Introduction

Campylobacteriosis is a zoonosis of worldwide distribution, caused by bacteria of the genus Campylobacter, the most frequent being C. jejuni and C. coli, characterized by causing invasive diarrhea, with abdominal pain, fever and general malaise. The main reservoirs are poultry, pigs and cattle. This pathogen is transmitted to humans by ingestion of raw or poorly cooked, contaminated food, mainly affecting children, the elderly and immunocompromised people¹. It is also associated with the increase in the consumption of chicken meat, due to its low cost per kilo, compared to meat from other animal species in our environment (pork and beef). In Bolivia, its per capita consumption demand is 44 kg².

Currently, there are 66 species and 16 subspecies of Campylobacter recognized, most of which are associated with diseases in humans and animals³. Only 4 species are pathogenic to humans, the most frequent being C. jejuni and C. coli, and less frequently C. fetus and C. upsaliensis^1-4.

The Campylobacter genus is characterized by being Gram-negative, S-shaped bacilli, 0.2-0.8 μm wideand 0.5-5 μm long, with generally monotric polar flagella, they have a characteristic mobility, like that of a corkscrew or corkscrew, however, small thermophilic campylobacters have a rapid movement, are microaerophilic, therefore they need small amounts of oxygen (5 %) and capnophilic, that is, they need high amounts of carbon dioxide (10 %) and 85 % nitrogen⁵. Their metabolism is characterized by not oxidizing and not fermenting carbohydrates since they obtain their energy from amino acids and the 4 to 6 carbon intermediates of the Krebs cycle⁶.

In the period from 2005 to 2013, Campylobacter was the most frequently reported gastrointestinal bacterial pathogen in humans in the European Union (EU). The number of confirmed cases of campylobacteriosis was 214779, with an EU notification rate of 64.8 per 100000 inhabitants, which was at the same level as in 2012, but the severity in terms of reported case fatality was low (0.05 %)⁷. Most recent data from 2021 from the European Centre for Disease Prevention and Control (ECDC) shows that gastrointestinal campylobacteriosis was the most reported. That year, 30 countries that are part of the EU and the European Economic Area (EEA) reported 129690 confirmed cases, the overall notification rate in the EU/EEA was 44.5 cases per 100000 inhabitants. This decline in the notification rate occurred since 2020 due to the pandemic caused by the SARS CoV-2 virus⁸.

In Bolivia, in 2009, a study was published on the monitoring of antibacterial resistance of Campylobacter spp. in four hospitals in La Paz. 370 stool samples were processed: 44 samples (12 %) were positive for Campylobacter spp., 28 were C. jejuni, 13 were C. Coli and 3 were C. lari⁹.

In Bolivia, there is no published data on the presence of Campylobacter in raw chicken meat (RCM). There is a lack of work on the subject, and food microbiology laboratories (FML) have not yet detected this pathogen in food, which is why regulatory agencies do not take preventive measures to avoid food contamination, which becomes a vehicle for the transmission of diseases.

This research work aimed to evaluate the presence of Campylobacter in RCM at points of sale in the city of La Paz to obtain data on contamination factors such as: type of market, type of sale and origin that contribute to establishing a baseline for the prevention and control of foods susceptible to contamination with this bacterial agent.

Materials and methods

The work was carried out in the city of La Paz-Bolivia, which is divided into 9 macrodistricts, 2 rural and 7 urban, part of the Pedro Domingo Murillo province of the department of La Paz, located in the geographical position of South 16º 30' 0'' (latitude) and West 68º 09' 0'' (longitude). The city has a mountain climate and an average annual temperature of 14° C, with an average rainfall of 512 mm¹⁰.

A cross-sectional observational study was carried out, by observing and recording events without intervening in their natural course, the measurement in relation to time was unique¹¹.

The samples were collected and processed from June 2018 to February 2019, and were processed at the FML of the Institute of Diagnostic Laboratory Services and Health Research (SELADIS) dependent on the Major University of San Andres. 108 whole RCM samples were taken in a non-probabilistic manner for convenience due to inclusion criteria in the sampling of 6 macrodistricts of the city of La Paz, taking into account the following study factors: i) origin (Cochabamba, Santa Cruz and La Paz), ii) type of market (popular and supermarket) and iii) mode of sale (refrigerated and non-refrigerated).

For the detection of Campylobacter spp. in RCM matrix, the Standard (ISO 10272-1:2017)¹² “Microbiology of the food chain - Horizontal method for the detection and enumeration of Campylobacter spp. Part 1: Detection method” was used (Method verified in SELADIS FML). The method applied consists of 4 stages which were: i) selective enrichment with a 1/10 dilution of the homogenized sample with Preston broth, and incubation in microaerophilia at 41.5º C for 24±2 h. ii) sowing of 10 μL on the surface by exhaustion in Modified charcoal cefoperazone deoxycholate agar (mCCD), and incubation in microaerophilia at 41.5º C for 44±4 h. iii) confirmation of characteristic colony-forming units (CFU), selection of suspected Campylobacter colonies for confirmation by oxidase test, morphology, motility and seeding of each of the selected CFU on 2 horse blood agar plates. To promote the development of the agent, one plate was incubated in microaerophilia at 41.5º C for 48 h and the other was incubated at 25º C for 48 h in aerobiosis. iv) differentiation of C. jejuni and C. coli species by biochemical tests of catalase activity, indoxyl acetate hydrolysis and hippurate hydrolysis.

For data analysis, the statistical package Microsoft Excel 2010 and InfoStat, version 2014, were used, and the results were expressed in percentages, contingency tables, frequency distribution and chi-square test (χ²).

Results

Of 108 RCM samples analyzed, 79.6 % (86) showed the presence of Campylobacter spp. (Figure 1), this being one of the first reports of the presence of the microorganism, object of study, in the city of La Paz. Analyzing the 86 positive samples, 93 % (80) were identified as C. jejuni and 7 % (6) as C. coli (Figure 2) (Table 1).

Figure 1
Growth of Campylobacter spp. on blood agar and mCCDA

A. Development of Campylobacter spp., in blood agar medium.

B. Development of Campylobacter spp., in Modified charcoal cefoperazone deoxycholate agar (mCCD) medium.

By origin (Table 2), significant statistical differences were evident (χ² (2) = 23.05, p˂0.05), indicating that the presence of Campylobacter spp. in RCM is dependent on its origin, at a level of 95 % reliability. The samples from Cochabamba and La Paz had a contamination percentage of 96.4 and 87.5 % respectively, figures higher than the 57.8 % observed in Santa Cruz.

Figure 2
Hippurate Hydrolysis Test for Differentiation between C. jejuni and C. coli

Negative (-), C. coli; Positive (+) C. jejuni

Regarding the type of market (Table 3), the RCM from Cochabamba did not show statistically significant differences (χ² (1) = 0.35, p>0.05) in relation to the presence of Campylobacter spp., regardless of whether it was sold in a popular market or supermarket. In contrast, the samples from Santa Cruz had a statistically significant difference (χ² (1) = 14.5, p˂0.05) depending on the type of market. It was observed that the presence of this microorganism was significantly lower in supermarkets (1 positive sample out of 11 analyzed), compared to popular markets (25 positive samples out of 34 analyzed).

Analyzing the type of sale (Table 4), the RCM that come from Cochabamba and La Paz, there were no statistical differences (p>0.05), therefore, the presence of Campylobacter in the samples under study, is independent of whether the samples were refrigerated (100 % contamination) or not refrigerated (93.3 % contamination).

In contrast, in the samples from Santa Cruz there is a significant statistical difference (χ² (1) = 16.79, p˂0.05), observing that the contamination of the RCM is dependent on the type of sale, with a percentage of 82.4 % contamination in non-refrigerated samples, compared to refrigerated samples which is 42.8 %.

Discussion

The detection of Campylobacter spp. in RCM was high 79.6 % (Table 1), results that agree with the data reported in Latin America, such as Mexico (89 %)¹³, Peru (97%) in markets in the province of Lima¹⁴, Chile (63.3 %)¹⁵ and Ecuador (71 %)¹⁶.

RCM contamination with C. jejuni was 93 % compared to C. coli 7 %, several works reported a lower percentage of C. jejuni in RCM, thus, in a work carried out in Chile in 2017 it was reported (63.3 %)¹⁵. In South America, data has been reported for more than 15 years, in Argentina (87.8 %)¹⁷, and Brazil (68.8 %)¹⁸.

C. jejuni is found in the digestive tract of poultry, for this reason, a high number of chicken carcasses are contaminated¹⁵. The presence of the microorganism is associated as one of the main causes of enteric zoonotic infections in the world. The World Health Organization (WHO) considers it as the first etiological agent of diarrhea in humans in developed countries, and the second or third in developing countries, such as Latin America¹.

Since 2008, it has been reported that campylobacteriosis is one of the most prevalent zoonoses in developed countries, due to the contamination of food of animal origin, mainly in poultry products¹⁹. The report by the European Food Safety Authority and the ECDC, noted the results of monitoring zoonoses in 36 EU countries, establishing that campylobacteriosis is in first place, salmonellosis in second and Escherichia coli, producer of Shiga toxin, in third place²⁰.

Campylobacter is prevalent in poultry intended for human consumption and generally does not cause disease in them¹. Due to this characteristic, campylobacteriosis is not detected in poultry farms, so contamination can start in the production area, continue to the processing plants (slaughterhouses) and reach the points of sale²¹.

In Bolivia, the Departmental Service of Animal Health and Food Safety (SENASAG) only has a National Program for the Eradication of Salmonellosis in chickens²². Currently, there is no regulation or control and surveillance program for Campylobacter, despite the increase in Foodborne Illnesses (ETAs) with this agent worldwide¹. It is considered important to implement a surveillance program for this agent throughout the food chain, from primary production to the consumer.

The WHO mentions that Campylobacter spp. is included among the 4 main causes of diarrheal diseases, with children under 5 years of age being the most affected, representing one third of foodborne diseases²³.

To avoid cross contamination, it is important to implement hygiene and handling measures for proper handling of chicken meat when cooking, since it is a vehicle for the transmission of diseases caused by Campylobacter¹.

When evaluating Table 2, it was evident that the origin of the RCM is related to the percentage of presence of Campylobacter spp., with the one from Cochabamba having a presence of 96.4 %. This data is alarming, because the 2 producing companies, according to their advertising, had ISO 9001:2015 certification, possibly their certification is not oriented to the safety of the product. In the meat samples from La Paz, the percentage of contamination with this agent was 87.5 %, and the suppliers were small and medium-sized producers from the north of La Paz. In comparison, the samples from Santa Cruz presented 57.8 % contamination by Campylobacter spp., which could be attributed to the fact that this poultry company is certified with ISO 9001 and 22000, which certifies food safety, implemented to ensure the quality of its product throughout its production chain.

Analyzing Table 3, the relationship between the type of market and the presence of Campylobacter spp. in RCM was evaluated. As previously discussed, the origin of the samples had a statistical relationship with the presence of the microorganism, so it was decided to analyze the type of market taking into account the origin, with the objective of evaluating individually if the type of market is related to the type of handling, and becomes relevant in the contamination of RCM. The analysis took into account only the meat from Cochabamba and Santa Cruz, because the samples from La Paz are distributed only in popular markets. The samples from Cochabamba showed that the contamination of the RCM is independent of the type of market, which could indicate that the samples could have been contaminated in one of the stages of the production chain²¹. This analysis contrasts with the results obtained in the samples from Santa Cruz, the type of market significantly influences (p<0.05) the contamination of the samples obtained, with the meat sold in popular markets having a significantly higher contamination (73.5 %) compared to that in supermarkets (9.1 %). This result may be due to the fact that in the on-site observation, there is a difference in the way sales and handling areas are organized. Supermarkets only sell whole packaged chicken and arranged in exclusive spaces by brand and in refrigeration. In popular markets the chicken was arranged together without differentiating the brand and without individual packaging, transported in plastic baskets and the handling was carried out without gloves, using the same knife to divide the chicken pieces and giblets, conditions that lead to cross contamination, affecting the safety of the food at the time of sale.

When analyzing the impact that the type of chicken meat sales has on the presence of Campylobacter spp. it was decided to evaluate this relationship of variables individually for each origin. In the samples from Cochabamba and La Paz, the statistical analysis revealed that the type of sales, whether refrigerated or not, did not have a significant impact on RCM contamination. This finding suggests that chicken contamination could occur before the sale of this food product, that is, at any point in the production chain.

In contrast, when examining the statistical relationship between the type of sales and the presence of Campylobacter spp. in the samples from Santa Cruz, it was clearly observed that refrigeration did have a statistically significant relationship. This result indicates that contamination is lower when the cold chain is maintained during distribution, while the presence of Campylobacter is favoured in places of sale where the meat is not refrigerated. This data is consistent taking into account that for the conservation of chicken meat it is essential to maintain the cold chain at less than 7° C, in order to avoid the multiplication of microorganisms due to the effect of temperature.

These findings are consistent with the need to maintain the cold chain below 7° C to prevent the multiplication of microorganisms due to temperature, which is crucial for the proper preservation of chicken meat.

The stalls in popular markets, where there is a greater attendance of consumers, do not meet the conditions for the sale of this product, since they do not maintain the cold chain. It was observed that the meat is exposed on counters and for several hours at room temperature, which conditions the reduction of the product's shelf life. Preservation by refrigeration is an ideal method for preserving food that consists of keeping it at a temperature of 4-6° C in order to reduce bacterial activity.

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Notes

Author notes

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