Cystic Echinococcosis in Romania: An Epidemiological Survey of Livestock Demonstrates the Persistence of Hyperendemicity

Abstract

An epidemiological survey of cystic echinococcosis (CE) in cattle and sheep was conducted from October 2009 to October 2011 in endemic areas of north-eastern and southern Romania. A total of 8569 animals (3043 cattle and 5526 sheep) were examined, and hydatid cysts were found in 984 (32.34%) cattle and 2756 (49.87%) sheep, respectively. The average number of cysts per animal was 11.3 (range, 1–47) in cattle and 4.2 (range, 1–16) in sheep. Of the CE-positive animals, 177 (17.9%) of the cattle and 358 (12.98%) of the sheep had hydatid cysts only in the lungs, 62 (6.3%) and 803 (29.13%) had only in the liver, and 723 (73.47%) and 1572 (57.04%), respectively, had hydatid cysts both in the liver and in the lungs. Very few animals had cysts in spleen (19 cattle and 23 sheep) and kidney (three cattle). From a sample of 422 cysts undergoing closer examination (258 from cattle, 164 from sheep), the highest fertility rate (38.41%) was found in sheep cysts, while from the cattle only four cysts (1.55%) were found to be fertile. Species identification of 13 animal isolates (nine from sheep, four from cattle) and one human isolate were done by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial nad1 gene and confirmed by partial sequencing of the cox1 gene. All showed the same RFLP band pattern (Echinococcus granulosus sensu stricto) and were identified as the “sheep strain” G1 by sequencing. The study emphasizes the hyperendemic presence of E. granulosus in Romania and outlines the necessity for the urgent development of sustainable surveillance and control strategies both in animals and humans.

Introduction

C ystic echinococcosis (CE), caused by the larval stages of Echinococcus granulosus sensu lato, is one of the most important parasitic infections in livestock in the world, as well as a major zoonosis (Eckert and Deplazes, 2004). The disease has a considerable impact on both human and animal health, with important economic consequences arising out of the costs of medical treatment and morbidity for human cases and losses in animal productivity (Torgerson, 2003). The annual CE-associated economic losses on a global basis have been recently estimated to exceed US$2 billion (Budke et al., 2006).

The parasite has an indirect life cycle, with dogs and other canids as definitive hosts for the intestinal tapeworm, and many herbivorous and omnivorous species, including wildlife and livestock, as well as humans, as intermediate hosts for the tissue-invading metacestode (larval) stage. Transmission to humans frequently results from close contacts with infected dogs excreting parasite eggs with their feces and carrying them on their fur, which may lead to accidental ingestion with contaminated water or food.

The wide variety of animal species, both domestic and wild, which act as intermediate hosts, has made this parasite to be widely distributed across the globe, and at least 10 genetically distinct taxa exist within the complex E. granulosus (Thompson and McManus, 2002; McManus and Thompson, 2003). Isolates of the parasite show considerable genetic variation in different intermediate hosts. Several genotypes and species are described in different eco-epidemiological settings (Romig et al., 2006).

In Romania, CE continues to pose significant problems of public health and cause important economic losses. As a result of the increased number of human and animal cases, Romania was framed in the forefront of the European countries and among the most affected countries worldwide, in 1995 (Neghina et al., 2010; Moldovan et al., 2012). Epidemiological data available showed that in 45.5% of Romanian localities (1367/3004) at least one person had undergone surgery for CE within only 4 years, from 1987 to 1991 (Stefanoiu, 1999). For animals, CE results in economic losses due to condemnation of parasitized organs, weight loss, and decreased productivity and fecundity. A survey performed throughout Romania (1983–1997) revealed prevalences ranging between 33% and 40% in sheep and cattle (Olteanu et al., 1997).

Despite the fact that incidence rates of CE in some areas are among the highest in the world, the epidemiology of cystic echinococcosis (CE) in Romania is rather poorly understood. Furthermore, the genetic variation of Echinococcus granulosus sensu lato in Romania has been little investigated and only in limited areas. Echinococcus granulosus sensu stricto was identified in sheep, cattle, and humans, while the pig strain (G7) of E. canadensis was found in pigs, all in western Romania (Bart et al., 2006). Additional epidemiological studies, including molecular genotyping of CE in different intermediate hosts and geographical areas, are needed to provide a comprehensive understanding of the nature and extent of the genetic diversity and its consequences for transmission and control.

Therefore, this study aimed at investigation of the epidemiology of CE in cattle and sheep from some endemic areas in northeastern and southern Romania and the genotypes existing in these regions using mitochondrial genes for cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) as genetic markers.

Materials and Methods

Epidemiological study

The study was conducted between October 2009 and October 2011 in two areas of north-eastern and southern Romania, where pastoral activities are predominant. In the Northeast, two counties (Suceava and Neamt) were investigated, located at the east side of the Carpathians, with two major relief units: the mountainous region and plateau region. The southern areas, with three counties selected (Valcea, Teleorman, and Constanta), are predominantly lowlands. In both areas, small mixed farms (with cattle, sheep, horses, pigs, and poultry) are well represented, but no recent data about CE in livestock are available.

A total number of 5526 sheep and 3043 cattle carcasses were examined for CE by visual inspection, palpation, and serial cuts of the organs to detect hydatid cysts. Numbers of cysts in each individual animal and affected organ were recorded. The animals were stratified according to age.

A sample of 422 cysts (258 from cattle, 164 from sheep) were examined macro- and microscopically in the laboratory. Cyst sizes were determined by measuring the diameter of each individual cyst using a measuring tape. Cysts were categorized as small (<2 cm), medium (2–5 cm), and large (>5 cm).

Fertility was determined by microscopic detection of protoscolices in aspirated cyst fluid. Cysts were classified as fertile (viable cysts with protoscolices), sterile (apparantly viable acephalocysts), or degenerated (calcified or caseous). Protoscoleces were examined for motility, appearance, and flame cell activity. Sterile or degenerated (calcified or caseated) cysts were classified as infertile.

Molecular study

Parasitic material

Protoscolices or germinal layer of 13 cysts from sheep (n=9) and cattle (n=4) were preserved in 70% ethanol and stored at room temperature for genetic characterization. A single human isolate obtained from a male CE patient operated in a hospital from Iasi was also included in the study.

DNA preparation

Either protoscolices (fertile cysts) or the germinal layer (sterile cysts) were used for DNA preparation. Single protoscolices or pieces of germinal layer were separated using a capillary pipette and lysed in 10 μL 0.02 M NaOH at 95°C for 10 min (Nakao et al., 2003). One microliter of the lysate was added directly as template to the PCR.

Restriction fragment length polymorphism (RFLP)–PCR of the nad1 gene

The PCR for differentiation of the known Echinococcus species was performed as described before (Hüttner et al., 2008, 2009). This method is based on the amplification of a 1073–1078 bp-long fragment including the complete nad1 gene with subsequent digestion of the amplification products with the restriction enzyme HphI.

The PCR reaction mixtures and amplification conditions were described previously (Hüttner et al., 2008). Briefly, for the first PCR, a 50-μL reaction mixture containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 μM of each dNTP, 12.5 pmol of each external primer, 1.25 U Ampli-Taq Polymerase (Applied Biosystems, Carlsbad, CA), and 1 μL of the lysate was prepared. The amplification conditions for 35 cycles were denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and elongation for 60 s at 72°C. For the nested PCR, 1 μL of the first PCR product was transferred to a reaction mixture consisting of the same components as in the first PCR, but using the internal primers. The conditions during reaction were the same as in the first PCR. The obtained PCR products were subsequently digested with the restriction enzyme HphI. The digestion mixture contained 10 μL of PCR product, 18 μL of H₂O, 2 μL of digestion buffer provided by the manufacturer, and 0.5 μL of the restriction enzyme HphI (Fermentas, Burlington, ON, Canada). PCR products were digested for 3 h at 37°C (followed by inactivating for 20 min at 65°C), and the fragments were separated on a 3% ethidium bromide–stained agarose gel.

Amplification and sequencing of partial cox1

Nested PCRs were performed using the cox1 primers, which had been described previously (Hüttner et al., 2008). The PCR reaction mixtures and conditions were the same as described for the nad1 gene. The obtained PCR products underwent confirmatory sequencing.

Statistical analysis

The statistical analyses were performed using Quantitative Parasitology 3.0 free software. The p-values by Fisher's exact test and chi-square test, and 95% confidence intervals (CI) were computed. A p-value of ≤0.05 was considered as statistically significat.

Results

Epidemiological study

Out of the 8569 animals (3043 cattle and 5526 sheep) examined for CE, 984 cattle (32.34%) and 2576 sheep (49.87%) were found infected (Table 1). The mean infection intensity was higher in cattle (11.3 cysts per infected animal) than in sheep (4.2 cysts per infected animal).

Table 1.

Prevalence of Cystic Echinococcosis (CE) in Cattle and Sheep, and Mean Number of Cysts per Animal

	Data on CE-positive			Data on of hydatid cysts
Animals	n	%	95% CI	Mean no.	Range
Cattle (n=3043)	984	32.34	30.70–34.02	11.3	1–47
Sheep (n=5526)	2756	49.87	48.56–51.19	4.2	1–16

Prevalence figures according to the geographic origin (Northeast-South) of both cattle and sheep showed small, but statistically significant differences (Table 2). The age of infected animals ranged from 8 month to 14 years in cattle, and from 3 months to 8 years in sheep. The animals were divided into three age groups (<2 years, 2–5 years, and >5 years). Prevalences increased with age in both cattle and sheep (Table 3), being highest in the oldest age groups (54.90% in cattle, 78.45% in sheep). Considering the number of slaughtered animals in each group, cattle of >5 years and sheep of 2–5 years yielded the highest number of infected animals. The chi-square test for differences of age class in both cattle and sheep was significant (χ²=602.247, p<0.01 for cattle; and χ²=2130.68, p<0.01 for sheep, respectively).

Table 2.

Prevalence of Cystic Echinococcosis (CE) According to Geographic Origin

	Cattle			Sheep
	Examined	CE positive		Examined	CE positive
Location area	n	n	%	n	n	%
Northeast	1184	343	28.97	2155	1014	47.05
South	1859	641	34.38	3371	1742	51.68
		p=0.002			p=0.000

Table 3.

Cystic Echinococcosis (CE) Frequency and Prevalence According to Age Groups

	<2 years			2–5 years			>5 years
Animals	Total	Infected	Percentage	Total	Infected	Percentage	Total	Infected	Percentage
Cattle	1037	65	6.27	802	258	32.17	1204	661	54.90
Sheep	1890	137	7.24	2114	1425	67.40	1522	1194	78.45

The distribution of cysts in the internal organs varied among animal species (Table 4). In cattle, the lungs were found to be the predilection sites for the hydatid cysts, whereas most of the infected sheep had hydatid cysts in the liver.

Table 4.

Frequencies and Prevalence According to Organ Localization of Hydatid Cysts

	Organ localization of hydatid cysts
Animals	Only lung, n (%)	Only liver, n (%)	Lung and liver, n (%)	Spleen, n (%)	Kidney, n (%)
Cattle (n=984)	176 (17.9)	63 (6.40)	723 (73.47)	19 (19.30)	3 (0.3)
Sheep (n=2756)	358 (12.98)	803 (29.13)	1572 (57.04)	23 (0.83)	—

The sizes of the cysts collected are presented in Table 5. In sheep, most of the cysts has sizes of <2 cm (66.46%), both in the liver (75.58%) and in the lungs (56.41), while those in cattle were predominantly small in the liver (61.29%), but medium in the lungs (49.70%).

Table 5.

Cyst Sizes (Numbers and Percentages) in Different Organs (Lungs and Liver)

		Cattle		Sheep
Organ localization of hydatid cysts	Cyst size (cm)	n	%	n	%
Liver	<2	57	61.29	65	75.58
	2–5	30	32.26	21	24.42
	>5	6	6.45	0	0
Lungs	<2	56	33.94	44	56.41
	2–5	82	49.70	31	39.74
	>5	27	16.36	3	3.85
Total=422		258		164

Fertile, sterile, or calcified/caseous cysts were found in both cattle and sheep (Table 6). The highest fertility rate was found in sheep cysts with 38.41% (63/164), while in cattle only four (1.55%; 4/258) cysts were fertile.

Table 6.

Cyst Viability In Different Organs

			Viability of cysts
Host species	Organs	Cysts, n	Fertile, n (%)	Sterile, n (%)	Calcified/caseous, n (%)
Cattle	Liver	93	0	74	96
	Lungs	165	4	45	39
	Total	258	4 (1.55)	119 (46.12)	135 (52.33)
Sheep	Liver	86	22	28	39
	Lungs	78	41	16	18
	Total	164	63 (38.41)	44 (26.83)	57 (34.76)

Molecular study

A total of 14 cysts (13 cysts collected during the slaughterhouse surveys from sheep [n=9] and cattle [n=4] and one cyst from a patient who underwent surgery procedure for CE) were determined to species level by RFLP-PCR. The banding patterns of the nad1 PCR products after digestion with the restriction enzyme Hph1 of all isolates were similar. All 14 (100%) belonged to Echinococcus granulosus sensu stricto (G1–G3 complex). Sequencing of partial cox1 gene of all 14 samples showed homology of 99–100% with the “sheep strain” G1 of E. granulosus when compared with data on GenBank^® (accession number M84661) (Bowles et al., 1992).

Discussion

Epidemiological and parasitological findings

Based on the results of this study, Romania remains a stronghold for CE in Europe, and the prevalence in livestock is exceeded by only a few countries worldwide. The findings also reveal that this situation is spread over a large part of Romania and has not improved over the last 20 years or more.

For the period 1983–1994, Neghina et al. (2010) cite prevalences of 24.3% and 92.9% in sheep, 31.2% and 43.6% in cattle, and 20.4% and 73.8% in swine. Olteanu et al. (1999), in a survey performed during 1994–1995 on CE in cattle, sheep, and swine based on field data collection from 19 counties, revealed a prevalence of 56.74%, 58.54%, and 17.8%, respectively. A similar study carried out between 1998 and 2003 in slaughterhouses from three counties (Timis, Arad, and Caras-Severin) in western Romania confirmed an active transmission of CE among cattle, sheep, and swine. Out of the three investigated species, cattle were the most intensely infected (12.31–22.36%), followed by sheep (5.83–11.51%), and swine (0.96–10.12%) (Morariu et al., 2011).

Our results update the above mentioned data and confirm the ongoing active transmission of E. granulosus in Romania, especially in areas where pastoral activities are prominent. This may be due to a sum of factors that act synergistically as predisposing factors for the transmission cycle of the parasite. Of these, traditional methods of animal husbandry, unsupervised home slaughtering of livestock that are still present in Romania, especially for sheep and pigs, and frequent absence of appropriate anthelmintic treatment of dogs are likely to be the most important.

Looking at other hyperendemic foci for CE in Europe, similar situations are reported in parts of Italy. In Sicily, the prevalence rates are up to 57.6% in sheep and 11.13–67.1% in cattle (Poglayen et al., 2003; Giannetto et al., 2004). In Sardinia, CE prevalences of 75.3% in sheep and 41.5% in cattle were reported (Garippa et al., 2008). In an endemic area of eastern Turkey, Simsek et al. (2010) found a prevalence in cattle as 33.9%. In Bulgaria, after failed control campaigns, re-emergence of CE is reported with prevalence increases in dogs and sheep from 4% to 7% and from 19% to 32%, respectively (Breyer et al., 2004). In Greece, recent surveys revealed a prevalence of CE of 23–39.2% for sheep, 7.6–14.7% for goats, but 0% in cattle and only 0.6% in pigs (Sotiraki and Chaligiannis, 2010).

The findings of our study are indicators for the infection risk for humans. In the recent Annual Epidemiological Report on Communicable Diseases in Europe (EFSA, 2010), Romania is listed with 119 cases of human cystic echinococcosis reported (the second highest number after Bulgaria) and a notification rate of 0.55 per 100,000. However, some discrepancies regarding prevalence and incidences of CE can be seen comparing data obtained by EFSA, the World Health Organization, and the scientific literature. For instance, a recent epidemiological and clinical investigation of 190 patients diagnosed with CE and hospitalized during 2004–2010 in two counties from south-western (Caras-Severin) and central (Hunedoara) Romania, reported a mean annual incidence of 3.3 cases/100,000 inhabitants (Moldovan et al., 2012). Similar studies from the same area, but different counties (Timis and Arad) reported also high levels of human CE with an annual incidence of 3.8 cases per 100,000 inhabitants in Timis county (Calma et al., 2011) and 2.4 cases per 100,000 inhabitants in Arad county (Calma et al., 2012).

Cretu et al. (2011), analyzing the incidence of CE in patients diagnosed during the period 2005–2008 at Colentina Teaching Hospital Bucharest, mention that 885 new cases were registered (230 cases in 2005, 231 in 2006, 241 in 2007, and 181 in 2008). Of these, 109 (12.30%) were from the five counties included in our study (57 from Teleorman, 27 from Constanta, 10 from Suceava, eight from Neamt, and seven from Valcea).

Looking at the manifestation of CE in livestock, our study demonstrated a marked prevalence increase with age in both cattle and sheep, which confirms the observation made in other studies from around the world (Torgerson et al., 1998; Kebede et al., 2011; Romig et al., 2011).

Here, we report for the first time data on organ localization, morphology, fertility, and viability of cysts in cattle and sheep in Romania. About 73.47% and 57.04% of the cattle and sheep, respectively, had hydatid cysts both in the lungs and the liver, but there were differences in the predilection organ. About 91.36% of cattle had cysts in the lungs, while 86.18% of the sheep had cysts in the liver. Very few cysts were found in spleen and kidney.

The sheep has been the most affected species in terms of both prevalence (49.87% vs. 32.34% in cattle) and cyst fertility (38.41% vs. 1.55% in cattle). These results are in accordance with most studies which compared the prevalence of fertile cysts in sheep and cattle of Echinococcus granulosus sensu stricto or in countries where this taxon is predominant (Yildiz and Tuncer, 2005; Rinaldi et al., 2008; Kebede et al., 2009). The findings revealed that, in Romania, the transmission is based mainly on sheep, while cattle, even though frequently infected, harbor predominant sterile cysts and play no significant role in the parasite transmission cycle; rather, they could serve as indicators for CE infection pressure in endemic areas.

Genetic characterization

Although only few samples were characterized in this study, all isolates were identified as the sheep strain G1. The findings are in accordance with a very recent molecular study on genetic variability of Echinococcus granulosus sensu stricto in the European countries, Romania included (near by Bulgaria and Hungary), that reported the predominance of Echinococcus granulosus sensu stricto (53 isolates/56 analyzed), and within this complex, the predominance of the G1 genotype (Casulli et al., 2012).

The one human cyst analyzed in our study was found to belong to the same taxon, highlighting its high potential for zoonotic transfer. As the most frequent strain associated with human CE appears to be the common sheep strain (G1) (Thompson et al., 1995; Eckert et al., 2000), we plan future extended analyses with parasite material of human and animal origin to confirm this hypothesis for Romania, based on a representative sample size.

Conclusion

The present study emphasizes the hyperendemic presence of CE in Romania and outlines the necessity for the urgent development of sustainable surveillance and control strategies both in animals and humans. With this respect, control can be achieved by strict supervision of slaughtering practices (destruction of infected offal), reduction of the numbers of stray dogs, combined with a deworming regime of dogs (e.g., 6-weekly dog dosing with praziquantel) as was applied in many parts of the world. Additionally, sanitary education, especially of people who professionally come in contact with dogs or work in the livestock sector should be part of the control program.

Footnotes

Acknowledgments

This work was supported by Romania UEFISCDI (project PNCDI2–Partnership; code 52-161/2008).

Disclosure Statement

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

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