Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique

The aim of this work was the achievement of microcysts formation from protoscoleces of E. granulosus of cattle origin using the in vitro vesicular culture technique. 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. On day 20, some microcysts with a complete laminated layer were observed. By day 48, microcysts completely developed could be observed. This is the first study where microcysts formation was obtained using protoscoleces of E. granulosus of cattle origin.     

Morphological characteristics of Echinococcus granulosus derived from buffalo in Iran

Cystic echinococcosis is a significant parasitic disease in Iran, where a variety of animals act as intermediate hosts. In this study, 25 isolates of Echinococcus granulosus obtained from water buffalo from various parts of Iran were characterized on the basis of the morphology of the metacestode and the adult worm. The characteristics of protoscoleces from the different studied areas were nearly similar. They showed 2 rows of alternating large and small hooks and their shapes were smooth in outline. In contrast to the protoscoleces, the adult rostellar hooks showed a rough outline. The results showed that the total length, the blade lengths of the large and small hooks and the number of hooks are almost similar to those isolated from sheep but significantly different from those isolated from camels. The growth rates of adult E. granulosus (total worm length, segmentation and maturation) of buffalo origin, at 35 and 41 days post-infection of dogs, were nearly comparable to the common sheep strain. The form of the strobila and the morphology of the reproductive system were also similar to those of sheep origin. This suggests that the common sheep strain (G1) of E. granulosus may also use buffaloes as its intermediate host.     

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.     

Comparative cytotoxicity of secondary hydatid cysts, protoscoleces, and in vitro developed microcysts of Echinococcus granulosus.

Infection with the metacestode of Echinococcus granulosus is characterized by a concomitant immunity. Survival of established and developing hydatid cysts in the intermediate host implies a mechanism to modulate its immunological reactions. In order to investigate this mechanism, secondary hydatid cysts were isolated from intraperitoneally infected laboratory white mice (strain NMRI) 12 months p.i. A number of hydatid cysts were freed from the surrounding host adventitial tissue. Monolayer cultures of non-stimulated peritoneal macrophages of NMRI mice were prepared and incubated in the presence of the hydatid cysts. By means of a trypan blue exclusion test and by measuring the incorporation of tritium labelled uridine, it was found that the presence of hydatid cysts reduced the viability of the macrophages in vitro. Toxic substances are probably secreted since the medium of cultured hydatid cysts also displayed cytotoxic activity. Hydatid cysts with adventitia, as well as culture medium of those cysts, were less toxic. When toxins, partially purified from hydatid cyst fluid, were previously incubated on a collagen coated surface, a reduced level of toxicity was found, suggesting that collagen of the host adventitia may play a role in controlling the liberation of toxins by the hydatid cyst. Virtually no toxicity was exerted by protoscoleces or by the medium of cultured protoscoleces, in contrast to in vitro vesiculated protoscoleces (so called microcysts). The results reveal a novel feature of hydatid cysts that may play a role in the survival of the parasite in the immunized host.     

Morphological variation of Echinococcus granulosus protoscoleces from hydatid cysts of human and various domestic animals in Jordan

, and 1988. Morphological variation of Echinococcus granulosus protoscoleces from hydatid cysts of human and various domestic animals in Jordan. International Journal for Parasitology 18: 1111–1114. Rostellar hook morphology of protoscoleces was employed to study the possible existence of Echinococcus granulosus strains in humans and various domestic animals in Jordan. A distinct form in the donkey was evident as the protoscoleces from this host did not share any characteristics with those from the other hosts examined. Sheep, goats and cattle appeared to be affected by another form since the protoscoleces from their hydatid cysts shared six out of nine characteristics studied. Protoscoleces of camel and human cysts shared seven out of nine characteristics studied and they were different in six characteristics from protoscoleces from other hosts. Differences observed among the three forms may reflect strain variation of E. granulosus in this country.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

Formation of Echinococcus granulosus laminated membrane in a defined medium

Echinoccocus granulosus protoscoleces were digested from brood capsule material using artificial gastric fluid, and were cultured for 60 days in medium NCTC 135. Vesiculation occurred within 7 days, and the first laminated membranes were observed after 17 days of culture. Some contaminating sheep antigens appeared to be lost after 21 days.     

Determination of the minimum time of praziquantel therapy required for the in vitro treatment of protoscoleces of Echinococcus granulosus

Ovine and equine protoscoleces of Echinococcus granulosus were cultured for 26 days with our without praziquantel and viability assessed, by eosin exclusion, for cultures in various drug concentrations (50, 250 and 500 micrograms/l) and periods of exposure (1, 3 or 7 days (d] before removing/'rescuing' to drug-free medium. Drug efficacy was proportional to drug concentration and to length of exposure. At higher drug concentrations shorter exposures were required to produce the effect of continuous drug treatment, 1d therapy at 500 micrograms/l killing 96% ovine protoscoleces by day 14 whereas 7d therapy at 50 micrograms/l was required to produce a similar effect. Equine protoscoleces appeared marginally less susceptible than those of ovine origin. The relevance of the results in the need for peri-operative prophylaxis against spilled protoscoleces in man is discussed.     

Modelling the age variation of larval protoscoleces of Echinococcus granulosus in sheep

In autumn 2006, a study of the age-dynamics of Echinococcus granulosus cyst abundance was undertaken from an abattoir study of 1081 sheep slaughtered in Naryn Province in central Kyrgyzstan, an area endemic for echinococcosis. The results demonstrated approximately 64% of sheep were infected with the prevalence increasing markedly with age. The mean abundance was 3.8 cysts per sheep. From established models, an infection pressure of 1.33 cysts per year was estimated. In addition all cysts were recovered from infected sheep and the numbers of protoscoleces was evaluated in each cyst. A new model was developed that examined the variation in numbers of protoscoleces per infected sheep with age. This demonstrated that young sheep aged 1-2 years had very few protoscoleces, but there was a massive increase as the sheep aged. The best-fitting model assumed that the number of protoscoleces in a sheep was proportional to the volume of the cysts. In this model, the radius of the individual cyst increased linearly with the age of the cyst and hence the volume increased with the cube of the cyst age. This combined with the linear increase in numbers of cysts with age resulted in a massive accumulation of protoscoleces with the age of sheep. When the model was parameterised it demonstrated that 80% of protoscoleces were present in sheep aged 4 years and older and this represented just 28% sheep slaughtered. An average sheep at 6 or more years of age has an abundance of over 9700 protoscoleces, whilst in a young sheep of 1 year of age an average of just 16 protoscoleces could be found. The average for the sampled population across all ages was 1562 protoscoleces per sheep. The maximum number of protoscoleces in a single cyst was just 482 for sheep aged 1 year rising to 92,000 for sheep aged 6 years or older. The mean volume of cysts containing protoscoleces increased from approximately 0.7 ml at 1 year of age to 8.8 ml at 6 years of age. Cysts containing protoscoleces ranged from a diameter of 0.5-8 cm with a volume of fluid ranging from 0.2 to 50 ml. It is hypothesised that removal of old sheep through a culling programme could substantially improve the control of cystic echinococcosis.     

Sequence Analysis of Attachment Gene of Lumpy Skin Disease and Sheep Poxviruses

In Egypt,protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy.In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected.Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared.The causative agent in the collected samples was identified using immunoflourescence (IF) and immuno...     

A bighorn sheep die-off in southern Colorado involving a Pasteurellaceae strain that may have originated from syntopic cattle

We investigated a pasteurellosis epizootic in free-ranging bighorn sheep (Ovis canadensis) wherein a Pasteurellaceae strain carried by syntopic cattle (Bos taurus) under severe winter conditions appeared to contribute to pneumonia in affected bighorns. Twenty-one moribund or dead bighorn sheep were found on the "Fossil Ridge" herd's winter range, Colorado, USA, between 13 December 2007 and 29 February 2008. Eight carcasses examined showed gross or microscopic evidence of acute to subacute fibrinous bronchopneumonia. All eight carcasses yielded at least one β-hemolytic Mannheimia haemolytica biogroup 1(±(G)) strain, and seven also yielded a β-hemolytic Bibersteinia trehalosi biogroup 4 (CDS) strain; evidence of Pasteurella multocida, Mycoplasma ovipneumoniae, and parainfluenza 3 and bovine respiratory syncytial viruses was also detected. Isolates of β-hemolytic Manneimia haemolytica biogroup 1(G) from a bighorn carcass and a syntopic cow showed 99.5% similarity in genetic fingerprints; B. trehalosi biogroup 4(CDS) isolates were ≥94.9% similar to an isolate from a nearby bighorn herd. Field and laboratory observations suggested that pneumonia in affected bighorns may have been caused by a combination of pathogens including two pathogenic Pasteurellaceae strains--one likely of cattle origin and one likely of bighorn origin--with infections in some cases perhaps exacerbated by other respiratory pathogens and severe weather conditions. Our and others' findings suggest that intimate interactions between wild sheep and cattle should be discouraged as part of a comprehensive approach to health management and conservation of North American wild sheep species.     

Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures

Proper tissue preservation from a wide range of animals of different species is of paramount importance, as these tissue samples could be used to reintroduce lost genes back into the breeding pool by somatic cloning. We aim to study the temporal and thermal post-mortem limits, tested in rabbits and pigs, within which there will be guarantees of obtaining living skin cells in goat, sheep, and cattle. We also intend to study the effect of vitrification on the ability of ear skin cells, stored at different times and temperatures, to attach to the substratum and grow in vitro after warming. Ears were stored either at 4 degrees C for 12, 252, and 348 h post-mortem (hpm), or at room temperature (22-25 degrees C) for 60 and 96 hpm. In all cases, skin samples from these ears were sorted into two groups: one group was in vitro cultured immediately after storage, and the other group was vitrified after storage and further in vitro cultured. In goat and sheep, no differences in attachment (100%: goat; 90-100%: sheep) or subconfluence (75-100%: goat; 70-100%: sheep) rates were observed between experimental groups. However, in days of culture to reach subconfluence, significant differences between non-vitrified and vitrified groups were observed when ears were stored at 4 degrees C for 12 and 252 hpm. In cattle, with respect to attachment rate, vitrified samples from ears stored at 22-25 degrees C for 60 hpm were different from non-vitrified control group (60 vs. 100%, respectively; P < 0.05). Also, days of culture to reach subconfluence were analysed by a non-parametric Cox Survival Analysis. In general, results from ANOVA and Survival Analysis were similar, because the proportion of censored data was quite low (9%), so the bias when using ANOVA is not too high. In spite of all the above, the lowest survival rates (75%: goat; 70%: sheep; and 40%: cattle) were sufficiently high to enable collection of skin samples from the majority of dead animals and their cryopreservation.     

Sequence analysis of attachment gene of lumpy skin disease and sheep poxviruses

In Egypt, protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy. In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected. Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared. The causative agent in the collected samples was identified using immunoflourescence (IF) and immunoperoxidase techniques. Of the 15 skin nodules suspected of LSD, 10 showed a positive reaction and 3 out of 5 skin scabs suspected of sheeppox were found to be positive. An antigenic correlation between field skin isolate of LSDV, tissue culture adapted LSDV/Ismailyia88 strain, field skin isolate of SPV and SPV/Kenyan vaccinal strain was studied using prepared hyperimmune sera. Also, nucleotide sequence of the PCR amplified attachment gene fragments of field skin isolate of LSDV, tissue culture adapted LSDV/Ismailyia88 strain, field skin isolate of SPV and SPV /Kenyan vaccinal strain were compared. The results revealed that the four used viruses were antigenically identical. Sequence analysis indicated that field skin LSDV isolate is more related to tissue culture adapted LSDV/Ismailyia88 strain than to vaccinal SPV/ Kenyan strain and the skin isolate of SPV is more closely related to field skin isolate of LSDV than to SPV/Kenyan vaccinal strain. Thus, further study should be applied on the advantage of a LSD vaccine prepared from LSDV in protection of cattle against LSD compared to the commonly used sheep pox vaccine.     

A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis

Macpherson C. N. L. and Mcmanus D. P. 1982). A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis. International Journal for Parasitology12: 515–521. The soluble enzyme extracts from protoscoleces obtained from hydatid cysts of human, camel, cattle, sheep and goat origin were compared on the basis of their isoenzyme patterns for GPI and PGM using isoelectric focusing. Consistent GPI and PGM isoenzyme patterns were obtained for larvae of human, camel and sheep material. Cattle material varied occasionally in having an additional cathodic band in some of the GPI patterns. Two distinct isoenzyme patterns were evident in the goat material for both enzymes. The more common goat patterns were similar to those of human, cattle and sheep (Kenya, U.K. and Argentina) material, which were similar to each other. The rare goat patterns were similar to those obtained for camel material. Cyst location in the various intermediate hosts had no effect on the zymograms obtained. Additionally, no alteration in the major banding patterns was observed between the larvae and homologous adults produced by experimental infections. Of 26 naturally infected dogs, 19 produced adult GPI zymograms resembling human/ sheep/goat (common form) experimental infection patterns, three were similar to experimental cattle infections and four had camel/goat (rare form) patterns.     

In vivo cultivation of Echinococcus multilocularis protoscoleces in micropore chambers

Micropore chambers containing unevaginated protoscoleces of E. multilocularis were implanted into the peritoneal cavity of AKR mice. Transformation from protoscoleces to fertile multivesicular cysts was obtained after 210 days. Ultrastructural observations of these morphological transformations indicate that a phase of histogenesis follows a phase of dedifferentiation. This morphogenetic process raises the question of the origin of new cell populations. The results reveal the potential role of protoscoleces in secondary echinococcosis and the value of this experimental model for further studies on the larval development.     

Echinococcus granulosus: possible formation of a shelled egg in vitro.

What appeared to be the early stages in the formation of a single egg with a striated embryophore was observed in an in vitro culture of Echinococcus granulosus protoscoleces isolated from sheep hydatid cysts in North Jordan. The 'egg' measured 19 x 19 microns in diameter and was formed in an intermediate vesicular/monozoic form which was never previously reported from a culture. This is the first report of an apparently shelled egg forming in an in vitro culture, but although promising, cannot be regarded as being unequivocal and will require confirmation by further work.     

Experimental infection of Culicoides brevitarsis from south-east Queensland with three serotypes of bluetongue virus

Laboratory-reared C. brevitarsis (biting midges) were fed on sheep which had been experimentally infected with bluetongue serotype 1 (CSIRO 156), bluetongue serotype 20 (CSIRO 19) or bluetongue serotype 21 (CSIRO 154), or on cattle experimentally infected with bluetongue serotype 20 (CSIRO 19). Approximately 77 000 C. brevitarsis were exposed to sheep and 9000 to cattle. The average percentage feeding on sheep was 54% and on cattle 47%. In attempts to transmit virus by bite 3360 C. brevitarsis which had fed on viraemic sheep were held for 11-15 days before exposure to susceptible sheep. Although 11% of these insects fed, transmission of virus from sheep to sheep was not demonstrated. Estimated infection rates of C. brevitarsis for each serotype from sheep and serotype 20 from cattle were similar at 0.4% or lower. These low infection rates are one of the factors which make it unlikely that C. brevitarsis could be an efficient vector of bluetongue viruses in sheep in the field.     

Tissue cryobanking for conservation programs: effect of tissue type and storage time after death

In this study, we investigated the temporal post-mortem limits, within which there will be guarantees of obtaining living cells from several tissues of sheep and cattle and the effect of vitrification on the ability of cells from tissue stored at different times. Muscle tissue and auricular cartilage were stored at 4°C for 5, 48, 72, 96 and 216 h post-mortem (hpm). Tissue samples were sorted into two groups: one group was in vitro cultured immediately after storage and the other was vitrified after storage and then in vitro cultured. In cattle and sheep, no differences in subconfluence rates were observed between the two experimental groups. At the same time, no significant differences were observed in the number of days required in culture to reach confluence between non-vitrified and vitrified groups when tissues were stored at 4°C for different times. In sheep, while the population doubling times (PDT) were similar in cartilage cells from vitrified and non-vitrified tissues and stored at 4°C for 5 and 216 hpm, PDT of muscle cells were longer in 216 hpm stored groups than in 5 hpm stored groups. In bovine, although the PDT of muscle cells were similar for 5 and 216 hpm and both vitrified and non-vitrified tissues and the PDT were longer in cartilage cells from vitrified than from non-vitrified tissues. In conclusion, although storage times and vitrification have different effects on tissues from cattle and sheep, this study showed that living cells could be obtained from all groups. Therefore, cartilage and muscle tissues can be stored at 4°C for 216 hpm and used for cyrobanking.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Epidemiology of Echinococcus granulosus in Arbil province, northern Iraq, 1990-1998

During the period 1990-1998, 99 cases of human cystic hydatidosis (12.4 cases per year) were surgically treated at the two main hospitals in Arbil province, northern Iraq, and from this the human occurence for the province was estimated to be 2 per 100,000 inhabitants. In the same area, 1270 sheep, 550 goats and 320 cattle were examined at slaughter for hydatid cysts and prevalence rates were found to be 15.0%, 6.2% and 10.9%, respectively. A decreasing tendency in livestock prevalences was found towards the end of the study period. As in humans, most of the hydatid cysts in livestock were located in the liver. Fertility of sheep cysts, i.e. those containing protoscoleces, was found to be significantly higher (64%) than that of goats (35.7%) and cattle (29.8%). The percentage of fertile cysts containing viable protoscoleces varied between 63 and 82% in the livers and between 72 and 79% in the lungs of the different animal species. A total of 97 stray dogs were examined post-mortem in the years 1991, 1992 and 1998, and Echinococcus granulosus worms were found in the intestines of 48 dogs (49.5%). High worm burdens (> 1000) were observed in 37% of the dogs, medium worm burdens (200-1000) in 41%, and low worm burdens (< 200) in 22%. In 1998, the prevalence of canine echinococcosis (24.3%) was found to be significantly lower than in 1991 (70.4%) and 1992 (60.6%). The prevalence of human hydatidosis did not differ significantly over the years, but the study confirmed that hydatidosis is endemic in northern Iraq, and that housewives, labourers and farmers appear to be at the greatest risk of infection.