Сельскохозяйственная биология, 2011, № 4, с. 82-89.

УДК 636.2.034:57.04:576.08

COMPARATIVE ESTIMATION OF CYTOMORPHOLOGICAL CHANGES OF LYMPHOCYTES IN CATTLE AND ANIMALS-BIOINDICATORS ON TERRITORIES POLLUTED WITH ¹³⁷Cs

V.V. Kalashnikov¹. Korovushkin², S.A. Nefedova²

In the conditions of the Ryazan oblast’, on territory of which the nuclear fallout was observed after Chernobyl AES accident (against the background of natural iodine endemia and partial pollution by industrial toxicant) by the use of Chic-reaction the authors determined the changes of marker and morphological parameters of lymphocytes (the size of  granules and their blocks in cytoplasm) in healthy and sick cows (mastitis, leucosis, hypothyroidism) (1996-2010), Microtus arvalis Pall, Clethorionomys glareolus Schreb and also Rattus norvegicus Berk (2003-2007). The possibility was proved of use of voles Scots as bioindicators reflecting the influence of the radionuclides, because this type of rodent dynamics deviations of phenotypical parameters of lymphocytes is identical to the same in leukemic cows.

Keywords: lymphocytes, Chic-response, environmental ecology, cattle, mastitis, leukemia, hypothyroidism, common vole, power fever with renal syndrome.

 The Chernobyl accident in 1986 has resulted in radioactive fallout in vast areas of Belarus, Ukraine and the Russian Federation. In Russia, the provinces of Belgorod, Bryansk, Voronezh, Kaluga, Kursk, Orel, Leningrad, Lipetsk, Ryazan, Tambov, Tula, Penza, Smolensk, Ulyanovsk and the Republic of Mordovia were subjected to it (1, 2). In the territory of Ryazan province, environmental monitoring should consider a combination of three factors: radioactive precipitation after the Chernobyl accident, industrial toxic contamination and the natural iodine deficit in the soil.
V.N. Mukhin (3) has shown the 10-years dynamics of cytochemical lymphocytograms in children living in the radiation-contaminated areas of the Krasnogorsk district of the Bryansk province, who had received different doses of the internal exposure. There have been also determined cytochemical characteristics of leukocytes in cows from contaminated areas. The studied cytograms were obtained from the peripheral blood smears subjected to Pearse stain with a-naphtylacetate esterase and Schiff reaction (McManus test for mucopolysaccharides). The comparative study has shown in children (and similarly in cows) the average number of lymphocytes not exceeding the norm established by the author. The data on Schiff reaction over the observation period indicate its new rise (1994-1995) after the first peak (1991) and subsequent reduce to normal levels (1992-1993). In recent years, there was detected the increase in cases of children with the critical rate of lymphocytes with intense granular Schiff reaction (the additional internal exposure of these children amounted to 0,5 mSv annually after the Chernobyl accident) (4). The increased rate of mutational processes was observed (5).
 The extent of damage to the thyroid gland and hormone levels in the blood of animals depend on the absorbed radiation dose (6). It necessitates searching for bioindication techniques allowing the control of ecotoxication effects in farm animals and wild inhabitants of contaminated areas, such as small rodents. In contrast to physical or chemical sensors, living organisms are the essential bioindicators interacting with a whole complex of factors and allowing to establish the degree of risk from even tracing amounts of pollutants or any other contaminants in the ecosystem, to predict the state of Biota and assess the cumulative effect (7). Small mammalians satisfy the basic requirements for indicator species: they are widely distributed in nature, provide a significant share of matter and energy in the ecosystem metabolism, highly sensitive to external impact, they rapidly respond to environmental changes, capable to be used in laboratory experiments and observations hardly performed on farm animals along, low cost of research (8).
The purpose of this work was to analyze cytomorphological changes in lymphocytes of healthy cows and in cows with leukosis, mastitis, hypothyroidism depending on the background radiation in the habitat area, to compare the obtained data with corresponding parameters of bioindicator species of small rodents and to substantiate the need in use of the Ca²⁺-antagonist enhancing the adaptive-compensatory reactions in cows.
Technique. Soil contamination with ¹³⁷Cs in the Ryazan province after the Chernobyl accident was characterized upon the archival materials of the State Agrochemical Service Station "Ryazanskaya" (1980-2000) and the data from the P.A. Kostychev Ryazan State Agrotechnological University (2000-2007; the results on ¹³⁷Cs content in 30 sites, soil samples collected twice a year from the depths of 0-20 cm and 20-40 cm).
The 20 species of small mammalians including common rat (Rattus norvegicus Berk), voles ordinary (Microtus arvalis Pall), voles red (Clethrionomys glareolus Schreb) were assessed for infestation with Hantavirus (Hantavirus, Bunyavirus), the agent of hemorrhagic fever with renal syndrome (HFRS) using the archival materials dated 2003-2007 of the Federal State Unitary Enterprise "Center for Hygiene and Epidemiology in the Ryazan Province".
Cytomorphological and interior parameters were examined in 2752 rodents (healthy ones and sick with HFRS) inhabitant in the agricultural land. To screen lymphocytograms considering ¹³⁷Cs territory contamination, the experimental groups of animals (10 individuals) were periodically formed: Black-and-White cows (healthy animals, sick animals with leukosis, mastitis, hypothyroidism, hypothyroidism against the use of Ca²⁺-antagonist providing adaptive-compensatory reactions to iodine deficit), voles (healthy and HFRS-carriers) and rats (healthy and HFRS-carriers). Feeding conditions and keeping the cows corresponded to standards accepted in the farms. Blood tests in cows were carried out in the years 1996, 2003, 2005 and 2007, in small mammalians - in 2003, 2005, 2007. Lymphocytograms were studied using the peripheral blood smears stained to reveal content of mucopolysaccharides (Schiff reaction). The proportions (%) of normal lymphocytes and cells with altered content of mucopolysaccharides granules (indicators of immuno- and oncotoxication) were accessed.
The adaptive-compensatory effects of Ca²⁺-antagonist against the natural iodine deficit was determined using nifedipine (the oil solution 0,25 mg/kg live body weight introduced per orally once a month during 6 months).
Statistical processing of data was performed using standard methods of variation statistics (9).
Results. Characteristics of post-Chernobyl radionuclide contamination in the Ryazan province landscape-geographical zones are shown on the map (Fig.). Between 1980 and 1985, the natural background radioactivity of 137Cs in the average for Klepikovsky, Kasimovsky, Ryazansky districts equaled to 0,80-7,40; in Sasovsky - 0,70-5,40; in Shilovsky and Kadomsky - 5,22; in Starozhilovsky, Chuchkovsky, Skopinsky and Putyatinsky - 2,60-5,30 and in Spassky district - 0,80-6,08 Bq/kg. After 1987, the background radioactivity of soil 137Cs has changed: in Klepikovsky and Ryazansky districts, it averaged to 28,50; in Shilovsky and Kadomsky – 51,43; in Starozhilovsky, Chuchkovsky, Skopinsky and Putyatinsky - 56,30-101,80; in Spassky - 34.20 and in Sasovsky – 12,50 Bq/kg.

Sasovsky district was the most demonstrative in respect to 137Cs radionuclide contamination (Table 1). Its territory was just partially subjected to radioactive fallout. Soil of the district is medium-sick layer of chernozem the low-podzol loamy with a humus content of 2,08-9,06% and acidity 0,58-5,85 mg-eq/100 g, pH 5,2-6,8. According to data of the State Agrochemical Service Station “Ryazanskaya, in soil from the JSC “Mayak truda”, ¹³⁷Cs content in the layer 0-20 cm corresponded to 24,4 Bq/kg, in the JSC "Bol’shevik" – 26,0 Bq/kg, which defines this part of the territory as clear (conditionally safe). In the JSC “Named after Clara Zetkin”, 137Cs content at the same depths were higher by, respectively, 15,8 and 14,2 Bq/kg, therefore, this part of the area was defined as contaminated. The soil layer 20-40 cm contains main pool of root systems of forage crops; in the forest-steppe zone of Sasovsky district, this soil layer showed 137Cs radioactivity equal to 15,0-17,1 Bq/kg in the clean area and 17,4-18,1 - in the contaminated area. Thus, there was observed a small radionuclide penetration into the soil owing to its properties and peculiarities of a structure.
In Spassky district, in the LLP “Delo Oktyabrya” located in the clean area of subtaiga zone  (humus content - 1,54-4,61%; soil acidity - 1,30-4,14 mg-eq/100 g; pH 5,1-6 3) radioactivity of the soil layer 0-20 cm was 43,8-51,0, the layer 20-40 cm - 32,0-33,3 Bq/kg. At the same time, in the LLP “Krest’yansky trud” (contaminated area), these indices were higher by, respectively, 51,1-111,8 and 2,4 – 37,6 Bq/kg. In these areas, soil is mainly sod-medium podzol loam providing rapid radionuclide penetration into deep layer and further accumulation in root systems of forage crops. In Putyatinsky and Chuchkovsky districts, radioactive fallout covered the part of the territory while the larger contaminated area than in Sasovsky and Spassky districts. In the contaminated areas, the maximum content of 137Cs at the depth 0-20 cm was 70,5-70,9 Bq/kg, in the layer 20-40 cm – 20,9-21,3 Bq/kg. The soil of these districts (forest loam; humus - 1,75-4,59%; soil acidity - 0,6-5,73 mg-eq/100 g; pH 5,1-7,0) provides quite easy radionuclide penetration to the depth of 20-40 cm. In the conventionally clear part of the district, soil radioactivity for 137Cs in the layer 0-20 cm amounted to 57,7-57,9 Bq/kg and in the layer 20-40 cm – to 31,9-32,3 Bq/kg.
Analyzing the data on Ryazansky and Klepikovsky districts, it should be noted the absence of radioactive fallout after the Chernobyl accident. In this territory, the background levels were considered upon the radioactivity of soil samples collected in the JSK “Dolgininsky” (Ryazansky district) and in the JSC “Makeevka” (Klepikovsky district) (humus 1,63-5,46%; acidity 2,24-5,48 mg-eq/100 g; pH 4,2-5,7). In the contaminated area, the content of 137Cs in soil at the depth 0-20 cm equaled to 25,0-29,3 and in the layer 20-40 cm - 15,2-18,0; in the conditionally safe area – respectively, 24,0-24,7 and 12,0-17,3 Bq/kg.
In Kadomsky and Shilovsky districts (soil density 1,35 kg/dm3; soil acidity 0,68-5,66 mg-eq/100 g; pH 5,1-6,0), the conditionally dangerous area showed 137Cs content in the soil layer 0-20 cm amounted to 57,3-67,2 Bq/kg, the layer 20-40 cm - 20,4-32,3 Bq/kg. In the conditionally safe zone, these indices were, respectively, 27,3-45,0 and 16,5-16,7 Bq/kg, so 137Cs almost didn’t penetrate deeper than 20 cm (the radioactivity of the layer 20-40 cm correspond to the allowable natural background).

During the analysis of lymphocytograms upon their Schiff-reaction, there were compared the data obtained from wild mammalians (rodents) and domesticated cattle. This work allowed more deeply assess the impact of 137Cs area contamination on the gene pool of animals, to identify bioindicator species, to determine the variation range of Schiff-reaction parameters from normal to critical levels. The obtained data can be used in further planning the activities aimed at improving the livestock health in environmentally harmful territories.
In cows from the areas with minimum radionuclide fallout, Schiff-reaction revealed the peripheral lymphocytes containing large granules and blocks: in healthy animals –0,1-0,3% cells, in cows with mastitis - 0,2-0,6%, in cows with hypothyroidism – 0,1-0,5%, in leukotic cows - 0,4-0,9% cells (Table 2). Against the conditionally medium 137Cs contamination, this type of Schiff-reaction (large granules and blocks) was found in 0,5-0,7% lymphocytes of healthy cows, in 0,4-0,6% cells of cows with mastitis and in 2,2-3,0% cells of cows with hypothyroidism. 

In the areas with maximum radionuclide contamination, the most pronounced effects of radionuclide exposure were observed: in healthy cows, large granules and blocks were observed in 0,3-0,5% lymphocytes, in cows with mastitis, hypothyroidism and leukosis – respectively, in 0,2 – 0,9; 0,7-1,0 and 4,0-4,9% lymphocytes. Interestingly that in hypothyroid animals the medium level of ¹³⁷Cs contamination (20,0-32,3 Bq/kg soil) provided more significant effects on Schiff-reaction as large granules and blocks than the high contamination degree (29,6-71,9 Bq/kg). In cows with leukosis, the number of large granules and blocks was sharply increased to be 3,7-4,4% higher than the corresponding characteristic of healthy animals and 3,6-4,0% higher than that in cows with leukosis from the clear zone.
The dynamics of cytochemical parameters in farm animals was found to coincide to that in wild species living in safe zone and in contaminated area (Table 2). Thus, in the ecologically safe zone (no radioactive fallout, natural background radioactivity15,2-18,0 Bq/kg),  voles with HFRS showed large granules and blocks in 0,1-0,7% lymphocytes, which level is comparable with the same parameter in cattle. The increase in radioactivity by 4,8-14,3 Bq/kg provided the shift of this parameter in HFRS voles by an average 0,9%. This trend persisted at the higher radiation background as well (29,6-71,9 Bq/kg): the increase in radioactivity by 14,4-53,9 Bq/kg contributed to 1,3% higher Schiff-reaction indices in lymphocytes of HFRS voles, which data are comparable with similar results in cows low resistant to leukosis.
Rats demonstrated very high resistance to radioactive contamination in the studied areas. Thus, HFRS rats living in clear zone (no radioactive fallout after the Chernobyl accident), in the medium contaminated zone and in the area with radioactive background 29,6-71,9 Bq/kg showed large granules and blocks in, respectively, 0,1-0,3; 0,2-0,6 and 0,4-0,6% lymphocytes, i.e. no reliable differences were found. Therefore, rats can’t be considered as proper bioindicators of environmental pollution. In adverse ecological conditions, voles have shown the cytochemical changes in lymphocytograms similar to those in cows, so voles can be used as test animals that help to predict the state of cattle health under the environmental contamination.
Nifedipine (the blocker of “slow” calcium channels optimizing the intracellular concentration of Ca²⁺) is the highly specific officially recommended Ca²⁺-antagonist (the applied doses allow neglecting its pharmacological properties). Using this pharmaceutical to regulate compensatory reactions to endemic iodine deficit in cows, the following data on lymphocytes with large granules and blocks were observed: 0,1-0,5% in clean areas, 2,2-3,0% in zones with medium contamination and 0,7-1,0% in high-contaminated areas.
In healthy cows, the shares of lymphocytes with Schiff-reaction as medium and small granules equaled to10-16, 26-32 and 20-28% in the districts with, respectively,  low, medium and high contamination for 137Cs (Table 2). Therefore, the most pronounced immunotoxicity for the  lymphocytes pool was observed at the medium radionuclide contamination of the territory (20,0-32,3 Bq/kg), which level was 10-12% higher than that in clear zone and 6,4% higher than in conventionally dangerous zone.
In 137Cs-clear zone, Schiff-reaction in cows with mastitis showed no reliable differences from that in healthy animals (Table 2). The content of such cells in the peripheral blood of cows amounted to 19-50% in medium contaminated areas and 14-48% in territories with high radioactive contamination.
In cows with leucosis, the share of cells with Schiff-reaction as small and medium-sized granules equaled to 10-19% in clear areas; the sharp increase in this parameter (by 51-60%) was detected in medium contaminated zone and the high levels (45-46%) – in territories with high radionuclide content in soil. Therefore, this blood characteristic can be suggested as the marker for low leukosis resistance trend in cattle.
In cattle with hypothyroidism, there were also observed the changes in number of lymphocytes with medium and small granules depending on the background radiation. Thus, in hypothyroid cows from ecologically safe area, this parameter averaged to 10-15%, while 28-44% at the medium soil radioactivity for ¹³⁷Cs (20,0-32,3 Bq/kg) and 32-41% in areas with high radioactivity for ¹³⁷Cs (the upper limit was slightly lower). Thus, the medium radioactive contamination was most harmful for the hypothyroid animals. To improve the animals’ adaptability to the dysfunction of thyroid gland at the endemic iodine deficit, using the Ca²⁺-antagonist nifedipine was established to be efficient: in the same territories, the animals receiving the pharmaceuticals showed lower rates of immunotoxicity parameters of lymphocytes (small and medium-sized granules) than the intact hypothyroid cows of the same age. In particular, in cows treated with the Ca²⁺-antagonist, this parameter amounted to 10-16% in clear zone,  15-16% - in medium-contaminated area (somewhat lower than in intact hypothyroid cows of this area). Under the high radioactive contamination, the nifedipine-treated hypothyroid cows showed 6-21% lower number of such cells than the intact ones.
In voles, the analysis of dynamics in number of lymphocytes with small and medium-sized granules has proved the suitability of using this parameter as the marker in environmental monitoring of areas. In clear zone, it amounted to 11-14%, at the medium level of ecotoxication – 14-18% and in high-contaminated area – 26-33%. In rats – the species resistant to radiation contaminated – the corresponding values were 9-12; 13-17 and 12-15% (Table 2).
Thus, the radioactive contamination with 137Cs in the Ryazan province has resulted in different effects on the gene pool of animals. In Klepikovsky, Ryazansky, Shilovsky and Kadomsky districts, the presence of small and medium granules in the peripheral blood lymphocytes of cattle revealed by Schiff-reaction can be considered as a marker indicator of immunotoxicity. In Spassky and Ryazhsky districts, the large granules and blocks in lymphocytes indicate oncotoxicity reflecting the low resistance to cattle leukosis. The data obtained from the lymphocytograms can be used in breeding work on dairy cattle in radionuclide-contaminated areas. Voles can be considered as bioindicators of radionuclides affecting the gene pool, because they demonstrate the changes in phenotypic parameters of lymphocytes identical to those in cows with leukosis. Rats have shown the greater radiation resistance than other studied animals.
TheauthorsthankthestuffoftheLaboratoryofEcology (The Chair of Bioecology and Animal Hygiene of P.A.Kostychev Ryazan State Agrotechnological University) for kindly presented data on ¹³⁷Cs contamination in soil of the Ryazan province dated 2000-2007.

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