Eberhard Schein

The piroplasms: life cycle and sexual stages

Publisher Summary The piroplasms are protozoa that are highly pathogenic to cattle, sheep, goats, and occasionally even to man. They comprise two genera—namely, Theileria and Babesia . The diseases they induce, known collectively as “theilerioses” and “babesioses,” cause fevers and lead to important economic losses in the tropics, subtropics, and southern Europe. This chapter highlights the differences between the life cycles of Babesia and Theileria species with respect to their morphology, studied by means of light and electron microscopy. The chapter describes the life cycle of piroplasms. They have a typical sporozoan life cycle comprising three phases: (1) Schizogony, an asexual reproduction phase in the vertebrate host. (2) Gumogony, the formation and fusion of gametes inside the intestinal cells of ixodid ticks. (3) Sporogony, an asexual reproduction in the salivary gland of the tick leading to the infectious, saliva-transmitted sporozoites. Comparative biological and morphological studies show that the economically important piroplasms comprise three groups: (1) Babesia species sensu strictu ; (2) Bubesia equi , B. microti ; and (3) Theileria species.

Detection of a new pathogenic Babesia microti-like species in dogs

Small babesiae in dogs are generally considered to belong to Babesia gibsoni. Here we describe the genotypic characterisation of small piroplasms found in the blood of a dog which suffered from clinical babesiosis. Pairwise identities as well as distance, parsimony and maximum likelihood analyses of the 18S rDNA clearly demonstrated that this isolate was only distantly related to the other canine piroplasms characterised genetically so far, including B. gibsoni. It was more closely related to B. microti, B. rodhaini, and Theileria equi. It is concluded that the small canine piroplasms described in this study represent a hitherto unknown species and that the fauna of piroplasms occurring in dogs is more diverse than assumed so far.

Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998

Abstract The horse-parasitizing species Babesia equi Laveran, 1901 was redescribed as Theileria equi Mehlhorn, Schein 1998 and, thus, transferred from one valid genus to another. This transfer was needed since it turned out that this horse parasite showed the relevant characteristics of theilerians with regard to biological data, morphological features, biochemical properties, and molecular biological relationships.

Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs

Abstract The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene were characterized in eight Babesia canis isolates of differing geographic origin, vector specificity, and pathogenicity to dogs. The genotypes determined by sequencing segregated into three clearly separated groups close to or near the species level and correspond to the previously proposed subspecies B. canis canis, B. canis vogeli, and B. canis rossi. The three genotypes can be distinguished by Sau96I digestion of the polymerase chain reaction (PCR)-amplified rDNA target.

‘ Babesia gibsoni ’ of dogs from North America and Asia belong to different species

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0.5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

Lichtmikroskopische Untersuchungen über die Entwicklung von Theileria annulata (Dschunkowsky und Luhs, 1904) in Hyalomma anatolicum excavatum (Koch, 1844)

SummaryA laboratory strain of H. a. excavatum was selected on high susceptibility for T. annulata through several generations. Giemsa-stained smears and wet smears of gut and gut content were studied.After engorgement of erythrocytic stages of T. annulata by the nymphs the following development was observed:1.Erythrocytic merozoites developed to slender, spindleshaped “microgamonts” in the gut 24 to 96 hours after repletion (p. repl.). Spherical stages with a conspicuous spike developed at the same time and earlier. The “microgamonts” then form up to 4 nuclei and several flagella-like appendices. Filiform “microgametes” obviously develop from the “microgamonts”. In addition, spherical stages, i.e. “macrogametes”, occur.2.Spherical “zygotes” with a vacuole-like center appear in the epithelial cells of the gut from day 5 p. repl. These “zygotes” increase steadily in size and then stain more intensely up to day 12 p. repl.3.From day 12 p. repl. the spherical “Zygotes” change to elongate forms by a continuing process of folding. Finally, from day 13 p. repl., they extend to clubshaped kinetes. These kinetes move actively by gliding within the gut cells and from day 17 p. repl. in the haemolymph. It could not be decided yet whether these kinetes are oo- or sporokinetes.ZusammenfassungDie Entwicklung von Theileria annulata in Nymphen der Überträgerzecke Hyalomma anatolicum excavatum wurde in giemsagefärbten Ausstrichen und im Phasenkontrast untersucht.Durch Selektion über mehrere Generationen wurde ein Laborstamm von H. a. excavatum isoliert, der eine hohe Empfänglichkeit für T. annulata aufwies. Nach der Aufnahme erythrozytärer Stadien stellt sich der Entwicklungsablauf in den Nymphen in folgender Weise dar:1.Im Darmlumen infizierter Nymphen entwickeln sich aus den erythrozytären Merozoiten sowohl schlanke, spindelförmige „Mikrogamonten“, die nach Kernteilung und Ausbildung geißelartiger Fortsätze in fadenförmige „Mikrogameten“ zerfallen, als auch Rundformen, die als „Makrogameten“ angesehen werden.2.In den Epithelzellen des Zeckendarmes treten ab dem 5. Tag nach der Repletion (p. repl.) runde „Zygoten“ mit aufgehelltem Zentrum auf. Eine stetige Größenzunahme und Plasmaverdichtung werden bis zum 12. Tag p. repl. beobachtet.3.Ab dem 12. Tag p. repl. entwickelt sich innerhalb der „Zygote“ durch einseitige Einfaltung ein abgewinkeltes, würmchenförmiges Stadium, das sich ab dem 13. Tag zu einem Kineten entfaltet. Dieser Kinet bewegt sich, aktiv gleitend, im Darm und wird ab dem 17. Tag p. repl. in der Hämolymphe angetroffen.

Seasonal dynamics of biting midges (Diptera: Ceratopogonidae, Culicoides spp.) on dairy farms of Central Germany during the 2007/2008 epidemic of bluetongue

The unforeseen outbreak of bluetongue in north-western Europe in August 2006 raised the question, which Culicoides spp. were involved in the transmission of bluetongue virus (BTV). Based on the decision 2007/20/EU of December 2006, a large-scale entomological surveillance programme was initiated in the five affected EU member states including Germany. This paper reports on the entomological findings obtained from March/April 2007 to May 2008 at 15 sampling sites in the federal states of Lower Saxony (eastern region), Mecklenburg-Western Pomerania, Brandenburg and Saxony-Anhalt: The number of captured biting midges in one trap varied from none or few Culicoides during winter (December 2007 to April 2008) to up to more than 12,500 individuals during summer and autumn. Catches of the C. obsoletus group were consistently higher than those of the C. pulicaris group. C. imicola, the principal afro-asiatic vector of BTV, was not detected. High numbers of midges were caught inside the cattle sheds. Eleven pools of biting midges were RT-PCR-positive to BTV-8 including pools of non-engorged midges of the C. obsoletus and of the C. pulicaris groups. The first BTV-genome positive pool of midges was detected in August 2007; the remaining genome-positive pools were detected during October and November 2007.

A field study to estimate the prevalence of Trypanosoma equiperdum in Mongolian horses

From May to July 2000, a cross-sectional study was conducted to estimate the prevalence of Trypanosoma equiperdum in the horse population of the central province (Tuv aimag) of Mongolia. On average, four herds were selected from each of the 29 aimag subdivisions (119 herds). From each herd, 10 horses were sampled in proportion to sex and age categories in the respective herds (1190 horses). Sera from 1122 horses were analysed for T. equiperdum antibodies using two serological assays, the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA). The crude estimate of the CFT and the ELISA seroprevalence was 7.6 and 6.7%, respectively. Concordance between the CFT and ELISA results was high (96%). The highest number of CFT positive animals was detected in one herd in Möngönmorit (6/10), followed by herds in Bayandelger (5/10) and in Bayantsagaan (5/10). Poor body condition was significantly correlated with positive serological status in both CFT and ELISA. A history of abortion appeared to be a risk factor for both CFT and ELISA seropositivity. Blood samples of all horses belonging to herds with at least three (3/10) seropositive animals (CFT and/or ELISA) were analysed by light microscopy and by PCR using a Trypanosoma (Trypanozoon) brucei specific primer pair. No trypanosomes or any other haemoparasites could be detected in Giemsa stained thin blood smears. Eight out of the 130 samples (6.2%) analysed by PCR gave positive signals. Seven out of the eight PCR positive horses were also serologically positive. One PCR (and ELISA) positive stallion from Möngönmorit showed emaciation, scrotal and preputial oedema and an oedematous skin plaque. From the serological and DNA-based results it is concluded, that trypanosome infections occur in horses in the Tuv aimag of Mongolia. Since at present neither serological nor DNA-based tests allow a subspecies specific identification within the subgenus Trypanozoon, no definitive diagnosis can be given for T. equiperdum. Whether the examined herds are infected with T. equiperdum or with T. evansi, the causative agent of surra, remains an open question. However, based on the clinical findings, the negative parasitological results and the concentration of conspicuous seroprevalences in single herds, circumstantial evidence supports the existence of infections with the causative agent of dourine.

DNA measurements and ploidy determination of developmental stages in the life cycles ofTheileria annulata andT. parva

The relative DNA levels of different developmental stages ofTheileria annulata andT. 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 betweenT. parva andT. annulata in these stages. For bothTheileria 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. WhereasT. parva zygotes underwent a two-step meiotic division, a comparable reduction division could not be unequivocally detected inT. annulata. Differences could also be detected in the further development of kinetes, indicating thatTheileria species are not characterized by only one life cycle, which is specific for this genus.

Characterization and comparison of merozoite antigens of differentBabesia canis isolates by serological and immunological investigations

Merozoites of fourBabesia canis isolates from Hungary, France, Africa, and Egypt were purified. Antigens were compared in an enzyme-linked immunosorbent assay (ELISA) and by immunoblotting. In the ELISA, antigen from the highly pathogenic isolate from Hungary showed the highest sensitivity for homologous and heterologous immune sera. This was confirmed by immunoblotting. Protein bands of the Hungarian isolate were strongly recognized by allB. canis immune sera, whereas the antigens from the other isolates showed only weak reactions with homologous and heterologous immune sera. Significant was a protein band of about 12 kDa appearing in all pathogenic isolates from Hungary, France, and South Africa but not in the apathogenic Egyptian isolate. This protein band may determine the virulence. For serological tests, theB. canis isolate from Hungary seems to be the one most suitable for detection of even mild infections.