Characterization of avipoxviruses from chickens and domestic pigeons in Tanzania
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Date
2016
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Publisher
Sokoine University of Agriculture
Abstract
Avipoxviruses (APVs) are double stranded DNA viruses belonging to the family
Poxviridae, sub-family Chordopoxvirinae and genus Avipoxvirus. APVs cause pox
in birds; in chickens the disease is known as fowl pox, caused by Fowlpox virus
(FWPV). In the recent years the incidence and prevalence of fowl pox in chickens
has increased in Tanzania, characterized by high mortalities of chicks and growers.
The main research question among researchers has been “are there variant strains of
FWPV or other APVs circulating in domestic birds that pose a threat to chickens in
Tanzania?’’ The general objective of this study was to investigate the characteristics
of APVs from chickens and domestic pigeons in Tanzania, focusing on development
of appropriate fowl pox vaccine. The specific objectives were to determine genetic
characteristics of APV isolates from chickens and domestic pigeons in Tanzania, to
investigate integration of Reticuloendotheliosis virus (REV) genes in the Tanzanian
FWPV field isolates and the imported commercial FWPV vaccines currently used in
the country, and to evaluate virulence characteristics of Tanzanian strains of FWPV
and pigeonpox virus (PGPV) in chickens. Samples of cutaneous nodular lesions
were collected from chickens (n = 154) and pigeons (n = 17) suspected to have pox,
followed by virus isolation. Genetic characterization involved DNA extraction;
polymerase chain reaction (PCR) amplification of FWPV and PGPV P4b gene, REV
envelope (env) gene and REV 5' long terminal repeat (LTR); gel electrophoresis of
PCR products; purification of PCR products; sequencing of purified PCR products
and analysis of the sequence data using standard procedures. Biological
characteristics, particularly virulence characteristics of Tanzanian strain(s) of FWPV
and PGPV in chickens, were investigated by inoculating 10-day old embryonated
chicken eggs and susceptible chickens with either REV-integrated Tanzanian FWPV
strain, REV-free Tanzanian FWPV strain, or Tanzanian PGPV strain. Sixty six (66)
out of the 154 samples (42.86%) analyzed for the presence of FWPV were found to
contain FWPV, indicating that the 66 chickens from which the samples were
collected had fowl pox as a result of FWPV infection. Analysis of sequences of the
P4b gene (open reading frame [ORF] 167) revealed that the Tanzanian FWPV
isolates were 99.65 – 100% identical to each other and 99 – 100% identical to
several published sequences of FWPV isolates from various countries in different
continents of the world, including Europe and Asia. Phylogenetic analysis revealed
that all Tanzanian FWPV isolates belong to clade A subclade A1. This implies that
based on sequences of the P4b gene the FWPVs currently prevalent in Tanzania are
genetically and phylogenetically closely related. However, analysis of selected
FWPV isolates in other genomic regions (between ORFs 201 and 203) revealed
integration of various genomic fragments of REV in the genome of FWPV. Out of
55 field isolates of FWPV analyzed for integration of REV env gene and REV
5'LTR, 96.36% (n = 53) were found to have genomic fragments of REV. Most of
them, 69.09% (n = 38) contained REV env gene and REV 5'LTR fragments, 18.18%
(n = 10) contained fragments of REV env gene only, and 9.09% (n = 5) contained
fragments of REV 5'LTR only. Two isolates (3.64%) were found not to be
integrated with either REV env gene or REV 5'LTR. None of the screened vaccine
strains from the imported commercial fowlpox virus vaccines was found to be
integrated with REV env gene and/or REV 5'LTR. Analysis of sequences of a PCR
product with fragment size 807 bp showed 95 – 100% identity to sequences of
several REV env gene obtained in the GenBank and 100% identity to sequence of
env gene of REV provirus integrated in a FWPV isolate. The sequence of a PCR
product with fragment size 370 bp showed 88 – 99% identity to sequences of several
REV LTR obtained in the GenBank, and 99 – 100% identity to sequences of LTR of
REV provirus integrated in several FWPV isolates from other countries. Of the 17
samples of cutaneous nodular lesions collected from pigeons, two (both from
Morogoro region) were found to contain PGPV. Sequence analysis revealed that the
Tanzanian PGPV isolate derived from this study was 90 - 99% identical to several
APV isolates from birds belonging to different species from several countries. The
Tanzanian PGPV isolate showed 91% identity to each of the Tanzanian FWPV
isolates, also derived from the present study, and 99% identity to all three PGPV
isolates obtained in the GenBank. Phylogenetic analysis revealed that the Tanzanian
PGPV isolate belongs to clade A, subclade A2, sharing a recent common ancestor
with APVs belonging to subclade A3. Biological characterization revealed that
unlike a PGPV isolate from a Norwegian wild pigeon, Palumbus palumbus, that
could infect and cause pox in chickens; the Tanzanian strain of PGPV can infect but
does not cause pox in chickens. The study also revealed high mortality rate (57%) in
chickens inoculated with REV-integrated Tanzanian FWPV strains as compared to
zero mortality observed in chickens inoculated with REV-free Tanzanian FWPV
strains, or a Tanzanian PGPV strain currently circulating in Morogoro region, and
chickens in the control group. Based on the findings from this study the following
conclusions are drawn: (a) Currently there is a heterogeneous population of FWPV
in Tanzania comprising of REV-integrated FWPV strains and REV-free FWPV
strains. (b) REV-integrated FWPV strains are more virulent in susceptible chickens
than REV-free FWPV strains. (c) The increased incidences and prevalence of fowl
pox currently experienced in Tanzania, characterized with high mortality rates of
chicks and growers, could be attributed to the emergence of variant strains of FWPV
which are REV-integrated. (d) The imported commercial fowl pox vaccines
currently used in Tanzania are not contaminated with REV, therefore the vaccines can safely continue to be used in the country. (e) Integration of genomic fragments
of REV in the genome of field strains of FWPV currently prevalent in Tanzania is
not attributed to imported commercial fowl pox virus vaccines currently used in the
country. It could be attributed to recombination of field strains of REV and field
strains of FWPV. (f) As opposed to a PGPV isolate from a Norwegian wild pigeon,
Palumbus palumbus, that could infect and cause pox in chickens; this study has
revealed that the Tanzanian strain of PGPV currently circulating in Morogoro region
is not pathogenic in chickens, therefore it does not pose a threat to chickens in the
country. On basis of the findings from this study the following future studies are
recommended: (a) More studies aiming at detection and characterization of PGPV
isolates from other regions and geographical locations of Tanzania should be
conducted in order to establish genetic and antigenic characteristics of PGPV
currently circulating in the country. This recommendation is based on the fact that in
the present study only two isolates of PGPV could be obtained from pigeons in
Morogoro region, Eastern Tanzania. (b) Studies to determine pathogenicity and
lethality of PGPV and FWPV in different host systems are required. (c)
Epidemiological features and risk factors for FWPV and PGPV transmission ability
and spread should be investigated. (d) Evolutionary characteristics of FWPV, PGPV
and REV should be systematically studied to unravel possible factors that could be
linked with their genetic and antigenic diversity.
Description
PHD THESIS
Keywords
Avipoxviruses, Chickens, Domestic pigeons, Tanzania, Chordopoxvirinae, Fowl pox, APV characterisation, Reticuloendotheliosis virus