Animal echinococcosis in Italy: epidemiological update

Cystic Echinococcosis (CE) is one of the most widespread parasitoses in the Mediterranean Region (MR). This is due to various factors, the most important being the close association between man, sheep and dogs in areas where open farming is practised. Although this disease has been known for several years and many studies have been carried out, nowadays in Italy there are no complete epidemiological data on its diffusion and distribution. The available data show that CE is mainly diffused in those districts where the sheep-dog cycle can be perpetuated, such as central and southern Italy, and the islands. Furthermore, no data are available on biomolecular characterisation of the strains of Echinococcus granulosus in Italy, apart form those in Sardinia, where the G1 (sheep-dog) and G7 (pig-dog) strains were recently isolated. One of the reasons why CE is a problem with no easy solution is undoubtedly the difficulty of making a certain diagnosis in the dog, the principal definitive host of E. granulosus.     

Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.     

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.     

Biochemical profiles of hydatid cyst fluids of Echinococcus granulosus of human and animal origin in Libya.

A comparative study on the biochemical parameters in hydatid cyst fluids of sheep, goats, camels, cattle and human cystic forms of Echinococcus granulosus has been made in Libya. Quantitative variations in the levels of sodium, potassium, calcium, cholesterol, glucose, urea, creatinine and gamma glutamyl transpeptidase (gamma GT) were found in the cystic fluids of different host origins although these differences were statistically insignificant compared with the hydatid fluids of sheep. However, the concentration of triglycerides and proteins were significantly elevated in the cyst fluids of sheep compared with the other fluids studied. Similarities in the biochemical composition of different hydatid cyst fluids suggest the existence of sheep strains of E. granulosus in human and other domestic animal intermediate hosts in Libya.     

A reconsideration of the Echinococcus granulosus strain situation in Australia following RFLP analysis of cystic material.

Restriction fragment length polymorphism (RFLP) analysis, previously shown to be a relatively simple and reproducible method for distinguishing discrete strains of E. granulosus, could not discriminate between E. granulosus originating from central Queensland macropod marsupials, Australian mainland sheep or United Kingdom sheep. Furthermore, sheep cyst material from Tasmania and the Australian mainland was indistinguishable using this approach. As a result of this DNA analysis, the existence of three distinct strains of E. granulosus in Australia, previously described on the basis of morphological, biochemical and developmental differences, may require re-evaluation.     

ITS-1 ribosomal DNA sequence variants are maintained in different species and strains of Echinococcus

This study investigated sequence heterogeneity in the first internal transcribed spacer (ITS-1) of ribosomal DNA within and among species and strains of Echinococcus. Different ITS-1 sequence variants exist in Echinococcus granulosus and Echinococcus multilocularis, which represent at least four evolutionary lineages: (1) a sheep strain-lineage of E. granulosus, (2) a sister lineage of a cervid and camel E. granulosus ITS-1 variants, (3) a lineage including the ITS-1 variants representing horse, bovine and camel strains of E. granulosus, as well as variants from E. multilocularis, Echinococcus oligarthrus and Echinococcus vogeli and (4) a distinct lineage of ITS-1 variants including E. granulosus strains from sheep and cervid, and E. multilocularis. At least two of the species (E. granulosus and E. multilocularis) were paraphyletic for ITS-1. Divergent ITS-1 variants from these two species shared distinct evolutionary lineages. The sequence data provided evidence that at least two turnover mechanisms, namely slippage and unequal crossing over/transposition, have led to the divergence and maintenance of sequence variants in Echinococcus species and strains.     

A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species

D evriese , L.A. 1984. A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. Journal of Applied Bacteriology 56 , 215–220.
A biotyping system for Staphylococcus aureus strains is proposed which is a simplified version of biotyping procedures described in the literature. It differentiates Staph. aureus strains from man and animals into host-specific ecovars and biotypes which are not host-specific. With the help of tests for βhaemolysin, staphylokinase, coagulation of bovine plasma and the crystal-violet reaction, the origin of many but not all Staph. aureus strains can be determined: 604 of 809 strains from man. poultry, cattle, pigs, goats, rabbits and foods could be alloted to four ecovars which are typically associated with man, poultry, sheep and goats and cattle. The other strains belonged to five non-host specific biotypes.     

Immunochemical characteristics of the ovine polymeric haptoglobin HpC

It was found that haptoglobins of camel, cattle, horse, pig, rat, guinea pig and man form upon immunoelectrophoresis precipitation arcs with antibodies against polymeric sheep haptoglobin C, corresponding to alpha 2-globulins. The immunocrossreactivity of haptoglobins of man and various animal species towards antibody to sheep haptoglobin (100, 88.0, 75.2, 72.1, 56.3, 51.0, 41.3 and 28.0% for haptoglobin of sheep, camel, pig, cattle, man, rat, guinea pig and horse, respectively) was determined. The intensity of crossreactions between sheep haptoglobin and the proteins under study towards antibody to haptoglobin C reflects the similarity of their primary structure and, consequently, the immune homology of their molecules. Using quantitative titration, the antigenic valency values for human (6), sheep (5), cattle (4) and horse (3) haptoglobins were determined.     

Growth and genotypes of Echinococcus granulosus found in cattle imported from Australia and fattened in Japan

At the abattoir on study in Miyazaki, Japan, 9537 imported cattle from Australia in average were slaughtered annually in the last 5 years (2006 to 2010) and hydatid cysts were constantly detected in about 1.8% of the cattle. In order to assess the risk of Echinococcus granulosus delivered to Japan by imported cattle, 250 cysts found in 103 cattle at the abattoir were examined for their biological characteristics and genotypes. The cattle slaughtered were imported from Australia at an age of 10-12 months old and fattened for 17-18 months in Japan. The cysts showed their size ranging from 4 to 108 mm and were mainly found in the lung. Mature protoscoleces were detected in the three largest cysts, all were of the G1 genotype. Most of the other cysts contained clear cyst fluid and had thin laminated layer with no protoscoleces. The finding implies a potential risk of E. granulosus being established in Japan, thus strict and proper meat inspection and consequent offal condemnation are requisite at abattoirs that deal with imported cattle. Genotyping based on partial fragments of mitochondrial cox1, rrnS and nad1 genes were performed on the 66 cysts, showing that most of the cysts were G1 genotype (common sheep strain). However, two and four cysts were considered as G2 (Tasmanian sheep strain) and G3 (buffalo strain) genotypes, respectively. Since it has been widely recognized that G1 is the only genotype distributing in mainland Australia and that G2 genotype has been eradicated from Tasmania, the finding of those genotypes from Australian cattle indicated that certain genotypes other than G1 genotype are distributing in mainland Australia.     

Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development

The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1). There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.     

Echinococcus granulosus strain differentiation in Iran based on sequence heterogeneity in the mitochondrial 12S rRNA gene

Parasite strain characterization is essential for the establishment of a prevention and control strategy in any endemic area. The aim of this study was to characterize different Echinococcus granulosus isolates from Iran by using DNA sequences of the mitochondrial 12S rRNA gene. Thirty livers and lungs of cattle, sheep and goats naturally infected with E. granulosus were collected from abattoirs in northern and western Iran between June and October 2007. These samples yielded 18 fertile cysts which we used for the genetic work. We designed and tested two new primer pairs which specifically amplify portions of the mitochondrial 12S rRNA gene of the two strains (G1 and G6) of E. granulosus known to occur in Iran. One primer pair amplified a fragment of 259 base pairs (bp) from only the G1 strain. The second pair amplified a fragment of 676 bp from the G6 strain. The G1 genotype was identified in all fertile cyst samples, in agreement with previous studies in Iran. Ten of our samples and a single reference sample of the G6 strain were sequenced and compared with the G1 and G6 sequences deposited in GenBank.     

Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues

The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain).     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

A biochemical study of adult and cystic stages of Echinococcus granulosus of human and animal origin from Kenya

A comparative biochemical study was performed on adult and cystic stages of Echinococcus granulosus. Basic quantitative differences in metabolism were apparent between the cystic forms of sheep origin from the UK and Kenya which suggest that each may represent a different geographical strain or sub-strain. The biochemistry of the human and sheep forms from Kenya was very similar, which probably reflects a certain close affinity between the two. The fact that the cattle, goat and camel forms of E. granulosus, from that country, were distinct biochemically, both from each other and from the sheep and human types, suggests the existence of an unusually complex strain picture there, and that these organisms are either non-infective or only poorly infective to man. The differences in biochemical composition and metabolism observed between adults produced experimentally and those obtained from naturally infected dogs, may have been as a result of their original hydatid source and/or their differing stage of development.     

Microquantitative determination of lactate dehydrogenase isoenzymes in the myocardium and the conducting system

Summary A newly developed technique was used for the electrophoretic separation of lactate dehydrogenase (LDH) isoenzymes from lyophilized tissue samples in the nanogram range. In this study portions of 10–200 ng from the myocardium and the conducting system of cattle, sheep, pig and man were microdissected and analysed.In the heart tissues of cattle, sheep and pig, the isoforms LDH1, LDH2 and LDH3 were detected in species-specific varying amounts. In all these animals, the conducting system is marked by high LDH1 activity, which is present at a ratio of about 2:1 compared with the myocardium. The values in man, however, differ from these values, but this might be due to post-mortem changes. The findings are discussed with respect to possible aerobic-anaerobic functions.     

Experimental infection of the baboon (Papio cynocephalus) with Echinococcus granulosus of camel, cattle, sheep and goat origin from Kenya

In different areas of the world, strains of Echinococcus granulosus have been described which appear to vary in their infectivity, and laboratory primates have been used as indicators of their infectivity to man. This phenomenon was evaluated in Kenya for hydatid material of human, camel, cattle, sheep and goat origin. Viable eggs, produced by experimental infections in dogs with larval material from all the above intermediate hosts, were fed separately to four baboons (Papio cynocephalus) in each case. Baboons were autopsied between 373 and 501 days following infection and the liver, lungs, heart, spleen and kidneys were thoroughly inspected. Hydatid cysts were recovered from two baboons in each of the camel, sheep and goat groups, three baboons in the cattle group and none of the baboons in the human group. Fertile cysts were found in the cattle and goat groups and it is suggested that the baboon could be used as an experimental model for this parasite.     

Investigation of methaemoglobin reduction by extracellular NADH in mammalian erythrocytes

The effect of extracellular NADH on the rate of reduction of nitrite-induced methaemoglobin in erythrocytes from man, cattle, dog, horse, grey kangaroo, pig and sheep was investigated. Extracellular NADH was found to enhance the rate of methaemoglobin reduction in man, dog, pig and kangaroo erythrocytes, but had essentially no effect on the rate of methaemoglobin reduction in erythrocytes from cattle, horse and sheep. In erythrocytes of those animals affected by extracellular NADH the rate of reduction of metHb in the presence of NADH was the same or greater than that observed in the presence of nutrients such as glucose and inosine. The combination of nutrient and NADH produced a more profound increase in the rate of methaemoglobin reduction. The rate of methaemoglobin reduction in all cases was significantly less than that observed with methylene blue, the standard treatment of methaemoglobinaemia. Extracellular NADH was found to indirectly increase the intracellular NADH concentration through displacement of the pseudo-equilibrium of the intracellular LDH reaction and relied upon the presence of sufficient LDH activity released into the extracellular medium through haemolysis. The lack of response of cattle, horse and sheep RBCs to extracellular NADH was found to derive mainly from their low extracellular LDH activity, but also correlated with their lower NADH-methaemoglobin reductase activity compared to the other species.     

Cystic echinococcosis in Italy from the 1950s to present

In Italy the epidemiological pattern of cistic echinococcosis (CE) is incomplete and the information for most regions is out of date, contradictory, and almost exclusively limited to the intermediate hosts. The disease is found most frequently in particular social and economic conditions: widespread use of extensive or semi-extensive sheep farming, illegal slaughtering, and high numbers of sheepdogs and other types of dogs. The highest incidence in sheep is found in Sardinia (70.6-92.8%), Sicily (6.5-36.5%), Basilicata (5-28%), Abruzzo (22%) and Tuscany (47%). It North Italy, it is never higher than 0.5% in slaughtered sheep. No data are available on the biomolecular characterization of the strains of E. granulosus in Italy, apart from Sardinia and recently Lazio. G1 (Sheep strain), G7 (Pig strain) G2 (Tasmanian sheep strain) have been identified in Sardinia and G1 and G3 (Buffalo strain) have been recently isolated in Lazio. In Italy, CE has was also found in buffaloes (2.63-9.8%) and horses (<1%). However, further epidemiological surveys and genotyping study are necessary. The small quantity of up to date information on the diffusion of E. granulosus in dogs (Abruzzo 4%, Sardinia 6-10% and Sicily 19.3%) highlights the need for modern, fast, sensitive and low risk diagnostic methods which would provide a true picture of the pattern of the infection in this host.     

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.     

Genomic typing of the Echinococcus granulosus isolates from the areas of Southern Urals

Nine larvocysts of Echinococcus granulosus isolated from nine patients and one cyst derived from a naturally infested cattle have been examined. Genomic typing was carried out in order to identify strains of E. granulosus. All DNA samples were shown to have the same genotype, E. granulosus G1.