Assiut university researches
Molecular identification and enumeration of
Eimeria species in turkey
‫ت عري ف وعد أج ناس األي م يري ا ف ي ال رومي ب وا سطه‬
.‫ال ب يول وج يا ال جزي ئ يه‬
Shiem Mohammed Ibrahim El-Sherry
‫ش يم محمد إب راه يم ال شرى‬
Nahed Abdel-Aziz Gad, Mostafa El-Bakri Sayfi El-Dein, John Robert
Barta
‫ جون روب رت ب رت ة‬،‫ م صط فى ب كرى س يف ال دي ن‬،‫ن اهد ع بدال عزي ز جاد‬
Abstract:
Multiple 18S r DNA sequences were obtained from two single
oocyst derived lines of each of Eimeria meleagrimitis and
Eimeria adenoeides. After analysingthe 15 new 18S rDNA
sequences from 2 lines of E. meleagrimitis and 17 new
sequences from 2 lines of E. adenoeides, there were clear
indications that divergent, paralogous 18S rDNA copies exist
within the nuclear genome of E. meleagrimitis. In contrast,
mitochondrial cytochrome c oxidase subunit I (COI) partial
sequences from all lines of a particular Eimeria species were
identical and, in phylogenetic analyses, COI sequences
clustered unambiguously in monophyletic and highly
supported clades specific to individual Eimeria species.
Phylogenetic analysisof the new 18S rDNA sequences from
E. meleagrimitis showed that they formed two distinct clades:
Type A with 4 new sequences; and Type B with 9 new
sequences; Both Type A and Type B sequences were
obtained from each of the single oocyst derived lines of E.
meleagrimitis. Together these rDNA types formed a well
supported E. meleagrimitis clade. Type A and B 18S rDNA
sequences fromE. meleagrimitishad a mean sequence
identity of only 97.4% whereas mean sequence identity within
types was 99.1-99.3%. The observed intraspecific sequence
divergence among E. meleagrimitis 18S rDNA sequence
types was even higher (about 2.6%) than the interspecific
sequence divergence present between some well recognized
species such as E. tenella and E. necatrix (1.1%). Our
observations suggest that, unlike COI sequences, 18S rDNA
sequences are not reliable molecular markers to be used
alone for species identification with coccidia, although 18S
rDNA sequences have clear utility for phylogenetic
reconstruction of apicomplexan parasites at the genus and
higher taxonomic ranks. Unlike with Eimeria species infecting
chickens, specific identification and nomenclature of Eimeria
species infecting turkeys is complicated and, in the absence
of molecular data, imprecise. In an attempt to reconcile
contradictory data reported on oocyst morphometrics and
biological descriptions of various Eimeria species infecting
turkey, we established single oocyst derived lines of five
important Eimeria species infecting turkeys, Eimeria
meleagrimitis (USMN08-01 strain), Eimeria adenoeides
(Guelph strain), Eimeria gallopavonis (Weybridge strain),
Eimeria meleagridis (USAR97-01 strain)and Eimeria dispersa
(Briston strain). Short portions (514 bp) of mitochondrial
cytochrome c oxidase subunit I gene (mt COI) from each
were amplified and sequenced. Comparison of these
sequences showed sufficient species specific sequence
variation to recommend these short mt COI sequences as
species specific markers. Uniformity of oocyst features
(dimensions and oocyst structure) of each pure line was
observed. Additional morphological features of the oocysts of
these species are described useful for the microscopic
differentiation of these Eimeria species. Combined molecular
and morphometric data on these single species lines
compared with the original species descriptions and more
recent data have helped to clarify some confusing, and
sometimes conflicting, features associated with these Eimeria
spp. For example, these new data suggest that the KCH and
KR strains of E. adenoeides reported previously represent
two distinct species, E. adenoeides and E. meleagridis,
respectively. Likewise, analysis of the Weybridge strainof E.
adenoeides, which has long been used as reference strain in
various studies conducted on the pathogenicity of E.
adenoeides, indicates that this coccidium is actually a strain
of E. gallopavonis. We highly recommend mtCOI sequencebased genotyping be incorporated into all studies using
Eimeria spp. of turkeys to confirm species identifications and
so that any resulting data can be associated correctly with a
single named Eimeria species. For the purpose of redescribing the Eimeria species that infect the turkey
(Meleagrisgallopavo) and to establish benchmark biological
information linked to genetic markers for each species, a
strain of Eimeria meleagrimitis Tyzzer, 1929 was obtained
from a litter sample from a turkey farm in Minnesota, USA in
2008. Multiple pure lines were derived by infecting turkey
poults with a single oocyst; one of these lines was then used
to re-describe biological and morphological features of E.
meleagrimitis in the turkey. Oocyst morphometrics of this line
matched those of this species as originally described by
Tyzzer (1929). Three asexual generations of merogony (the
first generation of meronts large in size and the second and
third generations small) were detected in the intestines before
the onset of gametogony; no developmental stages were
detected in the cecal pouches. No mortality was induced by
this line of E. meleagrimitiseven when turkey poults were
infected with high doses of oocysts (up to 5×105 oocysts/bird)
and despite the ability of E. meleagrimitis to induce severe
mucosal damage in the upper and middle duodenum.
Macroscopic lesions were characterized to provide a graded
lesion scoring guide that should assist assessment of the
severity of infections with this species in infected turkeys. The
pathogenicity of the strain was investigated and a significant
reduction in weight gain and feed conversion ratio was
observed with doses of 104 oocysts/bird or more. The
maximum yield of oocysts in the feces was obtained when
birds were inoculated with 5×103 oocysts. The Guelph strain
of Eimeria adenoeides was obtained from a commercial
turkey flock in Ontario, Canada; in approximately 1985.
Single oocyst derived linesofE. adenoeides were propagated,
and one of them used to re-describe biological and
morphological features of E. adenoeides in the turkey.
Oocysts of this strain are within the lower size ranges
reported by Moore and Brown (1951) in the original species
description but the average dimensions of the Guelph strain
is 19±1.4 µm by 14±0.9 µm. It is likely that the original
species description was based, at least in part, on a mixed
culture of two or more Eimeria species. First generation
meronts of E adenoeides Guelph strain were observed
histologically as early as 32 hours post infection in the ileum
and cecal neck. Early studies reported only two asexual
generations suggested that first asexual cycle, appeared at
32 hours post infection (HPI) was overlooked. In the present
study, three asexual generations were observed before the
start of gametogony. The Guelph strain is also characterized
by a prepatent period of 112 hours. The Guelph strain of E.
adenoeides is a highly pathogenic coccidiumthat forms
classic cecal lesions, including prominent caseous cecal
cores, during moderate to severe infections. The maximum
output of oocysts was obtained from birds inoculated with
1×103 oocysts and fecundity of 1.55×105 was obtained when
birds were inoculated with 1× 102 oocysts; fecundity dropped
dramatically as the inoculation dose increased.