When wildlife comes to town: interaction of sylvatic and domestic host animals in transmission of Echinococcus spp. in Namibia.

The present study was conducted in the isolated desert town of Oranjemund in the far south of Namibia. It is an extremely arid region where no livestock husbandry is practiced and only animals adapted to the desert can be found. However, in and around the city, artificial irrigation maintains lush green patches of grass that attract wild animals, in particular oryx antelopes (Oryx gazella). In 2015 four oryx antelopes were euthanised due to poor conditions and a post-mortem examination was conducted. Two were found positive for cystic echinococcosis and 16 cysts were collected for molecular analyses. In addition, faecal samples from black-backed jackals (n=5) and domestic dogs (n=9), which were regularly observed to feed on oryx carcasses, were collected and taeniid eggs isolated. Parasite species identification of the cysts and eggs was done by amplifying and sequencing the mitochondrial nad1 gene. Both oryx antelopes were found infected with E. ortleppi and one co-infected with E. canadensis G6/7. Both Echinococcus species were able to develop fertile cysts in oryx, making oryx antelopes competent hosts for these parasites. Therefore, the analysis of faecal samples was of high interest and although the numbers were quite small, taeniid eggs were found in three out of five faecal samples of jackals and in all nine dog samples. However, species determination was only successful with two jackal and one dog sample. All three were positive for E. canadensis G6/7. The absence of E. ortleppi may be due to the low number of faecal samples examined. In our small study, we discovered a rather unique lifecycle of Echinococcus spp. between jackals and domestic dogs as definitive hosts and oryx antelopes as intermediate hosts. Here, the presence of E. canadensis G6/7 is of particular concern, as it is the second most important causative agent of CE in humans.

Genetic characterization of Echinococcus species in eastern Ethiopia.

Cystic echinococcosis (CE) is a neglected zoonotic disease with considerable economic and public health burden worldwide, particularly affecting developing countries like Ethiopia. To initiate effective prevention and control of CE, comprehensive data on the local lifecycles of the various species/genotypes of Echinococcus are needed. In the present study, conducted in eastern Ethiopia, a total of 1106 livestock animals were examined at three slaughterhouses, which resulted in combined prevalence of morphologically and molecularly confirmed CE of 8.4% (75/891) in cattle, 1.1% (1/95) in sheep, 0.0% (0/95) in goats and 12.0% (3/25) in camels. All cystic lesions recovered during post mortem examination were assessed for cyst condition and underwent molecular characterization by PCR and sequencing of a 1081 bp fragment of the mitochondrial cox1 gene. A total of 175 cysts belonged to E. granulosus s.s. (n = 165), E. ortleppi (n = 6) and E. canadensis G6/7 (n = 4). Of all examined cysts, only 14 were fertile and contained protoscoleces, all from the lungs of cattle: 5 were E. granulosus s.s., 6 E. ortleppi and 3 E. canadensis G6/7. In sheep, only one sterile liver cyst of E. granulosus s.s. was found, while in camels seven sterile or caseated/calcified cysts of E. granulosus s.s. and E. canadensis G6/7 were found in liver and lungs. In conclusion, the prevalence of CE was rather low compared to other regions of Ethiopia, and, based on the number of fertile cysts, three Echinococcus spp. contributed almost equally to transmission. Cattle seem to be, epidemiologically, the most important livestock species. Our data provide a substantial basis for more detailed investigations of the transmission dynamics of CE in the study area.

A molecular survey of cystic echinococcosis in Sudan.

A survey of cystic echinococcosis in livestock was conducted from May 2001 to July 2003 in central, western and southern Sudan. Hydatid cysts were present in 59% (466/779) of camels, 6% (299/4893) of cattle, 11% (1180/10,422) of sheep and 2% (106/5565) of goats, with little variation among different geographical areas. 532 of these cysts were examined by PCR and could be overwhelmingly (98.7%) allocated to Echinococcus canadensis G6/7 (all of 215 cysts from camels, 112 of 114 cysts from cattle, 134 of 138 cysts from sheep, and all of 65 cysts from goats); the genotype G6 was identified by sequencing 13 of these isolates. Only 2 cysts from cattle belonged to Echinococcus ortleppi. The mean number of cysts per infected animal was much higher in camels (5.1) than in the other species (1.0-1.3), and cyst fertility was higher in camels and cattle (74% and 77%) than in goats and sheep (31% and 19%). Fertile cysts from five human patients from hospitals in Khartoum and Juba belonged to E. canadensis (G6). This study confirms the predominance of the 'camel strain' in Sudan and the infectivity of this strain for humans. This is the first genetic characterization of human CE in Sudan.

Genetic resistance to Sarcocystis miescheriana in pigs following experimental infection.

Clinical and parasitological traits of Sarcocystis miescheriana differ in Pietrain and Meishan pigs. For further description and characterization of the genetic basis of this variation a F(2) family based on Pietrain boars and Meishan sows as founders was generated. One hundred and thirty-nine F(2) pigs were challenged orally at an age of 100 days with 50,000 sporozysts to produce the typical clinical picture of a moderate dose Sarcocystis infection. Heritabilities were estimated for clinical and clinical-chemical traits, for specific antibody responses to the infection and for bradyzoite numbers found in skeletal (Musculus longissimus dorsi: M.l.d.) and heart muscles at necropsy 70 days post-infection (p.i.) Apart from several low to moderate heritabilities, high heritabilities were observed for bradyzoite numbers in the M.l.d. (0.68), IgM antibody levels (0.74) during the acute (14 days p.i.) and titres of specific IgG antibodies (0.42) in the early stage of cyst formation (42 days p.i.). Marked heritabilities of these traits, which are basic for acute phase of the disease (14 days p.i.) or chronic Sarcocystosis presume genes that explain sufficient shares of variance (QTL). The model is considered valuable for screening of gene variants associated with resistance/susceptibility to Sarcocystis infection. Such gene variants could then be used in susceptibility-scoring or selection programs in the future.

The Echinococcus multilocularis 14-3-3 protein protects mice against primary but not secondary alveolar echinococcosis.

Alveolar echinococcosis (AE), caused by the larval stage (metacestode) of the tapeworm Echinococcus multilocularis, exhibits very similar disease characteristics in humans and rodents. Recently, it has been shown that an over-expression of the parasite 14-3-3 protein could be associated to the proliferative growth of the E. multilocularis metacestode. We now demonstrate the expression of this protein at the E. multilocularis oncospheral stage as well. A recombinant E. multilocularis 14-3-3 protein (E14t) was used to vaccinate mice against either primary or secondary experimental E. multilocularis infection in BALB/c mice. Conversely to non-vaccinated but control infected mice, which developed a very weak anti-E14t response during infection, the response elicited in the E14t-vaccinated and subsequently infected animals exhibited a strong reactivity against the parasite 14-3-3 protein. Major differences became apparent between secondarily and primarily infected animals: whereas no protection against secondary infection was achieved by vaccination, vaccinated animals were protected by 97% against challenge primary infection with 2000 E. multilocularis eggs. Consequently, the parasite 14-3-3 molecule appears crucially involved in the early stage of the host-parasite interplay and exhibits potential to be used as target molecule for the development of protective tools against AE.

Binding of a monoclonal antibody to sporozoites of Sarcocystis singaporensis enhances escape from the parasitophorous vacuole, which is necessary for intracellular development.

Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation of a parasitophorous vacuole (PV). About half of the sporozoites left this compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.

Immunoglobulin subclass responses of wild brown rats to Sarcocystis singaporensis.

Immunoglobulin subclass responses of wild brown rats (Rattus norvegicus) from southeastern Asia to the endemic cyst-forming coccidian Sarcocystis singaporensis were characterised. The antibody response of brown rats to wild-type parasites (high reproductive capacity) showed a Th1 profile during acute infection, namely elevated concentrations of parasite-specific IgG2b and IgG2c and absence of IgG1. Chronic infection (bradyzoite development) resulted in a mixed Th1/Th2 pattern whereby significant concentrations of IgG1 appeared. A primary infection with 1000 sporocysts eight days before challenge induced protection, accompanied by significant concentrations of IgA and IgG2, particularly IgG2a. Western blot analysis of rat sera, using sporozoite and bradyzoite-extracts as antigen, revealed that IgG1, IgG2a, and IgG2b predominantly recognised molecules between 70-80 kDa in one or the other stage. Some of the antibodies were possibly directed against a 79 kDa heat shock protein of sporozoites. An apparent unresponsiveness to molecules in the low molecular weight range, particularly of bradyzoite antigens, was observed. This was abrogated by infection of rats with an avirulent strain of S. singaporensis (low reproductive capacity) indicating that a parasite that was less adapted to its host provoked a stronger immune response. These results suggest the existence of an immune evasion strategy used by Sarcocystis and show that wild rodents may be suitable as immunological research objects, reflecting the natural situation.

[Current spread and epidemiology of Echinococcus multilocularis]. / Zur aktuellen Verbreitung und Epidemiologie von Echinococcus multilocularis.

Recently, a wealth of new data was collected on the distribution and ecology of E. multilocularis. The parasite is now known to occur at surpisingly high prevalence rates in e.g. Belgium and northwestern Germany, new records exist for the Netherlands, and the parasite was found to be widespread in Poland and the Czech Republic. In addition, foxes in continental Europe have adapted their behaviour and are now common in many towns and cities where they are also known to carry the parasite. New data exist on endemicity regions in western Asia. In addition to new informations on the parasite's range, a summary is given of the current knowledge on prevalence of alveolar echinococcosis in man.

DNA measurements and ploidy determination of developmental stages in the life cycles of Theileria annulata and T. parva.

The relative DNA levels of different developmental stages of Theileria annulata and T. parva in the cow and the tick were measured by the cytophotometric DNA technique using the fluorochrome Hoechst 33258 as a staining dye. The results revealed that sporozoites, merozoites, gamonts, and gametes were haploid, whereas multinucleated intralymphocytic schizonts were polyploid. No difference was observed between T. parva and T. annulata in these stages. For both Theileria species, the DNA measurements revealed that fusion of gametes occurred in the gut of the final host, thus providing evidence of sexual reproduction. However, differences were observed between the two parasites in the tick. Whereas T. parva zygotes underwent a two-step meiotic division, a comparable reduction division could not be unequivocally detected in T. annulata. Differences could also be detected in the further development of kinetes, indicating that Theileria species are not characterized by only one life cycle, which is specific for this genus.

DNA measurements and ploidy determination of different stages in the life cycle of Sarcocystis muris.

The DNA contents of different stages within the life cycle of Sarcocystis muris were measured cytophotometrically using DNA-specific Feulgen staining. Stages of gamogony were obtained by the transfer of isolated cyst merozoites into cat kidney-cell cultures. For calculation of absolute DNA contents, the amounts of DNA in the parasites were compared with those in chicken erythrocytes, which are known. The measurements revealed that all investigated stages of S. muris contained haploid DNA except the early zygotes, which were diploid. The further development of the zygotes started with a nuclear division, resulting in two daughter nuclei that again revealed haploid DNA values. The results confirm the existence of zygotic meiosis; thus, a haplo-homophasic life cycle is proposed for the Sarcosporidia.