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We evaluated the growth inhibitory effect of triclosan, which has recently been reported to inhibit the growth of Plasmodium species and Toxoplasma gondii, on bovine and equine Babesia parasites in in vitro cultures The growth of Babesia bovis and B. bigemina was significantly inhibited in the presence of 100 microg/ml of triclosan, while B. caballi and B. equi were susceptible to as low as 50 microg/ml. Babesia bigemina and B. caballi were completely cleared as early as on the first and second day of the treatment, respectively. These parasites did not exhibit any growth in the subsequent five-day period of subculture without triclosan. Drug-treated parasites appeared pycnotic and atypically shaped, and ultrastructurally showed pronounced vacuolations, leading to complete destruction of parasites. Light microscopy showed that used concentrations of triclosan showed no toxicity against the host cells. The results suggest that triclosan can be used for chemotherapy of babesiosis.

ABSTRACT. The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T. gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle. KEY WORDS: ELISA, Neospora caninum.J. Vet. Med. Sci. 64(2): 161-164, 2002 Neospora caninum is a protozoan parasite of livestock and companion animals that causes abortion, neuromuscular paralysis and death [1,2]. Several studies have reported similarities and differences in the cyst wall morphology, tachyzoites ultrastructure, oocysts features, and life cycle of N. caninum, Toxoplasma gondii, Hammondia heydorni and Hammondia hammondi. The cyst wall of N. caninum bears resemblance to that of T. gondii and H. hammondi [12,13,17,18] [10,13]. In this paper, we investigate whether cross-reactivity occurs between N. caninum, T. gondii and H. heydorni using the surface protein NcSRS2 [5,7] and the soluble antigen (NLA) of N. caninum by using enzyme-linked immunosorbent assay (ELISA).Full-length NcSRS2 genes were cloned into baculovirus using the primer sets: 5'-ACG AAT TCA TGG CGA CGC ATG CTT-3', and 5'-GCG TCG ACT CAG TAC GCA AAG ATT-3'. The cDNA of N. caninum tachyzoites was used as the template in the polymerase chain reaction (PCR). PCR products were blunted by Klenow fragments, and ligated to a previously-digested Sma I baculovirus transfer vector pBacPAK8 (Clontech, Palo Alto, CA). Sf9 cells were co-transfected with a transfer vector and BacuoGold(r) Baculovirus DNA (PharMingen, San Diego, CA) using lipofectin TM reagent (Gibco BRL, Grand Island, NY). After 4 days of incubation at 27°C, the culture supernatant containing recombinant viruses was harvested and subjected to plaque purification. After three cycles of purification, recombinant viruses-expressing NcSRS2 were obtained. Autographa californica nuclear polyhedrosis virus and its recombinant products were grown in Spodoptera frugiperda (Sf9) cells in a TC-100 insect medium (Gibco BRL) supplemented with 10% FBS and 0.26% bacto tryptose broth (Difco, Detroit, MI).ELISA plates coated with the NLA antigen were prepared as described previously by Nishikawa et al. [15]. Monolayers of Sf9 cells that were grown in a 75 cm 2 flask and infected with 10 ml of recombinant baculovirus at 5 PFU/ cell in Sf-900II SFM medium (Gibco BRL). A...

ABSTRACT. The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-len gth), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliabl e reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.