**2.3 Parasitological analysis**

Aliquots of 10 µL of concentrated material were employed to prepare smears in two series of two slides each using the modified Ziehl-Neelsen-stain technique and the Kinyoun hot staining method, fixed in alcohol 70%, and processed for specific detection of Coccidea (*Cryptosporidium* sp., *Isospora belli*, and *Cyclospora caytanensis*).In order to detect enteral Microsporidia, the modified hot-chromotrope technique was used (Kokoskin et al., 1994). All the slides were analyzed in duplicate using a common optical microscope with a 100x oil immersion objective.

### **2.4 DNA extraction and amplification**

The modified method of Boom et al., (1990) was used to extract the genetic material, based on cationic exchange resin processes, simultaneously with the phenol/chloroform method of Sambrook & Russel (2001).

The detection of DNA was performed using Nested-PCR, a variation of the polymerase chain reaction (PCR). The literature was searched to find primers flanking site-specific regions of these opportunistic protozoan genomes (Table 1). The Nested-PCR method was applied only to the positive and/or doubtful samples detected by parasitological methods.

Three primer pairs were used: XIAF/XIAR (*Cryptosporidium* sp. and *C. parvum*), flanking a region of approximately 1325 bp; AWA995f/AWA1206R *(Cryptosporidium* sp.*),* amplifying a region of approximately 211 bp; LAX469F/LAX869R (*C. parvum),* amplifying a chromosomal region of approximately 451 pb.

A conventional PCR was carried out using primers XIAF/XIAR and two aliquots were taken from the resulting product, one for detection of protozoan genera via Nested-PCR, using primers AWA995f/AWA1206R, (Awad-el-Kariem, 1994) and the other for the detection of *C. parvum/C. hominis* using primers LAX469F/LAX869R.

PCR using primers XIAF/XIAR and 28 μL extracted DNA was performed in a final volume of 50 μL with the following reagents: 5.0 μL buffer 10X, 2.0 mM Mg, 200 μM dNTP (dATP, dCTP, dTTP, and dGTP), 0.5 μM of each primer, and 1.25 U Taq DNA polymerase. The reaction conditions were an initial denaturation step for 4 min followed by another denaturation step of 35 cycles of 94°C for 1 min, annealing at 55°C for 45 s, extension at 72°C for 1 min, and final extension at 72°C for 7 min (Xiao, et al., 1999).


Table 1. Primers selected to be used in confirmation/specification of protozoa detected by parasitological methods

PCR using primers AWA995f/AWA1206R and 14 μL DNA amplified by primers XIAF/XIAR was performed in a final volume of 25 μL with the following reagents: 2.5 μL buffer 10X, 1.5 mM Mg, 200 μM dNTP (dATP, dCTP, dTTP, and dGTP), 0.5 μM of each primer, and 1.25 U Taq DNA polymerase. The reaction conditions were an initial denaturation step for 7 min followed by another denaturation step of 40 cycles of 94°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 3 min, and final extension at 72°C for 7 min.

PCR using primers LAX469F/LAX869R Laxer, (1991) and 14 μL DNA amplified by primers XIAF/XIAR was performed in a final volume of 25 μL with the following reagents: 2.5 μL buffer 10X, 2.0 mM Mg, 200 μM dNTP (dATP, dCTP, dTTP, and dGTP), 0.5 μM of each primer, and 1.25 U Taq DNA polymerase. The reaction conditions were an initial denaturation step for 7 min followed by another denaturation step of 40 cycles of 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 7 min.

The PCR products were separated by electrophoresis on 8% acrylamide gels stained with silver nitrate and on 1.5% agarose gels stained with ethidium bromide. Samples presenting 211-bp and 451-bp bands were considered positive.

#### **2.5 Direct immunofluorescence assay kit**

One aliquot of each sample concentrate was tested employing the MERIFLUOR® direct immunofluorescence assay kit using homologous monoclonal antibodies for the detection of *Cryptosporidium* sp. and *Giardia* sp. Each sample was analyzed in duplicate; however, due to a shortage of reagents, this technique was applied to 50% (36/72) of the samples taken at random and the positive samples detected by parasitological methods.

#### **2.6 Statistical analyses**

The results obtained in this study were digitalized in spreadsheets using the software Microsoft Office Excel 2007. Statistical analyses were performed using the chi-squared test and the logistic regression analysis. Statistical significance level was set at p < 0.05 using the Statistical Package for the Social Sciences (SPSS) version 10.0.
