Detection of Campylobacter spp. in Cloacal Swab of Hens Reared in Different Breeding Conditions in Slovakia

Poultry meat is reported as the most common transmission factor of alimentary disease – Campylobacteriosis [1]. Campylobacter spp., especially Campylobacter jejuni and C. coli, are the main cause of human bacterial gastroenteritis in the developed world [2,3]. Epidemiological investigations of commercial flocks indicate that naturally acquired flock colonization is age-dependent. Newly hatched chicks appear to be free of Campylobacter. In Europe, this negativity persists until at least 10 days of age (the so-called lag phase), and most flocks become infected only 2 to 3 weeks after the placement of chicks into a broiler house [4]. The duration of colonization and shedding of Campylobacter in poultry has not been fully determined. It is generally accepted that colonization in chickens persists at least for the life span of a broiler, without a clinical form of infection [5]. Moreover, after 8 weeks, colonization may gradually reduce in terms of both the number of organisms recoverable from cecal contents and the number of colonized birds [6]. Elderly hens can be antibody positive without colonization, suggesting that an antibody response may be associated with elimination of infection [7]. Several changes in stock management may occur during altered feed composition even in organically produced birds. The prevalence of flock positivity is also dependent on flock size, type of production system [8], and age of birds [9]. There is generally a higher rate of infection in summer than in winter, and the timing of this peak also appears to vary with latitude. Some studies, on the other hand, have failed to demonstrate seasonality in the prevalence of C. jejuni in poultry [7,9]. The reason for seasonal variation is unknown but may reflect levels of environmental contamination. Certainly, poultry houses have more ventilation in the summer, increasing the contact of the birds with the outside environment. Individually cages hens also have a seasonal variation in excretion rates [10].

Due to the fact that the beginning of the chain of Campylobacter spp. infection in poultry relates with their breeding conditions and age of flocks the aim of this study was to detect enteropathogenic bacteria Campylobacter spp. in cloacal swab of hens reared under different breeding conditions.

Materials and Methods

In this study were used 180 hens of laying breeds placed on six farms in range from 6 to 12 month of age. For the identification of Campylobacter spp. in cloacal swab one battery-cage farm, three indoor farms, and two free range farms were chosen. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Samples were collected in the spring period (May-June). For collection of samples 30 hens were randomly chosen from each defined farm. Cloacal swab from individual hen was collected by swab stick and ELISA (enzyme-linked immuno sorbent assay) method was used for analysis - commercial ProspecTtm Campylobacter kit (Oxoid, USA) - to identify enteropathogenic Campylobacter spp. Diluted samples were added into pre-designated wells of microplate strips at a dose of 200μl. Ninety six well-microtiter plates were coated with rabbit polyclonal anti-Campylobacter SA (sialic acid) antibody. After the incubation of microtiter plates (20-25 °C, 60min), the content of the wells was aspirated and washed three times with wash solution (component of ELISA kit). Then 200μl of diluted enzyme-antibody conjugate binding with horseradish peroxidase in stabilizing buffer was applied into the plate wells, and incubated at 20-25 °C 30 minutes. After incubation, the plate was 3 times washed and 200μl of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution was added into each well. The reaction was stopped with 50μl stop solution and absorbance was measured spectrophotometrically at 450nm on microplate reader (Revelation Quicklink, Opsys MR, Dynex Technologies, USA). Interpretation was made using a calibration curve prepared according to the manufacturer’s protocol.

One-way ANOVA with Tukey post-test by Minitab 16 software was used (SC&C Partner, Brno, Czech Republic) for statistical analysis.

The results demonstrated low Campylobacter spp. positivity in hens reared in different types of examined poultry farms (Table 1). The presence of Campylobacter spp. was confirmed in one indoor poultry farm in 1 of 30 analyzed samples, representing 3.40%. Another two indoor poultry farms demonstrated the presence of Campylobacter spp. in 2 respectively in 3 of 30 analyzed samples, representing 6.70% and 10%. Battery-cage farm showed the presence of Campylobacter spp. in 2 of 30 samples, representing 6.70%. The presence of bacterial antigen was confirmed in both free range farms in 1 of 30 analyzed samples, representing 3.40%. However, differences between mean values for various rearing hence were not statistically significant.

Table 1:Seropositivity of poultry farms to Campylobacter spp.

Percentage of Campylobacter spp. positive hens was low in all monitored flocks. No notable difference in positivity of hens was found among farms with difference flock sizes. Barrios et al. [11] and Rushton et al. [12] demonstrated that environmental occurrence of Campylobacter spp. is related to its seasonal character with the peak in the flock prevalence during the warm summer months (July-August). In our study, the percentage of bacterial positivity in examined hens in spring months was low. On the other hand, the comparative monitoring during winter months was not done. However, low percentage of positive hens in spring months suggests no evident seasonality [2] in monitored flocks. Similarly, no significant differences in terms of hens positive to Campylobacter spp. indicate that contact of hens with contaminated water and soil from the environment did not play important role [13] in the Campylobacter infection. In our experiment, the lowest values were demonstrated in free range farms. This finding could be related to the time of sampling - the occurrence of pathogenic bacteria Campylobacter spp. in the environment in the spring months is low. The diversified feed intake during the growing season could be also the possible impact on the lower frequency of positivity to Campylobacter spp. It would be useful to examine a higher number of poultry farms to obtain more extensive database of results.

The duration of colonization and shedding in poultry has not been fully determined. It is generally accepted that colonization in chickens persists at least for the life span of a broiler. However, there is evidence that colonization may gradually reduce when the birds get older and individual birds excrete Campylobacters intermittently [6]. In retail chickens, prevalence of Campylobacter spp. was found to be at 75% [14]. Finally, more recently obtained results in our laboratory show that experimental infection of chickens with C. jejuni induces IgA mucosal antibody response [15] and increase of IgM+ cells in the intestinal mucosa [16], which demonstrates immune response development in Campylobacter spp.-infected chickens. Moreover, elderly hens can be antibody positive without colonization, suggesting that the antibody response may be associated with elimination of infection [7] Self-limitation of infection has been reported in other naturally colonized birds, for example, gulls can become negative for Campylobacter spp. within a period of 4 weeks [17]. Comparing to high positivity to followed bacteria in chickens [14,18] our results suggest self-limitation of infection in majority of examined birds.

In conclusion, our results demonstrate positivity to Campylobacter spp. in low number of monitored hens without evident effect of rearing in different farming modes on prevalence of positive hens. Self-limitation may be one of the reasons of low Campylobacterial numbers. The factors involved in the selflimitation of colonization are unclear but may involve acquired immunity.

The authors are grateful to E. Husáková for assistance and samples analyses. This work was supported by the Grant Agency for Science of Slovak Republik VEGA 1/0562/16, Slovak Research and Developmental Agency APVV 15-0165.

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