First molecular evidence of ocular transmission of Encephalitozoonosis during the intrauterine period in rabbits.

Many reports have been published on the suspected vertical transmission of Encephalitozoon cuniculi; however, prior to 2003, these reports were based on circumstantial evidence, such as histopathological, immunohistochemical, or serological diagnosis of the infection. In 2003, vertical transmission of the parasite was confirmed by detection of E. cuniculi DNA in fetuses with the nested polymerase chain reaction (PCR) technique. However, the passage of the parasite to eyes of fetus during the intrauterine stage still requires verification. In the current study, natively infected with parasite spores female rabbits were mated with non-infected males. All resulting offspring that died before ten postpartum days were investigated using molecular techniques to confirm the intrauterine transmission of the parasite to the offspring' eyes. In total, 119 DNA samples from rabbit offspring tissues were collected from blood, kidney, brain, eye (both eyes were used as single samples), lung, placenta, liver and heart were used for PCR. Parasitic DNA in the eyes of offspring was detected (54%) 6 of 11 naturally seropositive mother rabbits. PCR results were found to be positive for the eyes of 63% (19/30) of the offsprings from seropositive rabbits. Therefore, mother rabbits naturally infected with E. cuniculi showed the molecular presence of the parasite in their offspring' eyes. Sequence analysis confirmed the partial DNA sequence data of E. cuniculi and blast analysis identified the agent as genotype I. These results confirm transmission of E. cuniculi to rabbit offspring' eyes in the intrauterine period. This is the first molecular evidence to show ocular transmission of the infection via an intrauterine route in rabbits.

Evidence of transplacental transmission of Encephalitozoon cuniculi genotype II in murine model.

Microsporidia are obligate intracellurar unicellular parasite of wide range of vertebrates. Although ingestion or inhalation of microsporidian spores is the main route of infection, assumed vertical transmission was described in some mammals. The present study was focused on proof of vertical transmission in mice under experimental conditions. Mice were infected with E. cuniculi genotype II intraperitoneally after mating, or perorally followed by mating in acute or chronic phase of infection. Fetuses were delivered by Caesarean section or mice were kept up to the parturition. Some of cubs were immediately after birth transferred to uninfected surrogate mothers. Group of cubs was immunosuppressed. All cubs were examined using polymerase chain reaction for the presence of Encephalitozoon after birth or in their age of 3 or 6 weeks, respectively. All fetuses delivered by Caesarean section, which were intraperitoneally or perorally infected were negative as well as all neonatal mice and youngsters tested in age of 6 weeks. Only immunosuppressed cubs and cubs of immunodeficient mice in age of 21 days were positive for Encephalitozoon cuniculi genotype II. Present results provided the evidence that transplacental transmission of Encephalitozoon cuniculi in mice occurs, but the mechanism of these transport is still unknown.

DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence.

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.

Encephalitozoonosis in New Zealand rabbits and potential transmission risk.

Encephalitozoon cuniculi is a small protozoan parasite in the phylum Microspora. It has been shown to naturally infect several host species, including humans. Encephalitozoonosis is routinely diagnosed in vivo by serological examination or post mortem by histopathology. In a conventional rabbit colony, two animals suddenly showed clinical signs (torticollis and asthenia of limbs). Serum samples of these rabbits were seropositive for E. cuniculi after definitive diagnosis (Toxoplasma gondii and Listeria monocytogenes). The animals in the same breeding facility were also clinical examined, and the present study evaluated the prevalence of specific anti-E. cuniculi antibodies using serological testing, both in animals and in people working with animals, after two clinical cases. The rabbits showed no clinical symptoms of the disease. Blood samples were taken for E. cuniculi infection from 50 clinically healthy rabbits. Anti-E. cuniculi antibodies were found in two asymptomatic and two clinically affected animals belonging to the same rabbit colony. Finally, the present study found that the 7.7% (4/52) prevalence of CIA, test positive in rabbits. E. cuniculi spores were detected in the urine of one clinically affected rabbit, and one seropositive animal caretaker after staining with the modified trichrome stain. In conclusion, the presence of seropositive, but apparently healthy rabbits indicates the need for screening examinations to detect the anti-E. cuniculi antibody in rabbits, especially considering the potential zoonotic risk. Therefore, persons should avoid contact with the urine of infected or healthy animals, and always use good personal hygiene when handling animals.

Prevalence of antibodies to Trypanosoma cruzi, Toxoplasma gondii, Encephalitozoon cuniculi, Sarcocystis neurona, Besnoitia darlingi, and Neospora caninum in North American opossums, Didelphis virginiana, from southern Louisiana.

We examined the prevalence of antibodies to zoonotic protozoan parasites ( Trypanosoma cruzi, Toxoplasma gondii, and Encephalitozoon cuniculi) and protozoans of veterinary importance ( Neospora caninum, Sarcocystis neurona, and Besnoitia darlingi) in a population of North American opossums ( Didelphis virginiana) from Louisiana. Samples from 30 opossums were collected as part of a survey for T. cruzi in Louisiana. Frozen sera from these 30 opossums were examined using an indirect immunofluorescent antibody test (IFAT) against in vitro-produced antigenic stages of these protozoans. Additionally, 24 of the 30 samples were examined using hemoculture, and all 30 were examined in the modified direct agglutination test (MAT) for antibodies to To. gondii. The prevalences of reactive IFAT samples were as follows: 60% for T. cruzi, 27% for To. gondii, 23% for E. cuniculi, 17% for S. neurona, 47% for B. darlingi, and 0% for N. caninum. Hemoculture revealed that 16 (67%) of 24 samples were positive for T. cruzi, compared to 18 of 30 (60%) by IFAT. The sensitivity and specificity for the IFAT compared to hemoculture was 100% for each. The modified direct agglutination test revealed that 9 (30%) of the 30 samples from opossums had antibodies to To. gondii , compared to 8 (27%) using the IFAT. The sensitivity and specificity of the IFAT compared to the MAT was 100% and 72%, respectively.

Serological survey for antibodies to Encephalitozoon cuniculi in pet rabbits in Italy.

Pet rabbits (n = 125) from Southern Italy were submitted to a serological screening for Encephalitozoon cuniculi, using an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA). Seventy-eight examined rabbits showed clinical signs suggestive of encephalitozoonosis (head tilt, ataxia, paralysis, cataracts, uveitis, polyuria and polydipsia), whereas 47 were healthy rabbits. Antibodies anti-E. cuniculi were found in 84/125 (67.2%) sera analysed. The results of the chi-squared test showed that sex and health status had no significant effect (P > 0.05) on E. cuniculi seropositivity; however, rabbits older than 4 months had a seropositivity for E. cuniculi significantly (P < 0.05) higher than that of rabbits aged up to 4 months. The results of the present survey reinforce the assumption that rabbit may be indicated as the main reservoir of E. cuniculi; therefore, routine screening examinations in pet rabbits are strongly advised considering the zoonotic potential of this parasite.

First diagnosis of Encephalitozoon intestinalis and E. Hellem in a European brown hare (Lepus europaeus) with kidney lesions.

Encephalitozoon intestinalis and Encephalitozoon hellem were diagnosed in the kidneys of a free-ranging European brown hare (Lepus europaeus) with multifocal wedge-shaped chronic interstitial nephritis using real-time PCR and microarray. This is the first description of these microsporidia species in a European brown hare, which are both potential zoonotic agents.

Microsporidian species known to infect humans are present in aquatic birds: implications for transmission via water?

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 x 10(5) spores/g) than nonaquatic bird droppings contained (mean, 4.4 x 10(4) spores/g) (P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 x 10(8) microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.

Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi.

High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.

Prevalence of antibodies to Encephalitozoon cuniculi in horses in the Israel.

Infection with the intracellular microsporidium Encephalitozoon cuniculi can cause a serious disease--encephalitozoonosis in various animals and people. Several species of mammals, including the horse, were seem to be potential sources of encephalitozoonosis for animal as well as human hosts. The disease diagnosis is based on clinical signs, pathological findings, and the detection of E. cuniculi or circulating antibodies directed against the parasite. This study investigates the seroconversion to E. cuniculi in horses admitted to the Veterinary Teaching Hospital of the Hebrew University of Jerusalem and 3 different private horse-riding farms across Israel. Antibodies to E. cuniculi were determined using the IFA test in the sera from 102 horses. Of 72 asymptomatic horses, 60% were seropositive and 19% of the positive samples showed a titter of 1:512. Of 30 horses with various clinical signs, 80% were seropositive and 68% of the positive samples showed a titer of 1:512. High titers were associated with colic and neurological signs. This could prove to be interesting if the high percentages of prevalence of antibodies level in horses are an indication of health risk in humans.

Serological screening of occurrence of antibodies to Encephalitozoon cuniculi in humans and animals in Eastern Slovakia.

Encephalitozoon cuniculi is one of the mamalian microsporidian pathogens that can affect a number of different species of animals as well as humans. The presence of specific serum antibodies to Encephalitozoon cuniculi was studied in a group of animals and humans from Eastern Slovakia by the indirect immunofluorescence of antibodies (IFA). 456 people, 571 rabbits, 457 mice, 193 dogs, 72 cats, and 59 sheep were examined. Specific anti-E. cuniculi antibodies were found in 26 out of 456 human sera examined (5.7%). The highest occurrence of antimicrosporidial antibodies was found in the group of immunodeficiency patients - 37.5%. In the group of animals, the highest positivity was observed in rabbits - 41.7%, and in dogs - 37.8. The relative high prevalence, especially in rabbits and dogs as potential sources of microsporidial infection for humans, indicates the importance of performing the screening examinations in animals with aim of reducing or halting the spread of this disease.

In utero transmission of Encephalitozoon cuniculi strain type I in rabbits.

Pregnant rabbits were serologically diagnosed as having been infected with Encephalitozoon cuniculi. At necropsy at 28 days of gestation, does, placentas and fetuses were found to be infected with E. cuniculi strain type I as evidenced by using the nested-polymerase chain reaction (PCR) technique, thereby confirming vertical transplacental transmission.

A single genotype of Encephalitozoon intestinalis infects free-ranging gorillas and people sharing their habitats in Uganda.

Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.

Dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient.

This report describes the first dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient. In view of clinical and epidemiological findings, our E. cuniculi isolate was deduced to be of the dog strain. The patient's occupational involvement with dogs indicates that canines should be considered as a reservoir of human infections for both microsporidial species. Furthermore, our report provides detailed clinical and radiological information on a rare case of a symptomatic pulmonary infection by E. cuniculi and its improvement after treatment with albendazole.

[Natural transmission of microsporidia (Encephalitozoon cuniculi) by way of the chicken egg]. / Natürliche Ubertragung von Mikrosporidien (Encephalitozoon cuniculi) über das Hühnerei.

100 chicken embryos were examined for naturally occurring infections with Encephalitozoon cuniculi (E.c.). Two embryos were found to be dead, the others were killed at an age of 18 days. Samples from the oesophagus, intestine, liver, kidneys, heart and brain were collected and examined by immunohistochemical methods. E.c. was found in about 40% of the embryos. This microsporidian was observed in all organs, particularly, however, in the oesophagus, intestine, brain and heart. The number of E.c. was significantly larger in the two embryos that died than in the others. The investigations demonstrated, that E.c. may be transmitted naturally to the chicken egg. Further, the results suggested that E.c. may be a cause of death in chicken embryos. In most cases, however, E.c. infections are inapparent in embryos. Such inapparent infections may be an important way of contamination of chicken production units with E.c.

The pathology of subclinical infection of Encephalitozoon cuniculi in canine dams producing pups with overt encephalitozoonosis.

The macroscopic, microscopic and clinical pathology and the serology of 2 clinically normal Staffordshire Bull Terrier bitches, both of whom produced pups with confirmed encephalitozoonosis, is described. Mild histopathological changes, similar to those seen in the infected pups, were observed. The spores of Encephalitozoon cuniculi were seen in the renal tubules of the kidney of one of the bitches. The serum urea concentrations of one of the bitches was elevated. A positive titre against E. cuniculi was obtained in both of the bitches. A 10-year-old girl who had had close contact with one of the infected litters of pups, seroconverted to E. cuniculi. Her two siblings were serologically negative.