Prevalence and Morphological Characteristics of Taenia multiceps Cysts (Coenurus Cerebralis) from Abattoir-Slaughtered and Experimentally Infected Goats

One hundred and forty nine indigenous goats slaughtered at the Tete municipal abattoir, Mozambique, and fifty seven goats experimentally infected with Taenia multiceps eggs were inspected to determine the prevalence, predilection sites and morphological characteristic of cysts and scoleces of T. multiceps. The experimentally infected goats of mixed sexes were divided into Group one (28 animals slaughtered at 10 weeks post-infection) and Group two (29 animals slaughtered at 22 weeks post-infection). Each animal was experimentally infected with 3000 eggs of T. multiceps recuperated from experimentally infected dogs. The prevalence of infection was 14.8% in the naturally infected goats, 78.6% for Group 1 and 82.8% for Group 2 of the experimentally infected goats. The muscle and subcutaneous tissues were the most common anatomic location of cysts in both the naturally and experimentally infected (Group 1 and 2) goats with a prevalence of 9.1%, 82% and 75% respectively and this was followed by the brain in the naturally infected (8.5%), and Group 2 (11.4%) and the heart and mesentery (4.5%) in Group 2. In the abattoir-slaughtered goats, animals with one cyst were more frequent in both muscular and subcutaneous tissues (73.3%) and brain (75%) and fewer animals were found to harbor two or more cysts.

T he Journal of Neuroinfectious Diseases (formerly Journal of Neuroparasitology) is a scholarly open access, peer-reviewed, and fully refereed journal dedicated to developing a home for the reporting of biomedical studies that bridge the fields of infectious diseases and neuroscience. Neurological sequelae of infectious diseases are of critical importance. Infections such as those caused by eukaryotes including protozoa, helminths, and fungi as well as viral and bacterial infections will be considered for publication in this journal.
The journal provides scientists and clinicians with a unique format for peer-reviewed publication of basic science research findings, clinical studies, case reports, focused and comprehensive reviews, and topical commentaries and perspectives through a very genuine editorial tracking system. JNID is also presented with a vision of providing the young talents a platform to encourage them to develop and globalize their skills and talents for the welfare of the society they are associated with and ultimately to the whole world.

Introduction
Taenia multiceps (Leske, 1780) (syn. Multiceps multiceps) is a worldwide parasite which inhabits the small intestine of dogs, foxes, coyotes and jackals [22]. The larval stage, known as coenurus cerebralis, causes a central nervous system disease in sheep commonly known as coenurosis, gid or sturdy and may also affect other animal species such as domestic and wild ruminants including humans [5,14,23,26,29,30]. Apart from the cerebral form, which is usually fatal, intramuscular and subcutaneous connective tissues, liver, lung forms have been reported to occur [26].
Animal cerebral coenurosis is usually diagnosed based on a clinical examination protocol [16,24,25] and seldom includes imaging methods like radiology [28], ultrasonography [8] and computed tomography [12] which are mainly used in experimental situations. Immunodiagnosis tests such as skin test for immediate hypersensitivity, indirect haemaglutination antibody (IHA) test, immuno-electrophoresis (IEP), gel double diffusion (DD), immunoblot and enzyme linked immune-assay (ELISA) tests have been used experimentally [6,7,21,25]. Despite the availability of these tests which have their own practical challenges, post mortem findings of a thin walled cyst filled with transparent fluid and with numerous scoleces in the wall remain the definitive diagnosis [15,26].
The prevalence of T. multiceps larva in Gaza and Tete Provinces of Mozambique has been reported to range from 7.3% to 13.8% based on abattoir findings [3,32]. To the best of our knowledge, no other data exist on the occurrence of coenurosis in Mozambique and no references have been made to the site of predilection and the morphological characteristics of the larva and scoleces in goats. For this reason, the present study was conducted to determine the prevalence and predilection sites of T . multiceps cysts in goats slaughtered at a local abattoir and those experimentally infected and the morphological characteristics of the cysts and scoleces. The results will add new information on the predilection sites of T. multiceps in goats and assist meat inspectors in locating the cysts at abattoirs during meat inspection.

Meat inspection at abattoir
Various organs and muscle tissues from a total of 169 abattoir-slaughtered goats of local breeds of mixed sexes from a municipal abattoir in Tete Province, central west region of Mozambique, were inspected for the presence of T. multiceps cysts [22]. The number of organs and muscle tissues accessed for inspection varied as it depended on willingness of owners to access them for thorough inspection.

Goats
The experimental study was conducted at the Veterinary Faculty, Eduardo Mondlane University (EMU), Mozambique. Fifty seven male goats of local breed, Landin, aged between 8 and 18 months old were purchased from T. multiceps-free farms. In order to assess the morphological characteristics of T. multiceps cysts at different weeks post-infection, the animals were randomly divided into two groups (Group one, 28 animals and Group two, 29 animals) ( Table 1). Animals were housed at the Veterinary Faculty, EMU campus, under good clinical practice with free access to food (hay and grain ration) and water. The adaptation period, from purchase to infection, was four months. The study was approved by the Scientific Committee from the Veterinary Faculty, EMU.

Dogs
Six puppies of mixed breed (3 for each experiment) from the same breed and litter were selected for experimental infection with T. multiceps scoleces to generate eggs for experimental infection of goats. The puppies were weaned at 2 months and kept in separate kennels at the EMU veterinary hospital. They were fed commercial dog food, supplied with clean water ad libitum and treated following prophylactics measures established at the EMU veterinary hospital as follows: multiple vaccination against the common viral diseases including rabies and deworming with a combination of pyrantel, oxantel and praziquntel (Canex-4®) one month before the experimental infection. The health of the dogs was routinely monitored throughout the period of the study.

Experimental infection in dogs
Taenia multiceps cysts collected from naturally-infected goats from Tete municipal abattoir were immediately processed after collection by removing tissue surrounding the cysts. This was followed by washing the cysts in phosphate buffer saline (PBS), pH 7.3, preserving them in a sterile recipient and maintaining them at 4°C until time of infection.
The dogs were each orally infected with 100 scoleces at the age of four months. Patency was evaluated by collecting faecal samples from infected dogs once a week to detect taeniidae eggs using the McMaster flotation technique [15]. After the parasite had reached patency, shown by presence of taenid eggs in faeces, the dogs were euthanized with 20% pentobarbital (Eutha-Naze) at a dose of 200 mg/kg. Taenia multiceps adult parasites were removed from the small intestine and gravid proglotids were separated to recover the eggs.
Standard operating procedures regarding the safety of researchers were strictly followed at all stages of the experimental infection. The dog kennels were kept isolated and access was restricted to the researchers only. All faeces from the infected dogs were incinerated as from day one postinfection.

Experimental infection in goats
Gravid proglottids of T. multiceps were crushed in 2.5% potassium dichromate to release the eggs and the eggs were preserved in a refrigerator at 4°C before being subjected to the hatching process. In vitro hatching was performed using sodium hypochlorite [17,27,33]. Briefly, eggs were centrifuged at 1000 rpm for 5 minutes and 0.5 mL of sodium hypochlorite (0.5% of sodium hypochlorite in normal saline) and 10 mL of formol saline were added. The solution was centrifuged at 1000 rpm for 5 minutes. The sediment, containing the hatching eggs, was washed three times in normal saline and counted [17,27,33]. Each goat was orally infected with 3000 viable eggs via a cellulose and amide bolus.
Group one animals were slaughtered at 10 weeks postinfection and Group two at 22 weeks post-infection. The procedure for localization of cysts from the various organs and tissues was done following the anatomic classification according to following authors [19,20]. Cysts were removed from the tissue and then washed with phosphate saline buffer (PBS) pH 7.3 and placed in a Petri dish. The number of scoleces from each cyst was determined and the length and width of the scoleces were measured with a ruler under a stereomicroscope.

Data analysis
Descriptive statistics which included the determination of prevalence of T. multiceps cysts in abattoir-slaughtered and the experimentally infected goats were calculated. Data on the location of cysts from the abattoir-slaughtered and the experimentally infected goats were compared using a chisquare test with the level of significance set at P < .05. The computer software SPSS 13.0 SPSS Inc., Chigago, Il, was used for data analysis.

Results
The overall prevalence of T. multiceps cysts in abattoirslaughtered goats determined through meat inspection was 14.8% (22/149) and of experimentally infected goats was 78.6% (22/28) and 82.8% (24/29) for group one and two respectively ( Table 2). The muscle and subcutaneous tissues were the most common anatomic location of cysts in both the naturally and experimentally (Group one and two) infected goats with a prevalence of 9.1%, 82% and 75% respectively and this was followed by the brain in the abattoir-slaughtered group (8.5%), and Group two (11.4%) and the heart (7.0%) and diaphragm (5.0%) in Group one animals. A low number of cysts was observed in the brain (5% for Group one and 11% for Group two). Overall, T. multiceps cysts were distributed to a minimum of five anatomical sites in the experimentally infected goats whilst they were only observed at three sites in the naturally infected goats. It is worth mentioning that muscle and subcutaneous tissue included muscles from the neck, thoracic, lumbar and pelvic region and abdominal wall. A significant difference (P < .05) was observed between the number of cysts observed in the brain of abattoirslaughtered goats and experimentally infected animals with the abattoir-slaughtered animals having more cysts in the brain. There was no significant difference (P > .05) in the number of cysts observed in the muscles and subcutaneous tissue between the two groups.
The morphological characteristics of the T. multiceps cysts from naturally and experimentally infected goats were similar. Cysts were composed of a hyaline membrane with many scoleces on the inner surface and filled with a translucid fluid of varying volume. In both cases, cysts which were located outside of the brain were surrounded by a thick host membrane.
In the naturally infected group, animals with one cyst were more frequent in both muscular and subcutaneous tissues (73.3%) and brain (75%) and few animals were found to harbor two or more cysts. In the experimentally infected groups, animals with two cysts (11 animals) were the most common and only three goats had 10, 12 and 16 cysts each, respectively.
Size of larvae and number of scoleces per cyst increased with the age as observed at 10 and 22 weeks post-infection (Table 3). Young cysts of 10 weeks of age had fewer scoleces (51.7 ± 27.7) than the older ones of 22 weeks (92.2 ± 48.7).

Discussion
In Africa, T. multiceps cysts in the central nervous system (CNS) have been reported in domestic ruminants in Kenya, Ethiopia, Democratic Republic of Congo, Senegal, Sudan, Chad, Angola, Southern Africa [30] and Mozambique [32]. However, none of these studies contain information regarding the predilection sites of larval stages. The prevalence of T. multiceps cysts in naturally infected goats in this study (14.8%) was higher than the prevalence reported in a retrospective study in sheep in Ethiopia (2.34-4.54%) [2] and in goats in Gaza (8%) and Tete (7.3%) provinces of Mozambique [3] and comparable to the prevalence reported of (13.8%) in a study conducted in Tete abattoir, Mozambique [32]. Prevalence studies of T. multiceps infection in small ruminants in Southern African countries are very limited [32] and this study gives an indication that the parasite is wide spread, especially in the resource-poor rural communities and the zoonotic impact of the parasite is yet to be assessed.
The common predilection sites of T. multiceps cysts in sheep is the CNS, whilst in goats, apart from the CNS, the muscle and subcutaneous form have been reported [23,26]. Subcutaneous cysts found in an infected goat were classified as Multiceps gaigeri [13] and in subsequent studies similar cysts were found in several organs including muscles and subcutaneous tissue and were re-classified as T. multiceps [10,18,23].
Results from our study shows that a larger proportion of T. multiceps cysts were found outside the CNS and mostly in muscle and subcutaneous tissues in the experimentally infected goats than the naturally infected. This difference is likely to be due to the fact that the cysts in the muscles of the experimentally infected animals were located in the medium and deep layers of muscles which increase the possibility of becoming unnoticed at abattoir inspection. The low sensitivity of abattoir routine meat inspection for T. multiceps cysts is an important factor that may contribute in perpetuating the cycle of the parasite.
Previous experimental infections with T. multiceps larva were done in sheep using a high dose of 5500 eggs per sheep [4] and 6500 eggs [11]. In the two studies the infection rate was 100% in contrast to lower infection rates recorded in our study. This difference might have been associated to the combination of a relatively low infection dose, egg viability or regurgitation of the infected bolus.
Previous studies show that clinical coenurosis in sheep is common in young animals [1,22]. Age-related resistance to infection with T. multiceps in sheep has been suggested [31], however, the mechanism by which this may come about is not apparent and cross immunity within taeniidae cestodes has been discussed by some authors [9]. The increase with age in cyst size and the number of scoleces found in this study are in accordance with a previous study in sheep [34].
Our findings have shown the prevalence of T. multiceps cysts in goats is high in Mozambique and this might be the same situation in most countries in Southern Africa where goats are extensively reared. In addition, an important finding from the experimental infection study was that the predilection sites, apart from the brain include skeletal muscles and subcutaneous tissue, lungs, diaphragm, heart and mesentery and that the current routine abattoir inspection targeting only the brain is not a reliable method in detecting all cases T. multiceps infection in goats. Development and validation of a sensitive and specific diagnostic method for T. multiceps infection in goats should be investigated in order to improve the surveillance and control of the parasite.