BETWEEN GOAT-FARM BIOLOGICAL VARIABILITY
OF THE NEMATODE
MUELLERIUS CAPILLARISFIRST-STAGE LARVEA.
INFLUENCE OF ANTHELMINTIC TREATMENT
KULO A.*, CHARTIER С.* and CABARET J.**
Summary :
Seven dairy-goat farms from the centerwest of France were investi
gated for morphology and ecology of first-stage larvae (L1) of Muellerius capillaris before and after treatment of goats with febantel, a probenzimidazole anthelmintic. The lengths, survivals at 20 and -20°C, and infectivity of L1 to intermediate host Helix aspersa, were different between farms. The between farms diffe
rences in survivals were reduced after treatment of goats with febantel. The observed between farm differences in L1 did not seem to be related to farm characteristics (intensity of treatments, susceptibility to febantel, and intermediate host species).
KEY WORDS : Muellerius. lungworm. first-stage larvae. temperature. goat.
INTRODUCTION
Muellerius capillaris Muller, 1889 (Nematoda : Protostrongylidae) is the only species of lungworm recovered from dairy-goats in western France ; it is also highly preva
lent (Cabaret et al. 1986 ; Chartier and Reche, 1992).
The adult worms live in the lungs where eggs and first-stage larvae (L1) are excreted and then passed in the faeces. Survival of L1 in goat faeces had been stu
died (Cabaret et al, 1991 ; Morrondo-Pelayo et al., 1992) and desiccation of faeces was considered as a key factor at 20°C ; maintenance at temperatures over 30°C drastically reduced survivals. The length of L1 can vary from one place to another (see Boev, 1975) and thus could be an indicator of ecological diversity.
Land snails serve as the intermediate hosts : the L1 enters the foot of the snail, and then after two moults, becomes third-stage larvae. Among land snails, juve
nile Helix aspersa Müller 1774 might be used to monitor infectivity of L1 (Cabaret, 1992).
Dairy-goat farms do not exchange habitually goats and helminth populations are thus isolated and may evoluate in different ways in relation to general farm management (Cabaret and Gasnier, 1993) or anthel- minthic treatments. The purposes of the present work were to measure the between farm variability regar-
* C.N.E.V.A., Station régionale de Pathologie caprine, 60 rue Pied de Fond. B.P. 3081, 79012 Niort Cédex, France.
** Correspondence author : INRA, Station de Pathologie aviaire et de Parasitologie, Unité d'Ecologie des Parasites, 37380 Nouzilly, France.
Résumé : VARIABILITÉ BIOLOGIQUE INTER-FERMES CAPRINES DES LARVES DU PREMIER STADE DU NÉMATODE MUELLERIUS CAPILLARIS. INFLUENCE D'UN TRAITEMENT ANTHELMINTHIQUE.
Sept fermes caprines du centre-ouest de la France ont été étudiées pour la morphologie et l'écologie des larves de Muellerius capillaris du premier stade (L1) avant et après traitement des chèvres avec du febantel, un anthelminthique probenzimidazole. La longueur, les sur- vies à 20 et -20°C, et l'infestivité des L1 pour l'hôte intermédiaire Helix aspersa, sont différentes selon les fermes. Les différences inter- fermes, en ce qui concerne les survies, sont réduites après un traite- ment au fébantel: Les différences entre fermes des L1 ne semblent pas reliées aux caractéristiques de l'élevage (intensité des traite- ments, sensibilité au fébantel et espèces d'hôtes intermédiaires).
MOTS CLES : Muellerius. strongle pulmonaire, premier stade larvaire, tempé- rature, chèvre.
ding : i) the length o f L1 and its value as a marker o f biological variability, ii) the survival and infectivity o f L1, that might b e c o n s i d e r e d as important factors in establishing future goat infection, a n d iii) modifica- t i o n s i n d u c e d w i t h a n t h e l m i n t i c t r e a t m e n t o n t h e a b o v e parameters.
MATERIALS AND METHODS
FARMS AND FAECAL SAMPLINGS
e v e n dairy-goat farms w e r e investigated, from February to J u n e 1 9 9 1 . T h e y utilized p e r m a n e n t pasture throughout the y e a r and w e r e located in Centerwest o f F r a n c e ( D e u x - S è v r e s ) . T h e m e a n tem- perature w a s 11.4 °C and annual rainfall ranged from 9 4 0 to 1 3 2 7 mm. T h e size o f flocks ranged from 3 5 to 180 g o a t s . Individual faecal sampling (7 to 1 0 g ) w e r e performed o n 3 0 to 4 0 goats per farm w h e n anthel- mintic treatments had not b e e n d o n e for m o r e than t w o months. A first sampling at day 0 (Dn) w a s d o n e at the s a m e time as an anthelmintic treatment (Rintal , B a y e r Pharma, Puteaux, F : febantel, 5 m g / k g b o d y - w e i g h t ) . A s e c o n d faecal sampling was d o n e 21 days later (D21). Malacological investigation w a s performed in N o v e m b e r : all snails c o l l e c t e d for 15 m n b y 3 c o l - lectors w e r e identified and the three most represen- t e d s p e c i e s a r e s h o w n in T a b l e I. T h e efficacy o f febantel, initial LPG ( l a r v a e p e r g r a m m e o f f a e c e s ) and r e c e n t history o f treatments are also s h o w n .
Parasite, 1994, 1, 65-70 Mémoire 6 5
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1994011065
K U L O Α., C H A R T I E R С. A N D C A B A R E T J .
Date o f sampling
Number o f LPG reduction Intermediate host fauna ( % )
Farm Date
o f sampling LPG anthelmintic treatments
o n day 21
% Helix aspersa Deroceras reticulatum
Cernuella sp
1 2 0 / 0 2 201 3 (0)* 6 9 5.8 38.2 2 9 . 9
2 1 8 / 0 3 147 5 ( 5 ) 32 0 100 0
3 2 0 / 0 3 182 5 ( 5 ) 23 21 3 3 . 9 0
4 1 5 / 0 4 105 3 ( 2 ) 3 0 3 3 . 8 36.5 0
5 1 7 / 0 4 2 9 4 2 ( 2 ) 51 6.6 54.7 0
6 1 2 / 0 6 158 6 ( 4 ) 3 4 28 5.4 58.1
7 2 5 / 0 6 140 2 ( 2 ) 54 0 86.2 0
": in brackets : Ix-nzimidazolc compounds
Tableau 1. - Parasite/logicai characteristics of 7 dairy-goat farms : larval excretion in faeces at day 0 (LPG), susceptibility to febantel treatment, number of anthelmintic treatments per year, main intermediate host snails on pasture.
FIRST STAGE LARVAE COLLECTION AND PRESERVATION PROCESSING OF DATA T h e LÍ were extracted from e a c h sampled host by a
modified B a e r m a n n t e c h n i q u e (Cabaret et al., 1 9 8 0 ) . T h e obtained larvae were pooled for each farm. T h e length o f LÍ was measured by means o f a semi-auto
matic image analysis device Vids IV (Sirehill Industrial Estate, Saffron Walden, UK) on at least 30 larvae per farm and per period o f sampling ( D0 and D21). T h e remainder o f larvae was stored at -20 °C in tap water for 4 to 8 months before use for snail infection.
SURVIVAL OF L Í AT VARIOUS TEMPERATURES
For e a c h farm six duplicated 20 g samples were res
p e c t i v e l y m a i n t a i n e d u n d e r t h e f o l l o w i n g t e m p e r a tures: 56 °C (2h or 3 h ) , 20 °C (1 m o n t h ) . 4-6 °C (2 or 4 months), - 2 0 °C during 4 months. Humidity o f faeces was also modified under these conditions; the relative water loss was respectively 3 7 . 1 , 4 8 . 4 , 68.7, 6 7 . 4 , 64.7, and 3 6 . 1 % . T h e temperatures w e r e c h o s e n as to mimic n o r m a l to h a r s h c o n d i t i o n s in w i n t e r ( 4 - 6 ° C a n d -20°C) or summer (20°C and 56°C). T h e LÍ were extra
cted as described a b o v e ; survival was estimated as : ( n u m b e r o f LÍ at the collection time - n u m b e r o f LÍ after test p e r i o d ) / n u m b e r o f LÍ at the collection time.
INFECTIVITY OF L Í : INFECTION OF HELIX ASPERSA The LÍ o f each farm preserved at -20°C were thawed and used to infect 9 to 16 H. aspersa laboratory reared and aged approximately o f o n e month. T h e larval sus
pension used for infection contained 2L1 / μι 1 and 30 LÍ were in contact with each snail for 3 hours; snails were thereafter put together and maintained in o n e Petri dish per tested farm. Snails were examined 3 w e e k s post- infection for larvae: the shell w a s r e m o v e d and the body directly examined for larvae at magnification 25.
A Stat-Itcf c o m p u t e r p a c k a g e ( M a n u e l d'utilisation.
Institut T e c h n i q u e d e s C é r é a l e s et d e s f o u r r a g e s , 1988, Paris) w a s used to perform analysis o f v a r i a n c e ( A n o v a ) , principal c o m p o n e n t , c o r r e s p o n d e n c e a n d segmentation analyses.
RESULTS
LENGTH OF L Í IN RELATION TO FARM ORIGIN AND FEBANTEL TREATMENT OF GOATS
h e a v e r a g e length o n D0 w a s 2 8 3 - 9 pm a n d standard-deviation 14.3 ( 2 5 5 L Í ) . T h e distribu- tion was normal: Pearson's coefficient o f sym- metry and kurtosis w a s respectively 0 . 0 0 3 and 2 . 5 . No significant difference was shown between farms and before/after treatment with febantel by Anova (respecti- vely P=0.32 and P=0.31). However segmentation analy- sis on length o f LÍ on Dq in relation to farm did show that two groups o f LÍ could b e considered (P <0.03):
less and over 280 pm, and that these two groups were differently represented between farms (Table II).
Tableau II. - Distribution of Muellerius capillari* first-stage larvae length in relation to farm origin
M¿mriirp
A h i g h e r f r e q u e n c y o f small larvae ( < 2 8 0 m p ) was recorded in farms 3 and 5 and to a lesser extent in farm 7. Large larvae (>280 mp) were predominant in farm 2.
L Í SURVIVAL IN FAECES AT 4 TEMPERATURES IN RELATION TO FARM ORIGIN AND FEBANTEL TREATMENT OF GOATS
T h e LÍ survival was tested on D0 and D21 after treat
ment. Significant differences ( A n o v a ) b e t w e e n test (P
< 0 . 0 0 1 ) and b e t w e e n farms ( P < 0 . 0 1 ) w e r e found in survival values assessed o n larvae sampled at Dn and D21 ( T a b l e I I I ) . T h e v a r i a b l e s farm a n d test w e r e significantly interacting ( P < 0 . 0 1 ) i.e. the LÍ survival at o n e particular t e m p e r a t u r e w a s different from o n e farm to another ; c o r r e s p o n d e n c e analysis was thus appropriate to characterize LÍ survivals in the 7 farms (Fig. 1 ) . T h e analysed data matrix w a s as follows : the c o l u m n s c o r r e s p o n d e d to e c o l o g i c a l tests (survivals at 56, 2 0 , 4 - 6 and - 2 0 ° C ) and the rows to the correspon
ding LÍ survivals at D0 ; the survivals at D2i w e r e introduced as s u p p l e m e n t a r y data i.e. they did not p a r t i c i p a t e in t h e c o n s t r u c t i o n o f c o r r e s p o n d e n c e analysis but w e r e only located afterward o n the plane determined b y a x e s 1 and 2. At Dn four groups (I to IV) o f farms w e r e distinguished :
* at 20°C and -20°C : i) farms 1 and 2 exhibiting high LÍ survivals,
ii) farms 4 and 5 with medium LÍ survivals,
iii) farms 3 and 6 with low LÍ survivals,
* at all conditions : iv) farm 7 with low LÍ survivals.
After a n t h e l m i n t i c t r e a t m e n t , t h e e s t i m a t i o n s o f LÍ survivals did not differ as m u c h as before treatment;
this w a s m o s t l y d u e to a r e d u c t i o n o f survivals at 20°C in farms 2, 4 and 5. T h e LÍ survivals at 20°C remained high ( 3 2 . 8 % ) in farm 1.
L Í INFECTIVITY IN RELATION TO FARM ORIGIN AND FEBANTEL TREATMENT OF GOATS (Table IV)
No b e t w e e n farm ( P = 0 . 8 8 ) o r b e f o r e / a f t e r febantel treatment ( P = 0 . 2 3 ) significant difference in infectivity was recorded. T h e infection o f H. aspersa was higher ( I . 3 versus 0 . 2 - 0 . 3 ) w h e n fresh larvae w e r e used (data not s h o w n ) .
L Í VARIABILITY IN LENGTH, SURVIVALS AND INFECTVITY IN RELATION TO FARM CHARACTERISTICS
A principal c o m p o n e n t analysis w a s p e r f o r m e d o n length (% under 2 8 0 p m ) , survivals at 5 6 , 2 0 , -20°C at D0 and infectivity o n D21 post-treatment (matrix c o l u m n s w e r e farms a n d rows w e r e t h e r e s p e c t i v e v a l u e s o f t h e a b o v e m e n t i o n e d p a r a m e t e r s ) . T w o clear groups, farm 1 and 2 (very g o o d LÍ survival at - 2 0 ° C ) , and farms 4 and 5 ( g o o d survival at - 20°C and best infectivity s c o r e s ) w e r e found ; an interme
diate g r o u p was constituted o f farms 3 and 6, and farm 7 remained isolated (Fig. 2 a ) . A similar analysis was performed o n farm characteristics ( s e e T a b l e 1) : n o o b v i o u s grouping was found (Fig. 2 b ) . Farm cha
racteristics w e r e not apparently related to LÍ variabi
lity as farm grouping was different b e t w e e n the t w o analyses presented in fig. 2.
Tableau III. - Mean survival rates of M. capillaris first-stage larvae in relation to farm origin and ecological test before (D0) and after (D2 1) febantel treatment of goats in seven dairy goat farms.
KULO Α., CHARTIER С. AND CABARET J .
Fig. 1. - Between farms variations in M. capillari* first-stage larvae survival in faeces at 4 temperatures before (Day 0) and after (Day 21) febantel treatment of dairy-goats.Correspondence Analysis using farms as variables.
Fig. 2. - C h a r a c t e r i z a t i o n o f farms b y P r i n c i p a l C o m p o n e n t A n a l y s i s :
similarities b e t w e e n farms in r e l a t i o n t o M. capillari* first-stage l a r v a e l e n g t h , survival a n d infectivity ( a ) ;
similarities b e t w e e n farms in r e l a t i o n t o LÍ o u t p u t in f a e c e s o f g o a t s , e f f i c a c y o f f e b a n t e l t r e a t m e n t a n d i n t e r m e d i a t e h o s t fauna ( b )
AA<=mnîrf=>
' BIOLOGICAL VARIABILITY OF MIMIMWS CAPILLARIS
* Average number of second or third stage larvae/snail I 1 Range
( ) № of snails
T a b l e a u IV. - I n f e c t i o n o f t h e land-snail Helix aspersa with M. capillaris l a r v a e o b t a i n e d b e f o r e ( D0) a n d a f t e r ( D >1) f e b a n t e l t r e a t m e n t o f g o a t s in s e v e n d a i r y - g o a t farm.
tested, a n d LÍ infectivity w a s average. It was as if the different m a n a g e m e n t a n d environment b e t w e e n farms c o r r e s p o n d e d t o various r e s p o n s e s in suscepti
bility t o treatment, larval survivals, b u t finally e n d e d to similar larval infectivity. T h e adaptative significance o f parasite susceptibility t o treatment and larval ability to surviving in different conditions remained unclear.
T h e average LÍ survivals w e r e not much altered after treatment, but the b e t w e e n farm variability was redu
ced. This feature was striking and w e may hypothesise that t h e p h e n o m e n o n is o f short duration as t h e reduction in LÍ output after treatment corresponded probably to a temporary depletion o f female worm fertility rather than t o a destruction o f adult w o r m s (Dakkak, Cabaret, Ouhelli, 1979). W e should reject the possibility o f founder effects (establishment o f very few worms in a flock) associated to genetic drift (ran
dom variation due to the small size o f population). W e could better search explanations in the modifications o f environment o f L Í , for e x a m p l e duration o f transit from lung t o f a e c e s a n d humidity o f faecal pellets w h e n emitted.
ACKNOWLEDGEMENTS
W
e a r e g r a t e f u l t o C o n s e i l R é g i o n a l o f Poitou-Charentes for financial support and to G o v e r n m e n t o f T o g o for funding Ph.D.grant to Α.Κ. T h e snails w e r e provided b y C. B o n n e t (INRA, Magneraud).
DISCUSSION
T
he b e t w e e n farms differences were recorded for L1 size and their survival capabilities at various t e m p e r a t u r e s , mostly 2 0 a n d -20°C. B e t w e e n dairy-goat farms differences in ecology o f trichostron- gyle third-stage larvae has b e e n also found in center o f F r a n c e ( G a s n i e r a n d Cabaret, u n p u b l i s h e d r e s u l t s ) , indicating that each farm has its own adaptated population. No obvious relation b e t w e e n the two parameters, size and survival o f L1, could b e found. T h e ability to infect an intermediate host was not significantly diffe
rent from o n e farm to another (Anova) but principal c o m p o n e n t analysis suggested that t h e L1 from t w o farms might b e m o r e i n f e c t i v e t o t h e i n t e r m e d i a t e hosts. T h e only clear discriminating parameter is thus survival at cold or temperate temperatures in faeces.
T h e b e t w e e n farms differences w e r e n o t related t o parasitological characteristics o f the farm (susceptibi
lity t o f e b a n t e l t r e a t m e n t a n d i n t e r m e d i a t e - h o s t fauna). T h u s larval output w a s strongly r e d u c e d in farm 7, survival o f L1 w a s p o o r at all temperatures
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Accepté le 9 novembre 1993
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