Isolation of Meyerozyma guilliermondii
The yeast M. guilliermondii was isolated from oocyst suspensions recovered from ATD-1Coccivac B (MSD Animal Health), a commercial vaccine of live oocysts (E. acervulina, E.mivati, E.maxima and E. tenella). This vaccine is produced by Merck, a company that is intended to use ethically resources to fulfill customers expectative, they are adhere to: the International Federation for Animal Health Statement of Principles; the US-bases Animal Health Institute Advertising Guidelines; The UK-based National Office of Animal Health Code of Practice for the Promotion of Animal Medicine.
A loopful of a suspension was used to inoculate Petri dishes (90 × 15 mm, Ruisanchez, Mexico) of YPDA medium (Yeast extract, Peptone, Dextrose, Agar) [20 g/L yeast extract (BD, USA), 5 g/L peptone (BD Difco, USA), 40 g/L dextrose (Fermont, Mexico), 10 g/L agar (BD, USA)]; the plates were incubated (LabTech 3016A, USA) at 28 °C for 16 h. After the incubation, yeast colonies were observed, picked and grown in liquid YPD (Yeast extract, Peptone, Dextrose) [20 g/L yeast extract (BD, USA), 5 g/L peptone (BD Difco, USA), 40 g/L dextrose (Fermont, Mexico)] under the same conditions for further identification. Solid YPD was used only to grow and preserve yeast for a short period of time at 4 °C on a Petri dish. A loopful from this plate was used to cultivate M. guilliermondii in liquid YPD to perform the bioassays.
Obtaining of oocysts used in bioassays
Eimeria tenella oocyst suspensions were obtained from infected chickens and maintained in potassium dichromate at 4 °C. E. tenella oocysts were kindly provided by SENASICA (Servicio Nacional de Sanidad Inocuidad y Calidad Agroalimentaria,). All protocols used in this study were approved by the animal ethics committee of National Commission on Bioethics (http://www.conbioetica-mexico.salud.gob.mx/) consistent with the recommendations of the Mexican Official Standard NOM-062-ZOO-1999, Technical Specifications for the production, care and use of laboratory animals.
Eimeria spp. oocysts were obtained from commercial vaccine Autocox (Eimeria, Mexico) that contains oocysts from E. acervulina, E. maxima, E. tenella and E. praecox. Eimeria S.A de C.V., is certified by Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA, México) since it fulfills the requirement of good practices in manufacture of the vaccines contained in NOM-022-ZOO-1995 y NOM-026-ZOO-1994.
Amplification by PCR of 5.8S-ITS rDNA region of M. guilliermondii
Yeast cells were grown on YPDA medium at 28 °C for 48 h. Cells were collected with the tip of a toothpick and directly used for polymerase chain reaction (PCR).
To amplify the 5.8S-ITS rDNA region and the D1/D2 domain, a PCR reaction was performed as reported by . Thermal cycling was conducted with an initial step at 95 °C for 5 min, 40 cycles of 94 °C for 40 s, 55 °C for 40 s and 72 °C for 30 s and a final extension of 10 min at 72 °C.
Amplification of the 5.8S-ITS rDNA region was achieved with the primers reported by : its1 (5′-TCCGTAGGTGAACCTGCGG-3′) and its4 (5′-TCCTCCGCTTATTGATATGC-3′). Amplification of the D1/D2 domain of the 26S rRNA gene was achieved with the primers NL1 (5′GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTG TTTCAAGACGG-3′) .
After PCR, the 620-bp amplification products were excised from a 1 % agarose gel and purified using a High Pure PCR Product Purification Kit (Roche, USA) according to the manufacturer’s instructions. The purified fragments were then sequenced (Unidad de Síntesis y Secuenciación de AND, Instituto de Biotecnología-UNAM), and a BLASTn analysis was performed (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In order to detect nucleotide mutations and to confirm the integrity of the sequence, we have sequenced five independent clones.
In silico restriction fragment length polymorphism (RFLP) of 5.8S-ITS rDNA sequences
Once the purified fragment 0f 620 bp were sequenced, an in silico enzyme digestion with CfoI, HaeIII and HinfI was performed with EnzymeX software and Restriction Mapper V3 (http://www.restrictionmapper.org/).
Amplification by PCR of 18S rRNA
To amplify a 1565-bp product corresponding to sequence of 18S rRNA of M . guilliermondii (GenBank accession number KJ126853), we designed the primers Mgfwd: 5′AAACTGCGAATGGCTCATTAAATCAGTTATCG-3′ and Mgrev: 5′-GCGACGGGCGGTGTGTACAAAGG -3′. Thermal cycling was conducted with an initial step at 95 °C for 5 min, 35 cycles of 94 °C for 40 s, 61 °C for 1 min and 72 °C for 1.5 min and a final extension of 10 min at 72 °C.
A phylogenetic reconstruction was performed with the 1565 bp sequence of the 18S rDNA from M. guilliermondii 01 (GenBank accession number KJ126853) and 20 sequences retrieved in a BLASTn analysis.
The phylogeny was constructed with the software tool MEGA (Molecular Evolutionary Genetics Analysis) 6.0 , with the method Neighbor-joining and the predicted model of Maximun Compositum Likelihood. The robustness of the trees was statistically evaluated by bootstrap analysis with 1000 iterations.
Growth of M. guilliermondii 01
A loopful of yeast cells was grown in a pre-inoculum of 20 ml YPD for 14 h at 30 °C and 200 rpm (LabTech 3016A, USA). Next, 100 ml of YPD was inoculated with a starting optical density at 600 nm (OD600) (Thermo Biomate 3 IV-Vis Spectrometer, USA) of 0.1 and grown until the OD600 reached 1.0 or 1.8 (approximately 8 h) under the same temperature and shaking conditions. An OD600 of 1.0 corresponds to 2.72 × 106 CFU/ml, and an OD600 of 1.4 corresponds to 6.72 × 106 CFU/ml.
The effect of different CFU/ml values of M. guilliermondii on oocyst integrity was assessed by bioassays.
Recovery of culture and supernatant of M. guilliermondii
A culture of M. guilliermondii was obtained as described above, and 200 μl of the culture was used in each bioassay. For bioassays using the supernatant, supernatants were obtained by centrifugation at 4 °C for 15 min at 3,500 g (17303-15, Cole Parmer, USA), and 200 μl was used for anticoccidial activity bioassays. 200 μl of non-inoculated liquid YPD was cultivated in the same mentioned conditions and used in bioassays reactions. The supernatant of YPD medium was recovered by centrifugation and also used as negative control.
Ethyl acetate extraction of supernatant
M. guilliermondii culture supernatant (100 ml) was extracted three times with ethyl acetate (High Purity, Mexico). Briefly, the culture was mixed with one volume of ethyl acetate and strongly shaken by hand in an extraction funnel to homogenize the mixture. The funnel was then placed in a universal mount until two phases were observed (approximately 3 to 5 min). The upper phase was recovered and further extracted twice.
The final extraction volume (approximately 300 ml) was evaporated in a rotary evaporator (R-205, Buchi, Switzerland) at 80 rpm and 41 °C. The extract was then recovered in amber vials, dried for 4 days in an extractor hood, weighed and dissolved in approximately 20 to 30 μl of dimethyl sulfoxide (DMSO, J.T. Baker, USA). Two milligrams of the supernatant extract was used in anticoccidial activity bioassays. Non-inoculated YPD was also extracted with ethyl acetate and concentrated by evaporation in the same conditions. 2 mg of the product of this extraction resuspended in DMSO were used as negative control in bioassay reactions. In order to assess the nature of the compound extracted with ethyl acetate we performed incubation with trypsin (aqui no se las condiciones de tiempo y cantidad de enzima en que lo hizo Mayra).
Intracellular proteins extraction
Yeast cell were grown in the conditions mentioned above. After this, the culture was centrifuged for 15 min at 9,000 g and 4 °C and the supernatant was discarded. The pellet was recovered and resuspended in 20 mM phosphate buffer (pH 7.0). The cells were lysed by sonication for 10 min and 32 % of amplitude (Ultrasonic processor, Sonic & Materials) and then centrifuged for 15 min at 9,000 g and 4 °C.
The recovered supernatant, that contained the intracellular crude extract, was filtered through a PVDF membrane (Millex GV filter unit 0.22-μm, Millipore) and then quantified by the Bradford assay  using a standard curve of bovine serum albumin (BSA).
Proteins precipitation with ammonium sulfate
The concentrated intracellular proteins were precipitated with 20, 40, 60 and 80 % of saturated solution of ammonium sulfate. This mix was stirred on ice for 2 h and then centrifuged at 15,000 g for 15 min at 4 °C. The supernatant was discarded and the pellet was dissolved in 20 mM phosphate buffer (pH 7.0) and dialyzed overnight (12 kD dialysis tubing membrane, Sigma-Aldrich). Finally, the dialyzed fractionated proteins were concentrated by centrifugation (Amicon Ultra Concentrator with 10 kD molecular mass cut-off, Millipore) and quantified by the Bradford assay  using a standard curve of bovine serum albumin (BSA). Each fraction was used in anticoccidial activity bioassays.
Proteins denaturalizing treatment
Five hundred micrograms of proteins precipitated with a 40 % saturated solution of ammonium acetate were treated with a 1/10 of trypsin (1 mg/ml, Sigma-Aldrich) diluted in 12.5 mM Tris-HCl (pH 9.0) for 12 h at 37 °C. After this, proteins were incubated for 10 min at room temperature and then 10 min on ice. To stop the reaction, 100 mM of PMSF (phenylmethanesulfonylfluoride) protease inhibitor (Sigma-Aldrich) was added in a 2 mM final concentration; the reaction was mixed by inversion and kept on ice until use.
For samples treated with trichloroacetic acid (TCA), 1,500 μg of 40 % fractionated proteins were precipitated with 1 volume of cold TCA 20 % and mixed by inversion before incubate 20 min on ice. The mix was agitated by inversion several times and then centrifuged at 15,000 g for 15 min. The pellet was washed three times with 500 μl of cold acetone with intervals of centrifugation at 15,000 g for 10 min. Finally, once the acetone was evaporated; the pellet was resuspended in 10 mM Tris-HCl to neutralize the pH and 20 mM phosphate buffer (pH 7.0) was added to a final volume of 1 ml.
Separately, 1,500 μg of the 40 % fraction recovered after ammonium sulfate precipitation were heated for 15 min at 95 °C and then kept on ice for 15 min.
Both samples, treated with TCA and heat, were quantified and assayed for anticoccidial activity.
In vitro anticoccidial activity bioassays
Bioassays were performed using E. tenella oocyst suspensions (SENASICA) and Eimeria spp. commercial oocyst suspensions (ATD-1Coccivac B). Both oocysts suspensions consisted of a heterogeneous mixture of unsporulated and sporulated oocyst, with a predominance of sporulated (infective).
Oocysts were washed and quantified in a Neubauer chamber (Improved Bright Line, Loptik Labor, Germany) before bioassays were performed. A 1 ml sample of oocysts was washed three times with 1 ml of sterile injectable water (Pisa, Mexico) until the medium was clarified; centrifugation at 850 g for 1 min (Thermo Electron IEC Micromax Microcentrifuge, USA) was performed for every wash.
Under sterile conditions, bioassay reactions were prepared in 1.5 ml Eppendorf tubes containing 100,000 E. tenella oocysts along with 200 μl of M. guilliermondii culture, 200 μl of supernatant, 2 mg of ethyl acetate supernatant extract, 300 μg of intracellular proteins, 300 μg of 40 % fraction precipitated with ammonium sulfate or 300 μg of 40 % fraction treated with trypsin.
For the bioassays performed with culture and supernatant the final volume was 1 ml adjusted with injectable water. Bioassay reactions with ethyl acetate supernatant extracts were prepared in 1.5-ml tubes with 100,000 oocysts and 2 mg of extract dissolved in DMSO (final concentration 2 %, 20 μl), and the reactions were brought to 1 ml with injectable water as is shown in Additional file 1. On the contrary, all bioassays performed with intracellular proteins and 40 % fractions (with or without trypsin) were adjusted to 1 ml with 20 mM phosphate buffer (pH 7.0). Bioassays reactions were incubated at 30 °C and 200 rpm (LabTech 3016A, USA) and then observed microscopically after 24 and 48 h of incubation. Oocysts from the reactions were counted in a Neubauer chamber at 0, 24 and 48 h. Oocyst integrity was assessed microscopically (Leica DM750), an oocyst wall without damage was considered as a viable and infective oocyst, whereas an oocyst wall with the slightest damage was considered as non-viable and therefore non-infective oocyst.
The amount, 2 mg of ethyl acetate extract allows observing clearly the damage of the extract on oocysts in a determined period of time. Higher amounts of compound causes that damage in oocysts is not accurately observable; lower amounts extends the time of observation and damage is barely noticeable in a determined period of time.
Depending on the case, control reactions were prepared with only 100,000 oocysts, 200 μl of YPD medium, or supernatant of YPD medium and injectable water (up to 1 ml). As regards to control reaction in bioassays containing ethyl acetate extract, it contained, 100,000 oocysts, ethyl acetate extract of YPD medium resuspended in DMSO and injectable water (up to 1 ml). Control reaction of intracellular crude extract and 40 % ammonium sulfate fractions contained 100,000 oocysts and phosphate buffer 20 mM pH 7.2 (up to 1 ml). Control reactions were incubated under the same temperature and shaking conditions.
All experiments were standardized with respect to controls, so the standard deviation is not plotted. Independent triplicates were performed for each bioassay.
All statistical analyses were performed with Minitab® 15 Statistical Software. A one-way analysis of variance was used to analyze the differences in mortality among the different bioassays. To determine significant differences among the means, a Tukey test was performed. The significance threshold was set at P < 0.05.