Risk factors with the occurrence of parasites and coliform in vegetables from an agroindustry

Submetido em 16/1 1/2015 e apr ovado em 07/1 1/2017. 1 Instituto Federal de Educação, Ciência e Tecnologia de São Paulo, São Roque, São Paulo, Brasil. chicosoto34@gmail.com; jessica.vilela5@gmail.com; laine.tecnoambiental@gmail.com 2 Universidade Federal de São Carlos, Sorocaba, São Paulo, Brasil. iolanda.duarte@gmail.com 3 Universidade Federal de Campina Grande, Campina Grande, Paraíba, Brasil. sandrogazzinelli@hotmail.com *Autor para correspondência: chicosoto34@gmail.com Short communication Risk factors with the occurrence of parasites and coliform in vegetables from an agroindustry


INTRODUCTION
Vegetables are an essential part of the world's population diet (Costa & Oliveira, 2011).In Brazil, the average consumption is of 50 kg per habitant/year (Teixeira et al., 2006).
Currently, there has been an increase in the concern with the sanitary quality of vegetables consumed by the population, as well the practices used to produce them, and the use of technologies aiming at the environment preservation (Oliveira et al., 2014).
It can be said that there are two large groups of vegetable producers: those who will be able to adapt and meet the new sanitary requirements and will be competitive and those who will be marginalized in the process for not following the sanitary and environmental norms (Costa et al., 2011).
Among the set requirements, the maintenance of good production practices, from the cultivation of the vegetables to its commercialization to the final consumer aiming to reduce or eliminate the presence of total coliforms (TC), thermotolerant coliforms (TtC), and intestinal parasites eggs (IPE) in vegetables, mainly those which are consumed raw, takes on economical and public health importance (Silva et al., 2014).
The water contaminated by fecal matter of human or animal origin used in the irrigation of the vegetable gardens, the presence of infected handlers, inadequate sanitization, and transport practices are factors which have been pointed out as causes of contamination by TC, TtC, and IPE in vegetables (Carvalho et al., 2003;Beraldo & Farache Filho, 2012).The investigation of TC, TtC, and IPE in vegetables is important in the public health area, especially considering the production, storing, transport, handling, and commercialization stages of these products (Ferreira et al., 2013).The prevalence and the food-borne infections by TC, TtC, and IPE caused by the ingestion of raw vegetables have been frequently reported (Silva, et al., 2011;França et al., 2013).
This work aimed to evaluate the risk factors associated with the occurrence of TC, TtC, and IPE in vegetables from an agroindustry in the city of Ibiúna -SP, Brazil.

MATERIAL AND METHODS
The work was carried out in a vegetable processing agroindustry located in the in Ibiúna -SP, in the period from 25th August to 7th December 2014.
To perform the diagnosis of the risk factors and their posterior association with the presence or not of TC, TtC, and IPE, a questionnaire with 17 closed questions was initially applied to 24 vegetable producers who deliver their products to the agroindustry.The questions addressed the following areas: production system, type of fertilizer used, origin of the water used for irrigation, sanitization of the vegetables, and the form of transport used to the agroindustry.
For the investigation of TC, TtC, and IPE, a collection was carried out at the moment each producer was delivering the vegetables, without any processing at the agroindustry.To compose the analysis sample, between 15 and 20 vegetable leaves were collected randomly in aseptic conditions, from various boxes delivered by the producer.The samples were stored in aseptic polyethylene packages and transported the same day under refrigeration.Posteriorly, they were processed in the microbiology and parasitology laboratory of the Federal Institute of Education, Science and Technology of São Paulo, São Roque campus.The analysis of TC and TtC were carried out using the technique of Vanderzant & Splittstoesser (1992) and Silva et al. (2007).The samples were diluted from the dilution of 10 -1 , 10 mL of the samples were plated in three tubes containing 10 mL of Lauryl broth with double concentration with inverted Durhan tubes, 1.0 mL of the sample in three tubes with 10 mL of Lauryl broth with simple concentration, and 0.1 mL in tubes with 10 mL of Lauryl with simple concentration.
The tubes were incubated at 35 ± 1 ºC for 24-48 h.After this period, Durhan tubes which presented gas in their interior were considered as positive and compared to the combinations present in the Most Probable Number (MPN) table for TC.The investigation and counting of TtC with the use of inoculation loop aliquots were taken from the positive tubes and reprocessed to a tube containing 10 mL of EC broth also containing inverted Durhan tubes in a kiln at 45 ± 2 ºC/24-48 h.After this period, the presence of TtC was confirmed by the formation of gas in the Durhan tube and compared to the MPN table for TtC.
For the verification of the presence or not of IPE in the vegetables, the spontaneous sedimentation technique was used (Hoffman et al., 1934).Two hundred and fifty milliliters of distilled water were added to the samples, being these posteriorly filtered in four-fold gauze and left to rest for 24 h.The following day, the supernatant liquid was discarded and about 30 mL of remaining were transferred to a 50-mL sedimentation calix.
From the sediment obtained from the final part of the tube, three drops were collected with the aid of a plastic pipette and then placed between glass slides.When necessary, to differentiate protozoan cysts, lugol was added to the analyzed sample.Each sample was analyzed in triplicate, in an optical microscope, at 10x magnification, and a scanning was performed to find and identify the parasites.The confirmation of the parasitic structures was carried out using a 40x magnification.The parasitic forms were identified by using the parasitological classification key (OMS, 1991).
For the risk factor analysis, an univariable analysis was performed by using nonparametric tests to compare proportions (Chi-square or Fisher's exact tests), with a 5% significance level (Zar, 1999).The analysis was carried out with the software SPSS for Windows, version 20.0.

RESULTS AND DISCUSSION
Of the 24 producers investigated, 16 (66.66%)practiced conventional agriculture; four (16.66%), hydroponic agriculture; three (12.5%),conventional and hydroponic agriculture; and one (4.16%),organic agriculture Table 1 presents the results regarding the investigated risk factors for the presence of TC, TtC, and IPE in vegetables from 24 producers in the municipality of Ibiúna -SP.
Tables 2, 3, and 4 present the results regarding the association analysis for the risk factors and the respective P-values for total coliforms, TtC, and IPE.
For the risk use of non-treated organic fertilizer, more than half of the investigated producers (54.2%) had this practice (Table 1).Silva et al. (2011), also evaluating the use of organic fertilizer and the sanitary quality of vegetables produced in Catu and Alagoinhas, in the State of Bahia, detected that, respectively, 100 and 80% of the producers used this type of fertilization in their vegetable gardens.These types of non-treated organic fertilizers used by the horticulturists may be characterized as means of contamination for the vegetables.However, Machado et al. (2006) highlighted that the fertilizer is not a determining factor for a possible contamination, also confirmed in this present work, in which there was no statistical association with the use of non-treated organic fertilizer and the occurrence of TC, TtC, and IPE (Tables 2,  3 and 4).Water for irrigation with a septic tank and with the release of wastewater represented 20.8 and 33.3%, respectively, of the total evaluated (Table 1).In relation to the sanitization of the vegetables at the moment of harvesting, 54.2% of producers performed it; however, 100% of them did not carry out the individual sanitization with chlorinated water, evidenced by the absence of high impact measures in the reduction of TC, TtC, and IPE in the vegetables (Jesus et al., 2015).Only 8.33% of the producers still transported the vegetables in non-sanitized wooden  (Lourenço & Katz, 2010).
In relation to the presence of TC in the vegetables, they were detected in 24 (100%) of the samples, values which oscillated from 0.3 to 240 CFU/mL.As for the investigation of TtC, they were detected in 23 samples (95.83%), with values oscillating from 0.3 to 46 CFU/mL.Despite the broad numerical variation of the TtC found in the samples, this result is of utmost importance in public health due to the potential of these foods as sources of infections and foodborne infections (Martins et al., 2010) In the correlation analysis with the presence of TtC, there was association for the risk factor use of reservoir water where there was release of wastewater and absence of sanitization of the vegetables at the moment of harvesting (Table 3).The results obtained in this work corroborate those obtained by Santos et al. (2010), who studied the hygienic and sanitary quality of vegetables and irrigation water in Salvador -BA, Brazil, and detected unsatisfactory conditions of the investigated areas and the elevated number of vegetables and the water used for their irrigation, which presented contamination by TC and TtC.Such a result was also confirmed by Mattos et al. (2009), who affirmed that the microbiological contamination in vegetables is due to the use of contaminated water for irrigation.
For the investigation of IPE, these were found in eight samples (33.33%), with values which oscillated from one to 100 eggs; however, a significant statistical association with the investigated risk factors was not detected (Table 4).Nevertheless, Ferreira et al. (2013), who evaluated the quality of water for irrigation in Teresópolis -RJ, Brazil, found the presence of IPE in 23.82% of the investigated samples.Nascimento & Alencar (2014), who investigated 24 vegetable samples in Natal -RN, Brazil, detected parasitic forms in 41.7% of the samples.More elevated frequencies of IPE in vegetables (60%) were detected by Oliveira & Perez (2014) both in those with or without sanitization.

CONCLUSIONS
In the conditions under which this work was carried out, it was possible to conclude that: Total coliforms were found in 100% of the investigated vegetable samples and thermotolerant coliforms were detected in 95.83%.
Intestinal parasite eggs were found in 33.33% of the samples, with values that oscillated from 01 to 100 eggs, but there was no significant statistical association for the investigated risk factors.
There was a significant statistical association for the risk factor use of reservoir water, in which there is the release of wastewater and absence of sanitization of the vegetables at the moment of harvesting in relation to the presence of thermotolerant coliforms.

Table 1 :
Number of producers (NP) and the respective obtained percentage (%) which presented risk factor associated to the presence of thermotolerant and total and intestinal parasite eggs in the vegetables from a total of 24 samples investigated

Table 2 :
Association analysis for the risk factors and the respective P-values for total coliforms (TC)

Table 3 :
Association analysis for the investigated risk factors in the 24 producers and the respective P-values for thermotolerant coliforms (TtC)

Table 4 :
Association analysis for the investigated risk factors in the 24 producers and the respective P-values for the intestinal parasite eggs (IPE)