Toxigenity of yersinia enterocolitica strains isolated from various objects

Abstract

Introduction. Yersinia enterocolitica is a psychotropic zoonotic pathogen that causes acute gastroenteritis [19] and sometimes a more serious disease in humans. In some countries, it competes with salmonella and Campylobacter as a food pathogen, and because it can grow at cooling temperatures [8], this is a food safety concern. Infection with Y. enterocolitica can cause different symptoms depending on the age of the infected person. Y. enterocolitica infection is sporadic, most common in young children under 5 years of age [10, 12]. The predominant symptoms in humans, especially in young children, are fever, abdominal pain, and diarrhea, often with blood. In older children and adults, the effects of yersiniosis are severe and include acute infections, pseudoapendicitis, and long-term complications such as reactive arthritis and erythema nodosum [11].

Yersinia enterocolitica is thought to be a significant food pathogen, although pathogenic strains are rarely isolated from food. It is assumed that pigs are the main reservoir of pathogenic Y. enterocolitica, because so far, the pig is the species from which pathogenic strains are often isolated [13]. Several species of domestic animals, such as dogs, cats, cows, sheep, and horses, and several wild species [15] of animals, such as rodents (mostly mice), monkeys, deer, and foxes, have also been identified as potential reservoirs [12].

The geographical distribution of Y. enterocolitica is diverse. There are more than 50 different serotypes of Y. enterocolitica (based on antigenic variations of cell wall lipopolysaccharide), and only some of them are pathogenic. O: 8 is the main infectious serotype in the United States, followed by O: 3, O: 5.27, O: 13a, 13b, O: 20, O: 9, etc. D [9, 16]. Serotype O: 3 is most commonly isolated in humans in Europe [3]. In China, serotype O: 3 is mainly found in infections followed by O: 9 and O: 8 [21]. In addition, different serotypes demonstrate geographical specificity; for example, the predominant serotype in Australia, Europe, and Canada is O: 3 [18], O: 8 in Japan [19], and O: 9 in Scandinavia and the Netherlands [14].

Despite the availability of effective diagnostic tools, recommendations for prevention, yersiniosis remains an urgent medical and veterinary problem, the solution of which requires further research [1, 3, 4, 5, 7].

Purpose. The aim of the study was to study the toxigenicity of Yersinia enterocolitica strains isolated from animals, livestock raw materials, products and environmental objects.

Methods. The research was performed on the basis of the Department of Epizootiology, Microbiology and Virology of NULES of Ukraine, molecular research - in the Department of Molecular Diagnostics of the Ukrainian Laboratory of Quality and Safety of Agricultural Products.

Materials for the isolation of Yersinia enterocolitica were rectal samples of calves, piglets, samples of slaughter products (pieces of lungs, liver, spleen, kidneys, lymph nodes), samples of minced meat and finished meat products, samples of milk and dairy products, as well as flushes from the walls of livestock facilities, wastewater.

Bacteriological studies of materials to isolate Y. enterocolitica were performed in accordance with the Guidelines [6]. In order to study the enzymatic properties used strips ARI-20E - systems (France). Selected on the basis of phenotypic characteristics of microbial cultures were investigated by PCR.

Serotyping of isolated strains of Y. enterocolitica was carried out by setting RA with diagnostic O-monovalent blood sera, the presence of plasmid virulence was determined using serum diagnostic for virulent yersinia ( St. Petersburg, Russia).

Toxicity (cytotoxicity), cytotoxic effect (CE) of selected Yersinia enterocolitica strains was determined by inoculating different dilutions of the supernatant on a monolayer of Vero cell culture. Toxicity assessment was performed according to the method of J. E. Vyalykh [1].

The enterotoxigenic properties of Y. enterocolitica strains were studied in isolated segments of the small intestine of guinea pigs. Y. enterocolitica strains were considered enterotoxigenic, which determined a dilatation index (DI) of 1 or higher.

In the process of determining the enterotoxicity of the strains, pathological and morphological studies of biological materials taken from experimental guinea pigs were also carried out, macro- and microscopic changes in organs and tissues were analyzed. Postmortem autopsy was performed by the method of partial evisceration in the generally accepted sequence.

Animal experiments were carried out in compliance with bioethical norms of the “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (Strasbourg, 1986).

Results. In the bacteriological examination of 1234 samples of materials taken from sick and clinically healthy calves, piglets, dogs and cats, from livestock raw materials and products derived from it, water for watering animals, as well as washes from the surface of walls and floors, from wastewater from livestock premises was 87 cultures of microorganisms that had phenotypic characteristics characteristic of Yersinia were isolated. As a result of identification using immunological and molecular genetic methods, 64 isolates were identified as Yersinia enterocolitica, 23 - Yersinia spp..

Y. enterocolitica was most often isolated from patients with signs of piglet diarrhea and from pig slaughter products.

In the process of serotyping 57 strains of Y. enterocolitica it was found that 19 (33.3%) belonged to serotype O: 3, 3 (5.3%) - to serotype O: 9 and 7 (12.3%) - to serotype A: 6.30, 28 (49.1%) strains were not agglutinated with the used diagnostic sera (O: 3; O: 9 and O: 6.30).

Eight strains (30.8%) isolated from pig slaughter products and 4 strains (26.7%) isolated from cattle slaughter products were agglutinated with serum, indicating the presence of virulence plasmids (pYV - рlasmid associated with Yersinia virulence).

In a study of the cytotoxic properties of 64 strains of Y. enterocolitica on Vero cell culture isolated from different sources, 45 (70.3%) were toxigenic. The intensity of the cytotoxic effect of strains on cell culture was different. 2 strains (4.4%) were highly toxigenic. The average degree of cytotoxicity was shown by 10 strains (22.2%), low-toxigenic were 22 strains (48.9%). All strains of Y. enterocolitica isolated from sick animals and most strains isolated from pig slaughter products (63.6%) and calves (55%), as well as from milk and dairy products (71.3%) were cytotoxic.

Among the 20 strains studied, 11 (55%) isolated from various sources (from sick animals, pig slaughter products, cattle and dairy products) showed enterotoxigenicity. Under the conditions of the experiment on guinea pigs, they caused an intensive accumulation of fluid in isolated areas of the intestines of animals (ID ≥ 1).

During the pathological and anatomical autopsy of experimental ants, the most pronounced macroscopic changes were observed in the intestine. They were characterized by signs of serous-catarrhal and catarrhal-hemorrhagic inflammation. The mucous membrane of the duodenum and hungry intestine was swollen, covered with a thick layer of mucus, there were hemorrhages. At histologic research the most expressive changes are registered in a small bowel.

Microscopic changes in the liver, thymus, heart, spleen, kidneys, pancreas, as well as in somatic and visceral lymph nodes were also significant.

Bacteriological examination of blood samples, internal organs and somatic lymph nodes of experimental animals did not reveal microorganisms, which indicates the toxigenic origin of the above pathomorphological (macro- and microstructural) changes.

Most enterotoxigenic strains of Y. enterocolitica were isolated from animal diarrhea patients, but more than 50 % of the strains isolated from raw meat and meat products were also enterotoxigenic.

Discussion. Among the many pathogenic factors of the causative agent of yersiniosis, an important pathogenetic significance belongs to the enterotoxins YST-1 TAYST-11 [1, 3, 4]. The latter are similar in mechanism of action to enterotoxins of other enterobacteria. Other extracellular factors and endotoxins of Yersinia enterocolitica also play an important pathogenetic role [3, 5, 7]. Once in the blood, Yersinia enterocolitica toxins are distributed throughout the body and cause profound morpho-functional disorders in organs and tissues, which are manifested in particular by the macro- and microscopic changes described above.

Further study of the ability of toxin formation by different strains of Y. enterocolitica, study of the circumstances that contribute to the accumulation of toxins in food, will contribute to the development of effective therapies and prevention, to establish a system of reliable control of Yersinia infection in humans and animals.