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.

A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised goat milk and cheese in Germany, May 2016.

In May 2016, two cases of tick-borne encephalitis (TBE) were confirmed by serology (positive IgM and IgG antibodies against TBE virus (TBEV) in serum), with a possible link to raw milk and cheese from a goat farm in a region in Baden-Württemberg, Germany not previously known as TBE-endemic. The outbreak investigation identified 32 consumers of goat dairy products (29 consumers, one farm employee, two owners) of whom none had IgM antibodies against TBEV 3-8 weeks after consumption. Of the 27 notified TBE cases in the State, none reported consumption of raw goat milk or cheese from the suspected farm. Five of 22 cheese samples from 18 different batches were RT-qPCR-positive for TBEV -genome, and two of the five samples were confirmed by virus isolation, indicating viability of TBEV in the cheese. Nine of the 45 goats had neutralising TBEV antibodies, two of them with a high titre indicating recent infection. One of 412 Ixodes ricinus was RT-qPCR-positive, and sequencing of the E gene from nucleic acid extracted from the tick confirmed TBEV. Phylogenetic analyses of tick and cheese isolates showed 100% amino acid homology in the E gene and a close relation to TBEV strains from Switzerland and Austria.

Genetic polymorphism and population structure of Echinococcus ortleppi.

The zoonotic cestode Echinococcus ortleppi (Lopez-Neyra and Soler Planas, 1943) is mainly transmitted between dogs and cattle. It occurs worldwide but is only found sporadically in most regions, with the notable exception of parts of southern Africa and South America. Its epidemiology is little understood and the extent of intraspecific variability is unknown. We have analysed in the present study the genetic diversity among 178 E. ortleppi isolates from sub-Saharan Africa, Europe and South America using the complete mitochondrial cox1 (1608 bp) and nad1 (894 bp) DNA sequences. Genetic polymorphism within the loci revealed 15 cox1 and six nad1 haplotypes, respectively, and 20 haplotypes of the concatenated genes. Presence of most haplotypes was correlated to geographical regions, and only one haplotype had a wider spread in both eastern and southern Africa. Intraspecific microvariance was low in comparison with Echinococcus granulosus sensu stricto, despite the wide geographic range of examined isolates. In addition, the various sub-populations showed only subtle deviation from neutrality and were mostly genetically differentiated. This is the first insight into the population genetics of the enigmatic cattle adapted Echinococcus ortleppi. It, therefore, provides baseline data for biogeographical comparison among E. ortleppi endemic regions and for tracing its translocation paths.

[The ecology of ticks, tick-borne diseases and biological tick control in Baden-Württemberg]. / Ökologie von Zecken als Überträger von Krankheitserregern in Baden-Württemberg und biologische Zeckenbekämpfung.

Ticks and tick-borne diseases are of great significance for the health of humans and animals. However, the factors influencing their distribution and dynamics are inadequately known. In a project financed by the Baden-Württemberg Ministry of the Environment, Climate and Energy Industry, as part of the program BWPLUS, interdisciplinary specialists work together to determine the influence of weather, (micro)climate, habitat, land use, human activities, and the population dynamics of host animals on the distribution and abundance of ticks and the diseases that they transmit in Baden-Württemberg. The project comprises four modules: the large-scale distribution of ticks in Baden-Württemberg (module 1), detailed studies of host-tick-pathogen interaction in relation to the microclimate (module 2), and the spatial occurrence of important tick-borne pathogens (module 3). The fourth module involves the comprehensive analysis and synthesis of all data in order to determine the relative importance of the factors studied and to develop a risk model. Recently, intensive investigations into tick control have been undertaken using various entomopathogenic fungi and nematodes as well as a parasitoid wasp. Our aim was to determine whether these natural enemies could be used to effectively reduce the number of free-living ticks.

Hidden diversity of cestodes in wild African carnivores: I. Non-taeniid cyclophyllideans.

Our knowledge of parasites in wildlife remains limited, primarily due to restricted access to samples, especially of parasites from protected species. This present study contributes to the comprehension of the enigmatic world of helminths of African wild mammals and cestode biodiversity by combining both molecular and morphological analysis. Cestode samples were opportunistically collected from 77 individual definitive hosts in South Africa, Namibia and Ethiopia, encompassing 15 different species of wild African carnivores and additionally domestic cats. The analysis revealed 32 different cyclophyllidean species of which 21 (65.6 %) represent previously unknown genetic entities. They belong to the families Mesocestoididae, Hymenolepididae, Dipylidiidae and Taeniidae. Here we cover the non-taeniid cestodes, while the taeniids will be addressed in a separate publication. Three of the non-taeniid species uncovered in this study could be assigned to the genus Mesocestoides and were isolated from servals and domestic cats. The white-tailed mongoose was found to be a suitable host for a species belonging to the Hymenolepididae, which was identified as Pseudandrya cf. mkuzii. Both feline and canine genotypes of Dipylidium caninum were detected in domestic cats, the canine genotype also in an African wolf. In addition to these, a novel species of Dipylidium was discovered in an aardwolf. Lastly, four distinct species of Joyeuxiella were found in this study, revealing a cryptic species complex and emphasizing the need for a taxonomic reassessment of this genus. Despite the limited scope of our study in terms of geography and sample size, the results highlight that biodiversity of cestodes in African wild mammals is grossly under-researched and follow-up studies are urgently required, in particular linking morphology to gene sequences.

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.

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.

Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship.

Filarial nematodes are important and widespread parasites of animals and humans. We have been using the African bovine parasite Onchocerca ochengi as a chemotherapeutic model for O. volvulus, the causal organism of 'river blindness' in humans, for which there is no safe and effective drug lethal to adult worms. Here we report that the antibiotic, oxytetracycline is macrofilaricidal against O. ochengi. In a controlled trial in Cameroon, all adult worms (as well as microfilariae) were killed, and O. ochengi intradermal nodules resolved, by nine months' post-treatment in cattle treated intermittently for six months. Adult worms removed from concurrent controls remained fully viable and reproductively active. By serial electron-microscopic examination, the macrofilaricidal effects were related to the elimination of intracellular micro-organisms, initially abundant. Analysis of a fragment of the 16S rRNA gene from the O. ochengi micro-organisms confirmed them to be Wolbachia organisms of the order Rickettsiales, and showed that the sequence differed in only one nucleotide in 858 from the homologous sequence of the Wolbachia organisms of O. volvulus. These data are, to our knowledge, the first to show that antibiotic therapy can be lethal to adult filariae. They suggest that tetracycline therapy is likely to be macrofilaricidal against O. volvulus infections in humans and, since similar Wolbachia organisms occur in a number of other filarial nematodes, against those infections too. In that the elimination of Wolbachia preceded the resolution of the filarial infections, they suggest that in O. ochengi at least, the Wolbachia organisms play an essential role in the biology and metabolism of the filarial worm.

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.

Reduction of transmission stages concomitant with increased host immune responses to hypervirulent Sarcocystis singaporensis, and natural selection for intermediate virulence.

Parasite virulence (pathogenicity depending on inoculum size) and host immune reactions were examined for the apicomplexan protozoan Sarcocystis singaporensis. This parasite is endemic in southeastern Asia and multiplies as a proliferation (merozoite) and transmission stage (bradyzoite) in rats. Virulence in wild brown rats of parasites freshly isolated in the wild (wild-type) was surprisingly constant within the endemic area and showed an intermediate level. In contrast, serially passaged parasites either became avirulent or virulence increased markedly (hypervirulence). Production of transmission stages was maximal for the wild-type whereas numbers were significantly reduced for hypervirulent and avirulent (shown in a previous study) parasites. Analyses of B and T cell immunity revealed that immune responses of WKY rats to the transmission stage were significantly higher for hypervirulent than for wild-type parasites. These results suggest that it is the immune system of the host that is not only responsible for reduction of transmission stages in individual rats, but also could act as a selective force that maintains intermediate virulence at the population level because reduction of muscle stages challenges transmission of S. singaporensis to the definitive host. Collectively, the presented data support evolutionary theory, which predicts intermediate rates of parasite growth in nature and an 'arms race' between host immunity and parasite proliferation.

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana.

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.

[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.

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.

Diagnosis of Sarcocystis spp. in cattle (Bos taurus) and water buffalo (Bubalus bubalis) in Northern Vietnam.

Our aim was to develop a method for species diagnosis and to obtain data on the prevalence of Sarcocystis infections in cattle and water buffalo in the Son La Province of Northern Vietnam. Meat samples of naturally infected animals were examined by light and electron microscopy as well as by molecular methods. A PCR of part of the 18S rDNA gene followed by RFLP analysis was modified to detect infections with different Sarcocystis spp. in cattle and water buffaloes slaughtered in the Son La Province. It showed to be an economical method to detect multiple infections with Sarcocystis spp. Sequence analysis of the PCR amplicons was performed with selected samples and the results were compared with published sequences. With these methods the following Sarcocystis spp. were identified in cattle: Sarcocystis hirsuta, Sarcocystis cruzi and Sarcocystis hominis. Water buffaloes were infected with Sarcocystis fusiformis, S. cruzi, S. hominis and S. hirsuta. The results indicate that Sarcocystis spp. infecting cattle are also able to infect water buffaloes. So the validity of certain Sarcocystis spp. of water buffalo is discussed. Bovine lifestock in Northern Vietnam were commonly infected with Sarcocystis spp.

Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds.

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R (2), 0.98-0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 microM) and monensin (0.00225 to 0.144 microM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10(5) oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 microM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 microM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 microM or more, inhibition was at least 90%; 0.05 microM still yielded 80% inhibition, whereas at concentrations below 0.00625 microM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.

Autoradiographic and morphological investigations on the uptake and incorporation of tritiated lysin by acanthocephalans.

Four palaeacanthocephalans and two eoacanthocephalans from fish were autoradiographically investigated for their in vitro uptake of tritiated lysin. All worms took up the labelled amino acid at their surface without showing a species-specific pattern. The tegument of the metasoma was more heavily labelled than the presomal tegument, suggesting that the worms predominantly absorb the nutrient from the food inside the host's gut in vivo. Within the metasomal tegument the nuclei revealed the most intense labelling. In the muscles the outer myogenic belt absorbed more radioactivity than did the enclosed cytoplasmic core. In female worms the ovarian balls and eggs that showed incomplete eggshell formation were highly labelled. In male worms the cement glands showed the most intense labelling of all organs inside the body cavity. We conclude that the investigated acanthocephalans require lysin for protein synthesis and for the coding of protein synthesis in the tegumental nuclei.

Autoradiographic and morphological studies on the uptake of the triglyceride [3H]-glyceroltrioleate by acanthocephalans.

Two eoacanthocephalans, four palaeacanthocephalans, including the infective larvae of one species, and one archiacanthocephalan were exposed in vitro to a labelled triglyceride. The nutrient was almost exclusively incorporated by the tegument of the presoma. The uptake of [3H]-glyceroltriolete started at the anterior half of the presoma, followed by a consecutive labelling of the entire presoma tegument and the lemnisci. In the archiacanthocephalan, however, the general uptake was comparatively low. In the final host of the acanthocephalans, the absorbed nutrient must derive from the host's intestinal wall, injured by the parasite. In the eoacanthocephalans and the palaeacanthocephalans, the labelled nutrient was most intensively taken up by the tegument of the hooks. Inside the hook tegument, the basal membrane and the outer membrane form a labyrinth of entangling crypts and protuberances. In the surrounding proboscis tegument as well as in the neck tegument, the lipid seemed to be transported along these two membranes. In addition to its absorption by the presoma tegument, the labelled nutrient was intensively incorporated by the apical organ and the paired lateral organ of the eoacanthocephalan presoma and by the terminal part of the uterine endothelium of all female eoacanthocephalans and palaeacanthocephalans. The use of the nutrient by the parasites is discussed.

A new method for isolation of the intraerythrocytic stages of Plasmodium and Babesia from their host cells.

A new method for the isolation of intraerythrocytic stages of Plasmodium berghei and Babesia divergens from red blood cells is described. The technique is based on hydrodynamic forces occurring in a flow channel containing a turbulent liquid current, which are capable of rupturing infected erythrocytes and removing their plasma membrane from the parasites' surface. The temperature and the concentration of cells were revealed as factors influencing the hydrodynamic forces. About 90% of the intact and apparently infectious parasites of both species were isolated from the lysed erythrocytes.

Genetic differentiation of pathogenic and nonpathogenic strains of Entamoeba histolytica by random amplified polymorphic DNA polymerase chain reaction.

The random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) method was used to compare pathogenic and nonpathogenic strains of Entamoeba histolytica. DNA polymorphisms were detected among the different strains and dendrograms were constructed by PHYLIP and PAUP analyses to study the relationship of the strains. Both analyses resulted in identical results, which indicated that pathogenic strains of E. histolytica are closely related and clearly separated from the nonpathogenic strains. The results of this study agree with classification of the strains based on isoenzyme analyses. This suggests that RAPD-PCR is a valuable method in differentiating between strains of this parasite, and the results are consistent with the concept that pathogenic and nonpathogenic Entamoeba represent two different species.