Survival of snails and characteristic lesions of Fasciola hepatica infection in four European species of Lymnaea

Veterinary Parasitology, 40 ( 1991 ) 47-58 Elsevier Science Publishers B.V., Amsterdam

47

Survival of snails and characteristic lesions of Fasciola hepatica infection in four European species of Lymnaea P. S i n d o u a, J. Cabaret b and D. Rondelauda' 1 aFacult# de MOdecine, 2, rue du Docteur Raymond Marcland, 87025 Limoges, France bInstitut National de la Recherche Agronomique, Station de Pathologie Aviaire et de Parasitologie, UnitO d'Ecologie des Parasites, 37380 Monnaie, France (Accepted 28 February 1991 )

ABSTRACT Sindou, P., Cabaret, J. and Rondelaud, D., 1991. Survival of snails and characteristic lesions of Fasciola hepatica infection in four European species of Lymnaea. Vet. Parasitol., 40: 47-58. The survival of snails and characteristic organ lesions were investigated in four different age categories (new-born, 1 and 2 mm shell height, adults) and in four species of Lymnaea (L. glabra, L. palustris, L. peregra ovata, L. truncatula ) infected by Fasciola hepatica. Mortalities were higher in infected groups on Days 30 and 45 post-infection ( +42% and +45%, respectively) than in controls. Lesions depended on the species and age of the Lymnaea. Epithelial necrosis of the kidney was recorded in L. glabra, L. palustris and L. p. ovata, whereas intralamellar deposits were found in the same organ ofL. truncatula. All categories ofL. truncatula had evolutive infections (snails with living parthenitae); the young snails of other species became infected, but the adults did not, as estimated at Day 45 post-infection. The lesions were related to the intensity of evolutive infections: necrosis of the digestive gland was found in moderate to low infections and, conversely, gonadal atrophy with epithelial necrosis was found in highly infected snails.

INTRODUCTION

Lymnaea truncatula is the most efficient host for the transmission of liver fluke (Fasciola hepatica) in western Europe (Kendall, 1965, 1970 ). Lymnaea glabra, Lymnaea palustris, Lymnaea peregra and Lymnaea stagnalis may also play a role, and have been experimentally infected when young (Kendall, 1970; Boray, 1978). Histopathological results of infection have been extensively described in L. truncatula (Rondelaud and Barthe, 1978, 1980, 1983) and to a lesser extent in L. glabra (Bouix-Busson et al., 1985), L. palustris or L. peregra ovata ~Author to whom correspondence should be addressed.

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© 1991 Elsevier Science Publishers B.V. All rights reserved.

48

P. SINDOU ET AL.

(Sindou et al., 1986; Sindou, 1989). Two important points should be borne in mind: (i) the histopathological response of Lymnaea to stress (infection by F. hepatica or intoxication, Moukrim et al., 1988) does not vary much; (ii) the ability to cope with infection depends a great deal on snail age. A study has never been performed to compare the histological reaction of snails to infection at various ages and in different species. The aim of the present work, therefore, was to assess, in four species of Lymnaea in different age categories: (i) the prevalence of successful infection; (ii) an eventual increase of mortality in infected groups of snails; (iii) characteristic response patterns to infection, with histological descriptions for each category of snails (species at various ages); (iv) the response pattern, within all categories, to abortive infection (lysis of sporocysts) versus evolutive infection (development of living parthenitae). MATERIALS AND METHODS

The snails The adults were all collected from the wild in populations known to be uninfected (F. hepatica) from previous sampling and histological examination of 70% of the collected snails. The sampling sites were located in the centre of France: L. glabra (Mign6, Indre), L. palustris (Magnac-Laval, HauteVienne; Tersannes, Haute-Vienne), L. p. ovata (Thenay, Indre) and L. truncatula (Mign6, Indre; Nuret-le-Ferron, Indre). The new-born snails, and the snails whose shell heights were 1 or 2 m m , were bred from the adults of the above described populations. The breeding procedures and maintenance of snails were standard and are reported elsewhere (Sindou et al., 1990). Snails were maintained in covered, closed-circuit aquaria for a m a x i m u m of 45 days with 10 individuals per d m 3 of water. The sediment consisted of gravel lying under a 10 cm layer of oxygenated water. The aquaria were kept at a constant temperature of 20 °C with a balanced photoperiod (light-dark 12-12 h) and a light intensity of 30004000 lux. The snails were fed with lettuce ad libitum. Control batches were of 20 snails in all experiments, whereas infected batches had a range of 45-900 snails according to species and age.

Infection of snails Eggs of F. hepatica were collected from the bile of naturally infected cattle in a Limoges slaughterhouse (centre of France). After washing and sieving on gradually decreasing meshes (75, 50, 38/tm), the eggs were recovered and incubated for 20 days at 20°C in darkness in order to obtain miracidia for infection of snails.

E4SCIOLA HEPATICA I N F E C T I O N A N D LYMNAEA SP.

49

The infections were all monomiracidial. The area of contact was a 35 mm diameter Petri dish in which 2 ml of aquarium water were deposited. The duration of contact between the miracidium and the snail was 4 h. The snails were then put back in the aquaria.

Histological techniques At necropsy, the snails were put in Bouin fixative and the shell was gently removed. After dehydration (Gabe, 1968 ), the snails were embedded in paraffin at 56°C. Serial sections, 5/~m thick, were obtained and stained with Harris' haematoxylin and Gabe's modified trichrome (Moukrim et al., 1988 ).

Experimental design The snails were kept for 45 days post-infection (p.i.). For each category, a control and an infected batch were maintained; the histological examinations were performed on Day 30 p.i. The following records were made: survival at Day 30 and Day 45, and histological examination at Day 30. The following types of lesions were scored (a number code is given in parentheses), as already described (Rondelaud and Barthe, 1978, 1980, 1981, 1983; Sindou et al., 1990).

Digestive gland. Multifocal epithelial necrosis (1); generalized necrosis (2); epithelial reconstitution (3).

Gonad. Atrophy of male line (4); atrophy of female line (5); organ disappearance (6); atrophy and epithelial necrosis ( 7 ).

Kidney. Intralamellar deposits ( 8 ); oedema ( 9 ); epithelial necrosis ( 10 ); epithelial reconstitution ( 11 ).

Amoebocyte-producing tissue. Fibrosis ( 12 ); amoebocytic proliferation ( 13 ); proliferation and fibrosis (14); not developed ( 15 ). Thirty-two batches (uninfected and infected) were thus established. Within the four species of snails, four age categories or types were used: new-born (less than 2 h old), 1 m m shell height, 2 m m shell height, pre-adult L. glabra, L. palustris and L. p. ovata ( 5-6 m m in height), or adult L. truncatula (4 mm in height). The total numbers of snails involved in these experiments were 2642 (infected at birth), 1259 (infected when 1 m m ) , 940 (infected when 2 m m ) and 555 (infected as pre-adults or adults) (Table I ).

50

P. SINDOU ET AL.

TABLE 1

Infection of four species of Lymnaea at four ages with F. hepatica Species

Age category ~

n

L. glabra

EI%

n

EI%

31

100 50

42 40 0

57 90 100 60

29 O 52 0

1 2 3 4

900 300 400 60

7 0 0 0

126 105 78 60

61 0 0 0

1

50 110

0

150

0

61 40

2 87 33

84 61 60

85 88 0

337 60 60 110

23 5 4 41

950 400 80 115

7 75 7 13

l 2 3 4

L. palustris

L. p. ovata

2 3 4

L. truncatula

Day 45 p.i.

Day 30 p.i.

1 2 3 4

72 ll0

LAges: 1 = infected when new-born; 2 = 1 m m shell height; 3 = 2 m m shell height; 4 = adult or pre-adult

snails. n = n u m b e r of snails tested. El% = percentage of snails with evolutive infections.

Processing of data Multivariate analyses were performed with a STAT-ITCF computer package ( 1988 ). They were either discriminant analysis or principal components analysis. No transformation of data was carried out except when specifically mentioned. In all cases, the data were centred (actual d a t a - a v e r a g e value of the variable) and divided by the standard deviation. R E S U LTS

Infection of snails Nearly all the snails became infected. Evolutive infections were unevenly distributed within the species of Lymnaea, occurring either on Day 30 or Day 45 p.i. (Table 1 ). In most cases, the prevalence was lower at Day 45 p.i. than at Day 30 p.i. The opposite situation was observed for L. palustris (newborns ), L. p. ovata ( 1 m m shell height) and L. truncatula ( 1 m m shell height ).

[:4SCIOLAHEPATICAINFECTIONANDLYMNAEASP.

51

Among pre-adult and adult snails, only L. p. ovata (at Day 30 p.i.) and L. truncatula were infected. Survival rates in infected vs. control snails

The control batches had a survival rate of 97.5% (standard error of the mean (SEM): 3.2%), whereas infected ones had a survival rate of 55.7% (SEM: 15%) on Day 30 p.i. Similar trends were found on Day 45 p.i.: 91.9% (SEM: 5.0%) vs. 47.0% (SEM: 11.4%). The survival rates were significantly reduced in infected batches, as shown by ANOVA ( P < 0.001 for both periods p.i. ). There were, however, differences due to the age at which infection occurred (Fig. 1 ). Excess mortalities (mortality of infected snails-mortality of control snails) were always higher than 20%, but reached 97% on Day 45 p.i. A discriminant analysis was carried out in order to combine the two survival rates into a more accurate variable to separate the infected from noninfected snails. This view was substantiated since the pseudo-F was 62 compared with the 27 and 49 F values of ANOVA. The discriminant equation (centred and reduced matrix of data) was y=0.37SD30+0.72SD45. This showed that survival at Day 45 (coded as SD45) was more discriminating than that which was recorded at Day 30 (coded as SD30). The discrimination was poor in two records and was apparently due to L. truncatula adults having a low survival rate both in infected or non-infected batches, and adult and 2 m m L. glabra having high survival rates both in infected and non-infected snails. Overmortality at 45 clays p.i. 100 80 60 40 20 0 234

1 2 3 4

1 2 3 4

1 2 3 4

Age and species of Lymnaea glabra

~

Dalustris

I

ovata

I I ~ truncatula

Fig. 1. Excess mortalities (mortality of i n f e c t e d - mortality of control snails) of four species of Lymnaea infected at four ages (new-born: 1; 1 m m shell height: 2; 2 m m shell height: 3; preadults or adults: 4).

52

P. S I N D O U ET AL.

truncatula ovata

tru

axis 2

ova

palustris

glabra

~" u4 *3 ~ "1 . 2 ru2

ova3 axis 1

/

ov,~1,2 trul ru3

~

3 ~r 4

\

(a)

•

Digestive gland

Kidney

Gonad

Amoebocytic organ AXIS 2

O 3 AXIS 1

(b) Fig. 2. Principal component analysis of lesion scores in four species of Lymnaea infected at four ages (coded 1 for new-born to 4 for adult snails): correlation circles (a); projections of lesions on Axes 1 and 2 (b). 2 =generalized necrosis of digestive gland. 3 = epithelial reconstitution of digestive gland. 5 = atrophy of female line in gonad. 7 = gonadal atrophy with epithelial necrosis. 8 = intralamellar deposits in kidney. 10 = epithelial necrosis in kidney. 11 = epithelial reconstitution in kidney. 13=amoebocytic proliferation. 14=amoebocytic proliferation and fibrosis. 15 = no development of amoebocyte-producing tissue.

Lesion characteristics ofF. species

hepatica

infection in the different L y m n a e a

T h e f o l l o w i n g m a t r i x o f d a t a w a s used: c o l u m n s r e p r e s e n t the snail s p e c i e s at the d i f f e r e n t ages a n d r o w s r e p r e s e n t t h e a f o r e m e n t i o n e d l e s i o n s . A principal c o m p o n e n t a n a l y s i s w a s p e r f o r m e d o n t h e s e data. A x e s 1, 2 a n d 3 rep-

FASCIOLA HEPATICA INFECTION AND LYMNAEA SP.

53

resent, respectively, 43, 16 and 13% of the variability. The circles of correlation of variables (snails of each species at different ages) and of the projection of lesions on Axes 1 and 2 are, respectively, shown in Fig. 2 (a) and 2 (b). The criteria for the interpretation of Fig. 2(a) and 2(b) are: (i) the variables (snail species ) are well described by the organ lesions that were recorded when their position is near the outer circle, and are poorly described by the lesions as they are located near the origin of the axes; (ii) the proximity of variables (snails) indicates that they are positively correlated, i.e. their organ lesions are similar. Thus, the organ lesions in older L. truncatula (Tru4) are closer to those in younger L. palustris (Pall, Pal2), L. glabra (Glal, Gla2) than to those of the adult L. glabra (Gla4), and even more so to adult L. palustris (Pal4) o r L . p. ovata (Ova4) (Fig. 2(a) ). The two groups of lesions were found on Axis 1 (Fig. 2 ( b ) ) . The first group represented the association of no development of amoebocytic tissue, epithelial reconstitution of renal lamellae, oedema of the kidney and atrophy of the male gonad line. The second group included generalized necrosis of the digestive gland and gonadal atrophy with epithelial necrosis. Similarly, on Axis 2, two lesions were in opposition: the intralamellar deposits and epithelial necrosis of the kidney. From these results (Fig. 2(a) and 2(b) ), it may be assumed that the least appropriate hosts (L. palustris and L. p. ovata and, to a lesser extent, L. glabra) developed epithelial necrosis of the kidneys, whereas the most susceptible snails contained only intralamellar deposits in the kidneys.

Type of histological lesions in relation to the evolution of infection Ten of the most significant lesions recorded on Day 30 p.i. were taken into account. Twenty-eight groups with abortive infections or evolutive infections were studied, including Lymnaea of the four species and four age categories. The average values of the most significant lesions are noted in Table 2. A discriminant analysis was performed and justified since the values of F in ANOVA were much lower (4.06 was the m a x i m u m ) than those of pseudoF (24.5). The discriminant equation of the axis was y = 0 . 1 8 x2 - 0 . 0 4 x3 - 0 . 5 3 x 4 + 0.20 x7 +0.39 x8 +0.34 x l 0

+0.70 xl l -O.44 x13-O.13 x 1 4 - 0 . 5 1 x15 with y--linear combination of variables that discriminated best between abortive and evolutive infections. The respective coordinates of the infections on the axis were 0.60 and - 0 . 8 0 . The relative importance of each variable was established on centred and reduced data, and thus each coefficient in the equation was weighed. The number code of x (lesions) was presented in Materials and Methods. Abor-

54

P. SINDOU ET AL.

TABLE2

Abortive and evolutive infection of four species of Lyrnnaea: the most significant lesions (selected from ANOVA ~ or principal components analysis) on Day 30 p.i. Lesion

Infections (% of snails_+ SEM ) Abortive

General necrosis of digestive gland Atrophy of male line Atrophy and necrosis of gonads ~ Lamellar deposits in kidneys Epithelial necrosis in kidneys Reconstitution in kidneys Proliferation o f amoebocytes Non-development of amoebocytic tissue L

Evolutive

57 _+ 12 1 +_ 1

48 _+ 17**

55 + 14

30_+ 20

28 +_ 10

2 8 _+ 13

36 _+ 16

28 +_ 13

5 _+ 3

I _+ 1

14 _+ 6

13 + 11

3.1 _+ 2.8

9 _+ 12

13.1 + I 0

'Analysis of variance. **Confidence interval at P = 0 . 0 5 .

tive infections were associated with a high degree of necrosis of the digestive gland, gonadal atrophy with epithelial necrosis, intralamellar deposits in the kidney, epithelial necrosis and reconstitution of the kidney. Conversely, the following lesions were found less frequently in abortive infections: epithelial reconstitution of the digestive gland and alterations of amoebocytic tissue.

Relationship between infection, survival and lesions The data obtained on Days 30 and 45 p.i. were averaged for the prevalence of evolutive infections, survival rates and three types of lesions (generalized necrosis of digestive gland (x2), gonadal atrophy with epithelial necrosis (x7) and developmental absence of amoebocytic tissue (xl 5 ) ). The arc sine transformation of all the prevalences was performed. The data were not illustrated, but principal component analysis (active variables are the frequencies of the three types of lesions, and supplementary variables are infection and survival rates) was performed on the averages obtained in the 16 groups (four snail species at four ages). The first axis was a combination of x2 and x l 5 (46% of variance), and the second was in relation to x7 (34% of variance). The correlations between evolutive infections and Axes 1 and 2 were, respectively, 0.53 and - 0 . 3 8 . The mortality within a group (a species of Lyrnnaea of a given age category ) could not be related to the lesions of the survivors.

FASCIOLA HEPATICA INFECTION AND L Y M N A E A SP.

55

Prevalence (%)

I00 lOva3

Ola3

f

Glc~w, Pall Glc

Ova 1

Tru3

Ova4 60

,

Tru 1 4O

Tru4

Pal2pal3

2O

A iZ .....

0 hi h

moderate

(40-86

I

Z)

(20-39

nil

lOW

z)

Infection

% of Evolutive inf~

[~i]

% DG necrosis

• G atrophy/necrosis

~

% Am. tissue not dvp

Fig. 3. Prevalences of evolutive infections and the frequencies of three types of lesions in four species of Lymnaea infected at four ages. Species names are abbreviated to the first three letters, and numbers refer to increasing age from 1 (new-born) to 4 (adult); Tru4 are adult L. truncatula. Type of lesions: gonad atrophy with epithelial necrosis; general necrosis of digestive gland; no development of amoebocyte-producing tissue.

The most significant data and facts are presented in Fig. 3. The high prevalence of evolutive infection (40-86%) was found in Ova3, Ova2, Gla3, Tru2 (Ova, Gla and Tru stand, respectively, for L. p. ovata, L. glabra and L. truno catula, and the code numbers represent increasing age at which the snails were infected, from newly hatched (1) to pre-adults or adults ( 4 ) ) . Moderate prevalence (20-39%) was found in Glal, Gla2, Pall and Tru4 (P for L. palustris). Low prevalence (0-19%) was found in Ova4, Trul and Tru3. No infection was recorded in Gla4, Pal2, Pal3, Pal4 or Oval. Gonadal atrophy with epithelial necrosis was particularly frequent among heavily infected snails. Digestive gland necrosis was common in moderate to low prevalence infection groups and on one occasion in snails that were not successfully infected (newly hatched L. p. ovata), The developmental absence of amoebocytic tissue was most frequent in moderately infected groups. DISCUSSION

The results we obtained under experimental conditions are subject to much variation under natural conditions. The infections were monomiracidial and

56

P. S1NDOU ET AL.

their success (i.e. evolutive) was monitored 30 and 45 Days p.i.; infections in the field are often plurimiracidial and repeated over time (Rondelaud, 1973; Smith, 1984), and the epidemiological significance of evolution at Day 45 p.i. should be investigated further. The infections were performed independently for each category of snails; we do not know about the decoy effect (i.e. the capture of miracidia by a non-susceptible snail) in populations of Lymnaea comprised of several different species at different ages. In our opinion, the factor that is most subject to criticism is the assessment of mortality in infected and non-infected snails; apparently, breeding conditions were not equally satisfactory for every group of snails (species X ages ). L. glabra or L. p. ovata are good potential hosts in this experiment if they are infected when their shell height is 1 and 2 mm. These results agree with the reports of previous authors on these species (Kendall, 1950; Berghen, 1964; Boray, 1969, 1978; Busson et al., 1982; Bouix-Busson et al., 1983). Conversely, our population of L. palustris is likely to be of very limited importance in the transmission of the parasite to ruminants since it can be infected only when new-born. Previous reports on the latter species are somewhat confusing; most of them concern laboratory experiments and field data are minimal. Parthenitae of F. hepatica developed only in young L. palustris (Kendall, 1950; Boray, 1978; Busson et al., 1982) or in young and adult snails (Czapski, 1962, 1977; Kobulej and Janisch, 1970). This variety of results may be explained by the origin of the snail population used for the experiments and by the shell height at miracidial exposure. A good host should harbour evolutive infections and survive normally during the course of infection. In that respect, L. glabra, L. p. ovata and, to a lesser extent, L. truncatula, are effective hosts. The observed lesions were very different in highly susceptible and non-susceptible snails. Within the former group, the percentage of gonadal atrophy with epithelial necrosis was high, whereas it was not observed in non-susceptible groups. In our opinion, this lesion does not exert any influence on the reproduction of snail populations because the fasciolid infection rate of L. truncatula was often less than 5% in the field and studies in population dynamics revealed roughly constant numbers ofL. truncatula in control stations in the field (Rondelaud, 1982). The role of amoebocyte-producing tissue is open to question. Its developmental absence was mostly recorded in moderate infections. The difficulty in interpreting its fluctuations might be due to the high correlation with digestive gland general necrosis. The experimental investigation had its own limits: a restricted number of populations and a histological technique to determine the susceptibility to infection. It would be of interest to assess the possible role of the age and species of snails in natural populations, and the possibility of the decoy effect in mixed-species clusters of Fasciola intermediate hosts.

FAS(7OLA HEPATICA IN FECTION AND L YMNA EA SP.

57

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pathologie visc6rale chez six esp~ces de limn6es soumises dbs leur naissance/a des infestations monomiracidiennes individuelles. Bull. Soc. Zool., Fr., 115: 331-340. Smith, G., 1984. The relationship between the size of Lymnaea truncatula naturally infected with Fasciola hepatica and the intensity and maturity of the redial infection. J. Helminthol., 58: 123-127. STAT-ITCF, 1988. Manuel d'utilisation. Institut Technique des C6r6ales et des Fourrages, Paris, 239 pp.