on our findings, the continued occurrence of outbreaks in nigeria may not be attributed to the choice of vaccine but rather on other factors such as inadequacies related to the control strategy.
indeed annatte et al.,  <cit>  have reported lapses in veterinary extension services in relation to ppr control in lagos state, the situation may not be different in other states.
a consensus phylogenetic tree based on the lineage specific  <dig> bp of f gene sequence was constructed with the help of mega  <dig> program.
in accordance with the previous studies  <cit> , all the nigerian strains including the vaccine strain nig 75/ <dig> and nig 76/ <dig> isolate clustered together.
all the asian and turkish isolates, clustered together into a separate branch from the nigerian isolates, and the branching was supported by bootstrap values of  <dig> .
none of the sequences clustered with the strain from cote d'ivoire which belongs to lineage  <dig>  since the sequence data for the african isolates belonging to lineage  <dig> were not available in the genbank, they were not included in the phylogenetic analysis.
thus, from the phylogenetic tree , it was evident that all the isolates of kaduna and plateau clustered together with nig 75/ <dig> and nig 76/ <dig> isolates in lineage  <dig>  reinforcing the findings that these isolates are homologous.
epidemiologically these data suggest that there has been no introduction of any new pprv strain into nigerian small ruminant populations.
CONCLUSIONS
the data from this study have shown that ppr is a problem in kaduna and plateau states of nigeria.
the strains involved were genetically closely related to the vaccine strain  used in the country.
based on these data, the continued outbreaks in the country is not due to the efficacy of the vaccine.
therefore, to achieve effective control and possibly eradication of ppr in nigeria, the current control strategies should be revisited.

 peste des petits ruminants disease, caused by ppr virus, is a highly contagious disease of sheep and goats that has been widely reported in sub saharan africa. the disease has been characterized by fever, erosive-ulcerative stomatitis, fibrino-necrotic tracheitis, brochointestitial pneumonia and diarrhea. morbidity and mortality can be as high as 100% and 90%, respectively, depending on the endemic status of the disease in an area. this disease was first described in west africa in 1940s and the first outbreak was describe in nigeria in by taylor and abegunde. the virus has since spread to other countries. ppr has been reported in southern asia, near east, arabian peninsula and in recent years outbreaks have been reported in turkey, tibet china, morocco, tanzania and uganda. since the earlier reports on nigeria in the disease has become endemic giving rise to economic loses to the rural poor who rely on these animals as a source of livelihood. earlier studies have suggested that ppr might have been around for quite some time in different countries but was wrongly diagnosed. it has also been confused with rinderpest because of the clinical similarity. laboratory techniques that were used in diagnosis were virus neutralization test, agar gel immunodiffusion test, counter immuno-electrophoresis and virus isolation which has been time consuming and laborious. recently, molecular biology tools have made it possible to diagnose this disease rapidly and with great sensitivity compared to earlier test. etiological agent of ppr is a member of the family paramyxoviridae and genus morbillivirus. the viral genome is nucleotides long and contains six genes encoding six major polypeptides: nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large rna-dependent polymerase protein. it is a linear, single stranded, non segmented, negative sense rna virus. although pprv has been known to occur as one strain or serotype, partial sequence analysis of the fusion protein gene, indicates occurrence of four lineages, of which three have been reported in africa. the fourth lineage is the only one that exists in the indian sub continent but east african lineage has been reported to coexist with the lineage this phylogenetic analysis is helping in the epidemiological understanding of the spread of the disease among animal populations. in nigeria there is continuing pockets of outbreaks of ppr in small ruminants necessitating studies to delineate the molecular details of the circulating field virus. the aim of the present study was to determine the phylogenetic relatedness of ppr viruses from outbreaks in the north central states of nigeria.  detection of pprv f gene of the clinical samples collected from sheep and goats, were positive for pprv by f1/f primer amplification. the remaining samples were negative for pprv. all the samples collected from kaduna were positive while out of the samples from plateau only gave the target bp amplicon. the reference vaccine strain also yielded the bp amplicon. sequencing the bp region of the f gene of pprv positive samples were obtained. of the ten positive pcr products, nine sequences were generated. one sample did not yield any sequence. the remaining positive samples were not sequenced. all the sequences were submitted to the genbank sequence similarity analysis a sequence comparison of the nine bp f gene sequence fragments showed 98-100% nucleotide homology. the two sequences from kaduna showed 99% nucleotide homology while the seven sequences from plateau showed 98-100% similarities. two sequences from plateau were entirely identical in their nucleotide. inter- state, one sequence sample from kaduna and one from plateau were also entirely identical among them. all the nine field sequences also showed 93-95% nucleotide similarity with the vaccine. identity levels of deduced amino acid sequences of the samples ranged from - %. phylogenetic analysis the results of phylogenetic analysis of the sequences obtained from this study based on the bp lineage specific sequences of f gene are shown in figure they were aligned with other available sequences from the genbank, including sequences from turkey, india, pakistan and china. all the nigerian sequences clustered into lineage together with the vaccine strain nig 75/ and nig 76/ separate from the turkey and indian strain which belong to the lineage none of the sequences clustered with cote d'ivoire which belongs to lineage there were no sequences of lineage in the genbank to be used in generating the tree. from plateau state, hq and hq showed close relationship. these findings suggested that the nigerian ppr viruses vary in the level of similarity but are homologous and similar to the vaccine strain isolated in used in the country. discussion ppr is a very serious economic disease that has persisted in nigeria for decades. one of the most important outbreaks occurred in and. since then the disease has continued with outbreaks occurring sporadically. due to the endemic situation in nigeria, control has been mainly hinged on vaccination. outbreaks are detected clinically and confirmed either serologically or using molecular techniques. the f gene based rt-pcr developed by forsyth and barrett has gained much popularity, though pcr tools based on other gene targets have become available. the ppr specific primers f and f developed by aforementioned workers give an amplification of bp between positions to nucleotides of pprv f gene. in the present study, pprv was detected by f gene based rt-pcr in of the clinical samples tested, which confirmed pprv in the two north central states. previous survey by obi and ojeh in nigeria reported a positivity rate of % and % from tissue homogenate using dot elisa and standard indirect elisa, respectively, from the southern states of nigeria. our findings suggest that ppr is less prevalent in the northern states as opposed to the southern. arguably, the differences in positivity rate may also be related to the detection method, sample size and the presence of other exacerbating diseases such as pasteurellosis, caprine contagions pleuropneumonia, blue tongue, contagious ecthyma and foot and mouth disease which can occur concurrently. nonetheless the ppr positivity rate we found was similar to that reported recently in an endemic region in india using rt-pcr. although ppr has been a problem in nigeria for a long time, studies did not go as far as sequencing of the isolates apart from the full genomes sequence of nig 75/ and nig 76/ which were done in pirbright. our study is the first to carry out sequencing and sequence analysis of f gene from ppr viruses involved in the recent disease outbreaks. a sequence comparison showed high level of homology of the circulating viruses suggesting that these viruses do not undergo rapid genetic changes in the f gene. our data appear to be in agreement with other studies who reported that the ppr virus is more prone to mutations on the n gene compared to the f gene. between states, the ppr viruses were also highly homologous. this suggests criss-cross movement of infected small ruminants between states. geographically, the two states share boundaries but also human related activities such as persons buying and transporting animals which may account for long distance spread of ppr. according to ezeibe et al., vaccinated animals can shed the virus from faeces for up to weeks post vaccination. arguably, naturally infected animals if moved from place to place or state to state can form a big source of infection to naive populations. approximately years ago, ppr was reported in nigeria from sheep and goats purchased from open markets at vom and potiskum. since then the tissue culture rinderpest vaccine was used to confer protection against ppr until recently when the homologous ppr vaccine was developed by diallo from the attenuation of nig 75/ pprv strain. interestingly, all the nigerian strains from this study demonstrated 93-95% homology to the latter vaccine strain currently used for the control of the ppr in nigeria. our study revealed that the nig 75/ vaccine is indeed the suitable one for use in nigeria. based on our findings, the continued occurrence of outbreaks in nigeria may not be attributed to the choice of vaccine but rather on other factors such as inadequacies related to the control strategy. indeed annatte et al., have reported lapses in veterinary extension services in relation to ppr control in lagos state, the situation may not be different in other states. a consensus phylogenetic tree based on the lineage specific bp of f gene sequence was constructed with the help of mega program. in accordance with the previous studies, all the nigerian strains including the vaccine strain nig 75/ and nig 76/ isolate clustered together. all the asian and turkish isolates, clustered together into a separate branch from the nigerian isolates, and the branching was supported by bootstrap values of. none of the sequences clustered with the strain from cote d'ivoire which belongs to lineage since the sequence data for the african isolates belonging to lineage were not available in the genbank, they were not included in the phylogenetic analysis. thus, from the phylogenetic tree, it was evident that all the isolates of kaduna and plateau clustered together with nig 75/ and nig 76/ isolates in lineage reinforcing the findings that these isolates are homologous. epidemiologically these data suggest that there has been no introduction of any new pprv strain into nigerian small ruminant populations.  the data from this study have shown that ppr is a problem in kaduna and plateau states of nigeria. the strains involved were genetically closely related to the vaccine strain used in the country. based on these data, the continued outbreaks in the country is not due to the efficacy of the vaccine. therefore, to achieve effective control and possibly eradication of ppr in nigeria, the current control strategies should be revisited. 
therefore, to achieve effective control and possibly eradication of ppr in nigeria, the current control strategies should be revisited. this study was carried out to characterize ppr viruses from outbreaks in and from kaduna and plateau states. ppr is still a problem in kaduna and plateau states of nigeria. the strains involved were genetically closely related to the vaccine strain used in the country.
 peste des petits ruminants is an endemic disease of sheep and goats in nigeria and vaccination has been the method of control but sporadic outbreaks have been reported. this study was carried out to characterize ppr viruses from outbreaks in and from kaduna and plateau states.  of the clinical samples analysed, % were positive for f protein gene primers. all the samples had a sequence similarity of 98-100% among them and 92-97% with the reference vaccine strain. the deduced amino acid homology ranges between - %. phylogenetically all the nigerian sequences cluster with nig 75/ and nig 76/ in lineage  ppr is still a problem in kaduna and plateau states of nigeria. the strains involved were genetically closely related to the vaccine strain used in the country. based on this study, the continued outbreaks in the country is not due to the efficacy of the vaccine. therefore, to achieve effective control and possibly eradication of ppr in nigeria, the current control strategies should be revisited. 