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A study of morphological aspects of cystic echinococcosis in sheep in Sardinia
Accepted Manuscript Title: A study of morphological aspects of cystic echinococcosis (CE) in sheep in Sardinia. Author: Margherita Conchedda Valter Seu Salvatore Capra Alessia Caredda Sergio Pino Pani Pier Giorgio Lochi Gianfranco Bortoletti PII: DOI: Reference:
S0001-706X(16)30163-2 http://dx.doi.org/doi:10.1016/j.actatropica.2016.04.003 ACTROP 3914
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
2-11-2015 15-3-2016 5-4-2016
Please cite this article as: Conchedda, Margherita, Seu, Valter, Capra, Salvatore, Caredda, Alessia, Pani, Sergio Pino, Lochi, Pier Giorgio, Bortoletti, Gianfranco, A study of morphological aspects of cystic echinococcosis (CE) in sheep in Sardinia.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A study of morphological aspects of cystic echinococcosis (CE) in sheep in Sardinia.
Margherita Concheddaa, Valter Seua, Salvatore Capraa, Alessia Careddaa, Sergio Pino Panib, Pier Giorgio Lochib, Gianfranco Bortolettia.
Department of Life and Environmental Sciences, Cagliari University, Cittadella Universitaria di
Monserrato, Strada Provinciale Monserrato-Sestu km 0.700, Monserrato (Cagliari), Italy; bServizio Veterinario Igiene degli Alimenti di Origine Animale ASL 6, Sardegna (Italy).
Corresponding Author: Margherita Conchedda tel. +39 0706754530; fax +39 0706754536 E-mail: [email protected]
Graphical abstract
Highlights ►Cystic Echinococcosis is persistently widespread in sheep in Sardinia. ► Morpho-structural aspects of >16,000 CE cysts are analysed during 2 surveys. ►Detailed parameters for cyst classification are established. ►Cysts are grouped into 5 main types. ►Reporting of cyst types is proposed in a surveillance and control perspective.
Abstract We have investigated the morphological and structural aspects of hydatid cysts recovered from sheep raised in southern Sardinia during two surveys conducted over the 20 years since the last control attempt at the end of the 1980s. In the first study carried out during 1995-1997, a few years after the last control attempt, a total of 10,334 cysts were examined, recovered from 1,029 infected sheep out of a total of 1,375 inspected (prevalence 74.84%). In the second survey, conducted ten years later during the period 2005-2010 in the absence of specific control measures, a total of 6,249 cysts were recovered from 916 parasitized sheep out of the 1,414 examined (prevalence 64.78%). Cysts were grouped into 5 different types: “Unilocular”, “Multisepted”, “Calcified”, “Caseous”, “Hyperlaminated”. Unilocular: fertile, consist of
a single fluid-filled cavity; multisepted:
generally sterile, fluid filled, with cavity divided into spheroidal chambers (3-dozen); calcified: sterile, small, with internal chambers almost virtual due to the thickening of internal septa; caseous: sterile, the cavity filled with a thick yellowish matrix; hyperlaminated: sterile, with a virtual cavity filled with extensively folded and overlapping sheets of hyperproduced laminated tissue.
A thorough knowledge and the precise characterization of each type of lesion, making it possible to establish detailed parameters for cyst classification, appears very useful for the harmonization of data collection. From a surveillance and control perspective it is proposed that, in addition to other relevant epidemiological information, data from meat inspection be further improved, including morpho-functional indications about cyst type.
Keywords Cystic Echinococcosis; Sardinia; Sheep; Morpho-functional classification; cyst types.
1. Introduction In spite of improved knowledge of Cystic Echinococcosis (CE) and the apparently easy control of the infection that have reduced its burden in some countries, the disease remains a major, though often underestimated, public health problem in many regions of the planet, emerging or remerging in vast areas (Eckert and Deplazes, 2004, Jenkins et al., 2005, Buishi et al., 2005 and WHO, 2013). One million or more people worldwide are estimated to be currently suffering from CE, and the global burden in terms of DALYs lost is reported to be similar to other better-known neglected tropical diseases (such as Chagas disease, dengue, onchocerciasis, or African trypanosomiasis) (Budke et al., 2006 and Craig et al., 2007). Moreover, the financial burden of CE on the world's livestock industry has been estimated at an annual loss of around $2 billion (Torgerson and Macpherson, 2011). As is well known the prevalence of the zoonosis is closely connected with the social and economic conditions of the population: widespread use of traditional techniques for raising small ruminants (extensive or semi-extensive grazing), illegal slaughtering of the animals, and presence of a large number of dogs, particularly strays. Sardinia, an island in the middle of the Mediterranean, with more than 3,000,000 dairy sheep reared in extensive/semi-extensive production systems, has 40 % of Italy’s total sheep population. The island historically registered one of the highest CE diffusion rates both in livestock and in humans, despite three tentative control programmes carried out over time (Conchedda et al., 1997, 2002, 2010 and 2012). Our recent study highlighted the modifications that have taken place in the epidemiology of CE in Sardinia both in humans (Conchedda et al., 2010) and in sheep (Conchedda et al., 2012), over the twenty years since
the last (interrupted) control attempt at the end of the 1980s and discussed the profound ongoing structural transformation that is changing the scenario of the zoonosis. An annual incidence rate in humans of about 6.6 per 100,000 inhabitants is still currently observable on the island (Conchedda et al., 2010), where the long term (decades) natural history of CE has been traced by an in-depth knowledge of the morphological and functional states of different cyst types (Bortoletti et al., 2002, 2004 and 2013). With regard to livestock, data describing the current CE status and the trend of prevalence in sheep, the most common intermediate host in Sardinia, show a persistently high and widespread diffusion (prevalence of 65% today). All sheep are infected with the G1 Echinococcus granulosus strain (Varcasia et al., 2006, Garippa et al., 2008), the most common in Europe and in the Mediterranean Basin, responsible for the majority of human infections (Romig et al., 2006, McManus and Thompson, 2003 and Thompson, 2008). The prevalence is particularly high in less favoured areas (Conchedda et al., 2012), in spite of the diminished CE burden and the improvements achieved over time. This is due to the persistence of factors traditionally associated with transmission (large numbers of dogs, home slaughtering and offal disposal), notwithstanding the significant socio-economic, demographic and structural transformations that have taken place on the island (Conchedda et al., 1997, 2002, 2010 and 2012; Scala et al., 2006, Scala and Mazzette, 2009, Varcasia et al., 2011). The availability of reliable and detailed base-line data able to accurately depict CE diffusion is basic to monitoring the effects of preventive measures to be put in place in a control plan. In particular, considering the present vaccine option developed as a result of Australian studies on EG95 (Lightowlers et al., 1996 and 2003), one of the most important aspects to be carefully evaluated, together with epidemiological base line data, is the evaluation of parasite burden with particular regard to the morpho-functional status of cysts in the major intermediate host. This provides precise information as to how the life cycle of the parasite is actually maintained for the area examined, allowing careful evaluation of immunization efficacy also in terms of cost-benefits. A standardized classification of ovine CE cysts may be useful for facilitating both the uniform reporting of results from epidemiological field studies conducted in different areas and monitoring any changes that have occurred over time. In addition this approach may be of particular interest considering the increasing importance of ultrasonography in intra vitam screening for CE in sheep (Maxson et al., 1996, Sage et al., 1998, Guarnera et al., 2001, Lahmar et al., 2007 and Dore et al., 2014) similarly to humans. In this case , as well known,
the WHO-IWGE classification of ultrasound images of CE cysts (WHO-IWGE, 2003), together with the improved knowledge of the natural history of infection (Bortoletti et al., 2002, 2004 and 2013) have proven useful for advancing the clinical management of patients (Brunetti et al., 2010). For this purpose the present study has been conducted on the structural and functional aspects of hydatid cysts recovered in sheep raised in the province of Cagliari or Oristano and slaughtered in abattoirs in southern Sardinia. The investigation covered two periods: the 1st survey carried out during 1995-1997, fairly soon (5–7 years) after the last control attempt, the 2nd survey conducted roughly 10 years later over the period 2005-2010, long after the control plan had been dropped.
2. Materials and Methods The surveys concerned a total of 16,583 hydatid cysts from 1,945 sheep infected out of a total of 2,789 animals inspected during an epidemiological study carried out in two phases (1995-1997 and 2005-2010) in abattoirs in southern Sardinia (Conchedda et al., 2012). In detail during the 1st survey 10,334 cysts were collected from 1,029 CE infected sheep out of the 1,375 inspected (prevalence 74.84%). During the 2nd phase a total of 1,414 sheep were inspected, recovering a total of 6,249 cysts from 916 parasitized animals (prevalence 64.78%).
2.1. Area of study Sardinia is an island of 23,821 km2 in the middle of the Mediterranean sea (Italy) (38°51′ and 41°15′N, 8°8′ and 9°50′E), mostly hilly and mountainous, sparsely populated (about 1,600,000 inhabitants), with a typical Mediterranean climate characterized by mild winters and hot summers. Extensive or semi-extensive sheep farming is widespread on the island, where 3 million dairy sheep are reared. The most important source of income for the 14,500 farms is represented by sheep’s milk, cheese and dairy products, in addition to the sale of one-month old lambs. Therefore, and also owing to the low commercial value of mutton, ewes are normally slaughtered old, at the end of their production cycle. Furthermore, in this agro-pastoral system characterized by extensive/semi-extensive grazing, dogs play a major role. The dog population in Sardinia is estimated to be more
than 380,000 with 70-80,000 sheepdogs: on average 3.8 per farm according to a recent survey (Varcasia et al., 2011). In addition many “stray-“ and “community-“ dogs are also present on the island, and these contribute to fostering the perpetuation of the CE biological cycle, considering that in the past a general infection rate of 16-20% has been reported in Sardinian dogs, rising to 25% in sheepdogs (Arru et al.,1993) . These aspects together with the uncontrolled practice of home slaughtering, and the improper disposal of carcasses, favour the persistence of CE on the island (Conchedda et al., 1997, 2002, 2010 and 2012; Scala et al., 2006, Scala and Mazzette, 2009, Varcasia et al., 2011).
2.2. Processing of samples and morphological analysis Analysis during the two survey periods was carried out following the same methodology, as in earlier surveys (Bortoletti et al., 1990, Conchedda et al., 2008, 2012). In detail, after collection of data on age, origin, ownership of sheep and type of farm, drawn from farm registers or directly supplied by vets at abattoirs, viscera of slaughtered sheep were examined by visual and palpatory inspection to detect the presence of any cystic lesions (Fig. 1), to be taken to the laboratory for further analysis.
For each sheep the CE cysts
were counted, measured and classified according to size, type, as well as location. In more detail, the external diameter of each cyst was measured and the content carefully examined. Cyst fluid, including protoscoleces (PSC), were collected by aspiration with a Pasteur pipette, laminated membrane including germinal layer were washed and gently scraped to recover remaining PSC and to harvest adhering brood capsules (BC). A sample of the total sediment was examined by optical microscope, PSC number was estimated by extrapolation to the entire sediment volume, and viability assessed by 0.1% methylene blue exclusion test and by checking the motility of flame cells (Casado et al., 1986). Small cysts removed intact were cut and repeatedly rinsed in a Petri dish and the contents examined under a stereoscopic binocular microscope to detect any PSC. Lastly the cysts were classified into 5 morphostructural types in accordance with our previous classification (Bortoletti et al., 1993) suitably revised. For some samples, as already described (Conchedda et al., 2008), after careful rinsing in 0.13M Millonig’s phosphate buffer at pH 7.4, the material was fixed in 10% formaldehyde
buffered with Millonig 0.13M at pH 7.4, partially dehydrated and finally kept in ethyl alcohol at 80◦ for further observations under the stereo microscope. For the structural and ultrastructural study, the specimens were fixed in Karnowsky’s solution (2% PF, 2,5% GTA in Millonig 0.13 M, pH 7.4) for 12 h, post fixed with 1% osmium tetraoxide in Millonig for 3 h, and then progressively dehydrated in alcohol and embedded in Araldite. Semithin sections of approx. 1 µm, cut with an LKB ultratome III ultramicrotome were stained with methylene blue and Azur II for light microscopic examination. Ultrathin sections of 600–900 Å were stained with uranyl acetate and lead citrate for transmission electron microscopy, performed using a JEOL 100S microscope (Jeol, Tokyo, Japan).
2.3 Trend analysis
Data for the different types of cysts recovered in the two survey periods were analysed according to sheep age, also considering specific prevalences in the two provinces of southern Sardinia (Cagliari and Oristano). In addition, any changes that may have occurred over the years in CE prevalence, particularly of fertile cysts, was investigated by comparing present data with previous surveys carried out on the island from 1980 onwards (Arru et al., 1980; Bortoletti et al., 1989; Bortoletti et al., 1990; Bortoletti et al., 1993, Gabriele et al., 1998).
3. Results
3.1. 1st phase
During the survey 7,012 hepatic cysts (68% of total), 3,314 pulmonary (32%), plus 8 splenic cysts were recovered (Table 1).
3.1.1. Cyst size Analysis according to cyst size showed that of the 10,334 recovered specimens 78% (8,110) had a diameter of <1cm, 18% (1,850) ranged from 1 to 3 cm, 312 (3%) from 3 to 5
cm, and just 62 (< 1%) of between 5 and 10 cm (Fig. 2a). Analysis of cyst size according to fertility, showed that this pattern of distribution was to be attributed entirely to sterile cysts (Fig. 2c), which accounted for 92% of total specimens (9,516/10,334). In detail, these mostly small sterile cysts, measured 83.3% (7,927/9,516) <1cm in diameter, 14.6% (1,387) between 1 and 3 cm, 1.9% (182) between 3 and 5 cm while only 20 cysts were larger than 5 cm. Vice versa the size of fertile cysts was generally larger (Fig. 2b): out of a total of 818 specimens, 182 (22.2%) had a diameter of <1cm, 464 (56.7%) from 1 to 3 cm, 130 cysts (15.8%) ranged from 3 to 5 cm, and 42 (5.1%) >5 cm.
3.1.2 Cyst typology The detailed morphostructural analysis of the recovered cysts allowed to group them into 5 different types: unilocular, multisepted, calcified, caseous and hyperlaminated (Table 2). The unilocular cysts (Fig. 3) accounted for 7.9% of cases (818/10,334) (Table 1), 8.7 % (287/3,314) of pulmonary and 7.6% (531/7,012) of hepatic cysts respectively. Globular, lobate or plurilobate in shape (Fig. 3), ranging from 1 to 10 cm or more in size, these cysts showed a single fluid filled cavity and were almost invariably fertile, often with numerous brood capsules, but with widely varying PSC viability (from 1 to 100 %). The structural organization of cysts at the micro and sub-microscopic level was as previously reported (Bortoletti and Ferretti, 1978) (Fig. 4). They consist of a germinal layer (GL) which represents the parasite sensu stricto, supported by an acellular, heavily glycosylated, laminated layer (LL) (Diaz et al., 2011a and 2011b), surrounded by a pericyst of fibrous adventitial layer (AL) produced by resolution of host cellular inflammation precociously initiated during early post-oncospheral stages.
In these cysts LL
and GL are generally thin with
sparse
microtriches along the inner surface. Numerous large vacuoles are present in the cytoplasm of tegumental syncytium (ts) that appears “fenestrated”. The proliferative cell layer (pcl) is thin and slightly compact. GL characteristics, in terms of density of microtriches, size of ts and compactness of pcl, varied widely both in the different specimens and within the same cyst (Fig. 4c,d). Due to asynchronous formation of brood capsules inside the cysts, PSC at different stages of development are present (Fig. 4b) and cysts may contain both viable and non-viable or dead PSC in varying percentages.
The multisepted cysts (Fig. 5) (3,794 of the 10,334 specimens examined), accounted for roughly 36.7% of total cysts, 34.7 % of hepatic (2,434/7,012) and 42.0 % of pulmonary cases (1,360/3,314) respectively (Table 1). They measured between 0.5 and 3-4 cm in diameter but mostly less than 1 cm, had cavity invariably divided into spheroidal chambers (3-dozen) that although fluid-filled were generally sterile or, in very rare cases with very few PSC (from just a few to some hundreds). Cyst wall showed a thick external adventitial layer and a thin laminated layer with residual germinal layer (Fig. 6). Frequently recovered in the present study (Table 1), this type of cyst represents a parasite degeneration stage. In fact in about 5% of multisepted
cysts
“laminated brood capsules” (LBC) were recovered
i.e.
“anomalous” brood capsules characterized by an ectopic inner peripheral layer of laminated tissue in intimate contact with the internal wall surface and containing only degenerated protoscoleces dispersed in a finely granulated matrix (Fig. 7). These LBC are thought to be the likely effect of degenerative involution of the parasite (Conchedda et al., 2008). Further degenerative evolution of multisepted cysts results in the calcified cysts, the most frequently recovered, accounting for 51.9% of cases (5,373/10,334), especially in the liver (3,972/7,012; 56.6%). Very small (generally
~1 cm) and always sterile,
the internal
chambers of these cysts are almost virtual due to the thickening of internal septa (Fig. 8), and typically produce a gritty sound/feeling upon incision (Soulsby, 1982). About 3% of total specimens recovered (342/10,334) (Table 1), but more than 8% of those found in the lungs (270/3,314), appeared as caseous cysts (Fig. 9). Similar in shape to the unilocular cysts with diameter of between 1 and 5 cm, the cavity of these specimens filled with a thick matrix of creamy or cheesy consistency, probably due to bacterial contamination or infiltration. Occasionally, caseous degenerative process may concern multisepted cysts, leading to complex “multisepted-caseous” cysts where chambers contain large amounts of caseous material (Fig. 10). In other rare cases another cyst type was recovered during the study, accounting for only 7 cases out of 10,334, less than 1% of the total (Table 1), termed hyperlaminated (Fig. 11). They are of moderate size (1-5 cm in diameter) and the virtual cavity is filled with extensively folded and overlapping sheets of hyperproduced laminated tissue (SLT), well detected at the histological level (Fig 12).
Where scattered intermediate stages between different cyst types were observed in sheep, e.g. caseous degeneration of multisepted or hyperlaminated cysts, for categorisation purposes these were included in the prevalent morphostructural type.
3.1.3.Cyst type according to sheep age. As previously reported (Conchedda et al., 2012) specific prevalence rates in Sardinian sheep increase with age. Analysis of data on cyst types according to animal age showed the highest prevalence, increasing with sheep age for multisepted and calcified cysts (Fig. 13a), while rates were much lower for the other types in all age groups. In particular, in the province of Cagliari the calcified specimens were always the most numerous for all ages, while viceversa in the province of Oristano multisepted cysts prevailed in young and up to 6 year-old sheep. On the other hand prevalence of calcified cysts increased with age, exceeding multisepted in the oldest animals (Fig. 13a).
3.2. 2nd phase During the 2nd phase of the study (2005-2010 years) 64% of recovered cysts were hepatic (3,979/6,249) and 36% pulmonary (2,265/6,249) (Table 1).
3.2.1 Cyst size The morphostructural analysis confirmed a significant difference in size between fertile and non-fertile specimens (Fig. 2b,c), with larger cysts less frequently degenerated/ not viable. Cysts from the 2nd survey appeared on average smaller than the 1st study (Fig. 2a-c). In particular frequency of small fertile cysts (< 1 cm) increased over time, with a simultaneous decrease of 1-3 cm cysts (Fig. 2b). A minor increase in frequency of small sterile cysts (< 1cm) was also observed, while that of larger specimens 1-3 cm decreased slightly.
3.2.2 Cyst typology
In total roughly 10% of the cysts were unilocular fertile (624/6,249), a slight increase compared with the 1st survey (Figs. 13a,b-14). No significant difference in frequency was noticed for cyst location: 10.3% of pulmonary (234/2,265) and
9.9% of hepatic cysts
(390/3,979) respectively. In addition the morphostructural analysis showed that 31.6% of cysts were multisepted (1,977/6,249) (Fig. 13b) slightly less than in the 1st survey: 28.3% of hepatic (1,128/3,979) and 37.5% of pulmonary cysts (849/2,265) respectively. Calcified cysts were commonly recovered,
accounting for around 52% of the total (3,236/6,249), particularly in the liver
where they represented 60.2% of the 3,979 recovered cysts (Table 1). Further, about 5% of all cysts (316 on 6,249), but more than 11% of pulmonary ones (264/2,265), were classified as caseous, a slight increase over the 1st survey. Lastly, 1.5% of the 6,249 recovered cysts were hyperlaminated, found more frequently in the lungs (3.7% on a total of 2,265) than in the liver (0.3% of 3,979) (Table 1).
3.2.3 Cyst type according to sheep age. During the 2nd survey multisepted and calcified cysts were again found to be the most common (Fig. 13b). In particular in the Cagliari province a general decrease in prevalence with respect to the 1st survey was observed at all ages for all cyst types (and particularly for multisepted), except for caseous and hyperlaminated cysts, only occasionally observed, that showed a more irregular trend. Viceversa in the province of Oristano minor variations were detected with respect to the 1st study, with a constant increase in prevalence of unilocular (fertile) cysts in sheep of all ages (Fig.13 b).
3.3. Trend analysis
A very significant reduction has been observed over the years in cyst fertility in sheep in Sardinia as emerged from comparison of data from the present study with previous surveys carried out on the island from 1980 onwards (Arru et al., 1980; Bortoletti et al., 1989; Bortoletti et al., 1990; Bortoletti et al., 1993, Gabriele et al., 1998) (Fig. 15). The frequency of ewes harbouring at least 1 unilocular cyst was, considering all Sardinian provinces, about 70% 30 years ago (Arru et al., 1980). By the 1980s frequency had decreased to between 25
and 40% (Bortoletti et al., 1989 and 1990), dropping to around 18% in the mid nineties (12.5% in southern Sardinia and 21% in the North) though remaining at around 30% in the most pastoral Nuoro province (unpublished) and 27% in the area of the Goceano region between Nuoro and Sassari provinces (Soro et al., 2002). More recently (1998-2003) a decrease in frequency to 10.25% was reported for northern Sardinia (Scala et al., 2006), while the present study found at least 1 fertile cyst in 14.0% of the ewes inspected in southern Sardinia during the 2nd survey (2005-2010) (vs 12.5% in the past): 9.6% from sheep with pulmonary cysts alone, 4.5% from sheep with hepatic cysts alone, and 21.0% from sheep infected with both. In general, as reported, cysts from the latter survey, particularly sterile ones, were found to be smaller in size than in the 1st study period, and the relative frequency of the smaller cysts, particularly those 1-3 cm in size, was observed to increase for all cyst types (Figs 2a,b,c).
4. Discussion
Since the last control attempt interrupted in the early 1990s, no specific preventive measures have been put in place in Sardinia, while significant socio-economic, demographic and structural transformations have profoundly modified the zoonosis scenario (Conchedda et al., 1997, 2002, 2010 and 2012) and innovation and tradition have intertwined. In this context the findings of the two surveys carried out in 1995-97 and 2005-2010, after the last control campaigns, indicate that despite a reduction in terms of intensity, abundance and frequency of heavily infected animals (Conchedda et al., 2012), CE prevalence in sheep in southern Sardinia is still one of the highest in the Mediterranean region (Eckert et al., 2001a). The aim of the present study was to characterize hydatid cysts collected from sheep in Sardinia by means of morphological tools.
A thorough knowledge and the precise
characterization of each type of lesion, making it possible to establish detailed parameters for cyst classification, appears very useful for the harmonization of data collection. It facilitates the visual and accurate evaluation of parasite status, during offal inspection, even in a large number of infected sheep, without over-extending inspection time. The morphofunctional approach makes the monitoring of viable cysts that ensure easier maintenance
of the parasite's life-cycle, making it possible to compare disease patterns in different areas and gain an overview of the trends and transformations that have occurred over time. Therefore it is of potential interest for monitoring a control programme, especially if the sheep vaccination option is envisaged, providing valuable information on base-line infection pressure in livestock, and making it possible to evaluate the benefits of treatment.
Compared to human infection little comparable data regarding the morphological variability of CE cysts in animals are available (Bortoletti et al., 1993, Bortoletti and Ferretti,1978 and Dueger and Gilman, 2001), and cysts are generally analysed in terms of fertility, at the most by differentiating sterile and calcified cysts (Ernest et al., 2009, Daryani et al., 2009 and Kebede et al., 2009). The results of the present study revealed a wide variability of cysts in the natural intermediate host even though, unlike infection in humans where a natural history of decades can be traced (Bortoletti et al., 2004 and 2013), cysts from sheep are at the most 8-10 years old. Based on the detailed analysis of the thousands of cysts collected we were able to distinguish five different types: unilocular, multisepted, calcified, caseous and hyperlaminated.
Specimens from the same host and the same organ showed
morphostructural variability, also with regard to size and viability. As is well known, parasite survival depends on efficient host evasion mechanisms that come into play during development toward the hydatid cyst stage (Conchedda et al., 2004). During the establishing phase, CE is susceptible to host immune killing, as studies on EG95 vaccine have definitively shown (Lightowlers et al., 1996, 2003, Heath and Koolaard, 2012 and Jabbar et al., 2011). The developing cyst elicits a local inflammatory response, which may resolve leaving behind a collagenous capsule, or, if the reaction is too intense, lead to degeneration and eventual death of the parasite. Across the spectrum of host response, cyst fertility correlates with resolution of the inflammation, while an intense response is associated with infertility and, more extremely, degeneration of the cyst (Thompson, 1995). Resolution nearly always takes place in humans, less generally in sheep, in agreement with our observation (Bortoletti et al., 1978,1993, 2002, 2004 and 2013). Degenerating or dead cysts recovered at various later stages suggest a possible killing during established phase, when the parasite loses integrity and undergoes various and complex degenerative processes. This is demonstrated by the variety of cyst types recovered and by the presence of the laminated brood capsules, an indication of profound
degeneration (Conchedda et al., 2008), though no conclusive evidence that cyst death is immunologically mediated has yet been found. Present results strongly suggest that the only fertile specimens able to ensure CE life cycle perpetuation in Sardinia, are the unilocular cysts (Fig. 3). Though not as old as analogous excised human specimens, this type of cysts in sheep proves not to flourish so well and, by comparison, LL and GL are generally much thinner, with sparser microtriches along the inner surface and a larger number of vacuoles (Bortoletti and Ferretti, 1978). In addition the number of PSC and their vitality varies very widely both in unilocular cysts recovered from different ewes, and in cysts from the same animal or even from the same organ. Data from natural (Torgerson et al., 2009) and experimental infection in sheep (Heath and Koolaard, 2012) indicate that cysts may grow 0.5-2 cm a year, varying significantly from case to case. Small calcified lesions of less than 1 cm are commonly recovered, especially in the liver, whereas larger cysts are less frequently calcified. Though the control of cyst growth rate is not well known, the survival of cysts has been related to the ability to reach a threshold size (about 1 cm) during the early stages of development (Rogan et al., 2015). Otherwise cyst fertility appears to be correlated with cyst size so larger cysts are more frequently fertile. Apart from Vogel's earlier report on lesions in cattle similar to E multilocularis metacestode (Vogel, 1957), aberrant cysts indicated as multilocular have been observed in the yak Bos grunniens (Xiao et al., 2003 and Heath et al., 2005) and in mouflon (Ovis gmelinii anatolica) (Simsek and Eroksus, 2009). Visually similar to alveolar cysts with small alveolar vesicles and necrotic areas, these types of lesions have actually been identified at histologic and molecular analyses as E. granulosus cysts. In the present study this type of cyst has been suitably named multisepted,
being characterized by septa that divide the cavity into
chambers differing in number and size. Morphologically, cyst destruction is expressed as the degeneration of the germinal layer, and occasionally the collapse of the cyst wall into the cyst cavity, or calcification of the lesion. In sheep, as opposed to humans, multivesicular
types (with mother cyst containing
endogenous daughter cysts), thought to be a defensive reaction of the parasite against traumatic micro ruptures or micro fissures and /or biliary communication or immunological effector mechanisms, were rarely recovered. Sometimes hyperlaminated specimens were observed, with sheets of laminar tissue repeatedly back-folded after expansion of detached endocyst into the virtual cavity.
While multivescicular cysts are generally fertile, in
hyperlaminated cysts, once the cyst cavity is completely filled, the germinal membrane is no longer able to produce PSC and paradoxically the inherent plasticity of cyst, with LL overproduction, is detrimental to the larva itself. As in humans, cyst calcification may represent the terminal stage of parasite degeneration, but unlike humans where scattered or coarse calcification and more or less calcified walls may be observed in various cyst types, in sheep the very commonly found calcified cysts (>50% of specimens) represent further degenerative evolution of the multisepted type with almost virtual internal chambers, the result of thickening and calcification of internal septa. The exact mechanism involved in cyst degeneration is not well known, but Th1-biased response usually detrimental to parasites is indicated as being involved in initiating calcification, through macrophages as a possible source of osteopontin, a potent regulator of calcium deposit in tissues (Peng et al., 2006). In addition, inhibition of TGF-β1 signalling has recently been reported to increase calcification of the pericyst wall in the animal model of CE, associated with decreased cyst load index and lower viability of protoscoleces, to the extent that pharmacological modulation of calcification in pericysts has been postulated as a possible new therapeutic target in the treatment of hydatid disease (Wu et al., 2011). Apart from the ambiguous role of immunity which may provide some protection against Echinococcus infection (Vuitton, 2003), the hypothesis has been advanced that cyst degeneration in livestock might possibly be related to anthelmintics such as benzimidazolic compounds, routinely administered against other parasitosis perceived by farmers as very dangerous (Garippa et al., 1998 and Eckert et al., 2001b, Cabrera et al., 1995 and Santos et al., 2008). As the natural history of infection may differ in different host species, the results of an ongoing investigation into the development of CE cysts in lambs experimentally infected with E. granulosus and albendazole drenched at intervals of months (Lightowlers et al., 2014), probably will shed light on this subject. The continuous monitoring of CE cysts, facilitated by standardization of cyst types, may help to infer changes in parasite transmission dynamics. In the present study multisepted and calcified cysts represent the prevalent types, increasing with age, in sheep in southern Sardinia, outnumbering multisepted cysts in younger animals and rise of calcified with sheep ageing. The most relevant feature is that unilocular cysts appear to have slightly increased in the 2nd survey conducted 10 years after the first, particularly in the most rural province of Oristano compard to the more “urbanized” Cagliari province, suggesting a possible reversal of the downward trend previously observed over time for this type of cyst.
The general economic crisis and particularly the impact of globalization on revenue from dairy products has in fact diminished capital expenditure, with the result that farmers are likely to reduce treatment to the bare minimum particularly in less-favoured, more rural areas (unpublished). These data are of particular concern considering the persistent practice of unauthorised or home slaughtering on the island. Improvements in meat processing, in line with EU regulations, has resulted in a drastic reduction in the number of abattoirs in Sardinia, from 1081 in 1969 (Roncaglioli and Sibani, 1970) to around 90 today (Ministry of Health, 2011). This has lead to a substantial increase in meat processing and animal transportation costs, regarded by farmers as uneconomical. Considering the number of dairy sheep reared in Sardinia and the mean culling rate (15-20% of flock), an estimated 3400,000 ewes are slaughtered each year in Sardinia, vs the official figure of 150,000 (ISTAT, 2011). Monitoring the relative frequency of various cyst types in different areas may thus improve the assessment of parasite pressure and contribute to evaluating the effectiveness of potential control measures.
4.1. Conclusion
In conclusion, it is proposed that data from meat inspection, apart from other relevant epidemiological information (species, sex, age, geographical provenance, other parasitic and not parasitic pathologies, number of cysts, organ location etc.) be further improved by providing morpho-functional indications about cysts as classified herein, in a surveillance and control perspective.
Acknowledgements
The research was funded by a grant from the Regione Autonoma della Sardegna (L.R. 7 agosto 2007, n.7 “Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna” Progetti di ricerca di base) CUP F 71J09000490002, prot. CRP2_134.
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Fig. Captions
Fig.1. Sheep liver infected with cystic echinococcosis. Fig. 2. CE cysts recovered in Sardinian sheep during two surveys (1995-97 and 2005-2010), according to cyst size. (a) Total cysts; (b) Fertile cysts; (c) Sterile cysts. Fig.3. Unilocular cysts. Whole cysts (left), open cyst (right). Fig. 4. Structural organization at micro and sub-microscopic level of unilocular cysts from Sardinian sheep. (a) Cyst wall under optical microscopy showing germinal layer (GL) with brood capsules (BC), protoscoleces (PSC), and outer acellular laminated layer (LL), surrounded by the pericyst of fibrous adventitial layer (AL). (b) PSC at different stages of development due to their asynchronous formation in BC inside the cysts. (c,d) Ultrastructure of GL adhering to LL. In (d) respect to (c) microtriches along the inner surface appear sparser and larger vacuoles are present in the proliferative cell layer (pcl) that appears less compact. These features may vary widely in different specimens or even within the same cyst. Fig. 5. Multisepted cysts with cavity divided by septa into spheroidal chambers of widely variable number (few-dozen). Fig. 6. Ultrastructural aspects of multisepted cyst showing residual germinal layer (rgl) adhering to a thin laminated layer (LL). Fig. 7. “Laminated” brood capsule from hydatid sand of a multisepted cyst, showing inner peripheral ectopic laminated layer (LL) and containing only degenerate protoscoleces (d PSC) dispersed in a finely granulated matrix. Fig. 8. Calcified cyst showing almost virtual internal chambers. Fig. 9. Caseous cyst with cavity filled with a thick matrix of cheesy consistency. Fig. 10. Multisepted-caseous cyst. Degeneration of multisepted cyst with chambers filled with large amount of caseous material. Fig. 11. Hyperlaminated cyst with the virtual cavity filled with sheets of laminated tissue (SLT). Fig. 12. Sheets of laminated tissue (SLT) hyperproduced into the cavity of a hyperlaminated cyst (O.M.). Fig. 13. Prevalence of different types of cysts recovered in Sardinian sheep by age group (a) 1st survey 1995-1997; (b) 2nd survey 2005-2010. Fig. 14. Frequency of different types of CE cysts recovered in sheep from south Sardinia during two survey periods 10 years apart (1995-1997; 2005-2010). Fig. 15. Frequency of sheep from southern Sardinia harbouring fertile cysts.
Dimension of images:
Fig.1 (1.5 columns) Fig 2 (single column) Fig 3 (1.5 columns) Fig 4 (2 columns) Fig 5 (2 columns) Fig 6 (single column) Fig 7 (single column) Fig 8 (single column) Fig 9 (single column) Fig 10 (single column) Fig 11 (single column) Fig 12 (single column) Fig 13 (1.5 columns) Fig 14 (single column) Fig 15 (single column)
Figure Click here to download high resolution image
Figure Click here to download Figure: Fig 2 (single column).xlsx
Fig. 2a 100 80 %
60 1995-97
40
2005-10
20 0 < 1 CM
< 3 CM
< 5 CM
< 10 CM
cyst size (upper limit)
Fig. 2b 100 80 %
60 1995-97
40
2005-10
20 0 < 1 CM
< 3 CM
< 5 CM
< 10 CM
cyst size (upper limit)
Fig. 2c 100 80 %
60 1995-97
40
2005-10
20 0 < 1 CM
< 3 CM
< 5 CM
< 10 CM
cyst size (upper limit)
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download Figure: Fig 13 a,b (1.5 columns).xlsx
Figure Click here to download Figure: Fig. 14 (single column).xlsx
Fig. 14 100 80 %
60 40
1995-97 2005 2010
20 0
cyst type
Figure Click here to download Figure: Fig. 15 (single column).xlsx
Fig. 15 80 60
% 40 20 0 1980
1987-88
1989-90
survey periods
1995-97
2005-10
Table 1. Morphological study of CE cysts recovered in Sardinian sheep. SOUTH SARDINIA 1st Survey (1995-1997) No. of examined cysts
2nd Survey (2005-2010)
TOT
Hepatic
Pulmonary
TOT
Hepatic
Pulmonary
10,334 a
7,012
3,314
6,249 b
3,979
2,265
818 3,794 5,373 a 342 7
531 2,434 3,972 72 3
287 1,360 1,393 270 4
624 1,977 3,236 b 316 96
390 1,128 2,397 52 12
234 849 834 264 84
Cyst type: “UNILOCULAR” “MULTISEPTED” “CALCIFIED” “CASEOUS” “HYPERLAMINATED”
Province of Oristano 1st Survey (1995-1997) No. of examined cysts
2nd Survey (2005-2010)
TOT
Hepatic
Pulmonary
TOT
Hepatic
Pulmonary
5,039 c
3,360
1,676
3,177 c
2,109
1,065
338 1,920 2,593 c 184 4
179 1,248 1,885 46 2
159 672 705 138 2
277 1,091 1,667 c 122 20
176 669 1,238 20 6
101 422 426 102 14
Cyst type: “UNILOCULAR” “MULTISEPTED” “CALCIFIED” “CASEOUS” “HYPERLAMINATED”
Province of Cagliari 1st Survey (1995-1997) No. of examined cysts
2nd Survey (2005-2010)
TOT
Hepatic
Pulmonary
TOT
Hepatic
Pulmonary
5,295 b
3,652
1,638
3,072 d
1,870
1,200
480 1,874 2,780 b 158 3
352 1,186 2,087 26 1
128 688 688 132 2
347 886 1,569 d 194 76
214 459 1,159 32 6
133 427 408 162 70
Cyst type: “UNILOCULAR” “MULTISEPTED” “CALCIFIED” “CASEOUS” “HYPERLAMINATED” a including 8 splenic cysts b
including 5 splenic cysts
c
including 3 splenic cysts
d
including 2 splenic cysts
Table 2. Characteristics of different cyst types.
Type
Shape
Size Range (cm)
Unilocular
Globular, lobate or plurilobate
1-10
single, fluid filled
fertile
range 1-100
Multisepted
irregular, acinous
0.5-3-4
divided into 3dozen spheroidal chambers, with hydatid fluid
generally sterile
in about 5% presence of “laminated brood capsules” (Conchedda et al, 2008)
Calcified
irregular, acinous
generally ~1
septed, with internal chambers almost virtual due to the thickening of internal septa
sterile
Caseous
Globular, lobate or plurilobate
1-5
generally single cavity, filled with a thick creamy or cheesy matrix
sterile
Hyperlaminated
globular, lobate
1-5
virtual cavity filled with extensively folded and over- lapping sheets of hyperproduced laminated tissue (SLT)
sterile
Internal cavity Fertility % viability of PSC
S0001-706X(16)30163-2 http://dx.doi.org/doi:10.1016/j.actatropica.2016.04.003 ACTROP 3914
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Acta Tropica
Received date: Revised date: Accepted date:
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Please cite this article as: Conchedda, Margherita, Seu, Valter, Capra, Salvatore, Caredda, Alessia, Pani, Sergio Pino, Lochi, Pier Giorgio, Bortoletti, Gianfranco, A study of morphological aspects of cystic echinococcosis (CE) in sheep in Sardinia.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A study of morphological aspects of cystic echinococcosis (CE) in sheep in Sardinia.
Margherita Concheddaa, Valter Seua, Salvatore Capraa, Alessia Careddaa, Sergio Pino Panib, Pier Giorgio Lochib, Gianfranco Bortolettia.
Department of Life and Environmental Sciences, Cagliari University, Cittadella Universitaria di
Monserrato, Strada Provinciale Monserrato-Sestu km 0.700, Monserrato (Cagliari), Italy; bServizio Veterinario Igiene degli Alimenti di Origine Animale ASL 6, Sardegna (Italy).
Corresponding Author: Margherita Conchedda tel. +39 0706754530; fax +39 0706754536 E-mail: [email protected]
Graphical abstract
Highlights ►Cystic Echinococcosis is persistently widespread in sheep in Sardinia. ► Morpho-structural aspects of >16,000 CE cysts are analysed during 2 surveys. ►Detailed parameters for cyst classification are established. ►Cysts are grouped into 5 main types. ►Reporting of cyst types is proposed in a surveillance and control perspective.
Abstract We have investigated the morphological and structural aspects of hydatid cysts recovered from sheep raised in southern Sardinia during two surveys conducted over the 20 years since the last control attempt at the end of the 1980s. In the first study carried out during 1995-1997, a few years after the last control attempt, a total of 10,334 cysts were examined, recovered from 1,029 infected sheep out of a total of 1,375 inspected (prevalence 74.84%). In the second survey, conducted ten years later during the period 2005-2010 in the absence of specific control measures, a total of 6,249 cysts were recovered from 916 parasitized sheep out of the 1,414 examined (prevalence 64.78%). Cysts were grouped into 5 different types: “Unilocular”, “Multisepted”, “Calcified”, “Caseous”, “Hyperlaminated”. Unilocular: fertile, consist of
a single fluid-filled cavity; multisepted:
generally sterile, fluid filled, with cavity divided into spheroidal chambers (3-dozen); calcified: sterile, small, with internal chambers almost virtual due to the thickening of internal septa; caseous: sterile, the cavity filled with a thick yellowish matrix; hyperlaminated: sterile, with a virtual cavity filled with extensively folded and overlapping sheets of hyperproduced laminated tissue.
A thorough knowledge and the precise characterization of each type of lesion, making it possible to establish detailed parameters for cyst classification, appears very useful for the harmonization of data collection. From a surveillance and control perspective it is proposed that, in addition to other relevant epidemiological information, data from meat inspection be further improved, including morpho-functional indications about cyst type.
Keywords Cystic Echinococcosis; Sardinia; Sheep; Morpho-functional classification; cyst types.
1. Introduction In spite of improved knowledge of Cystic Echinococcosis (CE) and the apparently easy control of the infection that have reduced its burden in some countries, the disease remains a major, though often underestimated, public health problem in many regions of the planet, emerging or remerging in vast areas (Eckert and Deplazes, 2004, Jenkins et al., 2005, Buishi et al., 2005 and WHO, 2013). One million or more people worldwide are estimated to be currently suffering from CE, and the global burden in terms of DALYs lost is reported to be similar to other better-known neglected tropical diseases (such as Chagas disease, dengue, onchocerciasis, or African trypanosomiasis) (Budke et al., 2006 and Craig et al., 2007). Moreover, the financial burden of CE on the world's livestock industry has been estimated at an annual loss of around $2 billion (Torgerson and Macpherson, 2011). As is well known the prevalence of the zoonosis is closely connected with the social and economic conditions of the population: widespread use of traditional techniques for raising small ruminants (extensive or semi-extensive grazing), illegal slaughtering of the animals, and presence of a large number of dogs, particularly strays. Sardinia, an island in the middle of the Mediterranean, with more than 3,000,000 dairy sheep reared in extensive/semi-extensive production systems, has 40 % of Italy’s total sheep population. The island historically registered one of the highest CE diffusion rates both in livestock and in humans, despite three tentative control programmes carried out over time (Conchedda et al., 1997, 2002, 2010 and 2012). Our recent study highlighted the modifications that have taken place in the epidemiology of CE in Sardinia both in humans (Conchedda et al., 2010) and in sheep (Conchedda et al., 2012), over the twenty years since
the last (interrupted) control attempt at the end of the 1980s and discussed the profound ongoing structural transformation that is changing the scenario of the zoonosis. An annual incidence rate in humans of about 6.6 per 100,000 inhabitants is still currently observable on the island (Conchedda et al., 2010), where the long term (decades) natural history of CE has been traced by an in-depth knowledge of the morphological and functional states of different cyst types (Bortoletti et al., 2002, 2004 and 2013). With regard to livestock, data describing the current CE status and the trend of prevalence in sheep, the most common intermediate host in Sardinia, show a persistently high and widespread diffusion (prevalence of 65% today). All sheep are infected with the G1 Echinococcus granulosus strain (Varcasia et al., 2006, Garippa et al., 2008), the most common in Europe and in the Mediterranean Basin, responsible for the majority of human infections (Romig et al., 2006, McManus and Thompson, 2003 and Thompson, 2008). The prevalence is particularly high in less favoured areas (Conchedda et al., 2012), in spite of the diminished CE burden and the improvements achieved over time. This is due to the persistence of factors traditionally associated with transmission (large numbers of dogs, home slaughtering and offal disposal), notwithstanding the significant socio-economic, demographic and structural transformations that have taken place on the island (Conchedda et al., 1997, 2002, 2010 and 2012; Scala et al., 2006, Scala and Mazzette, 2009, Varcasia et al., 2011). The availability of reliable and detailed base-line data able to accurately depict CE diffusion is basic to monitoring the effects of preventive measures to be put in place in a control plan. In particular, considering the present vaccine option developed as a result of Australian studies on EG95 (Lightowlers et al., 1996 and 2003), one of the most important aspects to be carefully evaluated, together with epidemiological base line data, is the evaluation of parasite burden with particular regard to the morpho-functional status of cysts in the major intermediate host. This provides precise information as to how the life cycle of the parasite is actually maintained for the area examined, allowing careful evaluation of immunization efficacy also in terms of cost-benefits. A standardized classification of ovine CE cysts may be useful for facilitating both the uniform reporting of results from epidemiological field studies conducted in different areas and monitoring any changes that have occurred over time. In addition this approach may be of particular interest considering the increasing importance of ultrasonography in intra vitam screening for CE in sheep (Maxson et al., 1996, Sage et al., 1998, Guarnera et al., 2001, Lahmar et al., 2007 and Dore et al., 2014) similarly to humans. In this case , as well known,
the WHO-IWGE classification of ultrasound images of CE cysts (WHO-IWGE, 2003), together with the improved knowledge of the natural history of infection (Bortoletti et al., 2002, 2004 and 2013) have proven useful for advancing the clinical management of patients (Brunetti et al., 2010). For this purpose the present study has been conducted on the structural and functional aspects of hydatid cysts recovered in sheep raised in the province of Cagliari or Oristano and slaughtered in abattoirs in southern Sardinia. The investigation covered two periods: the 1st survey carried out during 1995-1997, fairly soon (5–7 years) after the last control attempt, the 2nd survey conducted roughly 10 years later over the period 2005-2010, long after the control plan had been dropped.
2. Materials and Methods The surveys concerned a total of 16,583 hydatid cysts from 1,945 sheep infected out of a total of 2,789 animals inspected during an epidemiological study carried out in two phases (1995-1997 and 2005-2010) in abattoirs in southern Sardinia (Conchedda et al., 2012). In detail during the 1st survey 10,334 cysts were collected from 1,029 CE infected sheep out of the 1,375 inspected (prevalence 74.84%). During the 2nd phase a total of 1,414 sheep were inspected, recovering a total of 6,249 cysts from 916 parasitized animals (prevalence 64.78%).
2.1. Area of study Sardinia is an island of 23,821 km2 in the middle of the Mediterranean sea (Italy) (38°51′ and 41°15′N, 8°8′ and 9°50′E), mostly hilly and mountainous, sparsely populated (about 1,600,000 inhabitants), with a typical Mediterranean climate characterized by mild winters and hot summers. Extensive or semi-extensive sheep farming is widespread on the island, where 3 million dairy sheep are reared. The most important source of income for the 14,500 farms is represented by sheep’s milk, cheese and dairy products, in addition to the sale of one-month old lambs. Therefore, and also owing to the low commercial value of mutton, ewes are normally slaughtered old, at the end of their production cycle. Furthermore, in this agro-pastoral system characterized by extensive/semi-extensive grazing, dogs play a major role. The dog population in Sardinia is estimated to be more
than 380,000 with 70-80,000 sheepdogs: on average 3.8 per farm according to a recent survey (Varcasia et al., 2011). In addition many “stray-“ and “community-“ dogs are also present on the island, and these contribute to fostering the perpetuation of the CE biological cycle, considering that in the past a general infection rate of 16-20% has been reported in Sardinian dogs, rising to 25% in sheepdogs (Arru et al.,1993) . These aspects together with the uncontrolled practice of home slaughtering, and the improper disposal of carcasses, favour the persistence of CE on the island (Conchedda et al., 1997, 2002, 2010 and 2012; Scala et al., 2006, Scala and Mazzette, 2009, Varcasia et al., 2011).
2.2. Processing of samples and morphological analysis Analysis during the two survey periods was carried out following the same methodology, as in earlier surveys (Bortoletti et al., 1990, Conchedda et al., 2008, 2012). In detail, after collection of data on age, origin, ownership of sheep and type of farm, drawn from farm registers or directly supplied by vets at abattoirs, viscera of slaughtered sheep were examined by visual and palpatory inspection to detect the presence of any cystic lesions (Fig. 1), to be taken to the laboratory for further analysis.
For each sheep the CE cysts
were counted, measured and classified according to size, type, as well as location. In more detail, the external diameter of each cyst was measured and the content carefully examined. Cyst fluid, including protoscoleces (PSC), were collected by aspiration with a Pasteur pipette, laminated membrane including germinal layer were washed and gently scraped to recover remaining PSC and to harvest adhering brood capsules (BC). A sample of the total sediment was examined by optical microscope, PSC number was estimated by extrapolation to the entire sediment volume, and viability assessed by 0.1% methylene blue exclusion test and by checking the motility of flame cells (Casado et al., 1986). Small cysts removed intact were cut and repeatedly rinsed in a Petri dish and the contents examined under a stereoscopic binocular microscope to detect any PSC. Lastly the cysts were classified into 5 morphostructural types in accordance with our previous classification (Bortoletti et al., 1993) suitably revised. For some samples, as already described (Conchedda et al., 2008), after careful rinsing in 0.13M Millonig’s phosphate buffer at pH 7.4, the material was fixed in 10% formaldehyde
buffered with Millonig 0.13M at pH 7.4, partially dehydrated and finally kept in ethyl alcohol at 80◦ for further observations under the stereo microscope. For the structural and ultrastructural study, the specimens were fixed in Karnowsky’s solution (2% PF, 2,5% GTA in Millonig 0.13 M, pH 7.4) for 12 h, post fixed with 1% osmium tetraoxide in Millonig for 3 h, and then progressively dehydrated in alcohol and embedded in Araldite. Semithin sections of approx. 1 µm, cut with an LKB ultratome III ultramicrotome were stained with methylene blue and Azur II for light microscopic examination. Ultrathin sections of 600–900 Å were stained with uranyl acetate and lead citrate for transmission electron microscopy, performed using a JEOL 100S microscope (Jeol, Tokyo, Japan).
2.3 Trend analysis
Data for the different types of cysts recovered in the two survey periods were analysed according to sheep age, also considering specific prevalences in the two provinces of southern Sardinia (Cagliari and Oristano). In addition, any changes that may have occurred over the years in CE prevalence, particularly of fertile cysts, was investigated by comparing present data with previous surveys carried out on the island from 1980 onwards (Arru et al., 1980; Bortoletti et al., 1989; Bortoletti et al., 1990; Bortoletti et al., 1993, Gabriele et al., 1998).
3. Results
3.1. 1st phase
During the survey 7,012 hepatic cysts (68% of total), 3,314 pulmonary (32%), plus 8 splenic cysts were recovered (Table 1).
3.1.1. Cyst size Analysis according to cyst size showed that of the 10,334 recovered specimens 78% (8,110) had a diameter of <1cm, 18% (1,850) ranged from 1 to 3 cm, 312 (3%) from 3 to 5
cm, and just 62 (< 1%) of between 5 and 10 cm (Fig. 2a). Analysis of cyst size according to fertility, showed that this pattern of distribution was to be attributed entirely to sterile cysts (Fig. 2c), which accounted for 92% of total specimens (9,516/10,334). In detail, these mostly small sterile cysts, measured 83.3% (7,927/9,516) <1cm in diameter, 14.6% (1,387) between 1 and 3 cm, 1.9% (182) between 3 and 5 cm while only 20 cysts were larger than 5 cm. Vice versa the size of fertile cysts was generally larger (Fig. 2b): out of a total of 818 specimens, 182 (22.2%) had a diameter of <1cm, 464 (56.7%) from 1 to 3 cm, 130 cysts (15.8%) ranged from 3 to 5 cm, and 42 (5.1%) >5 cm.
3.1.2 Cyst typology The detailed morphostructural analysis of the recovered cysts allowed to group them into 5 different types: unilocular, multisepted, calcified, caseous and hyperlaminated (Table 2). The unilocular cysts (Fig. 3) accounted for 7.9% of cases (818/10,334) (Table 1), 8.7 % (287/3,314) of pulmonary and 7.6% (531/7,012) of hepatic cysts respectively. Globular, lobate or plurilobate in shape (Fig. 3), ranging from 1 to 10 cm or more in size, these cysts showed a single fluid filled cavity and were almost invariably fertile, often with numerous brood capsules, but with widely varying PSC viability (from 1 to 100 %). The structural organization of cysts at the micro and sub-microscopic level was as previously reported (Bortoletti and Ferretti, 1978) (Fig. 4). They consist of a germinal layer (GL) which represents the parasite sensu stricto, supported by an acellular, heavily glycosylated, laminated layer (LL) (Diaz et al., 2011a and 2011b), surrounded by a pericyst of fibrous adventitial layer (AL) produced by resolution of host cellular inflammation precociously initiated during early post-oncospheral stages.
In these cysts LL
and GL are generally thin with
sparse
microtriches along the inner surface. Numerous large vacuoles are present in the cytoplasm of tegumental syncytium (ts) that appears “fenestrated”. The proliferative cell layer (pcl) is thin and slightly compact. GL characteristics, in terms of density of microtriches, size of ts and compactness of pcl, varied widely both in the different specimens and within the same cyst (Fig. 4c,d). Due to asynchronous formation of brood capsules inside the cysts, PSC at different stages of development are present (Fig. 4b) and cysts may contain both viable and non-viable or dead PSC in varying percentages.
The multisepted cysts (Fig. 5) (3,794 of the 10,334 specimens examined), accounted for roughly 36.7% of total cysts, 34.7 % of hepatic (2,434/7,012) and 42.0 % of pulmonary cases (1,360/3,314) respectively (Table 1). They measured between 0.5 and 3-4 cm in diameter but mostly less than 1 cm, had cavity invariably divided into spheroidal chambers (3-dozen) that although fluid-filled were generally sterile or, in very rare cases with very few PSC (from just a few to some hundreds). Cyst wall showed a thick external adventitial layer and a thin laminated layer with residual germinal layer (Fig. 6). Frequently recovered in the present study (Table 1), this type of cyst represents a parasite degeneration stage. In fact in about 5% of multisepted
cysts
“laminated brood capsules” (LBC) were recovered
i.e.
“anomalous” brood capsules characterized by an ectopic inner peripheral layer of laminated tissue in intimate contact with the internal wall surface and containing only degenerated protoscoleces dispersed in a finely granulated matrix (Fig. 7). These LBC are thought to be the likely effect of degenerative involution of the parasite (Conchedda et al., 2008). Further degenerative evolution of multisepted cysts results in the calcified cysts, the most frequently recovered, accounting for 51.9% of cases (5,373/10,334), especially in the liver (3,972/7,012; 56.6%). Very small (generally
~1 cm) and always sterile,
the internal
chambers of these cysts are almost virtual due to the thickening of internal septa (Fig. 8), and typically produce a gritty sound/feeling upon incision (Soulsby, 1982). About 3% of total specimens recovered (342/10,334) (Table 1), but more than 8% of those found in the lungs (270/3,314), appeared as caseous cysts (Fig. 9). Similar in shape to the unilocular cysts with diameter of between 1 and 5 cm, the cavity of these specimens filled with a thick matrix of creamy or cheesy consistency, probably due to bacterial contamination or infiltration. Occasionally, caseous degenerative process may concern multisepted cysts, leading to complex “multisepted-caseous” cysts where chambers contain large amounts of caseous material (Fig. 10). In other rare cases another cyst type was recovered during the study, accounting for only 7 cases out of 10,334, less than 1% of the total (Table 1), termed hyperlaminated (Fig. 11). They are of moderate size (1-5 cm in diameter) and the virtual cavity is filled with extensively folded and overlapping sheets of hyperproduced laminated tissue (SLT), well detected at the histological level (Fig 12).
Where scattered intermediate stages between different cyst types were observed in sheep, e.g. caseous degeneration of multisepted or hyperlaminated cysts, for categorisation purposes these were included in the prevalent morphostructural type.
3.1.3.Cyst type according to sheep age. As previously reported (Conchedda et al., 2012) specific prevalence rates in Sardinian sheep increase with age. Analysis of data on cyst types according to animal age showed the highest prevalence, increasing with sheep age for multisepted and calcified cysts (Fig. 13a), while rates were much lower for the other types in all age groups. In particular, in the province of Cagliari the calcified specimens were always the most numerous for all ages, while viceversa in the province of Oristano multisepted cysts prevailed in young and up to 6 year-old sheep. On the other hand prevalence of calcified cysts increased with age, exceeding multisepted in the oldest animals (Fig. 13a).
3.2. 2nd phase During the 2nd phase of the study (2005-2010 years) 64% of recovered cysts were hepatic (3,979/6,249) and 36% pulmonary (2,265/6,249) (Table 1).
3.2.1 Cyst size The morphostructural analysis confirmed a significant difference in size between fertile and non-fertile specimens (Fig. 2b,c), with larger cysts less frequently degenerated/ not viable. Cysts from the 2nd survey appeared on average smaller than the 1st study (Fig. 2a-c). In particular frequency of small fertile cysts (< 1 cm) increased over time, with a simultaneous decrease of 1-3 cm cysts (Fig. 2b). A minor increase in frequency of small sterile cysts (< 1cm) was also observed, while that of larger specimens 1-3 cm decreased slightly.
3.2.2 Cyst typology
In total roughly 10% of the cysts were unilocular fertile (624/6,249), a slight increase compared with the 1st survey (Figs. 13a,b-14). No significant difference in frequency was noticed for cyst location: 10.3% of pulmonary (234/2,265) and
9.9% of hepatic cysts
(390/3,979) respectively. In addition the morphostructural analysis showed that 31.6% of cysts were multisepted (1,977/6,249) (Fig. 13b) slightly less than in the 1st survey: 28.3% of hepatic (1,128/3,979) and 37.5% of pulmonary cysts (849/2,265) respectively. Calcified cysts were commonly recovered,
accounting for around 52% of the total (3,236/6,249), particularly in the liver
where they represented 60.2% of the 3,979 recovered cysts (Table 1). Further, about 5% of all cysts (316 on 6,249), but more than 11% of pulmonary ones (264/2,265), were classified as caseous, a slight increase over the 1st survey. Lastly, 1.5% of the 6,249 recovered cysts were hyperlaminated, found more frequently in the lungs (3.7% on a total of 2,265) than in the liver (0.3% of 3,979) (Table 1).
3.2.3 Cyst type according to sheep age. During the 2nd survey multisepted and calcified cysts were again found to be the most common (Fig. 13b). In particular in the Cagliari province a general decrease in prevalence with respect to the 1st survey was observed at all ages for all cyst types (and particularly for multisepted), except for caseous and hyperlaminated cysts, only occasionally observed, that showed a more irregular trend. Viceversa in the province of Oristano minor variations were detected with respect to the 1st study, with a constant increase in prevalence of unilocular (fertile) cysts in sheep of all ages (Fig.13 b).
3.3. Trend analysis
A very significant reduction has been observed over the years in cyst fertility in sheep in Sardinia as emerged from comparison of data from the present study with previous surveys carried out on the island from 1980 onwards (Arru et al., 1980; Bortoletti et al., 1989; Bortoletti et al., 1990; Bortoletti et al., 1993, Gabriele et al., 1998) (Fig. 15). The frequency of ewes harbouring at least 1 unilocular cyst was, considering all Sardinian provinces, about 70% 30 years ago (Arru et al., 1980). By the 1980s frequency had decreased to between 25
and 40% (Bortoletti et al., 1989 and 1990), dropping to around 18% in the mid nineties (12.5% in southern Sardinia and 21% in the North) though remaining at around 30% in the most pastoral Nuoro province (unpublished) and 27% in the area of the Goceano region between Nuoro and Sassari provinces (Soro et al., 2002). More recently (1998-2003) a decrease in frequency to 10.25% was reported for northern Sardinia (Scala et al., 2006), while the present study found at least 1 fertile cyst in 14.0% of the ewes inspected in southern Sardinia during the 2nd survey (2005-2010) (vs 12.5% in the past): 9.6% from sheep with pulmonary cysts alone, 4.5% from sheep with hepatic cysts alone, and 21.0% from sheep infected with both. In general, as reported, cysts from the latter survey, particularly sterile ones, were found to be smaller in size than in the 1st study period, and the relative frequency of the smaller cysts, particularly those 1-3 cm in size, was observed to increase for all cyst types (Figs 2a,b,c).
4. Discussion
Since the last control attempt interrupted in the early 1990s, no specific preventive measures have been put in place in Sardinia, while significant socio-economic, demographic and structural transformations have profoundly modified the zoonosis scenario (Conchedda et al., 1997, 2002, 2010 and 2012) and innovation and tradition have intertwined. In this context the findings of the two surveys carried out in 1995-97 and 2005-2010, after the last control campaigns, indicate that despite a reduction in terms of intensity, abundance and frequency of heavily infected animals (Conchedda et al., 2012), CE prevalence in sheep in southern Sardinia is still one of the highest in the Mediterranean region (Eckert et al., 2001a). The aim of the present study was to characterize hydatid cysts collected from sheep in Sardinia by means of morphological tools.
A thorough knowledge and the precise
characterization of each type of lesion, making it possible to establish detailed parameters for cyst classification, appears very useful for the harmonization of data collection. It facilitates the visual and accurate evaluation of parasite status, during offal inspection, even in a large number of infected sheep, without over-extending inspection time. The morphofunctional approach makes the monitoring of viable cysts that ensure easier maintenance
of the parasite's life-cycle, making it possible to compare disease patterns in different areas and gain an overview of the trends and transformations that have occurred over time. Therefore it is of potential interest for monitoring a control programme, especially if the sheep vaccination option is envisaged, providing valuable information on base-line infection pressure in livestock, and making it possible to evaluate the benefits of treatment.
Compared to human infection little comparable data regarding the morphological variability of CE cysts in animals are available (Bortoletti et al., 1993, Bortoletti and Ferretti,1978 and Dueger and Gilman, 2001), and cysts are generally analysed in terms of fertility, at the most by differentiating sterile and calcified cysts (Ernest et al., 2009, Daryani et al., 2009 and Kebede et al., 2009). The results of the present study revealed a wide variability of cysts in the natural intermediate host even though, unlike infection in humans where a natural history of decades can be traced (Bortoletti et al., 2004 and 2013), cysts from sheep are at the most 8-10 years old. Based on the detailed analysis of the thousands of cysts collected we were able to distinguish five different types: unilocular, multisepted, calcified, caseous and hyperlaminated.
Specimens from the same host and the same organ showed
morphostructural variability, also with regard to size and viability. As is well known, parasite survival depends on efficient host evasion mechanisms that come into play during development toward the hydatid cyst stage (Conchedda et al., 2004). During the establishing phase, CE is susceptible to host immune killing, as studies on EG95 vaccine have definitively shown (Lightowlers et al., 1996, 2003, Heath and Koolaard, 2012 and Jabbar et al., 2011). The developing cyst elicits a local inflammatory response, which may resolve leaving behind a collagenous capsule, or, if the reaction is too intense, lead to degeneration and eventual death of the parasite. Across the spectrum of host response, cyst fertility correlates with resolution of the inflammation, while an intense response is associated with infertility and, more extremely, degeneration of the cyst (Thompson, 1995). Resolution nearly always takes place in humans, less generally in sheep, in agreement with our observation (Bortoletti et al., 1978,1993, 2002, 2004 and 2013). Degenerating or dead cysts recovered at various later stages suggest a possible killing during established phase, when the parasite loses integrity and undergoes various and complex degenerative processes. This is demonstrated by the variety of cyst types recovered and by the presence of the laminated brood capsules, an indication of profound
degeneration (Conchedda et al., 2008), though no conclusive evidence that cyst death is immunologically mediated has yet been found. Present results strongly suggest that the only fertile specimens able to ensure CE life cycle perpetuation in Sardinia, are the unilocular cysts (Fig. 3). Though not as old as analogous excised human specimens, this type of cysts in sheep proves not to flourish so well and, by comparison, LL and GL are generally much thinner, with sparser microtriches along the inner surface and a larger number of vacuoles (Bortoletti and Ferretti, 1978). In addition the number of PSC and their vitality varies very widely both in unilocular cysts recovered from different ewes, and in cysts from the same animal or even from the same organ. Data from natural (Torgerson et al., 2009) and experimental infection in sheep (Heath and Koolaard, 2012) indicate that cysts may grow 0.5-2 cm a year, varying significantly from case to case. Small calcified lesions of less than 1 cm are commonly recovered, especially in the liver, whereas larger cysts are less frequently calcified. Though the control of cyst growth rate is not well known, the survival of cysts has been related to the ability to reach a threshold size (about 1 cm) during the early stages of development (Rogan et al., 2015). Otherwise cyst fertility appears to be correlated with cyst size so larger cysts are more frequently fertile. Apart from Vogel's earlier report on lesions in cattle similar to E multilocularis metacestode (Vogel, 1957), aberrant cysts indicated as multilocular have been observed in the yak Bos grunniens (Xiao et al., 2003 and Heath et al., 2005) and in mouflon (Ovis gmelinii anatolica) (Simsek and Eroksus, 2009). Visually similar to alveolar cysts with small alveolar vesicles and necrotic areas, these types of lesions have actually been identified at histologic and molecular analyses as E. granulosus cysts. In the present study this type of cyst has been suitably named multisepted,
being characterized by septa that divide the cavity into
chambers differing in number and size. Morphologically, cyst destruction is expressed as the degeneration of the germinal layer, and occasionally the collapse of the cyst wall into the cyst cavity, or calcification of the lesion. In sheep, as opposed to humans, multivesicular
types (with mother cyst containing
endogenous daughter cysts), thought to be a defensive reaction of the parasite against traumatic micro ruptures or micro fissures and /or biliary communication or immunological effector mechanisms, were rarely recovered. Sometimes hyperlaminated specimens were observed, with sheets of laminar tissue repeatedly back-folded after expansion of detached endocyst into the virtual cavity.
While multivescicular cysts are generally fertile, in
hyperlaminated cysts, once the cyst cavity is completely filled, the germinal membrane is no longer able to produce PSC and paradoxically the inherent plasticity of cyst, with LL overproduction, is detrimental to the larva itself. As in humans, cyst calcification may represent the terminal stage of parasite degeneration, but unlike humans where scattered or coarse calcification and more or less calcified walls may be observed in various cyst types, in sheep the very commonly found calcified cysts (>50% of specimens) represent further degenerative evolution of the multisepted type with almost virtual internal chambers, the result of thickening and calcification of internal septa. The exact mechanism involved in cyst degeneration is not well known, but Th1-biased response usually detrimental to parasites is indicated as being involved in initiating calcification, through macrophages as a possible source of osteopontin, a potent regulator of calcium deposit in tissues (Peng et al., 2006). In addition, inhibition of TGF-β1 signalling has recently been reported to increase calcification of the pericyst wall in the animal model of CE, associated with decreased cyst load index and lower viability of protoscoleces, to the extent that pharmacological modulation of calcification in pericysts has been postulated as a possible new therapeutic target in the treatment of hydatid disease (Wu et al., 2011). Apart from the ambiguous role of immunity which may provide some protection against Echinococcus infection (Vuitton, 2003), the hypothesis has been advanced that cyst degeneration in livestock might possibly be related to anthelmintics such as benzimidazolic compounds, routinely administered against other parasitosis perceived by farmers as very dangerous (Garippa et al., 1998 and Eckert et al., 2001b, Cabrera et al., 1995 and Santos et al., 2008). As the natural history of infection may differ in different host species, the results of an ongoing investigation into the development of CE cysts in lambs experimentally infected with E. granulosus and albendazole drenched at intervals of months (Lightowlers et al., 2014), probably will shed light on this subject. The continuous monitoring of CE cysts, facilitated by standardization of cyst types, may help to infer changes in parasite transmission dynamics. In the present study multisepted and calcified cysts represent the prevalent types, increasing with age, in sheep in southern Sardinia, outnumbering multisepted cysts in younger animals and rise of calcified with sheep ageing. The most relevant feature is that unilocular cysts appear to have slightly increased in the 2nd survey conducted 10 years after the first, particularly in the most rural province of Oristano compard to the more “urbanized” Cagliari province, suggesting a possible reversal of the downward trend previously observed over time for this type of cyst.
The general economic crisis and particularly the impact of globalization on revenue from dairy products has in fact diminished capital expenditure, with the result that farmers are likely to reduce treatment to the bare minimum particularly in less-favoured, more rural areas (unpublished). These data are of particular concern considering the persistent practice of unauthorised or home slaughtering on the island. Improvements in meat processing, in line with EU regulations, has resulted in a drastic reduction in the number of abattoirs in Sardinia, from 1081 in 1969 (Roncaglioli and Sibani, 1970) to around 90 today (Ministry of Health, 2011). This has lead to a substantial increase in meat processing and animal transportation costs, regarded by farmers as uneconomical. Considering the number of dairy sheep reared in Sardinia and the mean culling rate (15-20% of flock), an estimated 3400,000 ewes are slaughtered each year in Sardinia, vs the official figure of 150,000 (ISTAT, 2011). Monitoring the relative frequency of various cyst types in different areas may thus improve the assessment of parasite pressure and contribute to evaluating the effectiveness of potential control measures.
4.1. Conclusion
In conclusion, it is proposed that data from meat inspection, apart from other relevant epidemiological information (species, sex, age, geographical provenance, other parasitic and not parasitic pathologies, number of cysts, organ location etc.) be further improved by providing morpho-functional indications about cysts as classified herein, in a surveillance and control perspective.
Acknowledgements
The research was funded by a grant from the Regione Autonoma della Sardegna (L.R. 7 agosto 2007, n.7 “Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna” Progetti di ricerca di base) CUP F 71J09000490002, prot. CRP2_134.
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Fig. Captions
Fig.1. Sheep liver infected with cystic echinococcosis. Fig. 2. CE cysts recovered in Sardinian sheep during two surveys (1995-97 and 2005-2010), according to cyst size. (a) Total cysts; (b) Fertile cysts; (c) Sterile cysts. Fig.3. Unilocular cysts. Whole cysts (left), open cyst (right). Fig. 4. Structural organization at micro and sub-microscopic level of unilocular cysts from Sardinian sheep. (a) Cyst wall under optical microscopy showing germinal layer (GL) with brood capsules (BC), protoscoleces (PSC), and outer acellular laminated layer (LL), surrounded by the pericyst of fibrous adventitial layer (AL). (b) PSC at different stages of development due to their asynchronous formation in BC inside the cysts. (c,d) Ultrastructure of GL adhering to LL. In (d) respect to (c) microtriches along the inner surface appear sparser and larger vacuoles are present in the proliferative cell layer (pcl) that appears less compact. These features may vary widely in different specimens or even within the same cyst. Fig. 5. Multisepted cysts with cavity divided by septa into spheroidal chambers of widely variable number (few-dozen). Fig. 6. Ultrastructural aspects of multisepted cyst showing residual germinal layer (rgl) adhering to a thin laminated layer (LL). Fig. 7. “Laminated” brood capsule from hydatid sand of a multisepted cyst, showing inner peripheral ectopic laminated layer (LL) and containing only degenerate protoscoleces (d PSC) dispersed in a finely granulated matrix. Fig. 8. Calcified cyst showing almost virtual internal chambers. Fig. 9. Caseous cyst with cavity filled with a thick matrix of cheesy consistency. Fig. 10. Multisepted-caseous cyst. Degeneration of multisepted cyst with chambers filled with large amount of caseous material. Fig. 11. Hyperlaminated cyst with the virtual cavity filled with sheets of laminated tissue (SLT). Fig. 12. Sheets of laminated tissue (SLT) hyperproduced into the cavity of a hyperlaminated cyst (O.M.). Fig. 13. Prevalence of different types of cysts recovered in Sardinian sheep by age group (a) 1st survey 1995-1997; (b) 2nd survey 2005-2010. Fig. 14. Frequency of different types of CE cysts recovered in sheep from south Sardinia during two survey periods 10 years apart (1995-1997; 2005-2010). Fig. 15. Frequency of sheep from southern Sardinia harbouring fertile cysts.
Dimension of images:
Fig.1 (1.5 columns) Fig 2 (single column) Fig 3 (1.5 columns) Fig 4 (2 columns) Fig 5 (2 columns) Fig 6 (single column) Fig 7 (single column) Fig 8 (single column) Fig 9 (single column) Fig 10 (single column) Fig 11 (single column) Fig 12 (single column) Fig 13 (1.5 columns) Fig 14 (single column) Fig 15 (single column)
Figure Click here to download high resolution image
Figure Click here to download Figure: Fig 2 (single column).xlsx
Fig. 2a 100 80 %
60 1995-97
40
2005-10
20 0 < 1 CM
< 3 CM
< 5 CM
< 10 CM
cyst size (upper limit)
Fig. 2b 100 80 %
60 1995-97
40
2005-10
20 0 < 1 CM
< 3 CM
< 5 CM
< 10 CM
cyst size (upper limit)
Fig. 2c 100 80 %
60 1995-97
40
2005-10
20 0 < 1 CM
< 3 CM
< 5 CM
< 10 CM
cyst size (upper limit)
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download Figure: Fig 13 a,b (1.5 columns).xlsx
Figure Click here to download Figure: Fig. 14 (single column).xlsx
Fig. 14 100 80 %
60 40
1995-97 2005 2010
20 0
cyst type
Figure Click here to download Figure: Fig. 15 (single column).xlsx
Fig. 15 80 60
% 40 20 0 1980
1987-88
1989-90
survey periods
1995-97
2005-10
Table 1. Morphological study of CE cysts recovered in Sardinian sheep. SOUTH SARDINIA 1st Survey (1995-1997) No. of examined cysts
2nd Survey (2005-2010)
TOT
Hepatic
Pulmonary
TOT
Hepatic
Pulmonary
10,334 a
7,012
3,314
6,249 b
3,979
2,265
818 3,794 5,373 a 342 7
531 2,434 3,972 72 3
287 1,360 1,393 270 4
624 1,977 3,236 b 316 96
390 1,128 2,397 52 12
234 849 834 264 84
Cyst type: “UNILOCULAR” “MULTISEPTED” “CALCIFIED” “CASEOUS” “HYPERLAMINATED”
Province of Oristano 1st Survey (1995-1997) No. of examined cysts
2nd Survey (2005-2010)
TOT
Hepatic
Pulmonary
TOT
Hepatic
Pulmonary
5,039 c
3,360
1,676
3,177 c
2,109
1,065
338 1,920 2,593 c 184 4
179 1,248 1,885 46 2
159 672 705 138 2
277 1,091 1,667 c 122 20
176 669 1,238 20 6
101 422 426 102 14
Cyst type: “UNILOCULAR” “MULTISEPTED” “CALCIFIED” “CASEOUS” “HYPERLAMINATED”
Province of Cagliari 1st Survey (1995-1997) No. of examined cysts
2nd Survey (2005-2010)
TOT
Hepatic
Pulmonary
TOT
Hepatic
Pulmonary
5,295 b
3,652
1,638
3,072 d
1,870
1,200
480 1,874 2,780 b 158 3
352 1,186 2,087 26 1
128 688 688 132 2
347 886 1,569 d 194 76
214 459 1,159 32 6
133 427 408 162 70
Cyst type: “UNILOCULAR” “MULTISEPTED” “CALCIFIED” “CASEOUS” “HYPERLAMINATED” a including 8 splenic cysts b
including 5 splenic cysts
c
including 3 splenic cysts
d
including 2 splenic cysts
Table 2. Characteristics of different cyst types.
Type
Shape
Size Range (cm)
Unilocular
Globular, lobate or plurilobate
1-10
single, fluid filled
fertile
range 1-100
Multisepted
irregular, acinous
0.5-3-4
divided into 3dozen spheroidal chambers, with hydatid fluid
generally sterile
in about 5% presence of “laminated brood capsules” (Conchedda et al, 2008)
Calcified
irregular, acinous
generally ~1
septed, with internal chambers almost virtual due to the thickening of internal septa
sterile
Caseous
Globular, lobate or plurilobate
1-5
generally single cavity, filled with a thick creamy or cheesy matrix
sterile
Hyperlaminated
globular, lobate
1-5
virtual cavity filled with extensively folded and over- lapping sheets of hyperproduced laminated tissue (SLT)
sterile
Internal cavity Fertility % viability of PSC