SERUM CONCENTRATIONS OF ACUTE PHASE PROTEINS AND IMMUNOGLOBULINS OF CALVES WITH ROTA- VIRUS DIARRHEA.*

The aim of the present study was to characterize changes in serum protein concentrations according to the occurrence of rotavirus diarrhea in calves throughout the first month of life. Blood and fecal samples were taken before colostrum intake (mo ment 0) and at 1, 2, 7, 15, 21 and 30 days of age from twenty-four Holstein calves allotted in three experimental groups with eight animals each: calves that did not present diarrhea neither rotavirus infection (group A), calves that presented diarrhea, but tested negative for rotavirus in feces (group B), and calves that presented diarrhea and tested positive for rotavirus in feces (group C) (Experiment 1). Also, whenever the animals presented episodes of diarrhea, blood and fecal samples were collected at 24-hour intervals until the end of clinical signs (Experiment 2). Serum proteins were electrophoretically separated in polyacrylamide gels containing sodium dodecyl sulphate (SDS-PAGE) and rotavirus in feces was detected by polyacrylamide gel electrophoresis (PAGE). Data obtained were analyzed by ANOVA and the differences between groups were compared by Tukey’s test, considered significant at P < 0.05. Data obtained during the moments of diarrhea manifestation were subjected to the HSD test. From birth to 30 days of age, total protein, globulins, and IgG concentrations were lower in group C than in groups A and B. Ceruloplasmin and transferrin levels were higher in group C than in groups A and B. Serum concentrations of haptoglobin and α 1-acid glycoprotein did not differ significantly among groups throughout the experimental period. Calves presented diarrhea between 10.4 and 16.4 days of age in group B, and between 10.3 and 16.3 days of age in group C. In the moments of diarrhea manifestation, least square means of IgA, haptoglobin and α1-acid glycoprotein concentrations did not differ significantly between groups B and C, but ceruloplasmin and transferrin concentrations were higher in group C than in group B, as opposed to what occurred with IgG levels. These findings suggest that optimizing passive immunity transfer of immunoglobulins decrease the likelihood of calves developing diarrhea caused by rotavirus. In addition, ceruloplasmin may be a marker of rotavirus infection in calves.


INTRODUCTION
Bovine neonatal diarrhea is one of the most important disease complexes that affect calves worldwide, and one of the main causes of morbidity and mortality in young animals, resulting in economic losses due to the costs of treatment and prophylaxis, augmented susceptibility to other infections, impaired development of animals, and death (Radostits et al., 2007).
This multifactorial process may be caused by many pathogens, such as bovine rotavirus (BRV), coronavirus, Escherichia coli K99 (E.coli), and Cryptosporidium parvum.García et al. (2000) reported that they detected these microorganisms in 75% to 95% of cases of intestinal infections in calves younger than one month old, and found a close association between the presence of these agents and the occurrence of diarrhea.Previous studies of the simultaneous prevalence of multiple etiological agents of bovine diarrhea in calves found a prevalence of 20% of bovine rotavirus in Belgium, 25.1% in Brazil, and 59% in Switzerland (De Graaf et al., 1999;Langoni et al., 2004;Uhde et al., 2008, respectively).These reports demonstrate the epidemiological importance of BRV as an etiological agent of bovine neonatal diarrhea.
In neonatal calves, the mortality rate of BRV diarrhea may reach up to 80%, but most reports suggest it is about 5-20%, mostly in calves that received an insufficient volume of high quality colostrum, animals affected by other pathogens such as enteropathogenic E. coli, or animals living in stressful conditions (Dhama et al., 2009).
The acute phase response occurs as an early reaction of the host to tissue damage or infection through the induction of cytokines, resulting in the production of specific serum proteins by the liver, known as acute-phase proteins (APPs) (Baumann and Gauldie, 1994).APPs may serve as an alternative means of monitoring individual animal or herd health, due to the increase in their concentration during acute phase response, making them useful tools to characterize and quantify reaction to inflammation or infection (Murata et al., 2004).Serum proteinogram is a reliable and valid method for the identification and quantification of different protein fractions, including immunoglobulins and APPs.Determination of APPs is one of the methods that can be used to identify animals presenting clinical or subclinical diseases, and among all the different techniques employed for this purpose, polyacrylamide gel electrophoresis containing sodium dodecyl sulphate (SDS-PAGE) offers the advantage of allowing for the identification and quantification of larger numbers of specific protein fractions (Cole et al., 1997;Fagliari et al., 2006).
The aim of this study was to characterize changes in serum protein concentrations pursuant to the occurrence of diarrhea in neonatal calves in the first month of life, as well as to verify the existence of marker proteins in animals affected by rotavirus diarrhea.

MATERIALS AND METHODS
The experimental design was approved by the Animal Research Ethics Committee of the College of Veterinary Medicine of São Paulo State University (Unesp) in Jaboticabal, under protocol number 015110/10.Twenty-four Holstein calves were allotted in three experimental groups, according to the occurrence of diarrhea in the first month of life, as follows: group A -eight calves that did not present diarrhea or rotavirus infection (control group); group B -eight calves that presented diarrhea, but tested negative for rotavirus in feces; group C -eight calves that presented diarrhea and tested positive for rotavirus in feces.
Blood samples were taken by jugular venipuncture before colostrum intake (moment 0) and at 1, 2, 7, 15, 21 and 30 days of age (experiment 1).Also, whenever the animals presented episodes of diarrhea, blood samples were collected at 24-hour intervals until the end of clinical signs (experiment 2).Blood samples from experiments 1 and 2 were centrifuged and the serum obtained was stored at -20 o C until the moment of analysis.Fecal samples were collected directly from the rectum on the same occasions as blood samples, and were stored in plastic bags at -20 o C until analysis.Whenever calves manifested diarrhea (experiment 2), the first day clinical signs observed was considered moment 1, and so on, regardless of the animal's age.

Serum
proteins were electrophoretically separated in polyacrylamide gels containing sodium dodecyl sulphate (SDS-PAGE), using the technique described by Laemmli (1970).After separation of the protein fractions, the gel was stained with a solution of 0.2% coomassie blue.Concentrations of protein fractions were determined by computer-assisted densitometry (Shimadzu CS9301, Tokyo, Japan).Reference markers (Sigma, St. Louis, MO, USA) with molecular weights of 24 kDa, 29 kDa, 36 kDa, 45 kDa, 55 kDa, 66 kDa, 97 kDa, 116 kDa, and 205 kDa were used to identify protein fractions as well as electrophoretic mobility of purified ceruloplasmin, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, and immunoglobulin G.
Concentrations of total protein and albumin were determined spectrophotometrically, using commercial reagents (Labtest Diagnostica, Lagoa Santa, Minas Gerais, Brazil).Globulin levels were determined arithmetically based on the difference between total protein and albumin concentrations in serum.
Rotavirus in feces was detected by polyacrylamide gel electrophoresis, as recommended by Herring et al. (1982), with a few modifications suggested by Pereira et al. (1983).The data obtained in experiment 1 were analyzed by ANOVA and the differences between groups were compared by Tukey's test, considered significant at P<0.05.The data obtained during the moments of diarrhea manifestation (experiment 2) were subjected to the HSD test using the Statistical Analysis System (SAS) software.

RESULTS
In experiment 1, although there was no significant difference between groups, the total protein concentration (Table 1) was lower in calves testing positive for rotavirus in feces (group C) than in groups A and B at all the evaluated moments -this tendency was also observed for globulin levels (Table 1).In addition, the globulin concentration was significantly lower in group C than in groups A and B on the second day of life.After colostrum intake, both parameters increased significantly in all the groups when compared to moment 0, followed by a gradual decrease until 30 days of age.
Immunoglobulin A (IgA) concentration (Table 1) did not differ significantly between groups; between moments, the levels of this immunoglobulin increased gradually from birth to 30 days of age.
Ceruloplasmin concentrations (Table 1) differed significantly between groups at 7 days of age, when a higher level of this acute phase protein was observed in group C than in groups A and B, and at 30 days of age, when a higher concentration of ceruloplasmin was observed in group A and a lower level in group B. Between moments, the lowest concentrations of ceruloplasmin was observed in the first days of life and the highest at 30 days of age in all the groups.
Transferrin levels ( Mean values followed by the same upper case letters in the same column and lower case letter in the same line do not differ significantly according to Tukey test (P<0.05).
Before colostrum intake, the groups showed no significant difference in immunoglobulin G (IgG) concentrations (Table 1).On days 1 and 2 of life, groups A and B showed significantly higher levels of this protein than group C. Between moments, the lowest IgG concentration was found immediately after birth, and the highest at 1 and 2 days of life, after colostrum intake.IgG levels then decreased gradually until 30 days of age.
Serum concentrations of haptoglobin (Table 1) did not differ significantly between groups throughout the experimental period.Between moments, the concentration of this protein increased after colostrum intake, with the highest levels found at 15 days of age in all three groups.α 1 -acid glycoprotein levels (Table 1) did not differ significantly between groups or moments in groups B and C in the first month of life.In group A, the lowest concentration of this APP was found at birth and the highest at 2 days of age.
When evaluating the period of diarrhea manifestation (days 1 to 7) -experiment 2 -it was interesting to note that although the total protein concentration (Table 2) in group B was significantly higher than in group C only on the second day of diarrhea manifestation, this tendency was observed at all the moments evaluated.From the first to the fifth days of manifestation of clinical signs of diarrhea, group B showed higher globulin concentrations than group C (Table 2).
Serum concentrations of IgA throughout the moments of diarrhea manifestation showed no significant difference between groups B and C. Least square means varied from 304 to 352mg/dL in group B and from 305 to 323mg/dL in group C.

Least
square means of ceruloplasmin concentrations (Table 2) from the second to the fourth day of diarrhea were higher in group C than in group B. On the fifth day of diarrhea, transferrin concentration was significantly lower in group B than in group C (Table 2).
Immunoglobulin G concentrations (Table 2) were significantly higher in group B than in group C from the first to the fourth days of diarrhea.Albeit not always significant, this difference remained constant throughout the experimental period.Groups B and C showed no significant difference in haptoglobin and α 1 -acid glycoprotein concentrations in the period of diarrhea manifestation.Least square means of haptoglobin levels varied from 13.1 to 20.2mg/dL in group B and from 15.2 to 19.2mg/dL in group C, while α 1 -acid glycoprotein concentrations varied from 11.3 to 13.4mg/dL in group B and from 10.4 to 13.7mg/dL in group C.
In experiment 2, calves presented diarrhea with an average age of 10.4 to 14.6 days in group B, and 10.3 to 14.6 days in group C (Table 3).There was no significant difference between groups in terms of IgA levels, using SDS-PAGE technique, from birth to 30 days of age.Between moments, there was a gradual increase in IgA concentrations after colostrum intake, but it was interesting to note that, in group C, IgA levels decreased at 15 days of age, coinciding with the period of diarrhea manifestation, while in groups A and B the IgA levels increased at this same moment.Similar to our findings, in a study of experimental infection of calves with Salmonella Typhimurium, Ávila (2009) reported no difference between control and infected calves with respect to IgA concentrations throughout the period of evaluation.Regarding acute phase proteins, throughout the first month of life, the higher concentration of ceruloplasmin in group C than in groups A and B at 7 and 15 days of age, coinciding with the period in which the animals manifested diarrhea, may be an indicator of increased stimulation for the production of this APP in rotavirus-induced diarrhea when compared to diarrhea caused by other pathogens.The fact that, in Experiment 2, ceruloplasmin levels were also found to be higher in group C than in group B corroborates this finding.The increase in ceruloplasmin levels in the 30 th day of life in group A, when compared to groups B and C, may be attributable to the occurrence of other pathological conditions not evaluated in this experiment, such as pneumonia.
The use of ceruloplasmin as a diagnostic tool in cattle is still less common than that of haptoglobin and serum amiloid A; however, some studies have shown that this ferroxidase is an indicator of infection in cows (Murata et al., 2004).In other studies evaluating blood serum proteinogram through SDS-PAGE, of calves affected by different infectious causes of diarrhea, Ávila (2009) and Silva et al. (2011) reported an increase in ceruloplasmin concentrations in Holstein calves experimentally infected with Salmonella Typhimurium and Salmonella Dublin, respectively.Hajimohammadi et al. (2013) also found a significant increase in ceruloplasmin levels in calves that presented diarrhea, when compared to healthy animals.Our findings suggest a relationship between the viral origin of diarrhea, the age of calves, and the dynamics of ceruloplasmin evaluated by the SDS-PAGE technique.
Transferrin concentrations in groups A, B, and C, from birth to 30 days of age, were higher in group C than in groups A and B, albeit not statistically significant.The same tendency was found in groups B and C in the period of diarrhea manifestation.Although transferrin is considered a negative APP -its concentrations tend to decrease in the presence of an acute inflammatory reaction -the increase verified in our study may be related to the viral etiology of diarrhea, since the mechanism of action of transferrin is more relevant in bacterial diseases, in which bonding to iron ions prevents the bacteria from using these ions for growth (Murata et al., 2004).
There was no significant difference in the haptoglobin levels of groups A, B, and C from birth to 30 days of age.The values found and the dynamics of this APP after birth are consistent with those reported by Orro et al. (2008), who investigated temporal changes in APP concentrations in newborn dairy calves.In the present study, the increase in haptoglobin concentrations in animals affected by diarrhea in the first month of life (groups B and C -Experiment 2) was very discrete, possibly indicating a non-bacterial origin of diarrhea in animals of group B -since Eckersall (2000) reported that haptoglobin is a useful indicator of bovine bacterial infections -or that the etiological agents that affected these animals did not inflict sufficient stimulus to initiate an acute phase response.Also, according to Hajimohammadi et al. (2013), who evaluated relationships between acute phase proteins and clinical findings in dairy calf diarrhea, calves that presented severe clinical signs showed a more significant increase in their haptoglobin levels than calves presenting moderate or no systemic clinical signs.All the calves in our study belonged to private farms in which early treatment with antibiotics was initiated as soon as animals present clinical signs, regardless of the cause of diarrhea.In such conditions, all animals in this experiment presented discrete to moderate clinical signs.
Concentrations of α 1 -acid glycoprotein did not differ between groups A, B, and C throughout the first month of life, nor did they differ significantly between groups B and C in Experiment 2. According to Orro et al. (2008), colostrum intake is the main stimulus for the increase in APPs concentrations in calves, since this secretion contains high concentrations of proinflammatory citokines, which are the main inducers of hepatic production of APPs, thus explaining the increase in haptoglobin, transferrin and α 1 -acid glycoprotein levels after colostrum intake in all the evaluated groups.
Based on these findings, an optimal passive immunity transfer of immunoglobulins decreases the likelihood of calves developing diarrhea caused by rotavirus.In addition, ceruloplasmin measured by SDS-PAGE presents characteristics of a biomarker of rotavirus infection in calves.Experimental infection studies are needed to confirm if this relationship exists for other etiological agents of diarrhea in neonatal calves.

Table 1 .
Mean values and standard-deviations of serum protein concentrations in Holstein calves not presenting diarrhea (group A), presenting diarrhea but testing negative for rotavirus in feces (group B) and presenting diarrhea and testing positive for rotavirus in feces (group C) before colostrum intake (moment 0) and at 1, 2, 7, 15, 21 and 30 days of life

Table 2 .
Least square mean values of serum protein concentrations in Holstein calves that tested negative (group B) or positive (group C) for rotavirus in feces from the first to the seventh day of diarrhea manifestation Least square mean values followed by the same letter in the same column do not differ according to the HSD test (P<0.05).

Table 3 .
Age of calves in the beginning and end of diarrhea manifestation period in groups B and C (experiment 2)