RETRACTED ARTICLE: Occurrence, spread and control measures of Bothriocephalus acheilognathi (Bothriocephalidae: Cestoda)

Abstract

Fish infections can be readily detected at autopsy, with the recovery of entire tapeworms followed by microscopic examination of the scolex. The examination of faecal material may reveal detached segments from adult tapeworms or eggs, which possess an operculum at the apex. The presence of Bothriocephalus acheilognathi may also be achieved by a squash plate method. Glass slides or plates are used to flatten the intestinal tract and the worms are detected by reflected light and low-power microscopy. Although mature parasites may be conspicuous within the intestine of infected fish, the detection of light infections or presence of juvenile parasites and plerocercoids requires microscopic examination. While these infections may hold little importance to the disease status of individuals, detection may be critical for effective disease control and limiting the spread of infected fish. The pathology may be divided generally into: (i) damage caused by scolex attachment; and (ii) damage caused by the presence of strobila within the intestine lumen. Other organs may exhibit signs of pathological change. For example, infected fish can show signs of nutritional deficiency, with atrophy of hepatocytes within the liver. In severe cases, these changes are consistent with starvation. B. acheilognathi causes a number of physiological changes in juvenile fish. These include: (i) protein depletion; (ii) altered digestive enzyme activity; (iii) elevated muscle fatigue in heavily infected hosts; and (iv) mortality of young fishes. B. acheilognathi can have pronounced detrimental effects on fish. These include severe damage to the intestinal tract, physiological disturbance, reduced growth, condition loss and death. Records of 100 % mortality in hatchery reared common carp (Cyprinus carpio) highlight the pathogenic potential of this parasite.

Introduction

The Asian Tapeworm Bothriocephalus acheilognathi Yamaguti 1934 (Cestoda: Bothriocephalidea), is the most important pathogenic cestode of cyprinid fish, which causes bothriocephaliasis and one of the most dangerous helminth parasites of cultured carp (Bauer et al. 1977; Nie and Hoole 2000). The parasite has also been recorded in a range of other freshwater teleost fishes, prompting concern of the disease in wild fish populations (Clarkson et al. 1997; Heckmann 2000). It is listed as a ‘Pathogen of Regional Concern’ by the US Fish and Wildlife Service (2010). B. acheilognathi has been reported under more than 20 different specific names and the most frequently used are Bothriocephalus gowkongensis Yeh 1955 and Bothriocephalus opsariichthydis Yamaguti 1934 (for a list of synonyms—see Kuchta and Scholz 2007). According to Pool and Chubb (1985) and Pool (1987), all descriptions of Bothriocephalus tapeworms from cyprinid hosts represent the same parasite, differing only in length and the shape of the scolex because different methods were used to fix the worm. B. acheilognathi is indigenous to East Asia, but has spread rapidly throughout the world with the trade of fish. The parasite has now been recorded from every continent excluding Antarctica. The ability to successfully colonize new geographical regions has been facilitated by its simple, two-host life cycle (involving common copepod species as an intermediate host) and euryxenous host specificity (very wide range of suitable fish hosts). This has led to the transmission and establishment of B. acheilognathi to many new host species in areas where it has been introduced (Scholz 1999; Salgado-Maldonado and Pineda-Lopez 2003). Once established it may endanger native fish populations, including ecologically sensitive species and fishes that are phylogenetically unrelated to those in which it was introduced (Font and Tate 1994; Dove and Fletcher 2000). B. acheilognathi can have pronounced detrimental effects on fish. These include severe damage to the intestinal tract, physiological disturbance, reduced growth, condition loss and death. Records of 100 % mortality in hatchery reared common carp (Cyprinus carpio) highlight the pathogenic potential of this parasite.

Description

Bothriocephalus acheilognathi typically measures between 3.5 and 8 cm in length and up to 4 mm in width (Yeh 1955), although specimens of 60 cm and even 1 m (Table 1) have been observed (Baer and Fain 1958; Granath and Esch 1983a) Size is a highly variable morphological parameter as it depends on: (i) Ecological conditions (Nedeva and Mutafova 1988); (ii) Host size (Davydov 1978); (iii) Host species (Molnar and Murai 1973a; Granath and Esch 1983a); (iv) Host age; and (v) Intensity of infection (Davydov 1978). Upon relaxation, parasites can also increase in length by a factor of 1.5–2 (Pool 1987). Consequently, parasite size is not a valid feature on which to base identification, despite earlier beliefs (Molnar and Murai 1973a). An important morphological characteristic of B. acheilognathi is its heart-shaped scolex, with a weakly developed apical disc and a pair of deep, slit-like grooves (bothria) positioned dorsoventrally along the scolex (Figs. 1, 2). The scolex is much wider than the first body segments (proglottides). The strobila (body) of the tapeworm consists of numerous proglottides each containing one set of reproductive organs. The shape of these segments differs with maturity. Immature segments lacking fully developed genital organs are always wider than they are long; whereas more developed gravid segments bearing eggs are rectangular and longer than they are wide. However, contraction and relaxation of the segments also causes extreme variation in the length and width ratio of the strobila (Brandt et al. 1981). The male reproductive system is formed by numerous spherical testes situated in the medulla (the region internal to the inner longitudinal musculature). A muscular cirrus sac localized anterior to the ovary opens on the dorsal side of segments into a common genital atrium, which is situated alongside the median line of the body. The female reproductive system is composed by a bilobed ovary situated near the posterior margin of each segment. The vagina, which is short and slightly sinuous, opens into the common genital atrium posterior to the male genital pore. Vitelline follicles are very numerous, circumcortical and confluent between segments. The uterus is saccular, spherical to oval, and opens on the ventral side in the anterior third of the segment. The eggs are thick-walled, operculate (i.e. with a cap—the operculum) on a narrower pole, and usually unembryonated (without a formed embryo) when released into the water.

Table 1.

Characteristics of Bothriocephalus spp. from freshwater Fishes (Measurements in micrometers)

	1	2	3	4
Species	Bothriocephalus pearsei	B. claviceps	B. formosus	B. acheilognathi
Host	Cichlasoma cichlasoma	Anguilla rostrata	Percopsis omiscomaycus	Eleotris Pimephales,
Locality	Mexico	Canada	USA	Hawaii, Canada
Feature/Authority	Scholz et al. (1996a)	Scholz (1997)	Scholz (1997)	Scholz (1997)
Total length (µm)	26–32	54–137	21–33	22–35
Maximum width (µm)	0.91	2.19	0.89–1.06	0.71–1.43
Mature segments (L)	288–496	232–448	325–530	200–508
Mature segments (W)	528–873	934–1,300	853–1,025	576–1,320
Length/width ratio	1:1.0–3.1	1:2.3–5.6	1:1.9–2.9	1:1.6–5.0
Gravid segments (L)	345–1,060	240–487	280–812	280–1,015
Gravid segments (W)	496–914	893–2,110	812–1,060	480–1,430
Length/width ratio	1:0.83–2.4	1:2.2–7.4	1:1.0–3.0	1:0.45–4.5
Scolex (L)	720–880	1,180–1,360	384–536	569–731
Scolex (W)	224–344	232–344	134–202	660–1120
Terminal disc (W)	173–224	200–344	99–131	232
Neck (W)	154–173	152–200	118–147	160–504
Testes number	28–51	39–80	24–60	33–56
Testes (L)	28–65	56–125	28–49	34–68
Testes (W)	21–46	40–86	26–49	26–57
Cirrus-sac (L)	55–80	74–154	59–86	35–81
Cirrus-sac (W)	55–80	68–93	65–81	39–84
Ovary (L)	50–84	234–751	179–288	336–576
Ovary (W)	Absent medially	77–131	58–122	70–144
Distribution of vitelline follicles	14–45	Absent medially	Confluent	Confluent
Vitelline follicles (L)	13–43	17–66	18–52	19–53
Vitelline follicles (W)	54–67	12–48	14–40	15–43
Eggs (L)	32–39	54–63	49–54	45–59
Eggs (W)		31–37	32–35	27–41

	5	6	7	8
Species	Bothriocephalus acheilognathi	B. claviceps	B. claviceps	B. acheilognathi
Host	Schizothorax and Cyprinus	Anguilla japonica	Anguilla rostrata	Schizothorax and Cyprinus
Locality	Srinagar (Jehlum)	Japan (Lake Biwa)	North America	Srinagar (Jehlum and Dal Lake)
Feature/Authority	Ara et al. (2000)	Scholz et al. (2004)	Scholz et al. (2004)	Akhter et al. (2008)
Body length (µm)	175	139–161	155	170
Maximum width (µm)	3.0	2.21–2.37	2.9	2–3
Scolex (L)	1.44–1.49	1610–1640	460	0.16
Scolex (W)	1.15–1.17	432–568	300	0.18
Bothria (L)	0.58–0.60	1,180–1,340	–	0.2–0.05
Neck (W)	1.0–1.03	220–316	–	0.06–0.05
Testes (L)	0.02–0.04	–	–	0.02–0.06
Testes (W)	0.01–0.02			0.02–0.03
Ovary (L)	0.25–0.27	–	–	0.26–0.28
Ovary (W)	0.04–0.07	–	–	0.04–0.07
Eggs (L)	0.049–0.051	52–70	58–63	0.058
Eggs (W)	0.021–0.024	34–43	37–40	0.062

L Length; W Width

Fig. 1.

Life cycle and Morphology of Bothriocephalus acheilognathi. a Scolex b Total view (segmented body) c Mature segments (proglottis)

Fig. 2.

Scanning electron micrographs of the scolex of Bothriocephalus acheilognathi (Bar = 100 µm)

Life cycle and transmission

B. acheilognathi has a simple two host life cycle, involving a planktonic copepod (Copepoda: Cyclopidae) as an intermediate host (Fig. 1). In favorable conditions, the life cycle may be completed in about 1 month. Eggs shed by adult parasites into the gut lumen are released into the water with faeces. Depending on water temperature, an embryo (six-hooked oncosphere or hexacanth) is formed within the egg in a few days. The larva (coracidium) is surrounded by ciliated cells which enable its active movement in the water after hatching from the egg. A number of copepods are suitable intermediate hosts in both experimental and natural conditions. These include species of Acanthocyclops, Cyclops, Macrocyclops, Megacyclops, Mesocyclops, Thermocyclops and Tropocyclops (Marcogliese and Esch, 1989) which are considered common in most fresh waters throughout the world (Pool 1984). Other planktonic crustaceans, such as diaptomids and cladocerans, are not suitable intermediate hosts (Molnar, 1977). After ingestion, the coracidium loses its ciliature and penetrates the gut into the body cavity where is develops from an oncosphere into a plerocercoid (previously also called procercoid Chervy 2002 for terminology of cestode larvae). Larval development is completed in a few weeks depending on the water temperature: within 21–23 days at 28–29 °C (Liao and Shih 1956), but 1.5–2 months at 15–22 °C (Davydov 1978). The life cycle is completed when fish ingest infected copepods. Once established within the intestine of a suitable fish, egg production may begin in as little as 20 days (Liao and Shih 1956). It has been shown that transmission of adult parasites can occur from fish to fish via predation by piscivorous fish on infected prey, a phenomenon known as postcyclic transmission (Odening 1976; Hansen et al. 2007). Local spread caused by aquatic birds, such as Anas platyrhynchos and Chlidonias niger, was assumed to take place based on experiments conducted by Prigli (1975) and field observations (finding of B. acheilognathi in Ixobrychus minutus) by Borgarenko (1981), but this mode of parasite dissemination needs verification. There are also records of the tapeworm in an amphibian (axolotl Ambystoma dumerilii—Garcia-Prieto and Osorio Sarabia 1991) and a snake Thamnophis melanogaster (Perez-Ponce de Leon et al. 2001) although these records may represent only accidental infection.

Definitive (fish) hosts

The most suitable hosts of B. acheilognathi are cyprinids, especially the common carp (C. carpio) and grass carp (Ctenopharyngodon idella). However, the parasite has been reported from approximately 200 species of freshwater fishes, representing ten orders and19 families (Salgado-Maldonado and Pineda-LOpez 2003; R. Kuchta—unpublished data). Nevertheless, maturity of the worm may be reached in only a proportion of these fish species (Dove and Fletcher 2000). Holmes (1979) identified three classes of host, in terms of their ability to allow the maturation of parasites: (i) ‘required hosts’; (ii) ‘suitable hosts’; and (iii) ‘unsuitable hosts’. In ‘required hosts’ parasites usually obtain full maturity. In ‘suitable hosts’ parasites may gain sexual maturity, but are only found in small numbers, while in ‘unsuitable hosts’ parasites may establish but do not reach maturity. Consequently, although a parasite may infect many fish species, the maintenance of the parasite population may rely on a much narrower range in which reproduction takes place (Riggs et al. 1987). Small fish are more commonly and intensively infected with B. acheilognathi than large hosts (Leong 1986). Brouder (1999) detailed a strong negative correlation between size of host and infection intensity of B. acheilognathi.

Geographical distribution

B. acheilognathi was originally described from a small cyprinid fish, Acheilognathus rhombeus (Temminck & Schlegel) (Cypriniformes: Cyprinidae), from Lake Ogura, Kyoto Prefec-ture, Honshu, in Japan (Yamaguti 1934). The parasite is endemic in China, Japan and the Amur River, Asia (Yamaguti 1934; Yeh 1955; Dubinina 1971). Initial movements of B. acheilognathi were linked closely with the spread of carp westwards from Japan and China to Europe during the 1960s and 1970s (Minervini et al. 1985). The spread of B. acheilognathi throughout most parts of the world has been well documented (Bauer and Hoffman 1976) and represents one of the best examples of parasite translocation through man-assisted activities (Bauer and Hoffman 1976; Dove and Fletcher 2000). The rapid spread of this parasite has been assisted by the trade in many cyprinid species for: (i) aquaculture (Minervini et al. 1985); (ii) the ornamental fish industry (Edwards and Hine 1974; Evans and Lester 2001); (iii) aquatic weed control (Maitland and Campbell 1992); (iv) mosquito control (Dove and Fletcher 2000); and (v) the fishing bait industry (Heckmann 2009). The tapeworm was introduced between 1954 and 1962 into the European part of the former USSR as a result of uncontrolled imports of grass carp and common carp from the River Amur and China. Infections of B. acheilognathi became widespread in farmed carp as well as in a variety of wild fishes (Malevitskaya 1958; Radulescu and Georgescu 1962; Bauer and Hoffman 1976). The increased importance of carp for food and weed control led to the rapid spread of B. acheilognathi throughout Europe. By 1970–1975, the tapeworm had colonized several countries of Central and Eastern Europe (Austria, Bulgaria, former Czechoslovakia, Germany, Hungary, Poland and former Yugoslavia—Buza et al. 1970; Petkov 1972; Bauer and Hoffman 1976; Minervini et al. 1985). The worms, originally described in Japan and China, were apparently already introduced populations, while the natural host and geographical origin of this tape worm is the grass carp (Ctenopharyngodon idella) of the Amur River. By 1954–1962, infections had become widespread in farmed fish (in European and Chinese carp) as well as in a variety of wild fish in both the Asian and European USSR (Bauer and Hoffman, 1976). By 1970–1975 records of infections came from Hungary, Yugoslavia, East and West Germany (Molnar and Murai 1973a; Korting 1974; Bauer and Hoffman 1976), and by 1980, worms had become prevalent in France (Denis et al. 1983), Britain (Andrews et al. 1981), South Africa (Van As et al. 1981), USA, and Mexico. Worms were introduced into Mauritius via South Africa (Van As, pers. comm.). New records also came from Asia; Israel, Iraq (Khalifa 1986), Malaysia (Fernando and Furtado 1964), Sri Lanka (Fernando and Furtado 1963) and Korea (Kim et al. 1985). Allied species: B. aegypticus, known only from Egypt (Amin 1978), and B. kivuensis, which occurs in Central and South Africa (Baer and Fain 1960; Mashego 1982). The origin of African populations of B. acheilognathi, first described as Bothriocephalus (Clesthobothrium) kivuensis from barbells (Barbus spp.) by Baer and Fain (1958) in Zaire (now the Democratic Republic of the Congo), is not clear. The tapeworm has since been reported from Egypt and South Africa (Rysavy and Moravec 1975; Brandt et al. 1981). The tapeworm was introduced to the New World in 1965 with grass carp imported from China to Mexico (Lopez Jimenez 1981), then to the USA (Texas) in 1975 and Canada (British Columbia) in 1983 (Hoffman 1999; Choudhury et al. 2006). It was suspected that bait minnows (Plagopterus argentissimus) represented the main source of the tapeworm infecting fish populations in the western USA (Heckmann 2000, 2009). Bothriocephalus acheilognathi has been reported in 13 states of the USA (Hoffman 1999; Choudhury et al. 2006), and from the Great Lakes area (Marcogliese 2008). There is only one published record of B. acheilognathi from South America (Rego et al. 1999), most probably as a result of the import of carp from Europe to Brazil (Cornelio Procopio, Parana). Records of B. acheilognathi from India, Iraq, Israel, Korea, Malaysia, the Philippines, Sri Lanka and Turkey confirm its wide distribution in Asia (Paperna 1996; Hoffman 1999). In Australia, B. acheilognathi was detected in goldfish (Carrasius auratus (L.)) and koi carp (Cyprinus carpio (L.)) (Langdon 1992). The tapeworm is now widely distributed in many native finfish species in eastern Australia (Dove and Fletcher 2000). The parasite was imported to New Zealand with grass carp but was then eradicated during quarantine (Edwards and Hine 1974). The ability of B. acheilognathi to colonize isolated geographical localities has been confirmed by records of the parasite in Puerto Rico, Hawaii and Mauritius and remote subterranean sink- holes (cenotes) in Yucatan (Bunkley-Williams and Williams 1994; Font and Tate 1994; Scholz et al. 1996a). B. acheilognathi has so far been recorded in six continents as a result of the introduction of cyprinids (grass carp, carp), and guppies (mosquito-fish - Gambusia and Poecilia) by man to control mosquito larvae (Hoffman and Schubert 1984; Salgado-Maldonado and Pineda-LOpez 2003). However, the distribution area is limited between 60°N and 40°S. The spread of B. acheilognathi further north or further south in the southern hemisphere is unlikely, because the cestode is thermophilic with an optimum temperature between 22 and 25 °C (Bauer et al. 1981; Granath and Esch 1983a; Hanzelova and Itrian 1986).

Importance of the disease

B. acheilognathi is an important pathogen in aquaculture in Asia and Europe (Bauer et al. 1981; Heckmann 2009). Losses of juvenile fish, with up to 100 % mortality, occur in hatchery ponds. In commercial carp farms, fry (length 38–42 mm) can be infected 28-29 days after hatching (Hanzelova and Itrian 1986). The susceptibility of fry is probably because copepods make up a large proportion of the diet of these fish, and the limited space within the intestinal tract to accommodate these large parasites. Heavy tapeworm burdens cause blockage of the intestine and severe pathological changes, leading to reduced growth, condition and survival (Scott and Grizzle 1979; Granath and Esch 1983b; Hoole and Nisan 1994; Hansen et al. 2006). The tapeworm has also been the cause of disease problems in ornamental fish farms in Australia and Central Europe, involving Poecilia reticulata and Xiphophorus maculates (Evans and Lester 2001; R. Kuchta, unpublished data) and mortality of koi carp (Han et al. 2010). Far less information exists on the impact of B. acheilognathi in wild fish populations. The introduction of this alien tapeworm to new localities can endanger native fish species (Dove and Fletcher 2000; Heckmann 2000, 2009; Salgado-Maldonado and Pineda-LOpez 2003). This may be particularly serious in fish that attain only a small size at maturity, with potential for reduced recruitment, growth, fitness and survival. However, equilibrium between host and parasite can develop in a relatively short period, limiting disease impacts (Hoffman 1999). It is recognized that identification and evaluation of the effects of parasites in wild fish populations is problematic, as sick fish are rapidly removed by predators, water flow and necrophages (Blanc 1977).

Materials and methods

The methodology was almost same in all helminths, but it was slightly modified for different taxonomic group in order to get best results as per international standards. SEM of cestodes, were fixed in laboratory and were then taken to USIC (University of Kashmir) for Scanning Electron Microscopy study. Specimens were washed using distilled water, then fixed for about 1/2 h at 4 °C in 1 % osmium tetroxide in 0.1 M sodium cacodylate–HCl. They were washed for about 1 h in several changes of cold buffer (0.1 M sodium cacodylate containing 3 % sucrose and 0.1 M CaCl₂). Post-fixation was carried out for about 2 h using 2.5 % glutaraldehyde buffered to pH 7.3. The specimens were left in washing buffer overnight and were then dehydrated using an ascending series of alcohol solutions. Critical point drying was carried out using a Hitachi Critical Point Dryer (HCP-2, Hitachi Koki Co., Ltd. Tokyo Japan) with carbon dioxide as the transition fluid. The specimens were then coated with gold and examined with a Hitachi Toyota Japan (Model-S3000H) at 80 kV.

For histopathological sections, standard methods (Galigher and Kozloff 1971) were employed for the examination of the infected host intestinal tissue. Specimens that have been stored in 70 % ethanol were transferred to 10 % buffered formalin (v/v). The infected host tissue was dehydrated and blocked in paraffin. The blocks were sectioned at 4–6 micrometers (µm), placed on glass slides and stained with Harris hematoxylin and eosin (H & E) and then viewed with an LSM laser (Carl Zeiss, Thornwoood, New York) equipped compound light microscope. Representative pictures were taken with an attached digital camera at various magnifications and stored in a memory disk for future reference. H and E is a standard stain for viewing pathological tissue while Mallory’s trichrome is used for viewing specific cells characteristic of tissue inflammation.

Results and discussion

Diagnosis of infection and clinical signs

Infections can be readily detected at autopsy, with the recovery of entire tapeworms followed by microscopic examination of the scolex (Figs. 2, 3). The examination of faecal material may reveal detached segments from adult tapeworms or eggs, which possess an operculum at the apex. The presence of B. acheilognathi may also be achieved by a squash plate method. Glass slides or plates are used to flatten the intestinal tract and the worms are detected by reflected light and low-power microscopy. Although mature parasites may be conspicuous within the intestine of infected fish (Fig. 3), the detection of light infections or presence of juvenile parasites and plerocercoids requires microscopic examination. While these infections may hold little importance to the disease status of individuals, detection may be critical for effective disease control and limiting the spread of infected fish. Infected fish may become sluggish and swim close to the water surface (Hoole 1994). This may be accompanied by in appetence, slow growth, condition loss, emaciation and signs of anaemia (Liao and Shih 1956; Edwards and Hine 1974; Scott and Grizzle 1979; Hoole and Nisan 1994; Sopinska and Guz 1997). Infected fish may be more susceptible to secondary bacterial infections due to the debilitating effects of the parasite (Clarkson et al. 1997) and destruction of the epithelial layer of the intestine (Bauer et al. 1981). Heavy tapeworm burdens can cause the body of infected carp fry to become noticeably distended and swollen (Scott and Grizzle 1979; Brandt et al. 1981). In very small fish, the movement of tapeworms may be seen through the body wall prior to dissection. The intestinal tract of infected fish is often grossly enlarged, very thin-walled and obviously occluded. In such cases the gut wall can be stretched to the point of transparency, revealing the white-coloured worms within (Fig. 3) and even allowing individual body segments of parasites to be distinguished. Internal organs of infected hosts may become enlarged and the gall bladder swollen and turgid. Parasites usually accumulate at the posterior part of the first loop of the intestine, posterior to the common bile duct opening. Small focal haemorrhages may be detected at the point of scolex attachment, extending in severity to full haemorrhagic enteritis (Hoole and Nisan 1994). In extreme cases, intestinal perforation may result with rupture of the intestinal tract (Scott and Grizzle 1979; Heckmann 2000). Intensity of infection may range from 30 to 156 mature, gravid worms per pond-reared carp (90–160 mm in length), and up to 20, although usually less than ten, in fully-grown grass carp (Scott and Grizzle 1979). The highest number of parasites recorded in a single fish was 467 worms (Liao and Shih 1956), but no data on their size were provided. However, small numbers of very large tapeworms can also cause pronounced pathological changes. These records suggest that disease results from the overall mass of parasites, rather than a defined intensity of infection (Davydov 1977). The pathogenicity of B. acheilognathi may also vary at different times of year due to changes in the number of parasites present, water temperature, metabolic rates, and nutritional status of the host and duration of infection (Scott and Grizzle 1979).

Fig. 3.

Juvenile common carp (Cyprinus carpio) with infection of Bothriocephalus acheilognathi

Macroscopic and microscopic lesions

Histopathological studies have been conducted on a wide range of fish species including the common carp (Sekretaryuk 1983; Hoole and Nisan 1994), grass carp (C. idella—Liao and Shih 1956; Scott and Grizzle 1979), spottail shiner (Notropis hudsonius), fat- head minnow (Pimephales promelas), wound- fin (Plagopterus argentissimus) and round tail Chub (Gila robusta—Heckmann 2000). The pathology may be divided generally into: (i) damage caused by scolex attachment; and (ii) damage caused by the presence of strobila within the intestine lumen (Scott and Grizzle 1979; Hoole and Nisan 1994). Other organs may exhibit signs of pathological change. For example, infected fish can show signs of nutritional deficiency, with atrophy of hepatocytes within the liver. In severe cases, these changes are consistent with starvation (C. Williams, unpublished data).

Pathological changes caused by attachment of B. acheilognathi

Bothriocephalus acheilognathi attaches to the gut wall by its bothria, which engulf the intestinal folds. The parasite attaches near the anterior portion of the intestine, just posterior to the bile duct. An accumulation of tapeworms in this area leads to digestive tract blockage that distends the intestinal wall leading to perforation. When attached, B. acheilognathi envelopes parts of the intestines and induces an inflammatory response. The inflammation can lead to hemorrhage and necrosis. Symptoms can also include, weight loss, anemia, and mortality especially in young fishes, Paperna 1996. Infections can be detected by the presence of eggs or body parts in feces, and by the presence of the tapeworm in the gut of the fish (Fig. 4). Scolex attachment may also be associated with increased mucus production (Scott and Grizzle 1979). Tapeworm attachment also provokes a localized inflammatory response, consisting mainly of lymphocytes. In heavy infections, increased numbers of lymphocytes may occur throughout the lamina propria. Lesions associated with the scolex depend upon the force exerted by the bothria to maintain attachment. The scolex is often pushed firmly against the gut wall causing compression and the formation of localized pits, extending as far as the muscularis (Figs. 4, 5). These attachment sites lead to pronounced thinning of the intestine at these points. Scolex attachment can also result in a loss of brush border and an overall reduction in thickness of the terminal web. In advanced cases of infection, scolex attachment can cause localized ulceration. Desquamative catarrhal enteritis and proliferation of connective tissue around the point of scolex attachment have also been recorded (Bauer et al. 1973). Heckmann (2000) provided unique accounts of advanced scolex penetration. Studies on wound fin and round tail chub revealed severe pathology associated with penetration of the gut wall up to the muscularis, resulting in a prominent inflammatory response, extensive haemorrhaging an necrosis. The scolex of some parasites even continued to penetrate the intestine wall into the body cavity, extending as far as the liver and gonads (Heckmann 2000). This represents an unusual and rarely reported consequence of B. acheilognathi infection. Scolex attachment causes considerable disruption to the intestine, including: (i) destruction of the desmosomal junctions; (ii) loss of the gut microvillous border; (iii) separation and loss of enterocytes; (iv) release of host-cell debris into the gut lumen; and (v) infiltration of leukocytes into the infected area. In hosts less than 4 cm in length, damage through attachment can be extensive and is consistent with disruption of gut enzymes (Hoole and Nisan 1994). In many places, the plasmalemma between the microtriches and that of the epithelial cells of the host intestine are lacking, so that the matrix of the tegument is in direct contact with the cytoplasm of the host cells. Lysozomes have been demonstrated surrounding the microtriches embedded in the host cytoplasm (Smyth and McManus 1989).

Fig. 4.

Scolex of Bothriocephalus acheilognathi engulfing the intestine of common carp causing compression of the mucosa (arrow) and localized haemorrhage

Fig. 5.

Marked thinning of the intestine, with formation of pits in the intestinal wall caused by the attachment of numerous tapeworms

Pathological changes caused by the strobila of Bothriocephalus acheilognathi

The pathological changes caused by the strobila of B. acheilognathi generally exceed those associated with scolex attachment. The extent and severity of this damage can vary depending on host size, parasite size and intensity of infection (Davydov 1978; Hoole and Nisan 1994). In small cyprinid fish, pathological changes are characterized by distension of the intestine, compression of the intestinal folds and pronounced thinning of the gut wall (Fig. 6). In very heavy infections severe distension may be accompanied by a complete loss of normal gut architecture, with occlusion of the intestine, congestion, compression, pressure necrosis, thinning and atrophy of the mucosa (Nakajima and Egusa 1974a) (Fig. 6). Separation and degeneration of the epithelium, with regions of complete epithelial loss can occur. Lysis of large areas of the mucosa with necrotic changes has also been observed in heavily infected carp fry (Davydov 1977; Sekretaryuk 1983). The presence of parasite eggs caught between the parasites and gut wall can lead to epithelial abrasion, exfoliation of host cells and indentation of the mucosa. This damage, combined with an already compressed gut wall, can lead to ulceration. Inflammatory changes may be pronounced during heavy tapeworm burdens, with increased numbers of lymphocytes and eosinophilic granular cells occurring throughout infected regions. Paradoxically, heavy parasite infections and marked pathological changes have been observed in apparently healthy fish (C. Williams, unpublished data). Nakajima and Egusa (1974a) described no signs of mortality, despite serious histopathological changes in common carp fry. What is seldom clear in these cases are the metabolic and physiological costs of these infections and the energetic or behavioral demands upon infected fish to maintain condition.

Fig. 6.

Transverse section of common carp showing attenuation of the gut and partial occlusion from tapeworms within

Disease mechanism

Bothriocephalus acheilognathi causes a number of physiological changes in juvenile fish. These include: (i) protein depletion; (ii) altered digestive enzyme activity; (iii) elevated muscle fatigue in heavily infected hosts; and (iv) mortality of young fishes (Liao and Shih 1956; Davydov 1978; Scott and Grizzle 1979; Granath and Esch 1983b; Brouder 1999; Hansen et al. 2006). Bothriocephaliasis also reduces fat content and causes a decrease in kidney, liver and spleen weight (Balakhnin 1979; Zitnan and Hanzelova 1982). According to Clarkson et al. (1997), B. acheilognathi infection causes a reduction in reproductive capacity, depressed swimming ability and elevated muscle fatigue in heavily infected hosts. The findings of Granath and Esch (1983b) corroborated the reduced ability of mosquito fish to acclimatize with oscillations in water temperature resulting in the mortality of infected fish. Relatively little is known about the relationship between the fish immune response and B. acheilognathi infections, although inflammation occurs in intestines of infected fish and leukocytes were noted on the surface of the parasite (Hoole and Nisan 1994; Nie and Hoole 2000). The interaction between the parasite and pronephric lymphocytes of carp was studied by examining proliferation of lymphocytes isolated from both naive fish and fish injected intraperitoneally with cestode extract (Nie et al. 1996). Parasite extracts increased antibody production and pronephric antibody-producing cells in injected fish (Nie and Hoole 1999), and stimulated proliferation of pronephric lymphocytes in vitro after 5 and 10 days post-injection (Nie et al. 1996). Reduced haemoglobin and total blood volume were reported in infected carp (Kudryashova 1970; Par 1978; Svobodova 1978), but opinions vary as to the intensity of infection necessary to induce these pathogenic effects. Kudryashova (1970) found these effects in fish with more than five worms while Svobodova (1978) did not find any significant effect on various physiological indices in fish harboring between one and 21 worms and attributed the elevated leukocyte count to inflammation of the gut. According to Par (1978), fish with between one and 29 worms had an elevated leukocyte count and those with more than 15 specimens showed marked damage to the gut. Biochemical studies showed reduction in activities of enzymes, such as alanine and aspartate aminotransferase, intestinal trypsin and chymotrypsin, amylase and acid phosphatase (Kudryashova 1970; Matskasi 1984). Reduction in total serum proteins, disrupted carbohydrate and protein metabolism, and elevated oxygen consumption was observed in infected grass carp (Davydov 1978). Morbidity and mortality in winter were attributed to changes in alanine and aspartate aminotransferase activities, which interfered with protein synthesis (Lozinska-Gabska 1981). Inflammation of the gut, severe catarrhal- haemorrhagic enteritis at the parasite attachment point, with proliferation of the peripheral connective tissue, and thinning of the intestinal wall have been attributed to increased acid phosphatase activity during infections (Par 1978; Svobodova 1978; Scott and Grizzle 1979; Sekretaryuk 1983). There is some evidence to suggest B. acheilognathi secretes toxic materials (their composition is not known) leading to intoxication of the host (Degger and Avenant-Oldewage 2009) and damage to the epithelial lining (Hoole and Nisan 1994). According to Bauer et al. (1981) intoxication of the entire host can produce degenerative processes in organs. Hoole and Nisan (1994) revealed through ultra structural studies that electron- dense secretions are released from the surface of B. acheilognathi which may have a protective function for the parasite. Worms adhering to the host’s gut wall injure the lining epithelium, produce and secrete toxic materials, and obstruct the passage of the intestinal contents (Bauer et al. 1977).

Protective/control strategies

Due to the economic importance of Bothriocephalus acheilognathi, its global distribution and expanding host range, considerable efforts have been made to limit disease impacts. These include: (i) the intervention of stringent legislative controls; (ii) extensive prophylaxis; (iii) veterinary examination of fish stocks; (iv) reduction of intensive stock management; and (v) active therapy (Weirowski 1984). Control of the parasite can be directed at either the copepod intermediate hosts (drainage of the ponds in the spring to eliminate planktonic invertebrates) or the fish stage of the life cycle, although these measures are governed by economic and practical considerations. Fish ponds can be allowed to dry and disinfected with unslaked lime (Shcherban 1965). European fish farmers control bothriocephaliasis by drying the ponds annually or treating drained wet ponds with calcium chloride (about 70 kg/ha) or calcium hydroxide (about 2 t/acre) or calcium hypochlorite (HTH) to kill the copepod intermediate host, and treating the fish with anthelmintics. Insecticides employed as ectoparasiticides include Neguvon (Masoten or Dipterex – at 25 ppm, i.e. at 25 g of Masote per ton) or similar compounds (Bromex; Naled), can be used to reduce populations of copepods in ponds (Hoffman 1983). However, these are now banned in many countries as a result of environmental and health concerns. A wide range of Chemotherapeutic agents have been employed with varied success including natural products such as: (i) tobacco dust (Avdosyev 1973); (ii) lupin seeds (Balatski et al. 1976); (iii) conifer needles (Klenov 1969a); and (iv) horse radish leaves (Klenov 1969b). Also a number of compounds and insecticides have been used (Molnar 1970; Edwards and Hine 1974; Fijan et al. 1976; Par et al. 1977; Brandt et al. 1981). A comprehensive review of chemotherapeutic treatments used for the control and eradication of B. acheilognathi is in Bauer et al. (1981) and Williams and Jones (1994). Drugs are usually administered orally. Such preparations are often mixed in oil (corn, soy and fish) and sprayed on to pellets or mixed with feeds. Recent efforts have focused on water- borne chemotherapeutics, which alleviate some of the problems associated with Map- potency and dosage. It is important to distinguish the use of treatment to reduce parasite burden and treatment to achieve complete eradication of the infection. Tapeworms may shed segments during adverse conditions or periods of stress, regenerating when conditions become more favourable. Another important consideration is whether anthelmintics are ovicidal (i.e. kill parasite eggs). This is necessary to avoid the discharge of large numbers of infective eggs to the environment when the worm is evacuated from the fish. The eggs of B. acheilognathi can be killed rapidly by drying, freezing and ultraviolet rays. Among 11 chemicals tested for ovicidal effect, two chlorine-based compounds were found to be effective: (i) 3.1 ppm of sodium dichloroisocyanurate; and (ii) 9 ppm of bleaching-powder (Nakajima and Egusa 1974b). However, there are very few chemical treatments currently licensed for use for tape- worm infections. Furthermore, the use of chemicals for the control of parasites in open water bodies can be very difficult, ineffective, harmful, expensive and illegal.

Conclusions and suggestions for future studies

The Asian tapeworm is pathogenic to fresh- water fishes, especially young carp fry, and may cause great economic loss in hatcheries and fish farms. It has the ability to colonize new regions, and adapt to a wide spectrum of fish hosts. It represents one of the most impressive and deplorable examples of a parasite widely disseminated by man assisted movements of fish. The rate of dissemination and success of colonization has been aided by the cosmopolitan distribution of both intermediate and definitive hosts. However, the spread of Bothriocephalus acheilognathi to many parts of the world has also been the result of inadequate legislative controls, poor preventative measures and lack of appropriate health-checking procedures prior to fish introductions (Scholz and Di Cave 1993; Hoffman 1999; Heckmann 2000). Recent data indicate that the impact of the tapeworm in Europe may have decreased during the last decade. However, surveillance should be maintained to prevent its further expansion to new areas. Efforts are underway to identify the resistance of different strains of common carp used in European aquaculture. Hoole (1994) proposed the development of a vaccine against Bothriocephalus acheilognathi, although practical and economic constraints continue to limit this approach. Exported fish, especially cyprinids and ornamental species (like guppies), should be inspected by veterinarians before their translocation to prevent further dissemination of the tapeworm into new regions. Control measures are generally effective, including treatment of infected fish, but the use of some anthelmintics are no longer allowed because of their negative effect on human health or the environment. Future work must therefore seek to accommodate novel and effective treatments to minimize economic loss. Many aspects of the biology, ecology and pathology of Bothriocephalus acheilognathi are well understood and comprehensively documented. However, many of these observations are restricted to cultured fish populations. Due to the expanding host and geographical ranges of Bothriocephalus acheilognathi, the importance of the parasite to wild fish populations requires further assessment and documentation. This is an important consideration in view of declining global biodiversity and the growing conservation efforts to protect aquatic environments. Comparative studies are needed to understand differences in species susceptibility and disease potential in newly infected hosts and the consequences of the parasite in new environments. Sub lethal effects of the parasite on fish growth, fitness, fecundity, behavior or tolerance to environmental changes may also hold important ecological implications. The physiological and bioenergetics costs of the parasite under natural conditions also require clarification. This information is necessary to provide better understanding of future disease risks and to evaluate the role of this introduced parasite on the health and stability of fish populations.