acanthamoeba isolates and cultivation - clinical and environmental strains of acanthamoeba polyphaga ( ap2 - atcc 30461 and ap4 - atcc 30872 ) and acanthamoeba castellanii ( t4 - atcc 50492 and neff - atcc 30010 ) were used in this study . the organisms were grown at 30c in 2% proteose peptone , 0.2% yeast extract and 1.8% glucose containing antibiotics ( penicillin and streptomycin 40 l / ml ) to maintain axenic culture . the atcc strains used have been grown in this manner in the laboratory since 2006 . 
 in vivo infection model of acanthamoeba using intranasal inoculation - the stimulant solution ( ss ) for infection was prepared from cultures of different isolates of acanthamoeba . the medium containing 1 x 10 trophozoites / ml was centrifuged at 250 g for 10 min . the pellet was washed three times with phosphate buffered saline ( pbs ) and resuspended in 200 l of pbs . adult male wistar rats , with a mean age of three months , were first immunosuppressed by intraperitoneal injection with dexamethasone ( 5 mg / kg ) ( cappila et al . , the animals were anesthetised with ketamine hydrochloride ( 60 mg / kg ) and xylazine hydrochloride ( 8 mg / kg ) and were inoculated intranasally with 200 l of the ss . after this period , the rats were euthanised by depression of oxygen and the organs were removed and evaluated under a magnifying glass to observe the lesions . two animals were used as controls and were submitted to the same conditions as the test group . the parasites were re - isolated from the organ lesions and cultured in plates with non - nutrient agar 1.5% covered with inactivated escherichia coli ( atcc 25922 ) . only the t4r and ap4r re - isolates were re - inoculated into animals . ethics - this study had been previously approved by the ethical committee on animal studies at the federal university of rio grande do sul . histological analysis - the organs were removed from the infected animals and preserved in 4% paraformaldehyde . all the atcc isolates studied , i.e. , t4 ( 50492 ) , ap2 ( 30461 ) , ap4 ( 30872 ) and neff ( 30010 ) , were able to establish systemic infections in the rats . the lesions produced by ap2 ( 30461 ) and neff ( 30010 ) were disseminated in several organs and caused the deaths of the animals after an average period of 26 days . the lesions produced by ap4 ( 30872 ) and t4 ( 50492 ) were restricted to the lungs and these animals survived for the 30-day duration of the experiment ( table ) . for the rats infected with t4r and ap4r , the mean survival time after infection was 17 days ( table ) . generally , it can be assumed that the rats survived for shorter periods when the infections were more invasive . the two animals used as controls survived the 30-day period of the experiment and post - mortem examination revealed no injury to their organs . tableevaluation of in vivo model of acanthamoeba infection isolation in organsisolated lungs brain liver heart kidneysdays of survival 
 post - infection t4 + - - - - 30 ap2 + - + - + 22 ap4 + - - - - 30 neff + - + - - 30 t4r + + - + + 20 ap4r + + + + + 15ap2 : atcc 30461 ; ap4 : atcc 30872 ; neff : atcc 30010 ; t4 : atcc 50492 . ap2 : atcc 30461 ; ap4 : atcc 30872 ; neff : atcc 30010 ; t4 : atcc 50492 . all of the acanthamoeba isolates , whether clinical or environmental , were able to establish systemic infections in the immunosuppressed animals , where they mainly reached the lungs , as shown in fig . the animals infected with the standard strains , i.e. , ap2 ( 30461 ) and neff ( 30010 ) , suffered more invasive infections . the parasite was re - isolated from the lungs , liver and kidneys of rats infected with the atcc strain 30461 , as well as from the liver and lungs of rats infected with the atcc strain 30010 ( table ) . when the trophozoites that were re - isolated from lesions were used , i.e. , ap4r and t4r , the protists were found in several organs , including the brain ( table ) . the symptoms were perceived 10 days after infection and mainly consisted of weight loss , hair loss , shivering , diarrhoea and prostration . 1representative pictures of lungs of rats infected intranasally with acanthamoeba . a : lungs normal tissue , rats not infected with acanthamoeba ; b : lungs tissue with confirmed acanthamoeba infection ; c : histological analysis of tissue the lungs with acanthamoeba contamination . only the brains of animals infected with re - isolated ap4r and t4r showed altered morphology ( fig . 2representative pictures of brain of rats infected intranasally with acanthamoeba . a : brain normal tissue , rats not infected with acanthamoeba ; b : brain tissue with confirmed acanthamoeba infection ( t4r re - isolate ) ; c : histological analysis of tissue the brain with acanthamoeba contamination . during human infection , strains of acanthamoeba affect several organs in addition to the brain , including the skin , liver , lungs , kidneys , heart , diaphragm , eyes , adrenal glands , pancreas , prostate , lymph nodes and bone marrow ( mazur & jwiak 1993 , khan 2010 , young et al . ( 2009 ) studied acanthamoeba infections in locusts and observed that the insects developed disseminated infections in the haemolymph , fat body , muscle and brain tissues . in another study , acanthamoeba were found in the lungs and brain tissue of mice ( de jonckheere 1980 ) . the results of the present study showed that this infection model produced systemic infections in rats and that several organs were affected ( table ) . according to khan ( 2010 ) , the ability of acanthamoeba to cause systemic infections reflects its capacity to survive , attack the innate immune system and invade the cns , which is consistent with gae cases in humans . our results show that strains isolated from the environment can cause infections that are quite similar to those caused by the clinical strains of acanthamoeba ( table ) , which poses a potential risk to human health , especially in immunosuppressed individuals . this risk is consistent with the results of de jonckheere ( 1980 ) , who found that strains previously described as incapable of causing infections in mice were highly pathogenic and caused 100% mortality in animals that were inoculated intracerebrally . when recent isolates of acanthamoeba were used to infect rats , the animals developed more invasive and rapidly fatal infections . for leishmania spp , maintenance in vitro results in a progressive loss of virulence , which can be reversed by passage in a mammalian host . a significant correlation has also been found between higher parasite burdens in infected balb / c mice and fewer in vitro passages of the amoebae ( moreira et al . therefore , the different invasive capacities of acanthamoeba isolates and re - isolates can be explained by the expression of different virulence factors after the first infection . mazur et al . ( 1999 ) compared the pathological and morphological changes in the organs of rats caused by infection with strains of low and high virulence in vivo . these investigators reported that infections with each acanthamoeba isolate resulted in similar infections , but that the changes induced by the strain with low virulence developed later and were less extensive . they suggested that differences in the virulence of strains , due to the reactivation of factors , could explain why only the re - isolated amoebae reached the brain of the rats . another study demonstrated that parasites that had been grown for different periods of axenic in vitro culture induced different infections in rats ( moreira et al . studies in the 1970s demonstrated that when studying acanthamoeba , pathogenicity tests should be performed as soon as possible after isolation because the strains lose virulence with frequent transfers in axenic culture ( stevens & o'dell 1974 ) . moreira et al . ( 2012 ) noted that axenic culturing and long - term maintenance of the parasite can affect the outcome of in vitro infections . this influence was also demonstrated in in vivo studies by grnik and kuna - grygiel ( 2005 ) , who determined the degrees of virulence for six strains of acanthamoeba in mice based on the number of organs affected and the extent of histopathological changes due to systemic infections . using this criterion , the strains studied here were classified with varying degrees of virulence . as such , atcc strains ap2 ( 30461 ) and neff ( 30010 ) were more virulent and t4 ( 30492 ) and ap4 ( 30872 ) were less virulent . additionally , the t4r and ap4r re - isolates became more virulent after the first infection in animals . although in vitro models are more practical and efficient in restoring the virulence of acanthamoeba ( koehsler et al . 2009 ) , this study utilised an in vivo model because , even with immunosuppressed animals , the parasites must address different host defence mechanisms , which can result in the expression of different virulence factors and the selection of resistant forms . the capacity of the parasite to modulate macrophage activation decreases significantly with increasing numbers or intervals of in vitro passages ( borba 2002 , santos et al . the observed changes in the profiles of proteolytic activity between the isolates cultured in the laboratory for long periods and those recently passed through an animal model ( data not shown ) , as well as the increase in the severity of the infections caused by these re - isolates , indicate that the first contact with animals may have induced changes in the expression of the virulence factors . our results provide further evidence for the effect of prolonged axenic culture on the virulence of acanthamoeba . they indicate the importance of developing protocols to reactivate the virulence of acanthamoeba cultures in studies focusing on interactions with mammals , such as drug development or vaccine trial studies and pathogenicity .
prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity . 
 however , little is known about the changes in the pathogenic strains of acanthamoeba after long culture periods . 
 our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of acanthamoeba in an in vivo rat model . 
 atcc strains of acanthamoeba , isolates from the environment and clinical cases were evaluated . 
 the in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re - isolates . 
 the amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated , confirming the importance of passing acanthamoeba strains in animal models .