High seroprevalence of Encephalitozoon cuniculi in pet rabbits in Japan.

Infection with Encephalitozoon cuniculi in rabbits frequently exists as a chronic, latent infection, and only a percentage of infected animals develop clinical disease. This study presents a seroepidemiological study of E. cunicucli infection in 337 pet rabbits collected from 20 prefectures in Japan in 2006 and 2007, using enzyme-linked immunosorbent assay (ELISA) capable of measuring IgG and IgM antibodies. These rabbits were divided into the following four groups: healthy and isolated rabbits (n=74, group I), healthy and companioned rabbits (n=121, group II), neurologically diseased rabbits (n=105, group III), and other diseased rabbits (n=37, group IV). Using ELISA for IgG antibodies, the highest detection rate, 81%, was seen in group III, the second highest, 75.2%, in group II, and the lowest, 29.7%, in group I, which was significantly different to the other groups except for group IV (43.2%). On the other hand, when ELISA was used for IgM antibody detection, 14-40% of rabbits in the four groups were also observed to have anti-E. cuniculi IgM. This study demonstrated high seroprevalence of E. cuniculi in not only neurologically diseased rabbits but also healthy and other diseased rabbits.

Genetically unique microsporidian Encephalitozoon cuniculi strain type III isolated from squirrel monkeys.

Microsporidian spores were isolated from two squirrel monkeys (Saimiri sciureus) that had been bred at an animal-breeding colony in Japan. The spores were identified as Encephalitozoon cuniculi on the basis of nucleotide sequence analysis of the small-subunit (SSU) rRNA gene. The internal transcribed spacer (ITS) gene sequence revealed that these isolates were classified into genotype III because it contained tetrarepeats of 5'-GTTT-3'. However, the sequences of the polar tube protein (PTP) gene of the monkey isolates were not identical to a reported sequence of genotype III but were quite similar to a reported sequence of genotype II. On the other hand, sequence analysis of the spore wall protein 1 (SWP-1) gene revealed that the monkey isolates did not belong to any of genotypes I, II and III. These results suggest that the present E. cuniculi isolates of squirrel monkey origin are a new subtype of E. cuniculi ITS genotype III that can cause a disseminated infection.

Possible contribution of apoptosis-inducing factor (AIF) and reactive oxygen species (ROS) to UVB-induced caspase-independent cell death in the T cell line Jurkat.

We analyzed the mechanism of UVB-induced cell death using the Jurkat T cell line. Apoptosis was assessed by measuring phosphatidylserine (PS) externalization, caspase activity, the decrease in mitochondrial membrane potential (Delta Psi m), nucleosomal DNA fragmentation, and morphological changes such as chromatin condensation. The mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF) was evaluated by confocal laser microscopy. The cell death pattern of UVB-irradiated cells was similar to the Fas-induced cell death pattern. However, zVAD-fmk inhibited the nucleosomal fragmentation of DNA but not the externalization of PS, decrease in Delta Psi m, or mitochondrio-nuclear translocation of AIF. N-acetyl L-cysteine significantly inhibited the translocation of AIF induced by UVB. These results suggested that caspase-dependent and -independent pathways were involved in UVB-induced cell death in Jurkat cells, and the mitochondrio-nuclear translocation of AIF was associated with the latter pathway. In addition, reactive oxygen species generated by UVB might be involved in inducing the mitochondrio-nuclear translocation of AIF.

[Laboratory evaluation of commercial immunoblot assay kit for serodiagnosis of Echinococcus infections using sera from patients with alveolar hydatidosis in Hokkaido].

Using serum specimens from patients with alveolar hydatidosis (AH) in Hokkaido, we assessed the usefulness of "Echinococcus Western Blot IgG" (the French immunoblot assay, FIA), which has recently been launched from Ldbio Diagnostics (Lyon, France) as new commercial immunoblot assay kit of immunodiagnosis of Echinococcus infections. Eighty serum specimens were used for the present study: 64 preoperative sera and nine postoperative sera, which were taken from AH patients in Hokkaido, and seven sera from persons who were ELISA (enzyme-linked immunosorbent assay)--positive in mass screening which was conducted for checking on Echinococcus infections in Hokkaido since 1982. When the 64 preoperative sera were examined by the Western blotting method (the Hokkaido method of Western blotting, HWB) which had been carried out at Hokkaido Institute of Public Health between 1987 and 1993, it was found that 53 cases were positive and six cases were quasi-positive, i.e. the rate of the positive cases including quasi-positive cases was 92.2%. From immunostaining patterns, HWB-positive sera could be grouped in two types: the complete type, which showed a pattern of multiple bands containing the 55 and 66 kDa bands, and the incomplete type, which showed patterns of only few bands containing the AH-specific polysaccharide antigen named C antigen. Forty-three of the 53 HWB-positive sera were of the complete type and the residue was of the incomplete type. On the other hand, when the 64 preoperative sera were examined by FIA, 60 sera (93.8%) were judged to be positive and the others as negative sera. On the basis of the interpretation of immunostaining patterns described in the instruction manual, 47 (78.3%) of the 60 positive sera were regarded as pattern P3, five (8.3%) as pattern P4, and eight (13.3%) as pattern P5. All of the complete-type sera were regarded as P3, indicating high antibody titers. Contrarily, most of the incomplete-type or quasi-positive sera resulted in other patterns such as P4 and P5, indicating low antibody titers. Of 5 HWB-negative sera, two were FIA-positive (which showed P3 and P5 patterns respectively), however their immunoreactions were significantly low. Therefore, apart from interpretation of pathological conditions of cases with exceedingly low antibody titers, FIA may be able to give a serologically clear interpretation to HWB-quasi-positive cases, indicating that it is a highly sensitive and useful method for immunodiagnosis of Echinococcus infections.

Surveillance for an outbreak of Encephalitozoon cuniculi infection in rabbits housed at a zoo and biosecurity countermeasures.

An outbreak of encephalitozoonosis occurred in a rabbit colony at a zoo in Japan. Throughout the two years after the onset, all 42 rabbits were investigated clinically, pathologically and serologically for prevention and control of the disease. Eleven rabbits (11/42, 26.2%) showed clinical symptoms. Of 38 rabbits examined to detect specific antibodies against Encephalitozoon cuniculi, 71.1% (n=27) were found seropositive; 20 out of 30 clinically healthy rabbits (except for 8 clinical cases) were seropositive. The infection rate was 76.2% (32/42), including 5 pathologically diagnosed cases. The results of serological survey revealed that asymptomatic infection was widespread, even among clinically healthy rabbits. However, encephalitozoonosis was not found by pathological examination in any other species of animals kept in the same area within the zoo. Isolation and elimination of the rabbits with suspected infection based on the results of serological examination were carried out immediately; however, encephalitozoonosis continued to occur sporadically. Therefore, all the remaining rabbits were finally slaughtered. Then, the facility was closed, and all the equipment was disinfected. After a two-month interval, founder rabbits were introduced from encephalitozoonosis-free rabbitries for new colony formation. Since then, encephalitozoonosis has not been seen in any animals at the zoo. In this study, biosecurity countermeasures including staff education, epidemiological surveillance and application of an "all-out and all-in" system for rabbit colony establishment based on serological examination were successfully accomplished with regard to animal hygiene and public health for the eradication of E. cuniculi.

Spore-forming microsporidian encephalitozoon: current understanding of infection and prevention in Japan.

Microsporidia are spore-forming obligate intracellular parasites. They are known to cause opportunistic infections in humans through zoonotic, waterborne and foodborne transmission routes. This article reviews the present knowledge regarding microsporidian Encephalitozoon cuniculi infections in animals living in the human environment in Japan and discusses the basic measures required for the effective disinfection of Encephalitozoon. The article also discusses seroepidemiologic data from healthy people in order to shed light on the mechanisms of protective immunity and to identify potential strategies for preventive medicine.

Mouse monoclonal immunoglobulin E antibodies specific for the microsporidian Encephalitozoon cuniculi polar tube protein 1.

The microsporidian Encephalitozoon cuniculi is a spore-forming, obligate, intracellular parasitic pathogen with a unique organelle called a polar tube, the extrusion of which is essential for invading a host cell. The polar tube consists of three proteins: polar tube protein 1 (PTP1), PTP2, and PTP3. We established three mouse monoclonal antibodies (MAb1, MAb2, and MAb4) against E. cuniculi PTP1. An enzyme-linked immunosorbent assay (ELISA) indicated that all three MAbs reacted with the outer surface of extruded polar tubes and that they also strongly bound to an intracellular antigen in infected host cells. Two-dimensional (2-D) immunoblot analysis showed that MAb1 and MAb2 recognized PTP1 spots at 52 kDa and some spotty smears at molecular weights of less than 50 kDa, whereas MAb4 recognized only PTP1 spots at 52 kDa. Interestingly, all three MAbs were of the immunoglobulin (Ig) E class, suggesting that, in addition to the highly immunogenic or antigenic nature, the PTP1 antigen may have the potential to induce specific IgE antibody production in mice. These antibodies may be useful in the study of allergenic PTP1 as well in the purification and detection of the PTP1 antigen.

Detecting immunoglobulin M antibodies against microsporidian Encephalitozoon cuniculi polar tubes in sera from healthy and human immunodeficiency virus-infected persons in Japan.

Encephalitozoon cuniculi, a spore-forming obligate intracellular parasitic pathogen belonging to the phylum Microsporidia, has a unique and highly specialized organelle called the polar tube. Using an enzyme immunostaining assay in which germinated E. cuniculi spores were coated onto plastic surfaces, we tested healthy and human immunodeficiency virus (HIV)-infected individuals in Japan for anti-polar tube antibodies of each immunoglobulin (Ig) class. Anti-polar tube IgG was detected in just 4 of 380 healthy individuals; no anti-polar tube IgA was detected in any individuals; however, unexpectedly, anti-polar tube IgM antibodies were detected in 138 individuals (36%). When the healthy individuals were grouped by age, the highest rate of positivity to anti-polar tube IgM antibodies was seen in individuals aged 20 years old or younger. Fifty-nine percent (24/41) of the individuals aged 20 years or younger were anti-polar tube IgM antibody positive. This rate tended to decrease among individuals in older age groups. However, no anti-polar tube IgM antibodies were detected in 21 HIV-infected persons who were younger than 30 years of age and who had CD4 cell levels below 250/mul. These seroepidemiological results clearly indicate that circulating anti-polar tube IgM antibodies that are capable of strongly reacting with filaments extruded from geminated spores exist and suggest that such antibodies may play a part in protective immunity.