Effect of thermal treatment on free-living amoeba inactivation.

AIMS: To evaluate the effect of temperature on two amoeba strains of the genera Acanthamoeba and two amoeba strains of the genera Hartmannella separately treated depending on their life stage, trophozoite or cyst, when cells are directly exposed under controlled conditions. METHODS AND RESULTS: For thermal treatments, three temperatures were selected 50, 60 and 70°C, and a microcosm was designed using dialysis bags. The inactivation of each strain was determined using a method based on the most probable number quantification on agar plates. The results showed that for all amoeba strains, thermal treatment was more effective against trophozoites compared with cyst stages. The inactivation patterns showed statistical differences between the two genera analysed at temperatures above 50°C. The effectiveness of the thermal treatments at 60 and 70°C was higher for both life stages of Hartmannella vermiformis strains compared with Acanthamoeba strains, being the most resistant Acanthamoeba cysts. CONCLUSIONS: Free-living amoebae have been isolated in a wide range of environments worldwide due to their capacity to survive under harsh conditions. This capacity is mainly based on the formation of resistant forms, such as double-walled cysts, which confers a high level of resistance as shown here for thermal treatments. SIGNIFICANCE AND IMPACT OF STUDY: Free-living amoebae survival can promote a rapid recolonization of drinking water systems and is a likely source of emerging opportunistic pathogens such as Legionella. Because of that a better understanding of the factors that affect micro-organism inactivation in water systems would allow more efficient application of disinfection treatments.

Microfauna communities as performance indicators for an A/O Shortcut Biological Nitrogen Removal moving-bed biofilm reactor.

The microfauna communities present in the mixed liquor and biofilm of an Anoxic/Oxic Shortcut Biofilm Nitrogen Removal moving-bed biofilm process were characterised in order to optimise process control through the use of bioindicators. The system operated at high ammonium concentrations, with an average of 588 ± 220 mg N-NH4(+) L(-1) in the influent, 161 ± 80 mg L(-1) in the anoxic reactor and 74 ± 71.2 mg L(-1) in the aerobic reactor. Up to 20 different taxa were identified, including ciliates (4), flagellates (11), amoebae (4) and nematodes (1). Compared to conventional wastewater treatment processes (WWTPs), this process can be defined as a flagellates-predominant system with a low diversity of ciliates. Flagellates were mainly dominant in the mixed liquor, demonstrating high tolerance to ammonium and the capacity for survival over a long time under anoxic conditions. The data obtained provide interesting values of maximum and minimum tolerance ranges to ammonium, nitrates and nitrites for the ciliate species Cyclidium glaucoma, Colpoda ecaudata, Vorticella microstoma-complex and Epistylis cf. rotans. The last of these was the only ciliate species that presented a constant and abundant population, almost exclusively in the aerobic biofilm. Epistylis cf. rotans dynamics showed a high negative correlation with ammonium variations and a positive correlation with ammonium removal efficiency. Hence, the results indicate that Epistylis cf. rotans is a good bioindicator of the nitrification process in this system. The study of protozoan communities in unexplored WWTPs sheds light on species ecology and their role under conditions that have been little studied in WWTPs, and could offer new biological management tools.

Outbreaks of Ichthyophthirius multifiliis in redtail barbs Barbus haasi in a Mediterranean stream during drought.

In 2008, inland waterways in Catalonia (northeast Iberian Peninsula, Spain) experienced one of the worst droughts recorded in this region in recent decades. During this period, an epizootic of Ichthyophthirius multifiliis was detected for the first time in a Mediterranean stream, with 21% prevalence in a population of redtail barbs Barbus haasi. Environmental features and the fish population in this stream were compared during 2007-2009. Fish density and the average fish size were reduced significantly after the outbreak of I. multifiliis in this population. During 2008, parasitized fish were significantly larger than nonparasitized fish. In addition, a significant, positive correlation was found between parasite load and fish size. The origin of I. multifiliis is unknown, but an introduced species detected in April 2007 may have carried it. The combination of stress to the redtail barbs due to suboptimal conditions and favorable environmental conditions for parasite multiplication (e.g., suitable water temperature and low water flow) could have enhanced fish susceptibility to the parasite in April 2008. Further studies are needed to establish the incidence of freshwater fish diseases in Mediterranean watersheds, and water management policies should be reviewed to improve the conservation of native fish fauna.

Tetramicra brevifilum (Matthews & Matthews, 1980) (Microsporida: Tetramicriidae) in a new fish host, Lophius budegassa (Spinola, 1807) in Spain.

Tetramicra brevifilum, a microsporidian parasite of Scophthalmus maximus, was found in Lophius budegassa for the first time. This parasite was detected in 5 of 199 hosts captured in the coastal waters of Barcelona (Northwest Mediterranean), which enlarges the geographic distribution of this microsporidian. Affected fish did not show any external sign of disease, and cysts of T. brevifilum were found associated with the body musculature but were easily differentiated from those of Spraguea lophii, another microsporidian present in this host. A case of simultaneous infection by both T. brevifilum and S. lophii was found.

The pathologic cycle of the infection of the microsporidian Microgemma ovoidea (Thèl., 1895) Amigó et al. 1996 in the liver of the Red Band fish (Cepola macrophthalma L.).

Pathologic study of the lesions caused by Microgemma ovoidea has shown that after the formation of the xenoma (stage 1), the parasitized cell is infiltrated by host macrophages (stage 2) and quickly encysted by the activity of fibroblasts that form a xenoma wall composed of collagenous fibers (stage 3). The phagocytic activity of the macrophages leads to the formation of a granuloma (stage 4) in which the cyst contents comprise macrophages filled with phagocytosed spores. This phagocytic activity is limited by the fact that some parts of the microsporidian spores, such as the spore walls, cannot be lysed by macrophages, which leads to the formation of fused giant cells containing nondigestible spore remanants. The final step in the process is healing (stage 5), in which some cells may start proliferating to regenerate the damaged area. Nevertheless, the host occasionally fails to control M. ovoidea infections. This failure can take two forms: bursting of the granuloma, or the appearance of secondary infections in granulomas, probably through parasitism of macrophages.

Longevity and effects of temperature on the viability and polar-tube extrusion of spores of Glugea stephani, a microsporidian parasite of commercial flatfish.

Food contamination with respect to microsporidiosis in humans (associated with acquired immunodeficiency syndrome) and in marine fish farming deserves particular attention. For the first time, a study on the longevity and resistance to both heat and freezing of the spores of a vertebrate microsporidian, Glugea stephani, parasitizing a commercial flatfish was carried out. As judged on the basis of determinations of the extrusion rate, the resistance of the spores to temperature stress was remarkable. The extrusion rate, which can be directly related to infectivity, was always lower than the viability (membrane integrity). It should be pointed out that neither heat (60 degrees C for 30 min) nor freezing (-19 degrees C for 24 h) caused a complete reduction in the extrusion rate or viability. Consequently, the ingestion of poorly cooked or raw fish (even if previously frozen) represents a danger for aquaculture and, probably, for immunodepressed patients.

Comparative study of microsporidian spores by flow cytometric analysis.

Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis-benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono- and diplokaryotic populations of S. lophii identified. Staining of DNA by bis-benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.