The effects of inactive toxins of escherichia coli on hematological parameters in animals

Escherichiosis of calves and piglets is still a widespread infectious pathology on farms in different countries, including Russia, despite the vaccine prophylaxis of this disease. This fact testifies to the imperfection of present immunization means, the cause of which is discrepancy between antigenic composition of vaccines and etiological and pathogenetic factors responsible for the development of escherichiosis. In view of the above, the question of joint application of enterotoxins of Escherichia coli as a complex vaccine (anatoxin) becomes relevant. The question remains as to how they will affect the animal body after vaccine’s administration and what properties they will have, requiring special studies. In this regard, the aim of the work was to study the effect of the mixture of inactivated enterotoxins on the leukogram of animals. As a result of studies, it was found that after the introduction of anatoxin in rats, the primary response of the immune system was expressed in the form of an increase in the quantitative presence of physiologically mature neutrophils, followed by an increase in the number of immunocompetent cells – lymphocytes. The dose of injected anatoxin also mattered: the higher was the dose, the more pronounced were the changes in the leukogram, including the changes manifested by the increased presence of eosinophils in the bloodstream. Inactivated E. coli enterotoxins had no toxic and cytopathological effects when injected into the macroorganism; at the same time, they retained their antigenic and immunostimulatory properties, which allows them to be considered a candidate for a complex vaccine.


Introduction
Escherichiosis in calves and piglets continues to be a dreadful pathology both in in many countries. The predominant preventative measure for this disease has been found to be immunoprophylaxis [1,2]. The exotoxins of bacteria play a major role in the pathogenesis of escherichia diarrhoea, since they cause a number specific lesions [3].
The difference that exists between the antigen composition of the available vaccines and the etiological and pathogenic factors that cause the disease is one of the reasons accounting for this fact.  2 The particular emphasis that is placed on O-antigens and pili antigens in the development of vaccines is untenable in view of their considerable variation in pathogenic E. coli [8].
Lately, information about new toxins of E. coli has been gotten, which as well, play a significant role in the growth of pathologies, both in humans and animals [9,10]. It was established that some isolates of pathogenic E. coli isolated from calves and piglets contained several toxin genes, which denotes that they could concurrently produce more than one toxin [11].
Additionally, the use of famous commercial vaccines in escherichia-affected areas is not always effective for the control of infection. This is due to the fact that these drugs do not take into account the full set of pathogen antigens [12].
Since the mechanisms of developing escherichiosis do not only take into account the adhesion of classical fimbriae, but also non-fimbrial structures, specifically, intimin M agglutinins, AIDA-I adhesins. Meanwhile, toxin formation is at the heart of the pathogenic action in enterotoxigenic and enterohemorrhagic E. coli, and this should also be considered when producing a vaccine [13].
It has been confirmed that E. coli variants that produce shiga-like toxin (STX) are actively involved in the development of pathology in calves and piglets, as well as such variants that can produce various kinds of toxins at once, which have different toxic, immunosuppressive and immune-modulating properties and can affect the body in different ways [14].
The objective of the research was to study the effect of a complex preparation based on inactivated enterotoxins of E. coli on hematological parameters in animals.

Material and research methods
Through different cultivation of toxigenic strains of the causative agent of E. coli in test tubes with 10 ml volume of nutrient broth under thermostat conditions, a complex preparation including inactivated exotoxins of E. coli (anatoxin) was acquired. Used a temperature at 37 °C (98.6 F) for 6-8 hours until a slight turbidity appeared, indicating the growth of microorganisms. At the end of the culturing period, the obtained cultures were transferred into 200-300 ml. flasks with nutrient broth and incubated in the growth chamber at 37 °C (98.6 F), while the TLand TS-producing strains were incubated for 6 days and the STX-producing strain -for 7 days. Thereafter, broth cultures were inactivated by adding formalin to a concentration level of 0.3-0.4%. This procedure was performed for two weeks at 37 °C (98.6 F), and the microbial cultures were stirred twice daily. At the end of inactivation, the cultures were checked for sterility, their equal volumes were combined, and the culture medium and microbial mass were separated by sterilizing filtration. Consequently, the resultant complex solution was a clear liquid, light yellow in color, containing three types of inactivated enterotoxins.
After that, the prepared solution was poured into sterile vials under aseptic conditions and then capped with sterile rubber plugs and sealed with aluminum caps.
In studying the influence of inactivated toxins on the hematological indices, laboratory white rats, weighing 200±10 g were used. These animals were categorized into five groups of seventy animals each by the paired analysis method. Group 1 rats served as negative controls and were whole. Group 2 animals were positive controls and were administered with sterile nutrient broth. The remaining animals were injected with escherichiosis toxoid in different doses: Group 3 rats received 0.15 cm 3 ; Group 4 -0.3 cm 3 ; Group 5 -0.6 cm 3 . After the drugs had been administered, blood was drawn from the lateral tail vein in 10 animals of each group respectively, 1, 3, 6, 12, 24, 72 and 168 hours using an injection needle and syringe for hematological analysis.
The blood test (the number of red blood cells, white blood cells, and hemoglobin) was performed on an Abacus-3 hematological analyzer. The leukogram was derived separately, according to which the leukocyte index of intoxication (LII) was calculated to assess the intensity of endotoxemia and the extent of inflammatory response.

Research results
It was observed through the analysis of the results of the experiment that, the red blood cells index of lab rats remained stable and did not differ greatly from that in intact animals after the injection of both 3 sterile broth and anatoxin in all time intervals of the study. However, significant changes were seen in the white blood cells index.
A reduction in the total number of leukocytes was noticed in the animals of the experimental and positive control groups as compared to the animals of the negative control group (intact animals), after an hour of administering nutrient broth and anatoxin (table 1).
As a result, there was a change in the total number of white blood cells as against the registered leukopenia in animals. More specifically, a reduction in lymphocytes and a rise in neutrophil granulocytes were observed, but this process was different for animals from different experimental groups. If 85.0% of all leukocytes and 14.3% of neutrophils accounted for lymphocytes in the rats from the first group then, with the animals into which were injected anatoxin at doses of 0.15-0.3-0.6 ml from groups three through five, these cells amounted to 64.7; 67.0; 66.0 and 29.7; 29.3; 30.6% respectively.
An analogous change in leukogram was observed in the animals of the second group injected with sterile broth, but to a relatively lesser extent. Basically, in all instances, the observed effect can be explained by two reasons: first, by the response of the hematopoietic system to physical stress (fixation, intramuscular injection), and second, by the direct effect of the broth and anatoxin. The latter can be indirectly supported by an increase in the relative number of eosinophils in the blood of animals injected with broth and anatoxin. There were no toxic and irritating effects of broth and anatoxin, as observed by the absence of local changes at the place of injection.
The index of leukocyte intoxication was, however, increased by 0.2-0.3 units. This is negligible as compared to the indicators of severe toxicosis, when the index values can be more than 5 units.
The animals, after 3 hours of starting the experiment, exhibited an alteration in effect of toxoid on leukocytes (table 2). The drug dosages were of immense importance. Leukocytes in rats decreased by 0.7×10 9 /l at an immunizing dose of 0.15 ml of toxoid, as compared to the initial study. Increasing the dose of the drug, however, had the opposite effect. When 0.3 ml of toxoid was administered to the animal, the composition of leukocytes in the blood rose by 4.4×10 9 ×/l, and with a dose of 0.6 ml -by 7.4×10 9 /l, respectively.
In group 2 rats (positive control group) this parameter remained unchanged. The study of the leukograms in the animals of these groups likewise showed the dynamics of increase in neutrophilic granulocytes as against the decrease in the relative number of lymphoid cells. The animals, after the injection of the drugs, had no local inflammatory reactions and allergies. This was likewise noticed from the blood parameters that, the number of rod-shaped neutrophils and eosinophils remained at the same level as in the control animals. The total number of white blood cells in rats immunized with nutrient broth (positive control) did not differ from the same indicator in animals in negative control after 6-12 hours of the study (table 3  and table 4).
However, the number of lymphocytes in the leukograms of the rats from Groups 2 through 5 was still lower than that of the rats from Group 1. Thus, this difference was 10.  During the first 12 hours of the study, animals of both the experimental and positive control groups were activated by physiologically mature neutrophils in the blood, whose main function is to effect a phagocytic reactions and antigen processing to other cells of the immune system. Since anatoxin is a stronger antigen than nutrient broth, after its administration to animals, the production of segmented neutrophils in them was more active for 12 hours. Positive control animals had an enormous neutrophil count during the first 3 hours of the experiment. The number of white blood cells in the blood of rats did not change in the experimental and control groups after 24 hours of their immunization with drugs (table 5). Similarly, leukogram indicators in rats of positive and negative control groups did not differ. Concurrently, the animals of the experimental groups experienced a drop in lymphocytes and a rise in neutrophils in comparison with the negative control. Taking into consideration, the dependence on the dose of the administered toxoid, it was established that the higher it was, the stronger the antigenic stimulating effect. In the 5th experimental group of animals, the relative number of lymphocytes was 27.0% lower, and the number of neutrophils was 22.0% higher than in intact rats.
In 72 h after administration of anatoxin (table 6), the total number of leukocytes increased sharply in all lab animals. Thus, if in control group rats the number was at the level of 7.1±0.98 -7.3±0.13-10 9 /l, in experimental group rats it was 11.3±0.29-11.9±0.37-10 9 /l. Deductions from the leukogram revealed that the observed leukocytosis occurred due to a rise in the number of lymphocytic cells.