Haematological and biochemical parameters of blood and immune response of runt weaners

The study was carried out on 45 pigs weaned at 21 days of age with too low body weights, which is referred to as runting-stunting syndrome (RSS). The animals were divided into 3 groups depending on the cause of RSS (low weight at birth, coliform diarrhoea during sucking, or postnatal difficulties in moving). Haematological and biochemical (plasma levels of total protein, bilirubin, blood Ca 2 and Fe 3 contents) parameters were determined in each group on day 7 after weaning. The results were compared with the parameters determined in normal pigs. Some pigs were immunized with sheep erythrocytes (SRBC) on day 10 after weaning. Half the immunized weaners were treated with levamisole (1 mg/kg) for 4 consecutive days to determine the immune response of animals to this immunomodulating agent. Significant changes in biochemical parameters were observed only in pigs with RSS caused by coliform infections. The immune response of pigs with RSS to the immunomodulating dose of levamisole was limited.

So far, there have been no reports on haematological and biochemical parameters of pigs with RSS, taking into account its causes (low weight at birth, coliform diarrhea during sucking or postnatal difficulties in moving) and the subsequent immune response of the animals.
The aim of the present study was to determine the influence of runting-stunting syndrome on haematological and biochemical blood parameters and the immune response of RSS-weaners to levamisole (1 mg/kg) immunomodulating treatment.

MATERIAL AND METHODS
The study was carried out on 45 pigs weaned on 21 day of age.The animals were allocated to four groups.Each group of pigs was kept in a separate pen with free access to water and fed a commercial feed mixture (Prestarter).In group G-I the pigs (n=12) whose body weight at birth was below 1.1 kg (runt) were marked and observed during the entire sucking period until weaning.Group G-II consisted of 12 animals selected during the observation period, when RS syndrome developed as a result of colibacteriosis.The pigs in group G-II were collected from litters, where acute diarrhoea occurred and pathogenic strains of E. coli had been isolated from dead animals.Group G-III consisted of 12 pigs whose stunting condition was associated with difficulties in moving (parakinesia, congenital muscle hypoplasia, splayleg).The control group G-C consisted of 9 normal pigs randomly selected from the same litters as the affected animals.
Blood samples for haematological and biochemical examinations were collected once, after a 7-day adaptation period.The haematological determinations included: erythrocyte and leukocyte counts, haemoglobin (Hb), haematocrit (Ht), mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC).On the basis of the leukogram the neutrophil index NI (the ratio of band/segmented neutrophils) was determined.The following biochemical parameters were measured colorimetrically in plasma: the level of bilirubin using sulphanil acid and sodium nitrite (POCH), total protein content using copper sulphate and sodium potassium tartrate (Pointe scientific), iron (Fe +3 ) content using chromazurol B and cethyltrimethyl ammonium bromide (Analco) and calcium (Ca +2 ) content using o-cresolphtaleine complexone (Gesellschaft Human Fr Biochemical u. Diagnostica) When 3 days of observation elapsed (ten days after weaning), 16 piglets were selected at random from groups G-I, G-II and G-III and immunized intraperitoneally with 10% sheep red blood cells (SRBC) in a dose of 2 ml/animal.Eight immunized pigs were treated with levamisole hydrochloride (Biowet, Gorzow, Poland), i.m., in a dose of 1 mg/kg BW for 4 consecutive days, starting on the day of SRBC immunization.The blood samples were collected prior to immunization and on days 5 and 9 following immunization.The following immunological parameters were tested: (i) the number of neutrophils; (ii) phagocytic activity of neutrophils with nitroblue tetrazolium dye (percentage of NBT-positive cells) according to Park et al. (1968), (iii) the number of lymphocytes; (iv) percentage of T-cells forming E-rosettes according to Binns (1978), using dextran 110; (v) anti-SRBC haemagglutinin titers: total and 2-mercaptoethanol resistant (mainly IgG) by active haemagglutination test as described by Adler (1965).The data were analyzed using Statgrafics ver.5. 1.

RESULTS AND DISCUSSION
The body weight of normal 21-day-old weaners from group G-C averaged 4.96 kg (Table 1).On the weaning day the weight deficiency of RSS-pigs, as compared with GC was: 1.87 kg (G-I); 1.40 kg (G-II), and 0.93 kg (G-III), respectively (PO.01).The body weight gain of RSS-pigs (mean of G-I, G-II and G-III) was 36.7% lower than that of normal pigs in group G-C.Average daily gain (ADG) in G-I and G-II was nearly the same, but 17.6% lower than that in G-III.Pigs runt at birth (prenatal runting) are not able to reach normal body weight long after birth (Balenovic et al., 1994), but if they are separated from the litter and fed artificially, they grow faster and are able to achieve a body weight gain comparable to unaffected pigs (England, 1986;Muirhead, 1990).The results presented in this study show that infectious diseases of piglets with normal weight at birth (G-II) affect body weight gain to a similar degree as runting syndrome (G-I) whereas • noninfectious diseases (G-III) make the syndrome of stunting less pronounced.The haematological parameters in pigs from G-I, G-II and G-III were not significantly different, and they did not differ from the control group, either (Table 2), hence, the mean values did not exceed the physiological standards (Friendship et al., 1984;Klopocki et al., 1987;Vigo et al., 1992) examined haematological indices in runt pigs and compared the results with the respective values of normal pigs.They found slight differences between those groups in erythrocyte, leukocyte, Hb and Ht contents.No differences in MCV, MCH and MCHC were observed.
In group G-II, the total protein content was 10.8 % lower (P<0.05),bilirubin content was 25.1% lower (PO.05) and Ca +2 content was 8.0 % lower (P<0.05),as compared with G-C.The Ca +2 content in group G-III was 10.6% lower (PO.05)than that of the control group (G-C).No differences in the indices measured were found between G-I and G-C (Table 2).Correlation coefficients for the entire RSS population were as follows: body weight x total protein, r=0.40 (P<0.02);body weight x Ca +2 , r=0.45 (PO.01) and body weight x Fe +3 , r=-0.37 (PO.04).The differences in neutrophil index (NI) were found between the groups (Table 3).In group G-C the NI was the highest (0.141) and comparable to that in group GUI (0.134), while the lowest value was noted in G-I pigs (0.08).Bartnicka and Kondracki (1984) claim that the shift of the white cell picture toward immature forms may indicate higher granulopoesis ability or higher concentration of young neutrophils in the bone marrow.As far as the remaining values of the leukogram are concerned, no significant differences between the groups were found.The eosinophil populations in G-III and G-I were 36.3 and 29.3% higher than in group G-C, respectively.The most significant differences in haematological and biochemical parameters (Table 2) were found in the pigs which had been infected with colibacteriosis (G-II).Coliform diarrhoea leads to malabsorption and dehydration which in turn impair homeostasis (Depta, 1984;Wojcik, 1985).G-II pigs show that infectious diseases during the sucking period not only inhibit body weight gain, but also affect biochemical indices after weaning.In group G-II the correlation coefficient between Fe +3 content and the number of NBT-positive cells was significant (r = -0.71;P<0.01).This may suggest enhanced neutrophil activity in the pigs low in serum Fe +3 due to infectious diseases.Gherke (1989) reports that reduced Fe +3 concentration in blood does not allow the microorganisms to restore Fe +3 to levels sufficient for enzymatic processes in cells.
G-III pigs (having difficulties in moving) were exposed to chronic stress with limited access to feed, resulting in malnutrition and emotional disorders due to lower rank in the litter hierarchy.Fitko et al. (1988) indicate that this somaticemotional stress affects haematological and biochemical parameters to a lesser degree than acute stress (shift to different nursery groups, transportation).
Figure 1 shows immunological indices of SRBC-immunized RSS-pigs.There was a decrease in the number of lymphocytes (20%) and the increase (P <0.05) in neutrophils (40%) on day 9 following SRBC immunization.On day 5 following SRBC immunization, anti-SRBC haemagglutinin titer (IgG class) accounted for 50%o of total SRBC haemagglutinin titer and reached 60% on day 9 after immunization.The effects of levamisole on phagocytic activity of neutrophils (NBT), the percentage of T-lymphocytes forming E-rosettes and total anti-SRBC haemagglutinin titers in serum were insignificant, but levamisole significantly increased the class IgG titer of anti-SRBC haemagglutinins, which on day 9 accounted for 80% of specific haemagglutinins.
Levamisole is an immunomodulating agent commonly used in veterinary practice (0.5-2.0 mg/kg).It is well-known for its ability to restore T-lymphocytes and humoral response to T-dependent antigen, such as SRBC, and stimulate neutrophil activity.The mechanism of levamisole activity is well known (Renoux, 1985;Switala, 1992), therefore it is used in research as a reference agent.The enhanced production of IgG specific antibodies coinciding with the primary humoral response to T-dependent antigen is connected with the stimulation of T-lymphocyte activity, which is a typical effect of levamisole.
Our results indicate that levamisole action in RSS-pigs was seriously limited, as it did not affect the activity of neutrophils and percentage of T-lymphocytes forming E-rosettes.However, levamisole administered to older RSS-weaners increased the indices significantly (Switala, 1992;Kolacz et al, 1994).Hennesy et al. (1987) report that levamisole affects cellular immune response of piglets during the first four weeks of age.

CONCLUSIONS
The stunted growth of young pigs suffering from coliform infections during the sucking period corresponded with significant changes in biochemical parameters of blood.In pigs showing difficulties in moving, the stunting syndrome was less advanced than in the other groups.No changes in haematological and biochemical parameters of pigs with low body weight at birth were found, however, the runts were not able to reach the body weight of normal pigs.The effect of levamisole injected in a low, immunomodulating dose (1 mg/kg) to runts weaned 21 days after birth and SRBC-immunized on day 10 after weaning, was limited.

Figure 1 .
Figure 1.Immunological indices in runt pigs immunized with SRBC

TABLE 2
Haematological and biochemical parameters in weaners