Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection.

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.

Influence of zinc deficiency to the mice infected with Babesia microti.

Zinc (Zn) is an essential trace element for DNA synthesis and for cell growth and differentiation. The deficiency induces a wide range of disorders including immunodeficiency. In this study, the influence of Zn deficiency to the mice infected with Babesia microti was examined, and was compared with the influence in the rats infected with B. rodhaini previously reported. Experiments of B. microti infection were conducted using Zn-deficient (ZD; allowed to eat ad libitum on the ZD diet), Zn-adequate (ZA; allowed to eat ad libitum on the ZA diet), and diet-restricted (DR; supplied 2 g/day on the ZA diet) mice. It was suggested that the Zn deficiency exacerbated the infection dynamics of the mice with B. microti by the growth retardation, the reduction of immunity and the decrease in PCV. The results in the mice supported the consequences in the rats previously reported.

A new entodiniomorphid ciliate, Troglocorys cava n. g., n. sp., from the wild eastern chimpanzee (Pan troglodytes schweinfurthii) from Uganda.

Troglocorys cava n. g., n. sp. is described from the feces of wild eastern chimpanzee, Pan troglodytes schweinfurthii, in Uganda. This new species has a spherical body with a frontal lobe, a long vestibulum, a cytoproct located at the posterior dorsal side of the body, an ovoid macronucleus, a contractile vacuole near the cytoproct, and a large concavity on the left surface of the body. Buccal ciliature is non-retractable and consists of three ciliary zones: an adoral zone surrounding the vestibular opening, a dorso-adoral zone extending transversely at the basis of the frontal lobe, and a vestibular zone longitudinally extending in a gently spiral curve to line the surface of the vestibulum. Two non-retractable somatic ciliary zones comprise arches over the body surface: a short dorsal ciliary arch extending transversely at the basis of the frontal lobe and a wide C-shaped left ciliary arch in the left concavity. Because of the presence of three ciliary zones in the non-retractable buccal ciliature, the present genus might be a member of the family Blepharocorythidae, but the large left concavity and the C-shaped left ciliary arch are unique, such structures have never been described from other blepharocorythids.

The occurrence and ape-to-ape transmission of the entodiniomorphid ciliate Troglodytella abrassarti in captive gorillas.

Entodiniomorphid ciliates are often present in the colons of wild apes. In captive apes the infection tends to gradually disappear, with the exception of Troglodytella abrassarti. We used fecal examinations to screen the gorillas (Gorilla gorilla gorilla) in European (Czech Republic, UK) and Australian Zoos to explore the ape-to-ape transmission pattern of T. abrassarti. Gorillas from two out of three European Zoos were positive for T. abrassarti, while gorillas from the Australian Zoo were negative. We documented a horizontal transmission of T. abrassarti to a non-infected adult gorilla introduced into a Troglodytella-positive group in the Prague Zoo and traced the origin of the ciliate infection to the Paignton Zoo (UK) using serial fecal examinations. During this study, two infant gorillas born in the Prague Zoo (CZ) first became positive for T. abrassarti at the age of 9 mo. Ciliate morphology and the sequencing of the small subunit rRNA gene and the internal transcribed spacer rDNA spacer region revealed that T. abrassarti affects both captive gorillas and chimpanzees. We conclude that zoo transport plays a major role in the distribution of T. abrassarti among captive gorillas.

Influence of zinc deficiency to the rats infected with Babesia rodhaini.

Zinc deficiency induces a wide range of disorders including immunodeficiency. It is known that microbial infections occur with a high frequency in the zinc-deficient hosts, but the study on the correlation between parasitic infection and zinc status in hosts is scarcely performed. We observed that the influence of zinc deficiency to the rats infected with Babesia rodhaini. Experiments of B. rodhaini infection were conducted using zinc-deficient (ZD; eat ad libitum or 10 g/day on the ZD diet), zinc-adequate (ZA; eat ad libitum on the ZA diet), and diet-restricted (DR; eat 7 g/day on the ZA diet) rats. The findings in this study suggested that the zinc deficiency had deleterious effects on the hemodynamics and mortality of the rats infected with B. rodhaini.

Two new entodiniomorphid Triplumaria ciliates from the intestine of the wild African white rhinoceros.

Two new species of Triplumaria in the order Entodiniomorphida, T. alluvia n. sp. and T. grypoclunis n. sp., are described from the large intestine of the wild African white rhinoceros. T. alluvia has three bud-shaped caudalia, one broad skeletal plate with a wavy left dorsal edge, and an axe-shaped tail flap. T. grypoclunis has three short arched caudalia, two broad skeletal plates, and a pointed and ventrally curved tail flap. These two new species have a C-shaped adoral polybrachykinety, a slender perivestibular polybrachykinety, and paralabial kineties in their retractable adoral ciliary zone. In T. alluvia, the perivestibular polybrachykinety is joined to both ends of the adoral polybrachykinety and paralabial kineties along the ventral side of the adoral polybrachykinety, showing the same arrangement as in Cycloposthium species. In T. grypoclunis, the perivestibular polybrachykinety is joined only to the right end of the adoral polybrachykinety and paralabial kineties along the left ventral side of the adoral polybrachykinety, showing an arrangement analogous to the Tripalmaria species.

Lung worms of wild boars in the western region of Tokyo, Japan.

Prevalence of lung worms belonging to the genus Metastrongylus was surveyed on 42 Japanese wild boars (Sus scrofa leucomystax) captured officially for wildlife damage control in the western parts of Tokyo, Japan from April 2000 to April 2001. The number of parasites was the highest in the caudal lung lobes. Four species, M. elongatus (ME), M. salmi (MS), M. asymmetricus (MA) and M. pudendotectus (MP), were identified. All the boars were infected with 2 or more species, and 64.3% of the boars had all 4 species. The composition of species, ME:MS:MA:MP=1.3:3.4:1.0:1.4, was drastically different from the previous reports. The peak of the average number of the parasites was observed in the period of January to March 2001 because of the increase of MS.

New entodiniomorphid ciliates from the intestine of the wild African white rhinoceros belong to a new family, the Gilchristidae.

Gilchristia artemis n.g., n.sp. and Digilchristia draconis n.g., n.sp. in the order Entodiniomorphida are described from the large intestine of the African white rhinoceros, and a new family Gilchristidae is proposed to contain them. These new species have a C-shaped adoral polybrachykinety, a slender vestibular polybrachykinety, and paralabial kineties along the ventral side of the adoral polybrachykinety in their retractable adoral ciliary zone, showing the same arrangement as in the rumen ciliates in the family Ophryoscolecidae. G. artemis has two skeletal plates and D. draconis one plate. In both species the dorsal skeletal plate is bow-shaped, folded in half longitudinally, twisting in the anterior part, and lying along the dorsal left side of the macronucleus. The second plate of G. artemis is slender and lies along the ventral side of the macronucleus. G. artemis has three ciliary arches and D. draconis has four arches along the dorsal and ventral sides of the body. Their arches are long and non-retractable, closely resembling those of ciliates in the families, Spirodiniidae and Cycloposthiidae, and are not analogous to the single retractable ciliary arch of the rumen ciliates in the family Ophryoscolecidae.

Life cycle of Eimeria krijgsmanni-like coccidium in the mouse (Mus musculus).

Life cycle of Eimeria krijgsmanni-like coccidium isolated from the feces of naturally infected mice purchased from commercial sources was examined. The parasite was purified by single oocyst isolation and maintained by passage in the mice before experiments. The sporulated oocysts were ovoid or ellipsoid, measuring 19.3 x 14.8 microm on average. One or two small polar granules were present. Micropyle and oocyst residuum were absent. Sporocysts were ellipsoid, measuring 11.6 x 7.2 microm on average with a small Stieda body and sporocyst residuum. Six groups of respective 5 mice (4-week-old) were inoculated with doses varying from 2.0 x 10(1) to 10(6) oocysts. All the mice examined began to shed oocysts from 7 day postinoculation (PI) and their maximum number of oocysts per gram of feces were 10(6) on day 8 PI. Patency was 6 or 7 days. This parasite had severe virulence to the mice that is, the mice given 10(6) oocysts showed anorexia, diarrhoea and rough hair from 1 day and all of them died on day 3 PI. The mice given 10(3) or more oocysts showed the clinical signs described above from day 5 and 4 of them received 10(5) died on day 9 or 10 PI. The parasites occurred within the epithelial cells of cecum, colon and rectum of infected mice. Sporozoites, 13.9 x 3.0 microm, with two large refractil bodies on side of the nucleus located subcentrally were observed on day 1 and 2 PI. Merozoites were first observed at 24 hr PI, and sexual stages were found from 4 day PI. No parasites were detected in the small intestine and mecenteric lymph nodes.

Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to cerebral malaria in Myanmar.

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum malaria, and its pathogenesis leading to coma remains unknown. Heme oxygenase-1 (HO-1) catalyzes heme breakdown, eventually generating bilirubin, iron and carbon monoxide. The HO-1 gene promoter contains a polymorphic (GT)n repeat which may influence the expression level of HO-1. To explore the correlation between this (GT)n polymorphism and susceptibility to CM, we analyzed the frequencies of the (GT)n alleles in 120 Myanmarese patients with uncomplicated malaria (UM) and 30 patients with CM. The frequency of homozygotes for the short (GT)n alleles (<28 repeats) in CM patients was significantly higher than those in UM patients (P < 0.008, OR = 3.14). Thus, short (GT)n alleles represent a genetic risk factor for CM.

Induction of interferon-gamma (IFN-gamma) and T helper 1 (Th1) immune response by bitter gourd extract.

Mice were inoculated intraperitoneally wih 34 different types of vegetable juices, and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured as markers for the induction of Th1 and Th2 cells, respectively. Serum IFN-gamma level was markedly increased in mice inoculated with bitter gourd (Momordica charantia) juice, but IL-4 levels were not increased with any of the 34 vegetable juices. Testing of the various components of bitter gourd, including peel, pulp, and seed, showed that the pulp induced the highest levels of IFN-gamma. Trial immunogen including the heat extract of the pulp induced specific IgG(2a) antibody of the mice serum inoculated with this immunogen. These results demonstrate that bitter gourd pulp induced IFN-gamma production and show its promise as a means of effective immunostimulatory therapy specific for Th1 cells and IFN-gamma production.

Hematological findings and antibody responses in Syrian hamster (Mesocricetus auratus) infected with Babesia microti.

Hematological findings during the course of infection and the antibody response in Syrian hamsters infected with Babesia microti were examined. A macrocytic hypochromic anemia with an increase of the reticulocyte count was detected as a rise in the parasitized erythrocyte rate. White blood cell counts also remarkedly increased with the increases of both neutrophils and active-shaped monocytes, and thus they particularly play an important role in eliminating the parasite. In Western blotting with the sera from the hamsters infected with B. microti, a 38 kDa protozoan antigen reacted to the early-term sera, and additionally 28, 32, and 34 kDa antigens also reacted to the medium- and latter-term, and convalescent sera. These antigens were immunodominant and the antibodies against these antigens had also important roles for inhibition of this parasite.

Susceptibility of Chinese hamsters (Cricetulus griseus) to the infection of Babesia microti.

Chinese hamsters were examined for the susceptibility to the infection with Babesia microti based on the hematological parameters during the course of infection. A marked decrease in the RBC count, Ht value, Hb concentration, and an increase in WBC count due to the development of neutrophils or monocytes were recognized with the progress of parasitemia. Remarkable clinical findings were anemia and persistent infection with a low level of parasite burden in the chronic and convalescent stages. From these findings, it was concluded that Chinese hamsters were susceptible to infection with B. microti and would be useful for infection examination with the parasite.

Molecular cloning and sequences of interleukin-10 in the Djungarian (Phodopus sungorus), Chinese (Cricetulus griseus), and Syrian (Mesocricetus auratus) hamster.

Interleukin 10 (IL-10) genes of Djungarian, Chinese, and Syrian hamsters were cloned. The clones of IL-10 consisted of 537 bp nucleotides and 178 amino acids in full length, and the nucleotide and amino acid sequences exhibited a high degree of homology with those of the mouse and human. Since the number and position of signal sequences, N-glycosylations and cysteine sites in the IL-10 amino acid sequences of the hamsters were the same as those of the mouse, we suggest that the IL-10 molecular structures of the hamster are closer to that of the mouse than human.

High susceptibility of Djungarian hamsters (Phodopus sungorus) to the infection with Babesia microti supported by hemodynamics.

As the comparative study was carried out on the susceptibility by the pursuit of parasitemia among the Djungarian, Syrian, and Chinese hamsters as well as BALB/c mice infected with the Syrian hamster-adapted Babesia microti strain, and Djungarian hamsters showed the highest parasitemia among them. Then, the other hematological parameters were pursued in the Djungarian hamsters infected with the hamster-adapted B. microti strain. Remarkable symptoms observed were hemoglobinuria clinically, anemia hematologically, and splenomegaly macroscopically during all over the observation period for 24 weeks post infection (PI). Parasitemia began to rise at 2 weeks and peaked at 4 weeks PI. After that, parasitemia decreased gradually but was maintained with a level of about 10% on average until 24 weeks PI at the end of the experiment. A decrease in the RBC count, Hb, and PCV, and an increase in the reticulocyte and WBC counts due to the development of immature neutrophils, lymphocytes and monocytes were recognized together with a rise of parasitemia. The hamsters had macrocytic hypochromic anemia due to the increase of MCV and the decrease of MCHC in the growth phase of the parasite. It was considered that the Djungarian hamsters will be useful for the infection examination, isolation, maintenance, and passage of B. microti in laboratory.

The first report of the ante-mortem diagnosis of Ollulanus tricuspis infection in two dogs.

Ollulanus tricuspis is a small nematode parasite of the stomach, and its infection has been reported worldwide in cats but only one report in dogs as post-mortem diagnosis. Two dogs, kept in the Tokyo area, were presented for chronic vomiting. Chronic gastritis was diagnosed histologically, and many nematodes were detected in endoscopically-biopsied gastric samples and in the mucus of vomitus in both dogs. The parasites were small (<1 mm), and their morphological characteristics were consistent with those previously reported for O. tricuspis. The symptoms in one dog completely disappeared after anthelmintic therapy. To our knowledge, this is the first report describing ante-mortem diagnosis of spontaneous gastric O. tricuspis infection in dogs in which infectivity and pathogenicity of the nematode are suggested.

Molecular clonings and sequences of Djungarian (Phodopus sungorus) and Chinese (Cricetulus griseus) hamster interferon-gammas.

Djungarian (Phodopus sungorus) and Chinese (Cricetulus griseus) hamster IFN-gamma genes were cloned and sequenced. The Djungarian and Chinese hamster genes were both 525bp nucleotides, resulting in 174 amino acids in full length with a predicted molecular weight (MW) of 19,560 dal and 19,775 dal, respectively. The first 23 amino terminal amino acids consisted of a hydrophobic signal sequence when cleavaged, which would result in a mature 151 amino acid polypeptide with a predicted MW of 17,115 dal in the Djungarian hamster IFN-gamma and 17,255 dal in the Chinese hamster one.

Rumen ciliate protozoal fauna of reindeer in Inner Mongolia, China.

Rumen ciliate species and composition were surveyed on the reindeer kept in Inner Mongolia, China. As a result of survey, all the reindeer had the same 18 species of 8 genera of the ciliates in their rumen. Appearance of Entodinium parvum is the first record in this host species. Of the species detected, 15 species including Entodinium anteronucleatum, E. bicornutum, Enoploplastron confluens and Epidinium gigas which have been detected only from reindeer were common with those in the reindeer kept in the other areas, such as Alaska, Russia and Finland, indicating that every rumen ciliate faunae in reindeer has high similarity to each other, and suggesting that these hosts have been isolated from the other ruminants for long time and established characteristic ciliate fauna. The ciliate density was estimated as 1.1-2.5 x 10 (6)/ml, which value was fairly higher than those in domestic ruminants. Surface structures of characteristic species were observed by SEM.

Monoclonal antibodies preventing invasion of Neospora caninum tachyzoites into host cells.

Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells.

Susceptibility of Djungarian hamsters (Phodopus sungorus) to Neospora caninum infection.

Djungarian hamsters were examined for the susceptibility to Neospora caninum infection. After 29 Djungarian hamsters were intraperitoneally inoculated with 5 x 10(6) N. caninum tachyzoites of JPA1 strain, some animals showed symptoms such as ataxia, and many tissue cysts were detected in the brain and a cyst in the muscular tunics of stomach. Especially, more than 100 cysts per head were observed after 5 weeks post inoculation. It is suggested that the Djungarian hamster is a model useful to examine neosporosis.