USE OF ELECTROPHORESIS IN DIAGNOSIS OF CATTLE HUMORAL IMMUNITY

Małgorzata Szewczuk¹, Danuta Czernomysy-Furowicz², Ewa Czerniawska-Piątkowska¹

Departament of Ruminant Science¹ and Departament of Immunology and Microbiology², Agriculture University of Szczecin, Poland

ABSTRAKT: The aim of the study was to determine the differences in electrophoretic assays and concentration of total protein and its fractions in blood serum of Black-and-White calves with varied addition of Holstein-Friesian genes. The material comprised 148 samples of serum obtained from whole, non-homogenised blood of calves, collected before 5 days of age and at age 3 months.

In the calves that showed clinical symptoms of diarrhoea, a clear increase in alpha₂ fraction was observed and a slight increase in alpha₁. The concentration of albumin, and thus total protein, was reduced. Increased globulin fractions accompanied by decreased albumin concentration demonstrated a bacterial infection.

In a pneumonia-affected calf's serum, a significant increase in alpha1 and alpha₂ fractions was found with a slight increase in beta₂ fraction. The increase of both alpha fractions and beta₂ fraction is observed with enhanced cellular non-specific response to the disease process. Due to a slight increase in the gamma fraction increase, it can be concluded that M-class immunoglobulins have been synthesised.

An electrophoretic assay, which allows measuring concentrations of individual fractions and albumin-globulin ratio, represents a powerful that can be used in evaluation of immunity and health of livestock animals.

Keywords: calves, electrophoresis, health, acute phase proteins

INTRODUCTION

Electrophoresis, which allows us to determine the concentration of each faction and albumin to globulin ratio, is an important assay that can be used in assessment of immunity and health of livestock animals.

Very often, a lack of clinical symptoms in sick animals may lead to an epizootic, which is particularly dangerous within first months of life of calves managed in large herds. Electrophoresis of blood serum and the analysis of proteinograms allows us to picture the health condition of the animals, also in terms of therapeutic success. The proteins form particular fractions. As a result of inflammation, we observe an increase in the concentration of acute phase response proteins and, consequently, increased concentration of the fractions that contain acute phase proteins (APP). The role of APP in the acute phase response consist in quenching the inflammation process, eliminating the damage factor, healing the damaged tissues and organs, and thus restoring the organism homoeostasis (Kostro et al., 2002). Increased concentration of APP is reflected in the plasma electrophoretic assay in the form of increased concentration of, among others, alpha₁ and alpha₂ globulin fractions (Dembińska et al., 2002). Under a disease condition, the concentrations of haptoglobulin (HP), serum amyloid A protein (SAA), fibrinogen (Fb), alpha-1-acid glycoprotein (AAG), antitrypsin (AT), c-reactive protein (CRP), and alpha₂ macroglobulin (MG) increases (Czokała-Plichta, 2002), whereas the concentration of albumin (Alb) and transferrin (Tf) decrease (Czokało-Plichta, 2002, McNair et al., 1998, Kent, 1992).

Determination of the concentration of fractions and APP in the animal blood serum allows monitoring their health, which enables early detection of inflammations and post-clinical infections – the conditions that reduce weight gains, deteriorate the animal performance, and lead to an economic loss (Kostro et al., 1996; Heegaard et al., 1998; Włodarczyk-Szydłowska et al., 2000). Moreover, altered concentrations of these proteins in the blood serum of beef cattle represent an indication of faulty conditions in terms of welfare (Kostro, 2002).

MATERIAL AND METHODS

The studies took place during 2000-2003 in Western Pomerania, on farms Lubiana and Lubianka as well as Przewłoki and involved Balck-and-White calves with varied addition of the Holstein-Friesian breed.

The material comprised 148 samples of blood serum collected from calves. The serum was obtained from whole blood, non-homogenised, collected from the external jugular vein on the 5^th day after birth and at age 3 months. Total serum protein concentration was measured with burette method. The serum electrophoresis was performed in HR7 hydrogel using a Cormay Diagnostics chamber, whereas the concentrations of fractions were measured in a DS-3 densitometer (Cormay). The values of total protein content (in g/l) were used to convert the percentage of each fraction into their concentrations in g/l.

During the first 3 month of life, the calves were also examined for health. The recorded disease units were divided into three groups: airways diseases (pneumonia, bronchopneumonia), alimentary tract diseases (diarrhoeas of various origin), dermal diseases (mycoses).

RESULTS AND DISCUSSION

The following fractions were found in the studied calves: 1 albumin and 7 globulin fractions, i.e. alpha₁, alpha₂, alpha₃, beta₁, beta₂, gamma₁, and gamma₂. Albumin was the dominant fraction for healthy calves (Figure 1). No increased concentrations of globulin fractions were found, neither any additional peaks that would demonstrate enhanced synthesis of APP.

albumina α1 α2 α3 β1 β2 γ1 γ2

Ryc. 1. Proteinogram surowicy zdrowego cielęcia

Figure 1. Proteinogram of a healthy calf serum

The resulting proteinograms for ill animals showed changes in the fraction levels and additional proteins:

Changes at fraction level

The proteinograms of calves that underwent homoeostasis disturbances look entirely different (Figure 2). Calves with clinical diarrhoea symptoms showed an explicit increase in alpha2 and a slight increase in alpha2 fraction accompanied by reduced concentration of albumin and, consequently, total protein. In various diseases which run with increased permeability of capillaries, albumin tends to escape into the extravascular space, which is accompanied by a drop in the protein serum concentration. Increased globulin fractions accompanied by reduced albumin level may indicate a bacterial infection, which has also been confirmed on calves by Schneider (2003).

B

A

albumina α1 α2 α3 β1 β2 γ1 γ2 albumina α1 α2 α3 β1 β2 γ1 γ2

Ryc. 2. Proteinogramy surowicy cieląt z objawami biegunki

Figure 2. Serum proteinogram of a calf affected with diarrhoea

Increased concentration of both gamma-globulin fractions is typical for polyclonal hypergammaglobulinaemia (Figure 3). Changes of this kind occur during growth processes that are characterised by immunological complexes formed by homogeneous immunoglobulins with molecules of other immunoglobulins or with any other serum proteins. The fraction gamma2 revealed also an additional peak, characteristic for monoclonal hypergammaglobulinaemia. Enhanced synthesis of antibodies by a single B lymphocyte clone can be observed with viral infections. Bacterial infections, in response to which the synthesis of alpha fractions increases, represent a binding agent for viral infections. Therefore, it can be assumed that the calf which revealed clinical symptoms of bronchopneumonia had undergone through viral infection in the first place, followed by a bacterial infection.

albumina α1 α2 α3 β1 β2 γ1 γ2

Ryc. 3. Proteinogram surowicy cielęcia z objawami bronchopneumonii

Figure 3. Serum proteinogram of a calf with symptoms of bronchopneumonia

Figure 4 presents a proteinogram of serum collected from a pneumonia-affected calf. The blood was drawn on the day following the moment when the clinical symptoms had been observed. The serum of the calf contained a considerably elevated levels of the alpha₁ and alpha₂ fractions. The alpha₁ consists of alpha₁-antitrypsin and alpha₁-acid glycoprotein, while alpha₂ contains haptoglobulin and alpha₂-macroglobulin. A slight increase in beta₂ fraction, which contain beta-lipoprotein and C3 complement factor, was also observed (Bigoszewski et al., 2001; Dembińska, 2002; Kostro et al., 1996; 2001; 2002). An increase in both alpha fractions and beta₂ fraction accompanies an increase in non-specific cell response to the disease. Due to a small increase in the gamma fraction, which is formed from immunoglobulins, it may be concluded that synthesis of M-class immunoglobulins has occurred.

albumina α1 α2 α3 β1 β2 γ1 γ2

Ryc. 4. Proteinogram surowicy cielęcia w początkowym okresie pneumonii

Figure 4. Serum proteinogram of a calf in the initial stage of pneumonia

Presence of additional proteins

As a result of infection, the synthesis of immune proteins, which stimulate cellular immunity, accelerates. This enhanced synthesis is manifested in the electrophoretic assay not only through an increased concentration of the fraction, but with additional peaks as well.

albumina α1 α2 β1 β2 γ1 γ2

Ryc. 5. Proteinogram surowicy cielęcia z widocznym dodatkowym białkiem

Figure 5. Serum proteinogram of a calf with an additional protein

During the first 24 hours of pneumonia, serum revealed an increased concentration of c-reactive protein. This protein binds to pneumococcal C polysaccharide, and also activates the classic complement pathway. Serum electrophoretic assay of these calves has revealed an additional peak between beta and gamma fractions (Figure 5). An increase in the protein concentration sometimes precedes clinical symptoms (Bigoszewski et al., 2001; Dembińska, 2002; Kostro et al., 2001; 2002). It is also possible that the fraction also contains A immunoglobulins, whose increase is observed during the first days of infection.

Serum amyloid A protein (SAA) represents one of the first proteins of the acute phase (Bigoszewski et al., 2001; Dembińska, 2002; Kostro et al., 1996; 2001; 2002). An intensive increase in the concentration of this protein is observed within 20 hours after the traumatic agent activates. In a few day-old calf (Figure 6), which had not revealed any clinical symptoms, SAA could have originated from the dam, in which the concentration of this protein had increased during gestation.

albumina α1 α2 β1 β2 γ1 γ2

Ryc. 6. Proteinogram surowicy 3 dniowego cielęcia z widoczną zmianą frakcji albuminowej wywołane pojawieniem się białka SAA

Figure 6. Serum proteinogram of a tree day-old calf with a visible change in the albumin fraction resulting from the occurrence SAA

Local or general immunity suppression caused by endogenous factors (cancerous and autoimmunological diseases) or exogenous factors (infections, stresses, contamination of environment) leads to development of diseases caused by opportunistic microorganisms. Whether we succeed to prevent these diseases heavily depends on the proper health monitoring of the animals. One of the way to evaluate homoeostasis of the animals is to control the behaviour of the acute phase proteins (APP). Surveying the serum APP concentration within the monitoring of livestock animals health enables early detection of homoeostasis disturbances and a quick decision can be reached in order to its restitution (Bigoszewski et al., 2001; Kostro et al., 2001 after Hedstron et al.; Stefaniak, 2000). According to Kostro et al. 2001, measuring APP is particularly useful for identification of inflammations developing with non-symptomatic infections, which are difficult to diagnose. Nevertheless, proteinograms provide visualisation of both fractions concentration changes and APPs. This method may be applied before we assay specific APPs.

Plotting serum proteinograms for the animals, and thus monitoring of their health, allows us to detect disease conditions before clinical symptoms arrive, a quick veterinary treatment, and to avoid an economic loss. Monitoring the immunity of the animals by electrophoresis is of a great practical value and can be readily carried out. Blood may be collected along with official samplings, which does not disturb the farm operations; additionally, the ease of performance and readily available results represent another argument supporting a wider application of proteinograms in cattle disease diagnostics.

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Contact Address:

Dr. Małgorzata Szewczuk

Departament of Ruminant Science

Agriculture University of Szczecin

ul. Judyma 10

71-460 Szczecin

Poland

Tel. 091 4541-521 w. 349.

e-mail: małgorzata.szewczuk@ar.szczecin.pl