Epidemiological studies on fasciolosis in northern Tunisia

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1 FASCIOLOSIS IN NORTHERN TUNISIA 49 Epidemiological studies on fasciolosis in northern Tunisia N. HAMED 1, A. AYADI 1 & H. HAMMAMI 1 * 1 Fungal and Parasitic Molecular Biology Laboratory, Faculty of Medicine, Sfax, Tunisia. * Corresponding author: hayetfss@yahoo.fr ABSTRACT Epidemiological study on fasciolosis was carried out in northern Tunisia. The prevalence of animal liver infection was 3.6 % among 483 livers examined: 12.6% for cattle, 37.1% for sheep and 25.4% for goats. As Bulinus truncatus was an intermediate host of Fasciola hepatica in northern Tunisia, snail habitats were investigated from April 27 to March 28 to determine their ecological characteristics. The molluscs were adapted to a moderate temperature with a maximum of 28.2 C in summer. They also underwent a continuous rainfall and a basic ph of 7.5. During our study, the monthly average density of B. truncatus is 77/m 2. There are two annual generations for this snail. The infection of Bulinus with F. hepatica was observed at 3mm shell size. The percentages of snails containing the most differentiated larval forms ranged from 12.5 to 1% for rediae and from 15.4 to 1% for cercaria. KEYWORDS: Bulinus truncatus, Fasciola hepatica, Tunisia, Epidemiology. RÉSUMÉ Etude épidémiologique de la fasciolose dans le nord tunisien Une étude épidémiologique de la fasciolose a été réalisée dans le nord de la Tunisie. La prévalence d infection des foies d animaux était de 3.6% parmi 483 foies examinés : 12.6% pour les bovins, 37.1% pour les ovins et 25.4% pour les caprins. Bulinus truncatus est un hôte intermédiaire de Fasciola hepatica dans le nord de la Tunisie. Les habitats de ce mollusque ont été étudiés à partir d Avril 27 jusqu à Mars 28 afin de déterminer leurs caractéristiques écologiques. Les mollusques ont été adaptés à une température modérée avec un maximum de 28.2 C en été. Ils ont également subi les effets d une pluviométrie continue et d un ph basique de 7.5. Au cours de notre étude, la densité moyenne mensuelle de B. truncatus est 77/m 2. Ce mollusque a montré deux générations annuelles. L infection de Bulinus par F. hepatica a été observée à la taille de 3mm. Les pourcentages des mollusques contenant les stades larvaires les plus différenciés varient de 12.5 à 1% pour les rédies et de 15.4 à 1% pour les cercaires. MOTS CLES: Bulinus truncatus, Fasciola hepatica, Tunisie, Epidémiologie. Introduction Fasciolosis, an infection caused by the liver fluke Fasciola hepatica (F. hepatica), has been traditionally considered as an important veterinary disease because of the substantial damage and economic losses it causes in livestock, particularly sheep and cattle. The life cycle of liver flukes involves a freshwater snail intermediate host, in which an important asexual multiplication of the parasite occurs. The causative agent of endemic fasciolosis in the cattle, sheep and goat populations of south-western Tunisia is F. hepatica and Galba truncatula has been identified as its only intermediate host [3, 1]. In northern Tunisia, Bulinus truncatus (B. truncatus) is one of the larvae hosts of this trematode [9]. The objectives of this paper are firstly to study the definitive host infection as well as to identify the plants causing its contamination then at a later stage to determine the snail habitat ecological parameters, the density, the annual generation number of the intermediate host B. truncatus and the prevalence of its natural infection. Materials and methods Our investigations were carried out from April 27 to March 28 in Ghar Ettin lake (GPS: , , , ; area: 12m 2 and depth: ranging from 2cm at the edge and 1 cm at the center of the lake) in Sejnane delegation located in south-west of Bizerte governorate in northern Tunisia (fig. 1). p : habitats in Ghar Ettin Lake Figure 1 : Geographical location of Sejnane delegation ( ) in northern Tunisia. THE DEFINITIVE HOST INFECTION Sera and faeces animals were examined in November 27 to check the presence of fluke infection. These animals were living in Sejnane and they are grazing herbs near Ghar Ettin lake. Our investigations about the importance of animal fasciolosis due to F. hepatica in northern Tunisia are based on serological analysis using hemagglutination

2 5 HAMED (N.) AND COLLABORATORS and electrosyneresis techniques to check the specific arc two of fasciolosis: (41 animal s sera from 12 cattle, 18 sheep and 11 goats) and coprology analysis using concentration technique: (52 animal s feces from 6 cattle and 46 sheep). Feces examinations were performed with the qualitative method by simple sedimentation. The study of animal fasciolosis was carried out through the examination of 483 livers inspected in Sejnane slaughterhouse from April 27 to March 28 (from: 38 cattle, 2174 sheep and 2591 goats). The general liver aspect was initially examined, then the hepatic parenchyma was incised and pressed directly towards the bile duct with both hands and the flukes coming out of the ducts. PLANTS SUSPECT AT THE ORIGIN OF ANIMAL INFECTION All plant species growing in the water of the habitat and consumed by livestock were concerned. They were collected with snails in the same quadra of 1m 2 during our study period (April 27 to March 28). Then they were transferred to the Fungal and Parasitic Molecular Biology Laboratory, Faculty of Medicine, Sfax, Tunisia. The botanical identification was established by Pr. Mohamed Chaieb, Botanist at the Faculty of Science, Sfax, Tunisia. Metacercariae were sought by observation under a stereomicroscope. SNAIL HABITAT ECOLOGICAL PARAMETERS obtained from Sejnane meteorological station. The monthly water temperature was measured by using a thermometer at ten hour of the day each time. To determine the water chemical characteristics of B. truncatus habitats in Sejnane lake, samples were taken in November 27 at 25 cm from the lake bank and at 1 cm depth in standing water. They were then transported to the CRDA (Regional Office for Agricultural Development) laboratory located at Bizerte governorate for chemical analysis. The ph was measured with a ph-meter. The calcium (Ca ++ ) and magnesium (Mg ++ ) were measured by titrimetric method with a solution of disodium salt of acetic diaminététra ethylene acid (EDTA) at ph that comprises between 12 and 13 [18]. The determination of alkalinity (the content of free alkali in water, carbonates (HCO3 - )) is based on the neutralization of a sample volume with solution of hydrochloric acid.4 N [6]. The sulfate ion (SO 4 = ) and chloride (Cl - ) were detected by ion chromatography [21]. The sodium (Na + ) and potassium (K + ) were determined by atomic absorption spectrophotometry [17]. The monthly average temperature and the monthly average rainfall of Sejnane region in were April May June July August September October November December January February March Total cattle sheep (37%) (12%) (18.4%) (21.5%) (54.2%) goat (31.3%) (21.8%) (24.3%)(37.4%) (27.3%) 5 (16%) 188 (51%) 27 (2.7%) 6 (13%) 35 (26.2%) 42 (23.9%) 3 (27%) 25 (39%) 213 (28.2%) 145 (62.4%) 224 (22.8%) 13 (39.4%) 164 (15.6%) 2 (4%) 17 (52.7%) 162 (44.4%) 5 (32%) 24 (41.3%) 27 (16.6%) 38 (12.6%) 2174 (37.1%) 2591 (25.4%) Table I: The number of livers examined for cattle, sheep or goats and the prevalence of infection (into brackets) in Sejnane slaughterhouse from April 27 to March 28. ph Ca ++ (mg/l) Mg ++ (mg/l) K + (mg/l) Na + (mg/l) Cl - (mg/l) - HCO 3 (mg/l) SO - 4 (mg /l) Conductivity (µs/m) Total of soluble salts (mg/l) Dry residue (g/l) Table II: Chemical parameters of water in Ghar Ettin Lake, northern Tunisia, in November 27.

3 FASCIOLOSIS IN NORTHERN TUNISIA 51 DENSITY, ANNUAL GENERATIONS AND PREVALENCE OF INFECTION OF THE INTERMEDIATE HOST Monthly investigations on the intermediate host were carried out from April 27 to March 28 in Ghar Ettin lake. Snail collections were always made in the single area of the lake. They were collected by hand, under stones, herbs, leaves, branches and immersed plastic bags. They were collected at 25 cm from the lake bank and at a depth varying between 1 and 25 cm. Counting snails was performed on a surface of 1m 2 according to quadrat method. The snails collected were transported to the laboratory in isothermal conditions. These snails were identified and counted to determine their monthly density/m 2. The shell height was measured at the nearest.5 mm to identify the age category of snail and to determine their annual generations. All snails were measured from the apex to the anterior margin of the shell and were grouped with their shell length (l) according to Lahlou and Berrada [15]: juveniles (< 3 mm), preadults (3-6 mm) and adults (> 6 mm). The new generation is observed by the presence of juvenile class. SNAIL HABITAT ECOLOGICAL PARAMETERS The subsoil of the district studied was clayish. In the region of Sejnane, the climate was inferior humid. The mean monthly air and water temperatures and the mean annual rainfall of Sejnane region were presented in fig. 2. From April 27 to March 28 a total of 16 natural infected Bulinus truncatus were dissected and examined by microscopic technique to calculate the infection prevalence as well as the percentage of snails containing the most differentiated larval forms: sporocyst, redia and cercariae. Results THE DEFINITIVE HOST INFECTION The diagnosis of animal fasciolosis in November 27 by serology was positive for 21 animals among 41 (51.2%): 6 cattle among 12 (5%), 12 sheep among 18 (66.7%) and 3 goats among 11 (27.3%). The results of coprology analysis was positive for 25 animals among 52 (48.1%): 3 cattle among 6 (5%) and 22 sheep among 46 (47.8%). For each month, the number of livers examined for cattle, sheep or goats and the prevalence of infection were given in table 1. The prevalence of animal liver infection was 3.6 %. It was 12.6% for cattle, 37.1% for sheep and 25.4% for goats in Sejnane slaughterhouse from April 27 to March 28. Sheep and goats show continuous infection with two high infection periods. The first period is summer with a maximum of 54.2% for sheep in August and 37.4% for goats in July. The second period is winter with a maximum of 62.4% in December for sheep and 44.4% in February for goats. PLANTS SUSPECT AT THE ORIGIN OF ANIMAL INFECTION The major plants that have been collected in Ghar Ettin Lake during our study period were Apium nodiflorum, Phragmites communis and Ampelodesma mauritanica. The examination of leaves and steams of plants did not show the presence of metacercariae. Fig. 2: Ombrothermic diagram showing seasonal changes of monthly average air temperature ( ), water temperature (p) and monthly average rainfall ( ) in the region of Sejnane, northern Tunisia, throughout the year Sejnane region during our study period from April 27 to march 28 was characterized by a low air temperature ranging from 11.5 to 28.2ºC and frequent precipitation. The global quantity of precipitation was 74.3 mm. Water temperatures ranging from 9.5 to 24.2 C follows the same fluctuations as air temperatures, but they were always lower. There is a high dissociation between temperature and precipitation values. This is shown through the diagram of the first one which is under that of the second one; therefore there is a humid period throughout our study year. The water chemical characteristics of B. truncatus habitats were given in table 2. The magnesium content of water in Ghar Ettin lake was low (29.7mg/l) in comparison with that of calcium (134.4 mg/l). Sodium occurs in the form of chloride, sulfate and carbonate. In our water samples, the sodium concentration was 37.3 mg/l, the bicarbonate was 122 mg/l and the sulfate was higher with a concentration of 144 mg/l. A low value of 73.8 mg/l was determined for chloride in our water samples.

4 52 HAMED (N.) AND COLLABORATORS DENSITY, ANNUAL GENERATIONS AND PREVALENCE OF INFECTION OF THE INTERMEDIATE HOST The monthly average density of B. truncatus in Ghar Ettin Lake is 76.6/m 2 and their monthly density variations from April 27 to March 28 are represented in fig. 3. In Ghar Ettin Lake, we note the continuous presence of B. truncatus except in December. There are two peaks: the first one in October with a density of 169/m 2 and the second in January with a density of 131/m Bulinus infection was continued for preadults and adults during our study period except in January. The percentages of snails containing the most differentiated larval forms: sporocyst, redia and cercariae are given in table 5. Snails with both most differentiated larval stages rediae and cercariae were observed in two periods: from May to June then from October to November showing the presence of two F. hepalica parasitic cycles. Discussion THE DEFINITIVE HOST INFECTION In our study, serological and coprological investigations on animal specimen from Sejnane region revealed an approximately rate of 5% in cattle for both analysis, of 66.7% and 47.8% in sheep and 27.3% in goat. Fig. 3: Bulinus truncatus density per m 2 of habitat in the year There are two major peaks of juvenile class one at July and a second in November indicating the existence of two annual B. truncatus generations (fig. 4). The method of examination of liver in slaughterhouses is another way to know better the epidemiology of fasciolosis [2]. The liver infection rates in our study were lower than those reported by serology and coprology: 12.6% for cattle, 37.1% for sheep and 25.4% for goats. This could be explained by the fact that in recent infections, fluke shedding eggs may be present but the externally well-visible lesions are not developed [19]. Usually sheep and cattle are preferential hosts; goats are less receivers and less parasitized. In Kirkuk, Iraq slaughter house study, the rate of liver flukes was lower than our study: cattle (2.63%), sheep (.5%) and goats (.43%); for a respectively total of livers examined: 4233, 9223 and 2855 [13]. This lower infection rate in liver infestation was in accordance with other study such as in Malatya Province, Turkey: 5.45 % for a total of 513 cattle and 4.42% for a total of 1763 sheep [2]. PLANTS SUSPECT AT THE ORIGIN OF ANIMAL INFECTION Fig. 4: Bulinus truncatus generations during study periods (27-28). For each shell height class, the number of snails concerned by dissection, the number of infected snails and the prevalence of infection during 12 months from April 27 to March 28 were given in table 3. There was snail infection at 3mm size but later this infection didn t depend on snail shell length. The total number of infected snails, the number of infected juveniles, preadults and adults were shown in table Apium nodiflorum, Phragmites communis and Ampelodesma mauritanica, consumed by animals and growing in water of the habitat, were suspected to be at the origin of animal contamination. Apium nodiflorum was described as a metacercaria carrying plant in the oasis of Gafsa [11] and in the Limousin region in central France [7]. SNAIL HABITAT ECOLOGICAL PARAMETERS Information on the ecology of B. truncatus intermediate host of F. hepatica in northern Tunisia will certainly be useful as far as the control of this snail and the reduction of the prevalence of its infestation are concerned. The Ghar Ettin Lake was chosen for snail studies, as natural infection of B. truncatus with F. hepatica was detected there [9].

5 FASCIOLOSIS IN NORTHERN TUNISIA 53 Height of the snails (mm) Number or mean of Number of infected snails Infection prevalence % dissected snails : absence of snails Table III: The monthly prevalence (%) of snails with their heights in northern Tunisia during the study period from April 27 to March 28 Sejnane climate has a summer maximum temperature of 28.2 C and a continuous rainfall during our study period. These were considered as important factors to create favorable conditions for the biological life of snails. This result pertains to the findings of other researchers [1, 8]. The chemical composition of water is favorable for the survival of B. truncatus. The amount of calcium carbonate ions (Ca ++, HCO 3- ) is taken as a measure of the hardness of water and has an influence on shell constitution of snail. In our water samples, the sodium concentration of 37.3 mg/l is favorable for the survival of snail and this is maintained by Malek [16]. This author confirmed also that water with a high concentration of sodium ions compared with other cations, especially calcium, is not favorable for snails [16]. Bicarbonate occurs in the form of calcium, magnesium, sodium and potassium salts and is usually taken as a measure for the alkalinity [4]. Because of the high content of bicarbonate anion (122mg/l) in our samples, the ph of the water in Ghar Ettin was adequately buffered with a range of 7.5. The sulfate content (144mg/l) in Sejnane lake water was compared with that of Khuzestan south-western Iran and it contradicts with a lower sulfate content such as the Sardasht Stream (29.8 mg/l) and a higher sulfate content such as the Khorramshahr canal (19.6 mg/l). Thus, water in Sejnane might be within the tolerance limits of sulfate ions for B. truncatus. Therefore we have shown that B. truncatus in this lake isn t tolerant of high salinity (concentration of chlorides was73.8mg/l) which is confirmed by other studies that consolidated the fact that B. truncatus is absent in high salinity of their water [23]. The absence of this species from Lake Habbaniya in central Iraq may also be due to high salinity, which in the corresponding work has ranged in the past from 12 to 18 mg/l [23]. DENSITY, ANNUAL GENERATIONS AND PREVALENCE OF INFECTION OF THE INTERMEDIATE HOST According to theoretical studies, chance plays an important role in the variations of a species abundance in the edges of its distribution area. When environmental conditions (climate, etc.) are optimum, the regulation of the population density which is dependant on these factors plays a dominant role [22]. The total number of snails collected during the 12 months of the study is 919/m 2. The highest snail densities were observed from September to November and have disappeared at December whereas the lowest values were noted from June to August. These show that high temperature is not favorable for Bulinus reproduction. This result is compared with the one found in Egypt since March is the most favorable month for the population explosion of B. truncatus, and as weather conditions are very poor from May to August [5]. In Iraq the snails are most numerous during summer and autumn months and they are scarce during winter and spring months [23]. In spite of this finding, even if the presence of juvenile snails is noted during three separate months: July, September and November, the data presented in figure 3 and 4, show the presence of two annual generations. This interpretation is confirmed by the existence of two parasitic cycles for F.

6 54 HAMED (N.) AND COLLABORATORS Number of infected snails Months April May* June July August September October November December January February March juveniles 1 - Preadults Adults Total : absence of snails *: in May two snail samples were taken: at the beginning and at the end of the month. 6.5: is the mean number of these two samples. Table IV: Number of infected Bulinus truncatus: juveniles, preadults and adults collected in Ghar Ettin Lake, Sejnane, Months April May June July August September October November December January February March Total number of infected snails 4 The percentage (%) of snails containing the most differentiated larval form (9+4)/2= 6.5* Sporocyst - Rediae Cercariae 4/4 6.5/6.5 1/6.5 (15.4) 1/8 (12.5) 3/8 (37.5) 8/8 6/6 7/8 (87.5) 8/8 1 2/12 (16.66) 12/ /2 1/2 (5) 4/4 4/4 - : absence of snails; : sporocyst + germinative masses; : daughter redia. *: in May two snail samples were taken: at the beginning and at the end of the month. 6.5: is the mean number of these two samples. Table V: Total number of infected snails and the percentage of snails containing the most differentiated larval forms: sporocyst, redia and cercariae in natural infested Bulinus truncatus from April 27 to March 28

7 FASCIOLOSIS IN NORTHERN TUNISIA 55 hepatica shown by a high percentages of snails with major differentiated stages rediae and cercariae in spring and the beginning of summer (from April to July) and in autumn (from September to November) (table 5). two generations were noticed in this lake following the presence of juvenile class. These results correlated also with two most infection periods of animals in Sejnane especially goats and sheep in winter and summer. Besides, in Sejnane region, Jemli et al. demonstrated the presence of two parasitic cycles: one in spring (April) and another in autumn (November). These periods provided good conditions for a longger lifespan of metacercariae in pastures. Our results correlated with the Haouz area (Morocco); snails infected with F. hepatica were found in February, June, July and August but their infection rate did not exceed 3 %, and the main periods for transmission of fasciolosis were spring and autumn [12]. Our results differ from other studies which showed that the number of new Bulinus generation produced each year ranged between three and five [1]. It should be noted that in our results Bulinus hatching occurred in high temperature (26.2 C) of July and the low temperature (14.9 C) of November, which contradicts other authers claim that temperature had the greatest effect on the activity and productivity of Bulinus [1]. As B. truncatus acts as an intermediate host in the life cycle of F. hepatica, the existence of a two-generation pattern for this snail indicates that two parasitic cycles could develop within snails over the year. The surprisingly high prevalence of natural infection 38.8% with F. hepatica in B.truncatus [9] may be explained by environmental conditions favoring the development of snail populations and the transmission of the parasite in Sejnane region for the definitive hosts. We have determined the prevalence of natural infection with shell length of these populations of B. truncatus over the year to check the age at which snails were most infected with F. hepatica in order to implement control measures. Collected shells of B.truncatus ranged from 2 to 12 mm in height, however, we have found that its natural infection started from 3mm. Control measures could start from this age (3mm) to fight against both the parasite and the snail. The percentage of snails harboring the most differentiated larval stages redia and cercaria was higher than those harboring the earlier developmental larval stages sporocysts. This may be explained by the poor efficiency of microscopic technique. Therefore a DNA-based test may play an important role for the identification of snails infected with larval stages [14]. Molluscs with the most differentiated larval stages redia and especially cercariae were the most detected (1%) in the months of April, May, July, September, October and November. This may make any treatment easier at these periods of the year. These data will be a tool of utmost importance for the snail treatment and parasite control project against these parasitoses. Acknowledgements This study was funded by the Tunisian Ministry of Higher Education and Scientific Research. References 1. ARFAA F., SAHBA H., MASSOUD J.: Studies on the biology of Bulinus truncatus under laboratory conditions. Iranian J. Publ. Hlth., 1976, vol.5, N ASSOGBA M. N., YOUSSAO A. K. I.: Epidemiologie de la fasciolose à Fasciola hepatica (Cobbold, 1885) de la dicrocoeliose et de la paramphistomose bovine au Bénin. Ann. Méd. Vét., 21, 145, AYADI A., MAKNI F., BEN SAÏD M.: Etat actuel de la fasciolose en Tunisie. Bull. Soc. Fr. Parasitol., 1997, 15, CHU K. 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