Two hundred and seventeen captive great apes (150 chimpanzees, Pan troglodytes; 14 bonobos, Pan paniscus; 53 western gorillas, Gorilla gorilla) and 20 personnel from thirteen European zoos and two African sanctuaries were sampled and examined in order to determine the occurrence of Enterocytozoon bieneusi and species of Encephalitozoon in faecal specimens and to compare the epidemiological situation between zoos and sanctuaries. Microsporidia were detected at all sampling sites. Sequence analyses of ITS amplicons generated by using microsporidia-specific primers determined the presence of microsporidia in 87 samples including 13 humans; since two cases of simultaneous occurrence of Encephalitozoon cuniculi and Enterocytozoon bieneusi were identified, 89 full-length ITS sequences were obtained, namely 78 Encephalitozoon cuniculi genotype I, five E. cuniculi genotype II, two E. hellem 1A and four Enterocytozoon bieneusi. No Encephalitozoon intestinalis-positive samples were identified. This is the first report of Encephalitozoon species and Enterocytozoon bieneusi genotypes in captive great apes kept under various conditions and the first record of natural infection with E. hellem in great apes. A comparison of zoos and sanctuaries showed a significantly higher prevalence of microsporidia in sanctuaries (P<0.001), raising a question about the factors affecting the occurrence of microsporidia in epidemiologically and sanitarily comparable types of facilities.
Experimental activation of peritoneal macrophages by interferon gamma (IFN-γ) resulted in the inhibition of Encephalitozoon cuniculi replication. However, E. cuniculi could replicate either in a non-activated cell line of murine macrophages PMJ2-R or in IFN-γ-activated PMJ2-R cells. Moreover, activation with IFN-γ led to faster replication of E. cuniculi in these cells. Opsonisation of E. cuniculi spores with anti-E. cuniculi polyclonal antibody did not affect E. cuniculi replication in both, non-activated and activated murine macrophages. In contrast, opsonisation of E. cuniculi spores caused the most effective replication of E. cuniculi in activated PMJ2-R cells. However, production of nitric oxide by these cells was significantly more intensive than that in non-activated, infected cells, where the parasite replicated to a much lesser extent. Our results support the hypothesis that E. cuniculi uses phagocytosis for the infection of host cells. They also indicate that the mechanism by which spores of E. cuniculi are killed by macrophages is not dependent on nitric oxide and they reveal that PMJ2-R cells cannot substitute peritoneal murine macrophages in immunological studies on E. cuniculi.
Three strains of mice, BALB/c, IL-12 knock-out (KO) and INF-γ knock-out, were chosen as an experimental model for the study of intestinal immunity induction against Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 infection. Mice were infected perorally with 107 spores and re-infected with the same dose 70 days after the first infection. The anti-E. cuniculi IgA, IgG and IgM responses in sera and extracts of stool samples were determined by ELISA. Results have shown specific antibody production in the sera and intestinal secretions of all three strains of mice induced orally by E. cuniculi spores. BALB/c mice developed a stronger humoral immune response than IL-12 KO mice. The lowest antibody response developed in INF-γ KO mice that succumbed to the infection within 28 days post infection.
Parasites of the genus Cryptosporidium Tyzzer, 1910 are one of the most common protistan parasites of vertebrates. Faecal samples from 179 red foxes (Vulpes vulpes [Linnaeus]), 100 grey wolves (Canis lupus Linnaeus), 11 golden jackals (Canis aureus Linnaeus), and 63 brown bears (Ursus arctos Linnaeus) were collected in the Czech Republic, Poland and Slovakia. Samples were examined for the presence of Cryptosporidium spp. using microscopy and PCR/sequence analysis. Phylogenetic analysis based on the small subunit ribosomal RNA (SSU), actin and 60-kDa glycoprotein (gp60) genes using the maximum likelihood method revealed the presence of Cryptosporidium tyzzeri Ren, Zhao, Zhang, Ning, Jian et al., 2012 (n = 1) and C. andersoni Lindsay, Upton, Owens, Morgan, Mead et Blackburn, 2000 (n = 2) in red foxes, C. canis Fayer, Trout, Xiao, Morgan, Lai et Dubey, 2001 (n = 2) and C. ubiquitum Fayer, Santín et Macarisin, 2010 (n = 2) in grey wolves, and C. galli Pavlásek, 1999 in brown bears (n = 1) and red foxes (n = 1). Subtyping of isolates of C. ubiquitum and C. tyzzeri based on sequence analysis of gp60 showed that they belong to the XIId and IXa families, respectively. The presence of specific DNA of C. tyzzeri, C. andersoni and C. galli, which primarily infect the prey of carnivores, is probably the result of their passage through the gastrointestinal tract of the carnivores. Finding C. ubiquitum XIId in wolves may mean broadening the host spectrum of this subtype, but it remains possible this is the result of infected prey passing through the wolf - in this case deer, which is a common host of this parasite. The dog genotype of C. canis was reported for the first time in wolves.
Susceptibility of three strains of immunodeficient mice to two related microsporidian species Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 and Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) was compared. While both, severe combined immunodeficient (SCID) and interferon-gamma knock-out (IFN-γ KO) mice, succumbed to either intraperitoneal (i.p.) or peroral (p.o.) (natural) infection with both parasites, only i.p. infection with E. cuniculi killed interleukin-12 knock-out (IL-12 KO) mice. IFN-γ KO mice died earlier than SCID mice. Adoptive transfer of naive splenocytes from IFN-γ KO mice did not protect the SCID mice from a lethal infection with either of the Encephalitozoon species. However, reconstituted mice survived significantly longer (P<0.05), thus indicating the role of IFN-γ produced by host NK cells in the development of mechanisms of anti-microsporidial protective immunity. Non-lethal outcome of the infection always correlated with the increase in CD8+ T lymphocyte subpopulation. Both E. intestinalis-infected IFN-γ KO and IL-12 KO mice produced comparable levels of specific antibodies, suggesting that antibodies did not protect IFN-γ KO mice from lethal infection.