Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum

Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum Abstract This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism. oomycete, pythiosis, zoospores, hyphae, zoosporangia Introduction The aquatic oomycete Pythium insidiosum is the etiological agent of pythiosis, a disease which occurs in its chronic fibrovascular form in horses1 and, in its pyogranulomatous manifestation, in other mammalian species that inhabit tropical and subtropical areas.1,2 Pythiosis has been reported to affect man and animals, especially horses, in several countries.2,3 In humans, the disease has been predominantly reported in Thailand4 and is thought to be endemic in horses, particularly in the Brazilian Pantanal area3,5and in the State of Rio Grande do Sul, southern Brazil.6 In these areas, the highest number of cases occurs in the hottest months of the year (spring and summer);5–8 however, equine pythiosis has also been reported in seasons with lower temperatures (fall and winter).6 P. insidiosum performs its biological cycle in stagnant water in natural environments. In these environments, the microorganism reproduces asexually, yielding infective zoospores. Susceptible hosts are infected by getting in contact with contaminated environments.3,9 Several factors can influence P. insidiosum cycle in the environment. According to Miller and Campbell10, environment temperatures between 30°C and 40°C and water accumulation in marshes and ponds are essential factors that influence zoospore production, as well as are conditioning factors for the occurrence of the disease. Abdelzaher et al.11, upon evaluating the influence of different temperatures on the zoosporogenesis of different Pythium species, found that some of the evaluated species were able to produce abundant zoospores at temperatures of 15°C, 20°C, and 25°C. Nevertheless, P. insidiosum in vitro zoosporogenesis has only been evaluated at 37°C.12,13,14 In vitro studies have shown that temperature can influence P. insidiosum development. Krajaejun et al.15 demonstrated that the oomycete mycelial growth was inhibited at 8°C and that temperatures of 28°C and 32°C favored its development. However, these authors evaluated this influence on mycelial growth rather than the on the zoosporogenesis process that originates infective zoospores. This study aimed to evaluate the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. Methods Pythium insidiosum isolates The present study analyzed 26 P. insidiosum isolates from horses. The identification of each isolate was performed by macro- and micro-morphological characteristics and confirmed by polymerase chain reaction (PCR) and DNA sequencing, as described by Azevedo et al.16,17 Zoosporogenesis induction at different temperatures A 4-mm disk of each P. insidisoum isolate previously cultured on CMA (corn meal agar) was transferred to a Petri dish containing 0.1% yeast agar. Sterilized grass fragments (Paspalum notatum) were then placed around the disk. The cultures were incubated at 37°C for 72 hours. Subsequently, grass fragments of each isolate were transferred to Falcon tubes containing 10 mL induction medium containing solution 1 (K2HPO4 [1.0 mol/l], KH2PO4 [1.0 mol/l], (NH4)2HPO4 [3.66 mol/l], 500 ml distilled water) and solution 2 (MgCl2.6H2O [0.5 mol/l]), CaCl2.2H2O [0.5 mol/l], 250 ml distilled water). The induction medium final composition was 0.5 ml solution 1, 0.1 ml solution 2 and 1000 ml sterile distilled water.12 The experiments were divided into two stages. In the first stage, the tubes were incubated at 5°C (group A), 15°C (group B), 20°C (group C), and 37°C (control group). To evaluate P. insidiosum zoosporogenesis at different temperatures, grass fragments were removed from the tubes, placed between the blade and coverslip of microscope slides and evaluated under optical microscopy (10 to 20 × objective lens) at the first 4, 8, and 24 hours of incubation. In the second stage, groups A, B, C, and Control isolates were incubated at 37°C and retested as described in the first stage. The following morphological parameters for isolates under zoosporogenesis were evaluated for each period: hyphae development (short, developing, long and mycelia), presence or absence of vesicles, and zoosporangia and production of zoospores (absence or presence of mobile and immobile zoospores, and their Neubauer chamber count) as described by Pereira et al.14 and Azevedo et al.16 At the end of the second stage, the viability of immobile zoospores of P. insidiosum isolates was checked by seeding 100 μl induction medium on 0.1% yeast agar at 37°C for 72 hours. Results Table 1 and Table 2 show the results of the in vitro zoosporogenesis of 26 P. insidiosum isolates evaluated at different temperatures and different periods. Table 1. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours), during the first 24 h of incubation (1st stage of experiment).     Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)      Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large Table 1. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours), during the first 24 h of incubation (1st stage of experiment).     Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)      Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large Table 2. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated during the2ststage of experiment (all isolates from groups A, B, C and Control were incubated at 37°C).   Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)    Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large Table 2. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated during the2ststage of experiment (all isolates from groups A, B, C and Control were incubated at 37°C).   Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)    Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large At 5°C (group A), the presence of short hyphae was observed predominantly on the edges of grass fragments at all periods evaluated in the first stage of the study (Fig. 1A). There were no vesicles, zoosporangia and zoospores at this temperature. Figure 1. View largeDownload slide In vitro zoosporogenesis of Pythium insidiosum at different temperatures and periods evaluated. A (Group A, 5°C/24 h): Short hyphae (→). B (Group B, 15°C/8 h): Hyphae in different stage of growing: short hyphae (→), in developing (*) and long and mycelial hyphae (→). C (Group C, 20°C/4 h) - 1: Presence of vesicle (→) and zoosporangium (→) and 2 – release of zoospores. D (Group C, 20°C/24 h): Zoospores with the emission of germ tubes (→). E (Group Control, 37°C/48 h): Numerous immobile zoospores (→). A, B, C, D and E: 20 × Objective. F: Growth of P. insidiosum from Control group at 37°C/48 h. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide In vitro zoosporogenesis of Pythium insidiosum at different temperatures and periods evaluated. A (Group A, 5°C/24 h): Short hyphae (→). B (Group B, 15°C/8 h): Hyphae in different stage of growing: short hyphae (→), in developing (*) and long and mycelial hyphae (→). C (Group C, 20°C/4 h) - 1: Presence of vesicle (→) and zoosporangium (→) and 2 – release of zoospores. D (Group C, 20°C/24 h): Zoospores with the emission of germ tubes (→). E (Group Control, 37°C/48 h): Numerous immobile zoospores (→). A, B, C, D and E: 20 × Objective. F: Growth of P. insidiosum from Control group at 37°C/48 h. This Figure is reproduced in color in the online version of Medical Mycology. At 15°C (group B), there was hyphal development (Fig. 1B). In two P. insidiosum isolates (7.69%); long hyphae, vesicles, and zoosporangia were observed, as well as the production of zoospores in small quantities within 24 hours of incubation (Fig. 2A). Figure 2. View largeDownload slide Mean count of zoospores of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours). A. First stage of experiment: P. insidiosum isolates were incubated at 5°C (group A), 15°C (group B), 20°C (group C), and 37°C (control group) and evaluated at 4, 8, and 24 hours of incubation. B. Second stage of experiment: isolates of P. insidiosum from groups A, B, C, and control were incubated at 37°C and retested at 4, 8, and 24 hours. Figure 2. View largeDownload slide Mean count of zoospores of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours). A. First stage of experiment: P. insidiosum isolates were incubated at 5°C (group A), 15°C (group B), 20°C (group C), and 37°C (control group) and evaluated at 4, 8, and 24 hours of incubation. B. Second stage of experiment: isolates of P. insidiosum from groups A, B, C, and control were incubated at 37°C and retested at 4, 8, and 24 hours. It was evidenced that 92.31% (24/26) of the isolates submitted to 20°C (group C) showed mycelial growth on the edges of grass fragments within the first 4 hours of evaluation. The presence of vesicles, zoosporangia and zoospores was observed (Fig. 1C). As for zoospore count, 26.92% (7/26) of the evaluated isolates produced an average of 9.6 × 103 mobile zoospores/mL within this period (Fig. 2A). Additionally, at the other evaluated periods (8 and 24 hours), mobile and immobile zoospores with the development of germ tubes were detected (Fig. 1D). In the second stage of the study, group A and B isolates showed developing as well as long hyphae with the presence of vesicles, zoosporangia and zoospores after a 4-hour incubation period, with a mean zoospore count of 0.86 × 103 and 2.21 × 103 zoospores/ml, respectively (Fig. 2B). These characteristics were kept at the 8- and 24-hour evaluations; however, there was an increase in zoospore count (Fig. 2B). Control group isolates showed evident zoosporogenesis in the first 4 hours of evaluation, with the presence of mobile and immobile zoospores, and a mean count of 14.5 × 103 zoospores/mL (Fig. 2A). At the end of the second stage, these isolates included many immobile zoospores (Fig. 1E) which were nevertheless viable when their growth was evaluated in culture medium (Fig. 1F). Discussion In P. insidiosum biological cycle in aquatic environments, zoosporogenesis originates zoospores, which are the infective form of this oomycete for animals and humans that inhabit marshy areas of tropical and subtropical climates.2,3,9 In nature, temperature is one of the factors that directly influences P. insidiosum zoosporogenesis process. According to Miller and Campbell10, the development of zoospores is favored at environment temperatures between 30°C and 40°C. Pythiosis cases in animals are mainly reported during the hottest months of the year.3,5,6–8,10,18–20 However, cases of equine pythiosis have also been observed in the winter months in subtropical and temperate climates, such as in southern Brazil.6 The present study was carried out to verify the in vitro influence of temperature on the zoosporogenesis of P. insidiosum isolates from infected horses in the State of Rio Grande do Sul, Brazil, an important endemic region for equine pythiosis where cases of the disease have also been reported during the cold months of the year. In the present study, 96.15% of P. insidiosum isolates that were incubated at 37°C (Control group) showed mycelial growth, vesicles, zoosporangia and zoospores in the first 4 hours of evaluation, which is an expected result when the oomycete is incubated at this temperature. Previous studies12–14 evaluating the in vitro zoosporogenesis process in P. insidiosum isolates have reported that temperatures of 37°C favor the formation of zoosporangia and zoospores. Chaiprasert et al.13 and Mendoza et al.21 observed the presence of encysted zoospores after one hour of incubation. Mendoza and Prendas12and Pereira et al.14 showed that the asexual cycle began within 3 hours, with a zoospore production peak between 6 and 8 hours. In this study, the results at 37°C are in agreement with those reported by these authors. In addition, it was demonstrated that in cultures kept at 37°C for 48 hours, in vitro asexual cycle was maintained, although a small release of mobile zoospores as well as a large amount of immobile and encysted zoospores was observed. However, the latter were viable, evidencing pure P. insidiosum cultures grown on 0.1% yeast agar. Fonseca et al.22 demonstrated that in vitro zoosporogenesis from kunker clinical samples of horses infected by P. insidiosum also occurred within 8 to 48 hours of incubation at 37°C. Nevertheless, zoosporogenesis was not evaluated at different temperatures in these studies. Interestedly, when the 20°C temperature influence was evaluated (group C), there was the formation of zoosporangia and the release of zoospores within the first four hours of incubation. It is suggested that, in nature, a 20°C water temperature contributes to keeping the oomycete biological cycle, unlike what had been previously described by Miller and Campbell.10 In this study, P. insidiosum isolates kept at 5°C (group A) during the first 24 hours of incubation only showed the growth of short hyphae, with no formation of zoosporangia and zoospores. Nonetheless, isolates submitted to 37°C for another 24 hours showed zoosporogenesis with the release of zoospores in the first 4 hours of incubation. On the other hand, when P. insidiosum isolates were incubated at 15°C (group B), zoosporogenesis and a small number of zoospores were observed in two isolates within 24 hours of incubation. Krajaejun et al.15, upon testing the influence of different temperatures on mycelial growth of 13 P. insidiosum isolates on agar, observed that the temperatures of 28°C and 32°C favored the microorganism mycelial growth, while temperatures of 8°C and 42°C either inhibited or prevented its development in a 10-day incubation period. Differently from Krajaejun et al.15, the present study evaluated the influence of temperature on the zoosporogenesis process that originates the infective structures of the oomycete. It was shown that, although a temperature of 37°C favored the anticipation of the asexual cycle development, the temperature of 20°C also favored the formation and the release of zoospores in the first 4 hours of incubation. Conversely, temperatures of 5°C and 15°C did not kill the microorganism but rather delayed its development and the zoosporogenesis process. When the cultures maintained at these temperatures were transferred to 37°C there was zoospore production within a few hours. Although some authors have stated that refrigeration may result in P. insidiosum death3,23, the results in this study are in agreement with those by Grooters et al.24 who, on evaluating the storage of tissues from clinical lesions of infected animals at 4°C for 4 to 5 days, demonstrated the chances of P. insidiosum isolation increased. The influence of temperature on zoospore production by different Pythium species was evaluated by Abdelzaher et al.11 These authors evidenced that some species were able to produce zoospores in abundance at 15°C, 20°C, and 25°C between 4 and 6 hours of incubation. In the present study, seven P. insidiosum isolates produced zoospores at 20°C in the first 4 hours, while two isolates were able to produce these structures within 24 hours of incubation at 15°C. Marcolongo-Pereira et al.6 suggested that environment temperatures below 30°C may favor P. insidiosum zoosporogenesis in wetlands, once several factors such as soil depth, solar irradiance, cloudiness, environment temperature, daytime and wind can influence stagnant water temperature. In addition, water has the ability to retain heat, whether on days with high solar irradiation or on cloudy or rainy days.25 Based on in vitro results obtained, it can be inferred that at stagnant water temperatures of around 15°C and 20°C, the maintenance of the biological cycle of P. insidiosum with the production of infecting zoospores is viable. These findings could explain the appearance of pythiosis lesions in the cooler months of the year in Rio Grande do Sul State/Brazil, where average maximum and minimum temperatures in winter months are about 19°C and 10°C, respectively (http://agromet.cpact.embrapa.br/). Under the conditions in which the study was carried out, temperatures of 5°C and 15°C did not inhibit the microorganism development, and infective zoospores could be produced even at temperatures as low as 15°C. However, temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. Acknowledgments This work was financed by the Postgraduate Program in Parasitology/UFPel and CNPq (National Council for Scientific and Technological Development) (Research grants). The authors would like to thank CNPq and CAPES (Coordination for the Improvement of Higher Education Personnel) for the student scholarships and research grants. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Martins TB, Kommers GD, Trost ME et al.   A comparative study of the histopathology and immunohistochemistry of pythiosis in horses, dogs and cattle. J Comp Path.  2012; 146: 122– 131. Google Scholar CrossRef Search ADS   2. Gaastra W, Lipman LJA, De Cock AWAM et al.   Pythium insidiosum: an overview. Vet Microbiol.  2010; 146: 1– 16. Google Scholar CrossRef Search ADS PubMed  3. Mendoza L, Ajello L, Mcginnis MR. Infections caused by the oomycetous pathogen Pythium insidiosum. J Mycol Med.  1996; 6: 151– 164. 4. Krajaejun T, Sathapatayavongs B, Pracharktam R et al.   Clinical and epidemiological analyses of human pythiosis in Thailand. Clin Infect Dis . 2006; 43: 569– 576. Google Scholar CrossRef Search ADS PubMed  5. Santos CEP, Ubiali DG, Pescador CA, Zanette RA, Santurio JM, Marques LC. Epidemiological survey of equine pythiosis in the Brazilian Pantanal and nearby areas: results of 76 cases. J Equine Vet Sci.  2014; 34: 270—74. 6. Marcolongo-Pereira C, Sallis ESV, Raffi MB et al.   Epidemiology of equine pythiosis in southern of Rio Grande do Sul State, Brazil. Pesq Vet Bras . 2012; 32: 865– 868. [Epidemiologia da pitiose equina na Região Sul do Rio Grande do Sul]. Google Scholar CrossRef Search ADS   7. Meireles MCA, Riet-Correa F, Fischman O, Zambrano AF, Zambrano MS, Ribeiro GA. Cutaneous pythiosis in horses from Brazil. Mycoses . 1993; 36: 139– 142. Google Scholar CrossRef Search ADS PubMed  8. Leal ABM, Leal AT, Santurio JM, Kommers GD, Catto JB. Equine pythiosis in the Brazilian Pantanal region: clinical and pathological findings of typical and atypical cases. Pesq Vet Bras.  2001; 21: 151– 156. [Pitiose equina no pantanal brasileiro: Aspectos clínico-patológico de casos típicos e atípicos]. Google Scholar CrossRef Search ADS   9. Miller RI. Investigations into the biology of three ‘phycomycotic’ agents pathogenic for horses in Australia. Mycopathologia . 1983; 81: 23– 28. Google Scholar CrossRef Search ADS PubMed  10. Miller RI, Campbell RSF. Clinical observations on equine phycomycosis. Aust Vet J.  1982; 58: 221– 226. Google Scholar CrossRef Search ADS PubMed  11. Abdelzaher HMA, Ichitani T, Elnaghy MA. Effect of temperature, hydrogen ion concentration and osmotic potential on zoospore production by three Pythium species isolated from pond water. Mycoscience . 1994; 35: 377– 382. Google Scholar CrossRef Search ADS   12. Mendoza L, Prendas J. A method to obtain rapid zoosporogenesis of Pythium insidiosum. Mycopathologia . 1988; 104: 59– 62. Google Scholar CrossRef Search ADS PubMed  13. Chaiprasert A, Samerpitak K, Wanachiwanawin W et al.   Induction of zoospore formation in Thai isolates of Pythium insidiosum. Mycoses . 1990; 33: 317– 323. Google Scholar PubMed  14. Pereira DIB, Santurio JM, Alves SH, Argenta JS, Cavalheiro AS, Ferreiro L. In vitro zoosporogenesis among oomycetes Pythium insidiosum isolates. Cienc Rural.  2008; 38: 143– 147. [Zoosporogênese in vitro entre isolados do oomiceto Pythium insidiosum]. Google Scholar CrossRef Search ADS   15. Krajaejun T, Chongtrakool P, Angkananukul K, Brandhorst TT. Effect of temperature on growth of the pathogenic oomycete Pythium insidiosum. Southeast Asian J Trop Med Public Health . 2010; 41: 1462– 1466. Google Scholar PubMed  16. Azevedo MI, Pereira DIB, Botton SA et al.   Pythium insidiosum: morphological and molecular identification of Brazilian isolates. Pesq Vet Bras . 2012; 32: 619– 622. Google Scholar CrossRef Search ADS   17. Azevedo MI, Botton SA, Pereira DIB et al.   Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences. Vet Microbiol . 2012; 159: 141– 148. Google Scholar CrossRef Search ADS PubMed  18. Pérez RC, Luis-León JJ, Vivas JL, Mendoza L. Epizootic cutaneous pythiosis in beef calves. Vet Microbiol . 2005; 109: 121– 128. Google Scholar CrossRef Search ADS PubMed  19. Carrera MV, Peixoto RM, Gouveia GV et al.   Pythiosis in sheep from Pernambuco and Bahia States, Brazil. Pesq Vet Bras . 2013; 33: 476– 482 [Pitiose em ovinos nos estados de Pernambuco e Bahia]. Google Scholar CrossRef Search ADS   20. Pereira DIB, Botton SA, Azevedo MI et al.   Canine gastrointestinal pythiosis treatment by combined antifungal and immunotherapy and review of published studies. Mycopathologia.  2013; 176: 309– 315. Google Scholar CrossRef Search ADS PubMed  21. Mendoza L, Hernandez F, Ajello L. Life cycle of the human and animal oomycete pathogen Pythium insidiosum. J Clin Microbiol . 1993; 31: 2967– 2973. Google Scholar PubMed  22. Fonseca AO, Botton SA, Nogueira CE et al.   In vitro reproduction of the life cycle of Pythium insidiosum from kunkers equine and their role in the epidemiology of pythiosis. Mycopathologia.  2014; 177: 123– 127. Google Scholar CrossRef Search ADS PubMed  23. Newton JC, Ross PS. Equine pythiosis: an overview of immunotherapy. Compend Contin Educ Vet.  1993; 15: 491– 493. 24. Grooters AM, Whittington A, Lopez MK, Boroughs MN, Roy AF. Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues. J Vet Diagn Invest.  2002; 14: 288– 294. Google Scholar CrossRef Search ADS PubMed  25. Angelloci LR, Vila Nova NA. Water temperature variations during one year in a small lake at Piracicaba - SP, Brazil. Sci Agric . 1995; 52: 431– 438. [Variações da temperatura da água de um pequeno lago artificial ao longo de um ano em Piracicaba, SP]. Google Scholar CrossRef Search ADS   © The Author 2017. 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Abstract

Abstract This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism. oomycete, pythiosis, zoospores, hyphae, zoosporangia Introduction The aquatic oomycete Pythium insidiosum is the etiological agent of pythiosis, a disease which occurs in its chronic fibrovascular form in horses1 and, in its pyogranulomatous manifestation, in other mammalian species that inhabit tropical and subtropical areas.1,2 Pythiosis has been reported to affect man and animals, especially horses, in several countries.2,3 In humans, the disease has been predominantly reported in Thailand4 and is thought to be endemic in horses, particularly in the Brazilian Pantanal area3,5and in the State of Rio Grande do Sul, southern Brazil.6 In these areas, the highest number of cases occurs in the hottest months of the year (spring and summer);5–8 however, equine pythiosis has also been reported in seasons with lower temperatures (fall and winter).6 P. insidiosum performs its biological cycle in stagnant water in natural environments. In these environments, the microorganism reproduces asexually, yielding infective zoospores. Susceptible hosts are infected by getting in contact with contaminated environments.3,9 Several factors can influence P. insidiosum cycle in the environment. According to Miller and Campbell10, environment temperatures between 30°C and 40°C and water accumulation in marshes and ponds are essential factors that influence zoospore production, as well as are conditioning factors for the occurrence of the disease. Abdelzaher et al.11, upon evaluating the influence of different temperatures on the zoosporogenesis of different Pythium species, found that some of the evaluated species were able to produce abundant zoospores at temperatures of 15°C, 20°C, and 25°C. Nevertheless, P. insidiosum in vitro zoosporogenesis has only been evaluated at 37°C.12,13,14 In vitro studies have shown that temperature can influence P. insidiosum development. Krajaejun et al.15 demonstrated that the oomycete mycelial growth was inhibited at 8°C and that temperatures of 28°C and 32°C favored its development. However, these authors evaluated this influence on mycelial growth rather than the on the zoosporogenesis process that originates infective zoospores. This study aimed to evaluate the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. Methods Pythium insidiosum isolates The present study analyzed 26 P. insidiosum isolates from horses. The identification of each isolate was performed by macro- and micro-morphological characteristics and confirmed by polymerase chain reaction (PCR) and DNA sequencing, as described by Azevedo et al.16,17 Zoosporogenesis induction at different temperatures A 4-mm disk of each P. insidisoum isolate previously cultured on CMA (corn meal agar) was transferred to a Petri dish containing 0.1% yeast agar. Sterilized grass fragments (Paspalum notatum) were then placed around the disk. The cultures were incubated at 37°C for 72 hours. Subsequently, grass fragments of each isolate were transferred to Falcon tubes containing 10 mL induction medium containing solution 1 (K2HPO4 [1.0 mol/l], KH2PO4 [1.0 mol/l], (NH4)2HPO4 [3.66 mol/l], 500 ml distilled water) and solution 2 (MgCl2.6H2O [0.5 mol/l]), CaCl2.2H2O [0.5 mol/l], 250 ml distilled water). The induction medium final composition was 0.5 ml solution 1, 0.1 ml solution 2 and 1000 ml sterile distilled water.12 The experiments were divided into two stages. In the first stage, the tubes were incubated at 5°C (group A), 15°C (group B), 20°C (group C), and 37°C (control group). To evaluate P. insidiosum zoosporogenesis at different temperatures, grass fragments were removed from the tubes, placed between the blade and coverslip of microscope slides and evaluated under optical microscopy (10 to 20 × objective lens) at the first 4, 8, and 24 hours of incubation. In the second stage, groups A, B, C, and Control isolates were incubated at 37°C and retested as described in the first stage. The following morphological parameters for isolates under zoosporogenesis were evaluated for each period: hyphae development (short, developing, long and mycelia), presence or absence of vesicles, and zoosporangia and production of zoospores (absence or presence of mobile and immobile zoospores, and their Neubauer chamber count) as described by Pereira et al.14 and Azevedo et al.16 At the end of the second stage, the viability of immobile zoospores of P. insidiosum isolates was checked by seeding 100 μl induction medium on 0.1% yeast agar at 37°C for 72 hours. Results Table 1 and Table 2 show the results of the in vitro zoosporogenesis of 26 P. insidiosum isolates evaluated at different temperatures and different periods. Table 1. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours), during the first 24 h of incubation (1st stage of experiment).     Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)      Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large Table 1. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours), during the first 24 h of incubation (1st stage of experiment).     Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)      Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A (5°C)  4 h  92.31% (24/26)-SH  –  –  –      7.69% (02/26)-DH          8 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH          24 h  80.76%(21/26)-SH  –  –  –      19.22%(05/26)-DH        B (15°C)  4 h  38.46%(10/26)-SH  7.69% (02/26)  7.69% (02/26)  –      52.69%(15/26)-DH            8.85%(01/26)-LH          8 h  34.61%(09/26)-SH  76.92% (20/26)  76.92% (20/26)  –      57.70%(15/26)-DH            7.69%(02/26)-LH          24 h  19.23% (05/26)-DH  7.69% (02/26)  7.69% (02/26)  7.69%(02/26)      80.76% (21/26)-LH        C (20°C)  4 h  7.69% (02/26)-DH  7.69% (02/26)  42.30%(11/26)  26.92% (07/26)      92.31% (24/26)-LH          8 h  11.54%(03/26)-DH  84.61% (22/26)  42.30% (11/26)  30.76%(08/26)      88.46%(23/26)-LH          24 h  19.23%(05/26)-DH  61.53% (16/26)  30.76%(08/26)  65.38%(17/26)      80.76%(21/26)-LH        Control  4 h  100% (26/26) - LH  96.15% (25/26)  96.15% (25/26)  96.15% (25/26)  (37°C)    100% (26/26) – LH          8 h  100% (26/26) – LH  7.69% (02/26)  –  100% (26/26)    24 h  100% (26/26) – LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large Table 2. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated during the2ststage of experiment (all isolates from groups A, B, C and Control were incubated at 37°C).   Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)    Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large Table 2. In vitro zoosporogenesis of P. insidiosum isolates (n = 26) evaluated during the2ststage of experiment (all isolates from groups A, B, C and Control were incubated at 37°C).   Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)    Parameters evaluated  Group  Period  Hypha1 % ( )*  Vesicle  Zoosporangium  Zoospore  A  4 h  15.38%(04/26)-SH  23.07% (06/26)  15.38% (04/26)  15.38% (04/26)      69.23%(18/26)-DH          8 h  15.38% (04/26)-LH            11.53%(03/26)-DH  15.38% (04/26)  15.38% (04/26)  23.07% (06/26)    24 h  88.45% (23/26)-LH            7.69% (02/26)-DH  15.38% (04/26)  15.38% (04/26)  50% (13/26)      92.30% (24/26)-LH        B  4 h  19.23% (05/26)-DH  61.53% (16/26)  30.76% (08/26)  15.38% (04/26)      80.76% (21/26)-LH          8 h  15.38% (04/26)-SH  61.53%(16/26)  38.46% (10/26)  52.69% (15/26)      84.61% (22/26)-LH          24 h  15.38% (04/26)-SH  23.07% (06/26)  38.46% (10/26)  65.38% (17/26)      84.61% (22/26)-LH        C  4 h  8.85% (01/26)-DH  15.38% (04/26)  11.53% (03/26)  100% (26/26)      96.15% (25/26)-LH          8 h  3.84% (01/26)-DH  100% (26/26)  7.69% (02/26)  100% (26/26)      96.15% (25/26)-LH          24 h  3.84% (01/26)-DH  7.69% (02/26)  7.69% (02/26)  100% (26/26)       96.15% (25/26)-LH        Control  4 h  100% (26/26)-LH  15.38% (04/26)  15.38% (04/26)  100% (26/26)    8 h  100% (26/26) - LH  –  –  100% (26/26)    24 h  100 % (26/26) - LH  –  –  100% (26/26)  1P. insidiosum hyphae at different growth stages: DH, developing hypha; LH, long and mycelial hypha; SH, short hypha. ()*: no. of positive isolates / total number of isolates of P. insidiosum. -: Not observed. View Large At 5°C (group A), the presence of short hyphae was observed predominantly on the edges of grass fragments at all periods evaluated in the first stage of the study (Fig. 1A). There were no vesicles, zoosporangia and zoospores at this temperature. Figure 1. View largeDownload slide In vitro zoosporogenesis of Pythium insidiosum at different temperatures and periods evaluated. A (Group A, 5°C/24 h): Short hyphae (→). B (Group B, 15°C/8 h): Hyphae in different stage of growing: short hyphae (→), in developing (*) and long and mycelial hyphae (→). C (Group C, 20°C/4 h) - 1: Presence of vesicle (→) and zoosporangium (→) and 2 – release of zoospores. D (Group C, 20°C/24 h): Zoospores with the emission of germ tubes (→). E (Group Control, 37°C/48 h): Numerous immobile zoospores (→). A, B, C, D and E: 20 × Objective. F: Growth of P. insidiosum from Control group at 37°C/48 h. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide In vitro zoosporogenesis of Pythium insidiosum at different temperatures and periods evaluated. A (Group A, 5°C/24 h): Short hyphae (→). B (Group B, 15°C/8 h): Hyphae in different stage of growing: short hyphae (→), in developing (*) and long and mycelial hyphae (→). C (Group C, 20°C/4 h) - 1: Presence of vesicle (→) and zoosporangium (→) and 2 – release of zoospores. D (Group C, 20°C/24 h): Zoospores with the emission of germ tubes (→). E (Group Control, 37°C/48 h): Numerous immobile zoospores (→). A, B, C, D and E: 20 × Objective. F: Growth of P. insidiosum from Control group at 37°C/48 h. This Figure is reproduced in color in the online version of Medical Mycology. At 15°C (group B), there was hyphal development (Fig. 1B). In two P. insidiosum isolates (7.69%); long hyphae, vesicles, and zoosporangia were observed, as well as the production of zoospores in small quantities within 24 hours of incubation (Fig. 2A). Figure 2. View largeDownload slide Mean count of zoospores of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours). A. First stage of experiment: P. insidiosum isolates were incubated at 5°C (group A), 15°C (group B), 20°C (group C), and 37°C (control group) and evaluated at 4, 8, and 24 hours of incubation. B. Second stage of experiment: isolates of P. insidiosum from groups A, B, C, and control were incubated at 37°C and retested at 4, 8, and 24 hours. Figure 2. View largeDownload slide Mean count of zoospores of P. insidiosum isolates (n = 26) evaluated at different temperatures (°C) and periods (hours). A. First stage of experiment: P. insidiosum isolates were incubated at 5°C (group A), 15°C (group B), 20°C (group C), and 37°C (control group) and evaluated at 4, 8, and 24 hours of incubation. B. Second stage of experiment: isolates of P. insidiosum from groups A, B, C, and control were incubated at 37°C and retested at 4, 8, and 24 hours. It was evidenced that 92.31% (24/26) of the isolates submitted to 20°C (group C) showed mycelial growth on the edges of grass fragments within the first 4 hours of evaluation. The presence of vesicles, zoosporangia and zoospores was observed (Fig. 1C). As for zoospore count, 26.92% (7/26) of the evaluated isolates produced an average of 9.6 × 103 mobile zoospores/mL within this period (Fig. 2A). Additionally, at the other evaluated periods (8 and 24 hours), mobile and immobile zoospores with the development of germ tubes were detected (Fig. 1D). In the second stage of the study, group A and B isolates showed developing as well as long hyphae with the presence of vesicles, zoosporangia and zoospores after a 4-hour incubation period, with a mean zoospore count of 0.86 × 103 and 2.21 × 103 zoospores/ml, respectively (Fig. 2B). These characteristics were kept at the 8- and 24-hour evaluations; however, there was an increase in zoospore count (Fig. 2B). Control group isolates showed evident zoosporogenesis in the first 4 hours of evaluation, with the presence of mobile and immobile zoospores, and a mean count of 14.5 × 103 zoospores/mL (Fig. 2A). At the end of the second stage, these isolates included many immobile zoospores (Fig. 1E) which were nevertheless viable when their growth was evaluated in culture medium (Fig. 1F). Discussion In P. insidiosum biological cycle in aquatic environments, zoosporogenesis originates zoospores, which are the infective form of this oomycete for animals and humans that inhabit marshy areas of tropical and subtropical climates.2,3,9 In nature, temperature is one of the factors that directly influences P. insidiosum zoosporogenesis process. According to Miller and Campbell10, the development of zoospores is favored at environment temperatures between 30°C and 40°C. Pythiosis cases in animals are mainly reported during the hottest months of the year.3,5,6–8,10,18–20 However, cases of equine pythiosis have also been observed in the winter months in subtropical and temperate climates, such as in southern Brazil.6 The present study was carried out to verify the in vitro influence of temperature on the zoosporogenesis of P. insidiosum isolates from infected horses in the State of Rio Grande do Sul, Brazil, an important endemic region for equine pythiosis where cases of the disease have also been reported during the cold months of the year. In the present study, 96.15% of P. insidiosum isolates that were incubated at 37°C (Control group) showed mycelial growth, vesicles, zoosporangia and zoospores in the first 4 hours of evaluation, which is an expected result when the oomycete is incubated at this temperature. Previous studies12–14 evaluating the in vitro zoosporogenesis process in P. insidiosum isolates have reported that temperatures of 37°C favor the formation of zoosporangia and zoospores. Chaiprasert et al.13 and Mendoza et al.21 observed the presence of encysted zoospores after one hour of incubation. Mendoza and Prendas12and Pereira et al.14 showed that the asexual cycle began within 3 hours, with a zoospore production peak between 6 and 8 hours. In this study, the results at 37°C are in agreement with those reported by these authors. In addition, it was demonstrated that in cultures kept at 37°C for 48 hours, in vitro asexual cycle was maintained, although a small release of mobile zoospores as well as a large amount of immobile and encysted zoospores was observed. However, the latter were viable, evidencing pure P. insidiosum cultures grown on 0.1% yeast agar. Fonseca et al.22 demonstrated that in vitro zoosporogenesis from kunker clinical samples of horses infected by P. insidiosum also occurred within 8 to 48 hours of incubation at 37°C. Nevertheless, zoosporogenesis was not evaluated at different temperatures in these studies. Interestedly, when the 20°C temperature influence was evaluated (group C), there was the formation of zoosporangia and the release of zoospores within the first four hours of incubation. It is suggested that, in nature, a 20°C water temperature contributes to keeping the oomycete biological cycle, unlike what had been previously described by Miller and Campbell.10 In this study, P. insidiosum isolates kept at 5°C (group A) during the first 24 hours of incubation only showed the growth of short hyphae, with no formation of zoosporangia and zoospores. Nonetheless, isolates submitted to 37°C for another 24 hours showed zoosporogenesis with the release of zoospores in the first 4 hours of incubation. On the other hand, when P. insidiosum isolates were incubated at 15°C (group B), zoosporogenesis and a small number of zoospores were observed in two isolates within 24 hours of incubation. Krajaejun et al.15, upon testing the influence of different temperatures on mycelial growth of 13 P. insidiosum isolates on agar, observed that the temperatures of 28°C and 32°C favored the microorganism mycelial growth, while temperatures of 8°C and 42°C either inhibited or prevented its development in a 10-day incubation period. Differently from Krajaejun et al.15, the present study evaluated the influence of temperature on the zoosporogenesis process that originates the infective structures of the oomycete. It was shown that, although a temperature of 37°C favored the anticipation of the asexual cycle development, the temperature of 20°C also favored the formation and the release of zoospores in the first 4 hours of incubation. Conversely, temperatures of 5°C and 15°C did not kill the microorganism but rather delayed its development and the zoosporogenesis process. When the cultures maintained at these temperatures were transferred to 37°C there was zoospore production within a few hours. Although some authors have stated that refrigeration may result in P. insidiosum death3,23, the results in this study are in agreement with those by Grooters et al.24 who, on evaluating the storage of tissues from clinical lesions of infected animals at 4°C for 4 to 5 days, demonstrated the chances of P. insidiosum isolation increased. The influence of temperature on zoospore production by different Pythium species was evaluated by Abdelzaher et al.11 These authors evidenced that some species were able to produce zoospores in abundance at 15°C, 20°C, and 25°C between 4 and 6 hours of incubation. In the present study, seven P. insidiosum isolates produced zoospores at 20°C in the first 4 hours, while two isolates were able to produce these structures within 24 hours of incubation at 15°C. Marcolongo-Pereira et al.6 suggested that environment temperatures below 30°C may favor P. insidiosum zoosporogenesis in wetlands, once several factors such as soil depth, solar irradiance, cloudiness, environment temperature, daytime and wind can influence stagnant water temperature. In addition, water has the ability to retain heat, whether on days with high solar irradiation or on cloudy or rainy days.25 Based on in vitro results obtained, it can be inferred that at stagnant water temperatures of around 15°C and 20°C, the maintenance of the biological cycle of P. insidiosum with the production of infecting zoospores is viable. These findings could explain the appearance of pythiosis lesions in the cooler months of the year in Rio Grande do Sul State/Brazil, where average maximum and minimum temperatures in winter months are about 19°C and 10°C, respectively (http://agromet.cpact.embrapa.br/). Under the conditions in which the study was carried out, temperatures of 5°C and 15°C did not inhibit the microorganism development, and infective zoospores could be produced even at temperatures as low as 15°C. However, temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. Acknowledgments This work was financed by the Postgraduate Program in Parasitology/UFPel and CNPq (National Council for Scientific and Technological Development) (Research grants). The authors would like to thank CNPq and CAPES (Coordination for the Improvement of Higher Education Personnel) for the student scholarships and research grants. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Martins TB, Kommers GD, Trost ME et al.   A comparative study of the histopathology and immunohistochemistry of pythiosis in horses, dogs and cattle. J Comp Path.  2012; 146: 122– 131. Google Scholar CrossRef Search ADS   2. Gaastra W, Lipman LJA, De Cock AWAM et al.   Pythium insidiosum: an overview. Vet Microbiol.  2010; 146: 1– 16. Google Scholar CrossRef Search ADS PubMed  3. Mendoza L, Ajello L, Mcginnis MR. Infections caused by the oomycetous pathogen Pythium insidiosum. J Mycol Med.  1996; 6: 151– 164. 4. Krajaejun T, Sathapatayavongs B, Pracharktam R et al.   Clinical and epidemiological analyses of human pythiosis in Thailand. Clin Infect Dis . 2006; 43: 569– 576. Google Scholar CrossRef Search ADS PubMed  5. Santos CEP, Ubiali DG, Pescador CA, Zanette RA, Santurio JM, Marques LC. 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Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Dec 8, 2017

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