Gametocytes are essential for Plasmodium transmission, but little is known about the mechanisms that lead to their formation. Using piggyBac transposon-mediated insertional mutagenesis, we screened for parasites that no longer form mature gametocytes, which led to the isolation of 29 clones (insertional gametocyte-deficient mutants) that fail to form mature gametocytes. Additional analysis revealed 16 genes putatively responsible for the loss of gametocytogenesis, none of which has been previously implicated in gametocytogenesis. Transcriptional profiling and detection of an early stage gametocyte antigen determined that a subset of these mutants arrests development at stage I or in early stage II gametocytes, likely representing genes involved in gametocyte maturation. The remaining mutants seem to arrest before formation of stage I gametocytes and may represent genes involved in commitment to the gametocyte lineage.

In dogs, diuretic resistance developed after 14 days of furosemide, but not torsemide, administration; however, both loop diuretics were associated with increased BUN and plasma creatinine concentrations, compared with values before treatment.

Detection and analysis of

BackgroundOokinete is the form of the malaria parasite that invades the mosquito midgut epithelium to initiate sporogony. Differentiation of ingested gametocytes into ookinetes in the mosquito midgut lumen and subsequent interaction with the lumenal surface of the midgut epithelium in preparation for invasion is a complex and multi-stepped process. To facilitate the study of these events in detail it is necessary to produce sufficient numbers of pure, fully mature and functional ookinetes. However, production of even a small number of Plasmodium falciparum ookinetes in vitro has proven to be a daunting task. Consequently, over the past four decades our collective understanding of the biology of this parasite form remains sorely deficient. This article reports on investigations of five different ookinete media, in an effort to improve the in vitro transformation efficiency of P. falciparum gametocytes into mature ookinetes and their infectivity of the mosquito midgut.MethodsFive different ookinete media were evaluated for their ability to support the differentiation of gametocytes into gametes and further into mature stage V ookinetes. Moreover, infectivity of the in vitro-transformed ookinetes was evaluated by feeding them to vector mosquitoes and measuring their ability to traverse the midgut and form oocysts.ResultsOne of the five media (medium E) was clearly superior in that the cultured ookinetes produced the largest number of oocysts when fed to mosquitoes. Key components were additions of human serum, human red blood cell lysate and mosquito pupal extract, resulting in the production of larger numbers of ookinetes able to develop into oocysts when fed to mosquitoes.ConclusionThis simple and practical improvement over the prevailing methodology will facilitate the investigation of how this important human malaria parasite initiates its development in the mosquito and will contribute to the understanding of its transmission biology.

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The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated B. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.Babesia equi is a tick-borne hemoprotozoan parasite that causes piroplasmosis in horses. Equine piroplasmosis is an economically important disease that is characterized by fever, anemia, and icterus and that is mostly prevalent in tropical and subtropical areas as well as in temperate climatic zones (15). Areas of endemicity include many parts of Europe, Africa, Arabia, and Asia (15). Due to the almost worldwide distribution of various tick vectors, the introduction of a carrier into areas of nonendemicity should be prevented.The complement fixation test (CFT) and indirect fluorescent-antibody test (IFAT) have commonly been used to detect B. equi infection. However, these serologic tests are generally restricted by the antibody detection limits and cross-reactivity (4, 5). Besides CFT and IFAT, the enzyme-linked immunosorbent assay (ELISA) with B. equi lysate antigen has been used for detection of antibodies to B. equi (19). However, the ELISA is hindered by a limited antigen supply and poor specificity (4,5,19).Merozoite antigen 1 (EMA-1) is the major surface protein of B. equi (8). It is considered an important candidate with which to develop a diagnostic reagent for detection of antibodies to B. equi (9, 10). A competitive inhibition ELISA (CI-ELISA) that can detect antibodies to B. equi based on a monoclonal antibody to EMA-1 has been developed by Knowles et al. (11), who demonstrated that it can be more sensitive than CFT in detecting antibodies to B. equi. The CI-ELISA offers the advantage of a high degree of specificity but the disadvantage of the requirement of a complicated operating procedure. Therefore, there is a need to develop a simple ELISA method.Here, we established a highly specific, sensitive, and simple ELISA method using recombinant EMA-1 expressed in insect cells by baculovirus. Our data indicated that the recombinant baculovirus-expressed EMA-1 should be a useful diagnostic reagent for detection of antibodies to B. equi in horses. MATERIALS AND MET...

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ABSTRACT. An epidemiological study of Babesia canis in dogs in Nigeria was performed. Four hundred blood samples collected from dogs in Nigeria were investigated using nested PCR and sequence analysis. On nested PCR screening, nine samples (2.3%) produced a band corresponding to a 698-bp fragment indicative of B. canis infection. Sequence analysis of the PCR products identified eight samples (2.0%) as B. canis rossi and the ninth (0.3%) as B. canis vogeli. This is the first report of the prevalence of B. canis rossi and B. canis vogeli in dogs in Nigeria.