Description of Ostertagia ostertagi and Ostertagia leptospicularis Hybrids in

Experimentally Infected Sheep

Author(s): V. H. Suarez, M. C. Durette-Desset and J. Cabaret
Source: The Journal of Parasitology, Vol. 79, No. 6 (Dec., 1993), pp. 874-878
Published by: Allen Press on behalf of The American Society of Parasitologists
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 J. Parasitol., 79(6), 1993, p. 874-878
? American Society of Parasitologists 1993

DESCRIPTION OF OSTERTAGIA OSTERTAGI AND

OSTERTAGIA LEPTOSPICULARIS HYBRIDS IN
EXPERIMENTALLY INFECTED SHEEP

V. H. Suarez, M. C. Durette-Desset*, and J. Cabaret

INRA, Station de Pathologie aviaire et de Parasitologie, Unit6 d'Ecologie des Parasites, 37380 Nouzilly, France

ABSTRACT: Hybrids of Ostertagia ostertagi and Ostertagia leptospicularis, derived from experimental infections
in sheep, are described. Morphometrics of the hybrids were intermediate between those of parental lines for a
large range of parameters (distance from apex to cervical papillae and length of esophagus for males and females,
length of spicules for males, and length of vestibule, distance from vulva to the end of tail, width of tail at anus).
The morphological relationship between hybrids and their parent species was assessed by discriminant analysis
based on the relative values of these parameters (actual value/length of the worm). Each parental line, either
bred in sheep or in the natural host, was morphologically similar and differed from hybrids.

Hybridization in Trichostrongylidae is fairly
rare except for reports on Cooperia by Isenstein
(1971), Haemonchus by LeJambre (1979), and
Ostertagia by Suarez and Cabaret (1992). Hy-
brids between species have intermediate parental
morphological features. The most important in-
termediate features have been described, i.e., size
of spicules in Haemonchus contortus x Hae-
monchus placei (see Lichtenfels et al., 1988), but
the entire set of available characters was not taken
into account. The aim of the present description
was to combine all the usual measures by means
of multivariate analyses to determine if male hy-
brids were intermediate for the entire set of mea-
sures. The possible intermediate position of hy-
brid females was also estimated. Hybridization
experiments were performed in sheep with par-
asite strains maintained for several generations
in this abnormal host, and morphometrics of
species in their normal and experimental hosts
were compared.
MATERIALS AND METHODS
The interbreeding was performed between 310 fe-
males of Ostertagia ostertagi and 231 males of Oster-
tagia leptospicularis in lambs with abomasal fistulae as
described by Suarez and Cabaret (1992); 200 infective
larvae were obtained from fecal cultures. They were
given orally to a lamb and 10 hybrid males (accession
number Museum National d'Histoire Naturelle
119KK), and 12 hybrid females (MNHN 120KK) were
recovered. Some of the male specimens obtained were
deposited in the Museum d'Histoire Naturelle in Paris
(Laboratoire de Biologie parasitaire, Protozoologie,
Received 22 March 1993; revised 5 August 1993;
accepted 14 August 1993.
* M.N.H.N., Laboratoire de Biologie Parasitaire, Pro-
tozoologie, Helminthologie, 61 rue Buffon, 75235
Paris, France.
874
Helminthologie) and their accession numbers are given
in parentheses. Five males and 5 females of each of
the parental strains derived from sheep were examined
(0. ostertagi: accessions MNHN 124KK for males and
125KK for females; O. leptospicularis: 117KK for males
and 118KK for females). Five males and 5 females of
each of the following populations were also examined:
O. ostertagi experimental strain in calf (126KK, males
and females), O. leptospicularis (122KK, males and
females) originating from Rangifer tarandus and then
maintained in sheep, O. leptospicularis from natural
population in Capreolus capreoli (132KK, males only).
The file containing morphometric data is registered as
MNHN 442KK.
Bursal ray patterns were described using the system
of Durette-Desset and Chabaud (1981), and the char-
acters studied are shown in Table I for males and Table
II for females.
The measures were analyzed by discriminant anal-
ysis using a Stat-Itcf (Anonymous, 1988) computer
package. The measures were standardized as (actual
value minus average)/(standard error) before analysis.
The hybrids were introduced as supplementary vari-
ables, i.e., they were not used in the construction of
axes but simply located on the graph according to their
morphometrics. Each axis represented a combination
of morphometric measures such that differences be-
tween categories (species and hybrid worms) were max-
imized. Each axis accounted for a percentage of total
variance; groups of species/populations/hybrids close
together on the graph are positively correlated, whereas
negatively related taxa are opposed.
RESULTS
The descriptions of parental lines and hybrids
are shown in Figures 1-3. Hybrids were inter-
mediate in length of esophagus, position of cer-
vical papillae, distances between the extremities
of dorsal ribs 2 and 3, and dorsal ray. The mor-
phometrics of males and females, respectively,
are presented in Tables I, II. Teratological spic-
ules were found in 2 of 10 hybrid males.
Previous analyses demonstrated that relative
data (i.e., each parameter/length of worm) were

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 SUAREZ ET AL.-NEMATODE HYBRIDS IN SHEEP 875

TABLE I. Morphometrics (mean and range in parentheses) of male Ostertagia ostertagi (O.o.), Ostertagia lep-
tospicularis (0.1.), and their hybrid.

O.o. O.o. Hybrid O.1. O.1. O.1.

Character c* lp 1 lp 1 1p2 r

Body length (mm) 6.2 5.8 5.5 5.7 6.7 6.1

(5.8-6.8) (5.3-6.3) (5.1-6.0) (4.7-6.4) (5.2-7.6) (6.0-6.2)
Body widtht 68 65 75 54 62 51
(60-75) (60-75) (70-80) (40-60) (55-65) (50-55)
Cervical papillae4 308 275 316 261 286 256
(300-320) (250-295) (285-350) (255-272) (270-300) (245-280)
Esophagus length 603 533 671 682 732 694
(595-615) (510-560) (615-690) (627-690) (630-795) (620-725)
Number of cuticular ridges? 37 31 35 32 33 36
(35-40) (29-33) (32-37) (30-36) (31-35) (36-36)
Spicule length 216 185 176 176 184 166
(210-230) (175-200) (120-210) (170-180) (170-195) (150-170)
Distance between tips of bursal 8.0 7.6 9.1 9.9 8.3 9.7
rays 2 and 3 (right) (7.5-8.6) (5.5-9.2) (7.3-10.7) (8.5-11.0) (5.8-10.7) (8.6-10.5)
Distance between tips of bursal 8.9 7.0 10.1 10.7 8.7 9.9
rays 2 and 3 (left) (8.2-10.0) (5.9-7.8) (7.1-14.0) (9.0-12.1) (6.4-10.2) (8.2-11.6)

* c, calf; 1, lamb; lp, lamb passage (from roe deer lpl1 and from reindeer 1p2); r, roe deer.
t Width at distal end of esophagus.
$ Distance from anterior end.
? At midbody.

more efficient than absolute ones in discrimi-
nating the 3 categories (2 species and hybrid) in
each sex. Thus, the most effective character in
males was the distance from apex to cervical
papillae/length of worm (P < 0.01), coded cp,
and to a lesser extent length of esophagus/length
ofworm, coded es, and length ofspicules/length
of worm, coded sp. The result of discriminant
analysis is shown in Figure 4. Only the first axis
of discriminant analysis was taken into account
as it represented 99.5% of variance. The discrim-
inant equation (based on standardized data) was:

TABLE II. Morphometrics (mean and range in parentheses) of female Ostertagia ostertagi (O.o.), Ostertagia
leptospicularis (0.1.), and hybrids.

O.o. O.o. Hybrids O.1. O.1.

Character c* lp 1 lp1 1p2

Body length (mm) 7.8 7.1 6.8 7.1 8.7

(7.2-8.1) (6.8-7.6) (5.6-8.2) (6.6-8.4) (8.3-9.0)
Body widtht 63 65 61 54 63
(55-75) (60-75) (56-65) (40-60) (55-115)
Cervical papillae4 315 275 256 237 275
(290-330) (260-290) (210-300) (200-280) (250-310)
Esophagus length 638 550 566 646 757
(580-685) (480-605) (500-650) (610-720) (720-785)
Number of cuticular ridges? 35 29 29 30 29
(32-37) (27-31) (28-30) (28-31) (28-32)
Vulva-end of tail (mm) 1.17 1.12 1.20 1.30 1.50
(1.1-1.2) (1.1-1.3) (1.0-1.3) (1.2-1.6) (1.4-1.6)
Length of vestibule 153 132 132 99 96
(130-175) (105-160) (115-145) (82-130) (80-140)
Anus-end of tail 127 117 134 153 162
(120-140) (110-125) (125-140) (140-162) (140-180)
Width of tail|l 44 40 33 32 40
(40-45) (38-40) (30-40) (30-35) (35-45)

* c, calf; lp, lamb passage from cattle; lpl, from roe deer; 1p2, from reindeer; 1, lamb.
t Width at distal end of esophagus.
$ Distance from anterior end.
? At midbody.
II At anus.

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876 THE JOURNAL OF PARASITOLOGY, VOL. 79, NO. 6, DECEMBER 1993

-a'

b))
c(2

,e~o

2' ' 0~

3Q C
I) IC),C

5( '-

~~~/0 c 3 r
vc~~i-C) C(V)
bF3Ei

'3''I

?-oI. OS

~ c~C

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 SUAREZ ET AL.-NEMATODE HYBRIDS IN SHEEP 877

~,~,TJ Y: F?'?~'~t~:~

r. I. r.

I In

eO)rm

r.~Z~~

r.\ r.

eO)rm

:~? f~
a b c

FIGURE 2. Comparative morphology of Ostertagia

ostertagi and Ostertagia leptospicularis bred in sheep eO)rm
and their hybrid. Extremities of bursal rays 2 and 3 of
bursal lateral lobes. The right rays (r.) were observed
on ventral view and left ones (1.) on dorsal view. a, O.
ostertagi; b, hybrid; c, O. leptospicularis.

y = -0.89cp + 0.41es + 0.09sp. Negative values

of y corresponded to O. ostertagi and positive
values to O. leptospicularis. All specimens were
identified as O. ostertagi, O. leptospicularis, or
hybrids; the analysis did not permit correct iden-
tification of origins (sheep, cattle, from reindeer
passaged in sheep, from roe deer passaged in
sheep, from cattle passaged in sheep), as only
67% of strains were well identified.
The most effective characters in females were
distance from apex of cervical papillae/length of
worm (P = 0.03); length of vestibule/length of
worm (P = 0.04), coded ve; distance from vulva
to the end of tail of worm/length of worm (P <
0.01), coded vul; width of tail at anus/distance
from anus to the end of tail (P = 0.04), coded
tail; and to a lesser extent, length of esophagus/
length of worm. The first axis of discriminant
analysis represented 95.9% of variance for fe-
males. The discriminant equation based on stan-
dardized data was: y = 0.48cp - 0.13es - 0.46vul
+ 0.36tail - 0.1 lyve. Negative values corre-
:?
~
FIGURE 3. Comparative morphology of Ostertagia
ostertagi and Ostertagia leptospicularis bred in sheep
and their hybrid. Caudal bursa on ventral view. a, O.
ostertagi; b, hybrid; c, O. leptospicularis.
sponded to O. leptospicularis and positive values
to O. ostertagi. All specimens were identified cor-
rectly as O. ostertagi or O. leptospicularis, re-
gardless of strain or hybrid status. All male and
female hybrids presented a location intermediate
between those of both species for males and fe-
males on axis 1.
DISCUSSION
The fact that these species were maintained in
sheep instead of their normal hosts, cattle (0.
ostertagi) or roe deer (0. leptospicularis), did not

FIGURE 1. Comparative morphology of Ostertagia ostertagi and Ostertagia leptospicularis bred in sheep and
their hybrid. Anterior end, ventral view (a, b, c) and female tails, right lateral view (a', b', c'). a, a': O. ostertagi;
b, b': hybrid; c, c': O. leptospicularis.

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878 THE JOURNAL OF PARASITOLOGY, VOL. 79, NO. 6, DECEMBER 1993
Males
-1.4 1.1
Cerv.p. I II Eso.
Oos Oob H OIrOId Ols
Females
-1.3 1.3
Vest. , ,I I Cerv.p.
Ols OIr H Oos Oob
FIGURE 4. Comparative morphometrics of Oster-
tagia ostertagi from sheep (Oos) and cattle (Oob); Os-
tertagia leptospicularis from sheep (Ols), reindeer, and
then sheep (Olr), and roe deer (Old). Discriminant anal-
ysis of relative values (divided by body length) shown
on axis 1: cervical papillae (Cerv.p.) (distance from the
anterior end) - (length of esophagus [Eso.]) for males
and length of vestibule (Vest.) - Cerv.p. for females.
greatly modify their morphometrics, except that
they were of smaller size. Thus, the length of
worms was not used in the analyses and only
ratios based on length of the worms were com-
pared. Females of O. ostertagi and O. leptospicu-
laris, from the natural host or sheep, were easily
identified with certainty using more characters
than used by Lancaster and Hong (1990). Length
of esophagus was not among the most important
criteria in females in contrast to the latter au-
thors. This was primarily due to the use of a
combination of quantitative parameters in the
present work, whereas Lancaster and Hong (1990)
used qualitative parameters (i.e., short versus long
esophagus). Obviously, the host origin of species
does not modify significantly relative morpho-
metrics of males and females.
Few hybrid worms (22) were recovered in re-
lation to the 200 third-stage larvae (L3) used to
infect sheep, the egg output was very low (max-
imum of 0.2 egg/g of feces), hatchability of these
eggs was nil, and no L3 was obtained from cul-
tures of feces (Suarez and Cabaret, 1992), indi-
cating that under experimental conditions the
hybridization process was biologically unsuc-
cessful. Hybridization apparently is infrequent
(Suarez and Cabaret, 1992): hybrids were ob-
tained once, although 4 interbreeding assays were
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performed (twice as female O. ostertagi x male
0. leptospicularis and twice as female O. lepto-
spicularis x male O. ostertagi). The anatomy of
male and female hybrids was intermediate be-
tween parental lines for the entire set of mor-
phometrics and not just for spicule length of male
hybrids (see Lichtenfels et al., 1988). Hybrids of
the 2 species of Ostertagiinae were identified
readily by discriminant analysis and thus might
possibly be identified under natural conditions
of infection.
ACKNOWLEDGMENTS
We are indebted to M. B. Lancaster (Wey-
bridge Central Laboratory, England) and F. H.
M. Borgsteede (C.I.D., Lelystadt, The Nether-
lands) for providing the original strains that were
adapted to sheep. This study was funded in part
by "AIP Chevreuil" of INRA, and V.H.S. was
supported by a Ph.D. grant from INTA (Argen-
tina).
LITERATURE CITED
ANoNYMOUS. 1989. Stat-Itcf. Manuel d'utilisation.
Institut des Cirrales et des Fourrages, Paris, 238 p.
DURETTE-DESSET, M. C., AND A. G. CHABAUD. 1981.
Nouvel essai de classification des nimatodes
Trichostrongylidea. Annales de Parasitologie Hu-
maine et Comparre 56: 297-312.
ISENSTEIN, R. S. 1971. Hybridization of two species
of nematodes parasitic in ruminants, Cooperia on-
cophora (Railliet, 1898) Ransom, 1907, and Coo-
peria pectinata Ransom, 1907. Journal of Para-
sitology 57: 320-326.
LANCASTER, M. B., AND C. HONG. 1990. The iden-
tification of females in the subfamily Ostertagi-
inae, Lopez-Neyra 1947. Veterinary Parasitology
35: 21-27.
LEJAMBRE, L. F. 1979. Hybridization studies of Hae-
monchus contortus (Rudolphi, 1803) and H. placei
(Place, 1893) (Nematoda: Trichostrongylidae). In-
ternational Journal for Parasitology 9: 455-463.
LICHTENFELS, J. R., P. A. PILITT, AND L. F. LEJAMBRE.
1988. Spicule lengths of the ruminant stomach
nematodes Haemonchus contortus, H. placei and
their hybrids. Proceedings of the Helminthological
Society of Washington 55: 97-100.
SUAREZ, V. H., AND J. CABARET. 1992. Interbreeding
in the subfamily Ostertagiinae (Nematoda: Trich-
ostrongylidae) of ruminants. Journal of Parasitol-
ogy 78: 402-405.