LEVELS OF MINERALS IN SERA OF CHICKENS SUFFERING FROM MAREK ' S DISEASE

Jurajdova J.: Levels of Minerals in Sera of Chickens Suffering from Marek's Disease. Acta vet. Brno, 47, 1978: 189-196. The concentrations were studied of sodium, potassium, calcium, magnesium and phosphorus as well as the correlations and coefficients of their level ratios in sera of RIR chickens 56 days after infection with Marek's disease virus, and they were compared with the controls. In the infected chickens a reduction of the concentration of calcium was observed, with the exception of chickens suffering from tumors, and an increased concentration of magnesium which was the highest in chickens with tumors. In chickens with tumors a reduced phosphorus level was observed, while in those with macroscopically unchanged kidneys also a decrease in the sodium concentration was found. In infected chickens the coefficient of the Ca/Mg ratio was reduced and reached its maximum value in tumorous chickens. Correlations between calcium and phosphorus found in control chickens were not proved in the infected chickens and no other were observed. Marek's disease, chickens, serum, mineral substances. Properties of the internal milieu of the organism were maintained in values optimal for cel and tissue activity. The stability of the internal milieu is ensured through the activity of many complicated regulation mechanisms under the direct coaction of biocatalysts among which we include mineral substances as well. Studies dealing with the importance of mineral substances in proteosynthesis (Greger and Schwartz 1974; Rubin 1975), oncogenicity (Zyka 1972; Sula 1974; Anast et al. 1975; Brennan et al. 1976) or with the effect of chelates in in vitro experiments on the yield of Marek's disease virus (Aldinger and Calnek 1971) led us to studies of the metabolism ofminera\ substances in Marek's disease in in vivo experiments with chickens. Material and Methods Experimental chickens Experiments were carried out with one-day-old chickens of Sykes Line B Rhode Island Red (RIR) from the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague, susceptible to infection with the Marek's disease virus (Biggs et al. 1968). Chickens were infected aerogenically with dust from an enterprise with a permanent occurrence of acute Marek's disease (MD) and stored for 95 -118 days after collection underiaboratory temperature in a way described earlier (Jurajda and KIimeS 1970). The infectiousness of the dust was proved by in vitro examinations in cell cultures of chicken kidneYl! 7.25 X 102 PFU/g of dust (Hlozanek et al. 1976).


Preparation of the cutaneous MD-antigen
Feathers from the MD-positive chickens with tumors, infected with the MD virus earlier, were cut off as nearest to the skin as possible in such a way as to leave the quill shaft in the skin.
A 20 % (w/v) homogenate in a buffered physiological solution (BPS, pH 7.2) was prepared from the skin with an addition of 0.2 % of EDTA (Chelaton III) and antibiotics.After three repeated CYcles of freezing and thawing (mixture of solid CO. in methylalcohol and water bath at 37°C) the homogenate was centrifuged for 15 minutes at 1.000 g.The supernatant was divided into am~ pules of 0.5 ml each and stored at -60°C in dry ice and served as a positive MD skin antigen for preparation in agar gel test.

Agar gel precipitation (AGP) test
Used was a double diffusion according to Ouchterlony in Petri dishes of 10 cm in diameter filled with agar according to Okazaki et aI. (1970): 1 % of Noble agar (Difco) in a NaK phosphate buffer with 8 % of NaCl, pH 7.4.The central well was filled with the MD skin antigen and the marginal wells with sera tested.Petri dishes were stored in an atmosphere saturated with water vapours at a laboratory temperature and results were read after 24 and 48 hours.A serum from the MD-positive chickens with tumors earlier infected with a-MD virus served as a control.

Biochemical examinations
After finishing the experiments the chickens were killed by bleeding out and the concentrations of macro-elements (Na; K, Ca_ and Mg) in the serum were determined using the method_ of atomic absorption spectrophotometry on an Atomspek apparatus of the firm Hilger and Watts and photometrically (P) using Bio/Lachema tests as described in earlier studies (Juraj dovil 1978).From the values measured the coefficients were calculated of ratios of levels of the individual elements.Statistical evaluation of the results and correlations of the levels of the individual elements was done using a Hewlett Packard computer,9810A model, by means of Duncan's and Student's t-test.

Experimental procedure
Aerogenically infected were 264 chickens (i.e. 135 females and 129 males); 71 chickens served as controls (36 females and 35 males).Both groups were kept in isolation, fed complete mixtures for chickens BR I-start, and given water ad libitum.Chickens which died during the experiment were dissected and results of the dissections were recorded.After finishing the experimentt 56 days after infection, the chickens were examined for the presence of macroscopic changes or MD-precipitation antibodies; according to the results they were divided into 5 groups: (1) control, (2) infected but with no macroscopical changes and MD-precipitation antibodies (MD-neg), (3) with MD-precipitation antibodies (MD-Ab), (4) with macroscopical changes (AGP was not performed) (MD-tum), and (5) MD-positive (MD-pos), including groups (3) and (4).The features investigated were then mutually compared in all groups, also with regard to the effect of sex.

Results and Discussion
A survey of the occurrence of Marek's disease in infected RIR chickens is given in Table 1.The average levels of minerals (in mmol/l), coefficients, and correlations of their levels in sera of control and infected chickens are given in Tables 2-4.   3) Significant difference as compared with group with MD-precipitation antibodies (MD-Ab) at P < 0.01 §) Mean ± standard error of mean ' • Significant at P"< 0.05 The occurrence of MD in experimental RIR chickens after infection with dust containing the MD virus was similar as after infection with other strains of acute MD (Purchase and Biggs 1967;Eidson and Schmittle 1968;Bankowski et al. 1969, and others).In contradistinction to the affinity of the acute MD virus to the gonad tissue (Biggs and Payne 1967;Chomiak et al. 1967;Purchase and Biggs 1967) found most often were tumorous changes in the kidneys, liver and spleen.The total occurrence of tumorous changes was lower than described in the RIR chickens after infection with acute strains of HPRS (Biggs et al. 1965;Purchase and Biggs 1967).
The differences from Na concentrations in the serum of control groups were observed only in MD-tumorous bird.In females a decrease of 4.74 % (p < 0.01) was observed, and in chickens in total it was 3.05 % (p < 0.01).With regard to the importance of kidneys as the main organ of Na excretion, we observed the Na levels in the serum of these birds with regard to the macroscopical affection of the kidneys.It was found that the level of Na in the serum of birds with kidneys afflicted with tumors did not differ from the controls.On the other hand, the so- .)Significant difference as compared with group with MD-precipitation antibodies (MD-Ab) at P < 0.05 .)Significant difference as compared with group with MD-precipitation antibodies (MD-Ab) at P < 0.01 S Mean ± standard error of mean • Significant at P < 0.05 •• Significant at P < 0.01 dium concentration (137 and 139.7 mmol/l) in females arid in chickens in total, resp., with macroscopically unchanged kidneys but with tumors of other visceral organs was found to differ significantly from control values (P < 0.01).
The effect of the degree of affliction of kidneys was observed also in other features but with a negative result.
The level of potassium in the serum of infected birds did not reveal and deviations from the control values, the only exception being a lower concentration of K in chickens with MD-precipitation antibodies by 6.18 % as compared with controls (P < 0.05).
. The most evident changes of mineral concentrations in the serum of MD-infeeted birds were observed in calcium and magnesium.The Ca level was decreased in MD-negative birds (in chickens by 8.66 %) and bird with precipitation antibodies (in chickens by 7.53 %), while in birds afflicted with tumors the Ca concentration did not differ from that of controls.In magnesium, on the contrary, an increase was observed in the group of birds with MD-predicitation (in chickens by 6.67 %), the maximum occurring in birds afflicted with tumors.Significant was also the difference in Mg concentrations in the serum of females and males afflicted with tumors.Lansing et al. (1948) found a lower Ca content in the tumorous tissue as compared with a healthy tissue.The reduction of the level of serum albumin observed together with some kidney afflictions is sometimes accompanied with a reduction of the level of serum Ca (Mazur and Harrow 1971).Samadieh et al. (1969), Washburn and Eidson (1970), Bankowski et al. (1970), andFletcher et al. (1972) descri~ed the reduced level of serum albumin in MD--positive chickens.However, this does not correspond with our findings in birds with macroscopically changed kidneys in which the values of serum Ca did not differ from the controls.
The metabolism of calcium and magnesium is closely related with regard to their common mechanism of resorption in the intestine and renal tubules.Mutual competition of Ca and Mg in the organism appears with the insufficiency of one ion by increasing the concentration of the other.Besides a decreased capacity of the kidneys to excrete magnesium, also the affliction of liver and suprarenal gland leads to an increase of the plasmatic magnesium level.Hypermagnesemia occurs often due to the transmission of Mg between the intra and extra-cellular space, i. e. magnesium from the cells due to cellular catabolism (Fencl 1960;Kapitola and KiicheI1968).A more detailed explanation of the causes of hypocalcemia and hypermagnesemia in MD-positive birds calls for further investigations and studies.
The concentration of phosphorus in the serum of infected birds decreased only in males afflicted with tumors (by 11.53 %) and in chickens in total (by 9.05%).The effect of sex on the P level in serum was recorded only in the MD-negative group, even though the P concentrations found did not themselves differ from the control values.
With regard to the decrease of the Ca concentration observed and increase of Mg in the serum of infected birds, also the coefficient of the Ca/Mg ratio decreased, at the same time a maximum decrease was observed in birds afflicted with tumors (in chickens by 14.95 %).Alexandrowicz et al. (1970) observed a similar decrease of the Ca/Mg ratio coefficient in leukemic cows (2.68) as compared with healthy animals (3.20).
The increase of magnesium concentration in the serum probably affected also the decrease of the K/Mg ratio coefficient in the serum of birds with MD-precipitation antibodies (by 12.63 %), even though the change of K in the serum of this group of birds was not expressive.Close relationships between potassium and magnesium, attributed to their mutual intracellular deposition and to some common regulation mechanisms, are considerably variable and probably depend also on the condition of the organism as well as on the actual concentration of both ions (Kapitola and Kiichel 1968).According to Zyka (1972) the disturbance of the K/ Mg ratio is considered to be a carcinogenic factor.
Correlations of greater significance were observed in the control group only in females (r = 0.6738 at P < 0.01) and then in total in chickens (r = 0.5501 at P < 0.01) between Ca and P levels.In infected birds this correlation was expressed only in MD-negative females (r = 0.5643); in addition, in some cases we found positive correlations between Na/K (r = 0.5920) and K/Ca (r = 0.6381) levels.In the remaining groups of infected birds, the correlations between the studied levels of minerals were minimum to non-significant.
Results obtained in infected RIR chickens indicated .that in the course of Marek's disease the metabolism of mineral substances was also affected.A more detailed explanation of the deviations found (as compared with control values) calls for further investigations and studies to say nothing of the necessity of a thorough analysis of the metabolism of mineral substances in healthy poultry.

Table 1
Occurrence of Marek's disease (MD) in infected RIR chickens

Table 3
Average levels of minerals (mm31/1), coefficients and correlations of their levels ill sera of experimental RIR males -MineralS I Natrium (Na) I Kalium(K) I Calcium (Ca) I Magnesium (Mg) I ') Significant difference as compared with control at P < 0.05 ') Significant difference as compared with control at P < 0.01 ') Significant difference as compared with MD-negative (MD-neg) group at P < 0.05 ') Significant difference as compared with MD-negative group at P < 0.01 I I