Changes in serum protein electrophoresis profiles and acute phase proteins in calves with diarrhea

Abstract

Calf diarrhea leads to substantial economic losses in the livestock industry worldwide due to medical treatment costs, retarded growth performance, and even death. The objective of this study was to investigate changes in serum protein profiles and acute phase proteins in calves with diarrhea and identify the association between these changes and diarrhea. A total of 185 Korean beef calves were used and divided into 3 groups by age: 1 to 10 days (n = 46), 11 to 20 days (n = 65), and 21 to 30 days (n = 74). Blood and fecal samples were collected from each calf. Serum concentrations of total protein, protein fractions (albumin, α1-globulin, α2-globulin, β-globulin, and γ-globulin), haptoglobin (Hp), and serum amyloid A (SAA) were analyzed. Compared to calves without diarrhea, calves with diarrhea had significantly lower albumin concentrations at 11 to 20 days and 21 to 30 days of age (P = 0.017 and P = 0.000, respectively) and significantly higher α1-globulin fractions at 21 to 30 days of age (P = 0.01). Interestingly, α2-globulin fractions were significantly higher in diarrheic calves in all age groups, whereas γ-globulin fractions were significantly lower in calves with diarrhea aged 1 to 10 days, compared with normal animals. In calves with diarrhea, the concentration of Hp was significantly higher, whereas SAA levels were not different between normal and diarrheic calves. In addition, a positive correlation was found between α2-globulin and Hp (P = 0.0004). Taken together, these results provide useful information about the use of serum protein profiles and Hp as prognostic and diagnostic markers for animal health status.

Introduction

Diseases that occur during breeding of cattle can lead to substantial economic losses in the livestock industry due to medical treatment costs, retarded growth performance, and even death. Neonatal calves have a greater tendency to contract various diseases from birth, which in serious cases, may result in mortality (1,2). In particular, exposure to infectious agents through the oral route and the respiratory tract contributes to disease occurrence in calves, as newborn calves are easily exposed to external environmental contaminants during delivery. Therefore, delivery facilities housing neonatal calves should always be kept clean and isolated from adult cattle to prevent potential infections from pathogens. Overall, prevention and control of diseases during this period are very important because if these factors are neglected, poor animal management systems and inappropriate breeding methods can lead to increased disease incidence.

Neonatal calf diarrhea is associated with a high worldwide mortality rate within the first month of life (3). Calf diarrhea may be attributed to multifactorial etiologies, including various infectious agents, animal management systems, hygiene, and nutrition (4). Of the numerous infectious agents causing calf diarrhea, the focus is mainly on individual pathogens such as bovine viral diarrhea virus (BVDV), coronavirus, norovirus, rotavirus, torovirus, Clostridium spp., Escherichia coli, Salmonella spp., Cryptosporidium parvum, Eimeria spp., and Giardia duodenalis (5–9). In addition, because neonatal calves are highly vulnerable to these pathogens, management of animal facilities and care, appropriate feeding, and immunity status of calves during this period are very important in preventing major diseases such as diarrhea and respiratory disorders. Therefore, immediate and rapid colostrum intake after calving should be performed, as it is necessary for preventing diseases in neonatal calves that are completely dependent on passive immunity.

Acute phase proteins (APPs) are plasma proteins synthesized by hepatocytes that appear during the acute phase response, including infection, tissue injury, neoplasia, and inflammation (10). Acute phase proteins serve as the core of the innate immune response and are conserved across animal species (11,12). Changes in the concentrations of APPs are recognized as a useful tool for evaluating cattle health, and haptoglobin (Hp), serum amyloid A (SAA), fibrinogen, and α1-acid glycoprotein are the most common APPs in cattle (8,13). Several studies have shown that APPs have good properties as markers of respiratory infections in calves (14–17); however, there are limited data available on APPs as disease markers for calf diarrhea.

Immunoglobulin concentrations in serum can be evaluated through electrophoretic techniques. The determination of serum protein fractionation is of important diagnostic value in clinical biochemistry and serum protein electrophoresis is a laboratory test that separates serum proteins into albumin and globulin. The latter is then subdivided into α-, β-, and γ-globulins (18–21). Therefore, the objective of this study was to investigate the differences in serum protein profiles and APPs between healthy and diarrheic calves during the first month after birth and to identify any association between the changes in serum protein patterns and/or APPs and diarrhea. The results obtained in this study may provide valuable information for veterinary clinicians to predict and treat animal physiological status.

Materials and methods

Experimental animals

This study was conducted on 185 Korean beef calves (≤ 30 d of age) being raised in the Republic of Korea. Calves were classified by age into 3 groups: 1 to 10 d (n = 46), 11 to 20 d (n = 65), and 21 to 30 d (n = 74) (Table I). The physical activity of all calves used in this experiment was noted as “bright,” which is a state of normal response to stimuli. No dehydration was observed in diarrheic calves.

Table I.

Classification and total numbers of calves used in this study.

Classification of calves	Age
	1 to 10 d	11 to 20 d	21 to 30 d	Total
Normal	18	39	48	105
Diarrhea	28	26	26	80
Total	46	65	74	185

Blood and fecal sampling, and detection of diarrheal pathogens

Blood was collected from the jugular vein of each calf. Samples were divided into tubes and transferred to the laboratory on ice for a complete blood (cell) count (CBC) and a serum test. An experienced veterinarian directly collected feces from the rectum and fecal samples were transported to the laboratory. Feces were subdivided into solid, semi-solid, loose, and watery stools. Feces that were considered solid and semi-solid were classified as normal and those that were loose and watery were classified as diarrhea. DNA and RNA were extracted from 200 mg of each fecal sample using an AllPrep PowerFecal DNA/RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s directions and stored at −20°C until ready for use. Real-time polymerase chain reaction (RT-PCR) was performed to detect BVDV, coronavirus, rotavirus, E. coli and Salmonella spp., C. parvum, and G. duodenalis in the samples as previously described (22,23). For the detection of Eimeria species, fecal samples were analyzed for the presence of oocysts using the flotation method with Sheather’s solution (saturated sugar solution; specific gravity = 1.28) and examined microscopically (400 × magnification). One or more of the pathogens related to diarrhea were detected in diarrheic calves, whereas no pathogens under investigation were detected in normal calves.

Serum protein gel electrophoresis

Serum samples were separated from blood by centrifugation. Subsequently, agarose gel electrophoresis was performed to analyze 5 protein fractions (albumin, α1-globulin, α2-globulin, β-globulin, and γ-globulin) using a semi-automated agarose gel electrophoresis system (HYDRASYS 2; Sebia, Camberley, United Kingdom), following the manufacturer’s protocols. Briefly, 30 μL of serum was subjected to the microtechnique assay, electrophoresed for 35 min, stained for 5 min, de-stained for 5 min, and cleared for 30 s. Excess solution was removed with a glass rod and samples were dried for 10 min and then measured by optical density scanning (HYDRASYS; Sebia). Normal serum was used as a control for measurement accuracy. The results of the serum protein electrophoresis gel were reviewed and interpreted by a laboratory expert.

Acute phase proteins

Analyses for Hp and SAA were performed in serum. The concentrations of Hp were assessed using commercial colorimetric kits (Tridelta Development, Kildare, Ireland) based on the hemoglobin-binding assay. The SAA was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) kits (Tridelta Development). The optical densities were read on a microplate reader (BioTek Instruments, Winooski, Vermont, USA) at 630 nm for Hp and at 450 nm and 630 nm as a reference for SAA.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis (i.e., Wilcoxon signed-rank test) of protein fractions between healthy and diarrheic calves was performed using SPSS Statistics version 25 for Windows (IBM, Armonk, New York, USA). A P < 0.05 was considered significant. Correlations between Hp and a globulin selected from the diarrhea group were evaluated using Spearman’s correlation. Correlation coefficients (r) < −0.4 and > 0.4 were considered to be significant negative and positive correlations, respectively.

Results

Of the pathogens examined from 80 diarrheic calves, rotavirus, BVDV, E. coli, Eimeria spp., C. parvum, and coronavirus were detected in 36 (45%), 13 (16%), 13 (16%), 11 (14%), 4 (5%), and 3 (4%) calves, respectively. Giardia duodenalis and Salmonella spp. were not found in diarrheic calves. The prevalence of rotavirus was the highest, whereas the incidence of coronavirus was the lowest in pre-weaned Korean native calves.

Overall, the concentrations of total protein increased with age in calves with diarrhea, but there were no significant differences noted (Table II). Albumin concentrations steadily increased with age in both normal and diarrheic calves and this increase was much greater in normal calves than in diarrheic calves (Table II). Albumin concentrations in calves with diarrhea were significantly lower at 11 to 20 d (P = 0.017) and 21 to 30 d (P = 0.000) compared with normal calves at those ages. The globulin fractions between normal and diarrheic calves are shown in Table I. The α1-globulin fractions were slightly higher in calves with diarrhea after 10 d than in normal calves, although statistical significance was observed only in diarrheic calves aged 21 to 30 d (P = 0.001; Table II). The α2-globulin fractions were also significantly higher in calves with diarrhea compared with normal calves for all age groups (Table II). The β-globulin fractions decreased with age in normal calves, whereas β-globulin fractions gradually increased with age in calves with diarrhea and were significantly higher in calves with diarrhea aged 21 to 30 d (P = 0.000; Table II). When comparing γ-globulin fractions between the 2 groups, the γ-globulin fractions in normal calves gradually reduced with age, whereas there was not much difference across ages for diarrheic calves. The γ-globulin fractions were significantly lower in calves with diarrhea aged 1 to 10 d (P = 0.000; Table II).

Table II.

Concentrations of total protein and albumin and globulin fractions (α1, α2, β, and γ) in serum of calves according to age. Data are expressed as mean ± standard deviation. P-values were obtained using the Wilcoxon rank-sum test to compare normal calves with diarrheic calves.

Variables (g/dL)	Fecal consistency	Age
		1 to 10 d	11 to 20 d	21 to 30 d	1 mo
Total protein	Normal	6.6 ± 1.2	6.7 ± 0.7	6.1 ± 0.6	6.2 ± 0.7
	Diarrhea	6.1 ± 1.5	6.3 ± 0.9	6.4 ± 0.9	6.3 ± 1.1
Albumin	Normal	2.8 ± 0.3	3.3 ± 0.3	3.7 ± 0.3	3.4 ± 0.4
	Diarrhea	2.7 ± 0.7	3.0 ± 0.5**	3.2 ± 0.5***	2.9 ± 0.6
α1-globulin	Normal	1.1 ± 0.3	0.7 ± 0.1	0.5 ± 0.1	0.7 ± 0.2
	Diarrhea	1.1 ± 0.3	0.8 ± 0.4	0.8 ± 0.3***	0.9 ± 0.3
α2-globulin	Normal	0.3 ± 0.1	0.4 ± 0.1	0.4 ± 0.1	0.4 ± 0.1
	Diarrhea	0.9 ± 1.4**	0.9 ± 1.0*	0.7 ± 0.3***	0.8 ± 1.0
β-globulin	Normal	1.0 ± 0.4	0.9 ± 0.2	0.8 ± 0.2	0.9 ± 0.2
	Diarrhea	0.8 ± 0.3	0.9 ± 0.3	1.1 ± 0.3***	0.9 ± 0.3
γ-globulin	Normal	1.4 ± 0.9	0.8 ± 0.5	0.7 ± 0.4	0.8 ± 0.6
	Diarrhea	0.6 ± 0.5**	0.8 ± 0.4	0.6 ± 0.4	0.6 ± 0.4

^*P < 0.05;

^**P < 0.01;

^***P < 0.001.

The globulin composition ratio between normal and diarrheic calves is presented in Figure 1. The α1-globulin fractions were not different between the 2 groups, whereas the α2-globulin fractions were significantly higher in diarrheic calves of all ages compared to normal calves. The β-globulin fractions were significantly lower only in diarrheic calves aged 11 to 20 d. When compared to normal calves, the γ-globulin fractions in diarrheic calves were lower for all age groups and significantly lower in calves aged 1 to 10 d.

Figure 1.

The comparison of globulin composition ratios (α1, α2, β, and γ) between normal and diarrheic calves. The number in the box represents the mean of each globulin fraction.

The concentrations of Hp and SAA were compared between normal and diarrheic calves. As shown in Figure 2, the Hp concentrations in calves with diarrhea of all age groups were significantly higher than in normal animals. Haptoglobin concentrations in the serum were at least 5-fold higher in calves with diarrhea compared to clinically healthy calves. The concentrations of SAA were higher in calves with diarrhea compared to normal calves, but differences were not statistically significant. A positive correlation was found between α2-globulin and Hp in calves with diarrhea (P = 0.0004; Figure 3), whereas there were no correlations between α1-globulin and SAA in these calves.

Figure 2.

Haptoglobin (Hp) (A) and serum amyloid A (SAA) (B) concentrations (mean ± SEM) in the serum between normal calves and calves with diarrhea by age. The concentrations of Hp were significantly increased in calves with diarrhea. A P < 0.05 according to the Wilcoxon rank-sum test was considered significant.

N — normal; D — diarrhea. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Correlation between haptoglobin (Hp) and α2-globulin. The concentrations of Hp were positively correlated with α2-globulin (P = 0.0004) in calves with diarrhea.

Discussion

This study used electrophoresis analysis to evaluate changes in serum protein and APP profiles due to diarrhea. The results showed that the composition of albumin and globulin fractions were markedly different between normal calves and calves with diarrhea. In terms of the globulin composition, the most significant differences between the 2 groups were observed in α2-globulin and γ-globulin fractions. The concentrations of Hp in the serum were significantly elevated in calves with diarrhea and this increase was associated with α2-globulin. These findings suggest that the changes in serum proteins (especially α2-globulin and γ-globulin) and Hp may be used as prognostic markers to predict and diagnose the health status of animals.

Albumin is the major protein component of serum. It is mainly produced in the liver and is involved in the transport of water or metabolites from tissues. One of the main functions of albumin is to maintain colloid osmotic pressure (24–27). Several studies have shown that albumin is decreased in cases of malnutrition, infection, chronic liver disease, and kidney disease. According to our results, albumin was decreased in calves with diarrhea compared to normal calves, indicating that diarrhea causes a loss of albumin in the contents of the intestine. In addition, the loss of albumin in calves with diarrhea increases when large volumes of fluid, ingesta, and hypertonic solutions are present in the intestinal lumen (28). It is speculated that lower albumin may be associated with disturbed absorption of nutrients or water loss due to diarrhea and with increased levels of various inflammatory markers, such as APPs. Given our results, diarrhea in calves was accompanied by a reduction in albumin, suggesting that low levels of albumin may indicate a higher risk of disease and poor health of animals.

Globulins comprise a much smaller protein component and are divided into 4 categories: α1-, α2-, β-, and γ-globulins. In this study, of the 4 globulin fractions, α2-globulins were present at significantly higher levels in calves with diarrhea compared to normal calves. A previous study showed a significant association between diarrhea and α2-globulins in calves (21) and our findings are consistent with that result. The α2-globulins include some APPs, such as Hp, ceruloplasmin, and α2-macroglobulin, and are involved in inflammation (29). Diarrhea in calves usually causes inflammation of the intestines. As a result, the increase in α2-globulin supports the conclusion that inflammation had already developed in diarrheic calves.

The γ-globulin fraction is actively involved in the defense system against infectious agents (30,31). According to our results, overall γ-globulin concentrations were low in calves with diarrhea. The concentrations of γ-globulin also showed a decreasing trend with age in calves. The progressive decline of γ-globulin concentrations in normal calves may be attributed to the degradation of colostrum-derived immunoglobulins and a gradual initiation of production of immunoglobulins by their maturing immune system. Contrary to the explanation of this change in normal calves, the reason for γ-globulin reduction in calves with diarrhea may be the shift from γ-globulin to α2-globulins. As such, a decrease of γ-globulin may confer lower immune resistance when calves need protection from various infections, resulting in worsened clinical symptoms. Consequently, these results indicate that γ-globulin levels in serum may be influenced by the health status of calves.

In this study, the most marked difference between the 2 groups was in the concentrations of Hp. Haptoglobin concentrations in calves affected by diarrhea were more than 5-fold higher than in normal calves; in contrast, SAA levels did not exhibit changes with diarrhea. A previous study performed by our group showed that the concentrations of SAA increased in coronavirus-infected calves, but it was not to a statistically significant extent (10). The biggest difference between the current and previous experiments is the age of the calves used; consequently, with the results from the current study, SAA may be a less suitable diagnostic parameter for the assessment of cattle health. Moreover, Hp has been used in detecting disease status and inflammation (32). In several studies, Hp concentrations in serum were found to be significantly increased in calves with respiratory diseases, suggesting that there were associations between serum Hp concentrations and respiratory tract disease (14,32–34). Angen et al (35) reported that although SAA concentrations increased in calves with respiratory disease, the values did not show a significant difference when compared to changes in Hp. This result was consistent with the present study. Haptoglobin is a good marker to identify calves with respiratory disease. The concentrations of Hp were significantly higher in calves with diarrhea, indicating that this phenomenon may be associated with increased α2-globulin concentrations. In addition, there was a positive correlation among Hp, α2-globulin, and diarrhea. Taken together, these results suggest that Hp may be important as a sensitive marker that is preferable in the field and can be useful for monitoring the disease status of animals because of its larger and more prolonged response.

The present study showed that changes in serum protein electrophoretic profile and Hp levels are markedly influenced by the health condition of calves. Significant differences were found for α2-globulin and γ-globulin in calves with diarrhea. Moreover, Hp concentrations in calves with diarrhea were significantly increased and revealed a positive correlation between α2-globulin and diarrhea. These results provide valuable information for clinicians who can use serum protein profiles and Hp to evaluate the diagnosis and prognosis of calves with diarrhea.