Introduction

There have been several clinical entities affecting buffaloes all over the world especially in developing countries which could result in serious economic losses in milk and meat production, increase cost of treatment and fatal losses in pregnant affected animals (Ghanem et al. 2012; El-Ashker et al. 2013, 2014; Mohan et al. 2015). Among the commonly reported disease conditions affecting buffaloes, digestive troubles are becoming of utmost clinical significance and yet received a growing attention (El-Ashker et al. 2014). Disturbances of appetite and abnormalities of electrolytes remain the most common features of affected animals (Ghanem et al. 2012; Radostits et al. 2007). The main functions of alimentary tract include motility, secretion, digestion, and absorption. Any disturbance of these four functions could prone the animal to digestive troubles which vary according to the site and nature of the lesions (Radostits et al. 2007).

Hematological and serum biochemical reference intervals are essential for diagnosing diseases. The basic part of laboratory diagnostic tests is the measurement of hematological and biochemical variables that are used to establish the normality and to diagnose disease as well as physiological alterations (Mohapatra et al. 2005; Radostits et al. 2007; El-Ashker et al. 2014; Mohan et al. 2015). Several bovine diseases are often associated with acute inflammation, but most of the practicing veterinarian includes only a limited number of laboratory tests for the diagnosis of inflammation. Indeed, the total and differential white blood cell (WBC) counts and acute phase protein (APP) measurements are the most common tools that used for the diagnosis of bovine inflammatory diseases (Murata and Miyamoto 1993; Hirvonen and PyӦrӓl 1998; Petersen et al. 2004; Ceciliani et al. 2012).

The APPs are a group of plasmatic proteins of hepatic origin whose concentration changes during or after inflammation, infection, or surgical trauma or stress factors can affect the acute phase response (APR) (Murata and Miyamoto 1993; Petersen et al. 2004). APR is a component of the innate immune response and is observed uniformly across animal species. APPs can provide valuable diagnostic and prognostic information as well as detection and monitoring of diseases in several animal species (Ulutas et al. 2011). The leukocyte proliferation and synthesis of cytokines, immunoglobulin, and positive APPs can significantly contribute to protein change of APPs and hence considered as markers of systemic inflammation in bovine (Jafarzadeh et al. 2004).

Recently, there has been a great interest shown on the usage of APPs as a marker for animal health or, alternatively, as an indicator of disease severity in veterinary medicine (Ulutas et al. 2011). The present study was therefore delineated to provide a detailed hemato-biochemical profile as well as the measurement of selected APPs in buffaloes demonstrating some digestive troubles particularly stomatitis, acute traumatic reticuloperitonitis (TRP), acute rumen impaction, and enteritis. Our hypothesis was that diseased buffaloes might have significant hemato-biochemical alterations as well as detectable inflammatory reactions.

Materials and methods

Study population

The present study included 50 native breed water buffalo (Bubalus bubalis) with an average 3–5 years of age and 350–500 kg body weight (BW) in the period between October 2015 and April 2016. The studied buffaloes were raised at different farms and different localities in Dakahlia governorate, Egypt. The examined animals were kept in half-open shelter system and fed a mixture of corn silage, concentrates, and clover (Trifolium alexandrinum), while water was offered ad libitum.

Forty of the studied buffaloes showed various symptoms of digestive disorders. Collectively, the following manifestations exist including dullness, variable appetite, decrease of milk production, weight loss, tucked-up abdomen, lacrimation, and grunting with arched back. Some buffaloes demonstrated abdominal distention and voluminous diarrhea, while others exhibited profuse watery diarrhea with offensive odor. A detailed physical examination of each animal was carried out according to the standard methods described previously (Radostits et al. 2000) and the obtained clinical findings were recorded simultaneously. Based on the competent case history and the findings of physical examinations, the investigated animals were categorized into four equal sized groups including uncategorized stomatitis, acute TRP, acute rumen impaction, and undifferentiated enteritis. For comparison, ten apparently healthy buffaloes were randomly selected from the same farms and were considered as control group. Animals included in this study have had the following criteria: being adults and showed solely digestive disorder, with no concurrent ailments. Animals were enrolled with owner’s consent and were given a document containing information about the disease definition, its economic impact as well as the potential clinical consequences.

Sampling and sample processing

Ten milliliters of blood was collected from each animal via jugular vein puncture and was divided into two aliquots. The first aliquot was added to a tube containing EDTA for adopting hematological examination using automatic electronic cell counter analyzer (MINDRAY BC-3000plus, China), while the second aliquot was added to a plain tube for obtaining blood serum. Only clear non hemolysed sera were harvested and kept frozen until estimation of the following parameters: potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, albumin, blood urea nitrogen, glucose, haptoglobin (Hp), and C-reactive protein (CRP). With the exception of Hp and CRP, serum biochemical variables were measured spectrophotometrically using commercial test kits according to the standard protocols of suppliers. The following test kits were used for measuring K (Biomed; Hannover, Germany; Cat No: POT100100), Mg (Quimica Clinica Aplicada; Spain; Cat No: 99 29 85), Ca (Spinreact; Spain, Cat No: 1,001,061), Na (Biomed; Hannover, Germany; Cat No: SOD100100), Fe (Chema Diagnostica via Campania; Monsano, Italy; Cat No: FE 0100 CH), AST (Chemelex; Barcelona, Spain, Cat No: EZ012LQ), ALT (Chemelex; Barcelona, Spain, Cat No: EZ016LQ), ALP (Biomed; Hannover, Germany; Cat No: ALP101090), total protein (Biomed; Hannover, Germany; Cat No: TP116250), albumin (Diamond Diagnostics; Hannover, Germany; Cat No: 025445), blood urea nitrogen (Biomed; Hannover, Germany; Cat No: URE118100), and glucose (Spinreact; Spain; Cat No: 1001190). For Hp and CRP, their values were quantified by using ELISA kits supplied by Lifespan Biosciences, USA (Cat No: LS-F11659), and Biomed; Hannover, Germany; (Cat No: CRP301050), respectively.

Statistical analysis

Data were statistically analyzed using Statistical Package for Social Sciences (SPSS) version 17.0 (USA). Mean and standard deviation for each variable were determined. The differences between groups were compared by one-way ANOVA with post hoc Bonferroni multiple comparison test. P values at ≤0.05 were considered statistically significant.

Results

An overview of hematological and serum biochemical alterations in buffalo with some digestive disorders is illustrated in Table 1 and Figs. 1, 2, 3, 4, 5, and 6. Clinically, the studied buffaloes demonstrated common clinical findings including dullness, anorexia, decrease of milk production, rumen stasis, and signs of dehydration. Nevertheless, buffaloes with stomatitis exhibited profuse salivation, slow and painful mastication, fetid odor of oral cavity , swollen tongue and inflamed soft palate. Buffalo with acute TRP exhibited tucked-up abdomen, stiff gait, arching back, grunting, lacrimation, and history of recurrent tympany. Buffalo with acute rumen impaction had a characteristic nutritional history, anorexia, distended abdomen, staggering gait, and voluminous diarrhea, while buffaloes admitted with uncategorized enteritis had anorexia, rumen stasis, tucked-up abdomen, straining, and profuse watery diarrhea with clinically evident dehydration. The heart rate and respiratory showed no statistically significant alterations (P > 0.05) in all diseased buffalo compared with those of controls and the values were within the normal reference interval (data not shown). The rectal temperature was, however, significantly elevated in buffaloes showing stomatitis and those with enteritis compared with those of controls (P ≤ 0.05) (data not shown).

Table 1 Means ± SD of some hematological variables in buffalo with some digestive disorders compared with those of controls
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Fig. 1
figure 1

Mean values ± SD of serum potassium, magnesium, calcium in buffalo showing some digestive disorders compared with controls. Bars labeled with different letters are statistically significant (P < 0.05). TRP traumatic reticuloperitonitis, ARI acute rumen impaction

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Fig. 2
figure 2

Mean values ± SD of serum sodium (​mEq/l) and iron (​ug/dl) in buffalo showing some digestive disorders compared with controls. Bars labeled with different letters are statistically significant (P < 0.05). TRP traumatic reticuloperitonitis, ARI acute rumen impaction

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Fig. 3
figure 3

Means ± SD of serum activity of liver enzymes in buffalo showing some digestive disorders compared with controls. Bars labeled with different letters are statistically significant (P < 0.05). TRP traumatic reticuloperitonitis, ARI acute rumen impaction

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Fig. 4
figure 4

Means ± SD of protein profile in buffalo with some digestive troubles compared with controls. Bars labeled with different letters are statistically significant (P < 0.05). TRP traumatic reticuloperitonitis, ARI acute rumen impaction

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Fig. 5
figure 5

Means ± SD of serum activity of serum urea and glucose in buffalo with some digestive disorders compared with controls. Bars labeled with different letters are statistically significant (P < 0.05). TRP traumatic reticuloperitonitis, ARI acute rumen impaction

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Fig. 6
figure 6

Means ± SD of haptoglobin and CRP in buffalo with some digestive disorders compared with controls. Bars labeled with different letters are statistically significant (P < 0.05). TRP traumatic reticuloperitonitis, ARI acute rumen impaction

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Hematologically, all diseased buffaloes showed a statistically significant elevation of the total leucocytes (P ≤ 0.05) with significantly decreased values of total erythrocytes and hemoglobin (Hb) concentration compared with controls (P ≤ 0.05). However, the packed cell volume (PCV %) showed no statistical significant variations among all groups compared with controls (P ≤ 0.294) (Table 1).

Biochemically, serum K levels showed statistically significant high values in buffaloes showing enteritis (P ≤ 0.05) with no statistically significant alterations in other diseased buffaloes compared with control (Fig. 1). Serum biochemical variables including Mg, Na, and iron showed a statistically lower values in all studied buffaloes than those of controls (P < 0.05), while serum calcium levels were significantly (P ≤ 0.05) decreased in buffaloes showed stomatitis and those with TRP compared with those of control group (Figs. 1 and 2). Activities of liver enzymes including ALP, ALT, and AST along with serum globulins were significantly higher in all diseased buffaloes than those of controls (P ≤ 0.05) (Figs. 3 and 4). The diseased buffaloes showed a statistically significant low values (P ≤ 0.05) of serum albumin and A/G ratio compared with controls; however, serum total protein concentrations showed no significant variations among all groups (P > 0.05) (Fig. 4).

Values of serum urea showed a statistically significant higher values (P ≤ 0.05) in buffaloes with acute rumen impaction, enteritis and those with TRP than those of controls, while its value were significantly decreased (P ≤ 0.05) in animals with stomatitis (Fig. 5). For serum glucose, its values were significantly increased in buffaloes with stomatitis and those with acute rumen impaction (P ≤ 0.05) compared with controls with no significant variation (P = 0.176) in buffalo with TRP. Nevertheless, its values were significantly decreased in buffaloes with enteritis (P ≤ 0.05) compared with all groups (Fig. 5). Serum Hp and CRP showed statistically significant higher values in all diseased animals compared with those of controls (P ≤ 0.05) (Fig. 6). The highest values of serum Hp were recorded in buffaloes showing enteritis while buffaloes showing stomatitis had the highest values of serum CRP.

Discussion

Digestive disorders in buffaloes are of utmost clinical significance since they can result in serious economic losses. The present study set out an insight into the biochemical alterations as well as the inflammatory reaction in buffaloes exhibiting various digestive troubles including acute forms of stomatitis, TRP, rumen impaction, and enteritis. The presenting data clearly demonstrated that all investigated buffaloes showed marked hemato-biochemical alterations as well as various inflammatory responses that warrant further attention. Briefly, the studied buffaloes demonstrated low values of total erythrocytes and Hb concentration with no significant alterations of PCV%. These findings were in agreement with those reported in cattle and sheep showing stomatitis (Gokce et al. 2004; Mohapatra et al. 2005; Ghanem et al. 2010), and in buffalo calves demonstrating diarrhea (El-Dessouky and El-Masry 2005; Ghanem et al. 2012). The alterations of these hematological variables could be attributed to the reduction of the process of erythropoiesis or the endocrinopathy which occurs secondary to infection or inflammatory reactions (Ghanem et al. 2010) or might associate the long-standing cases of diarrhea (El-Dessouky and El-Masry 2005). For the PCV%, our findings were consistent with that given by Neamat-Allah (2015) in buffaloes with TRP who attributed the normal PCV% in anemic animals to the state of dehydration, i.e., anemia masked by dehydration.

In the present study, the total leucocyte counts were significantly higher in all diseased animals than those of controls. These findings were in harmony with those obtained in bovine with various clinical situation including stomatitis (Mohapatra et al. 2005), TRP (Kirbas et al. 2015), and diarrhea (Ghanem et al. 2012). The significant alterations of TLC might be attributed to the associating inflammation or probably to the coexistence of infection. Unfortunately, the differential leucocytes were not available for all cases and hence not provided.

Hyperkalemia was evident in buffaloes showing enteritis compared with controls. The obtained finding was in harmony with that reported in kids, neonatal calves, and buffalo calves showing diarrhea (Ghanem et al. 2012). Hyperkalemia has been reported to occur in association with a metabolic acidosis, particularly when the acidosis results from volume depletion complicated by renal shutdown as a result of movement of intracellular potassium into the extracellular fluid without change in external potassium balance (Kaneko et al. 2008).

Our findings regarding serum Mg and Na were in agreement with those given by several authors in buffaloes showing rumen acidosis (Nikolov 1998), or in cattle exhibiting TRP (Ghanem 2010), and buffalo calves demonstrating diarrhea (Ghanem et al. 2012). The resulting hypomagnesaemia could be attributed to the state of anorexia or to the increase renal excretion of Mg (Gokce et al. 2004; Ghanem 2010).

Hypoferremia was prevailed in all disease buffaloes compared with controls. That finding was in agreement with previous reports including acute inflammatory diseases in cattle (Baydar and Dabak 2014), and in buffalo calves showing diarrhea (Ghanem et al. 2012). The significant decrease of serum iron could be attributed to the decrease in the absorption of food nutrient through intestine and losses in feces or being a response of the state of APR. In that context, some authors have tested its potential significance for the diagnosis and prognosis of acute inflammatory conditions in dairy cattle (Baydar and Dabak 2014).

Our findings demonstrated a statistically significant low value of serum calcium in buffaloes showing stomatitis and those with TRP compared with those of controls (P ≤ 0.05). These findings were in harmony with those reported by several authors in similar clinical entities (Gokce et al. 2004, Mohapatra et al. 2005; Ghanem et al. 2010). The resulting hypocalcaemia could be attributed to the state of anorexia or to the reduction of calcium uptake as a result of illnesses which affect the appetite and decrease its absorption (Gokce et al. 2004). Collectively, hypocalcaemia observed in the present study could probably develop in association with gastrointestinal stasis and insufficient dietary uptake of calcium.

The activities of liver enzymes were significantly higher in all diseased buffaloes than those in controls (P ≤ 0.05). The highest values of liver enzymes were observed in buffaloes exhibiting acute rumen impaction. These findings were in line with other reports elsewhere (Gokce et al. 2004; Mohapatra et al. 2005; Ghanem et al. 2010, Ghanem et al. 2012). The alterations of these enzymes might indicate either liver or enteric damage.

The findings of proteinogram that was observed in our study were consistent with those obtained in several clinical situations including stomatitis (Ghanem et al. 2010), TRP (Kirbas et al. 2015), and diarrhea (El-Dessouky and El-Masrey 2005). In contrast, our findings were away from those reported by Bozukluhan and Gokce (2007). In the latter study, the authors have found that cattle exhibiting TRP often have high serum total protein concentrations. The obtained hypoalbuminemia could be attributed either to the severe anorexia, or to off food due to oral lesions or being a response of the animal to infection (Kaneko et al. 2008).

The values of serum urea were significantly higher in buffaloes demonstrating TRP, rumen impaction, and those having enteritis than those of controls (P ≤ 0.05). These findings were in line with those reported in buffaloes suffering from TRP (Neamat-Allah 2015), in goats exhibiting rumen acidosis (Lal et al. 1992) and in buffalo calves showing diarrhea (Ghanem et al. 2012). The significant elevation of serum urea levels has been considered an index of decreased glomerular filtration rate in acidotic animal, either due to renal damage or to the reduction in the effective renal flow and fall in arterial blood pressure which results in subnormal renal function (Lal et al. 1992). Interestingly, the values of serum urea were significantly decreased in animals exhibiting stomatitis (P ≤ 0.05). That finding was in agreement with that reported previously in cattle showing stomatitis (Gokce et al. 2004). The authors attributed that to the anorexic state of affected animals with a resultant less protein intake.

In this study, buffaloes with stomatitis and those with acute rumen impaction demonstrated hyperglycemia compared with control. This finding was coincided with that reported in cattle and sheep showing stomatitis (Gokce et al. 2004; Mohapatra et al. 2005; Ghanem et al. 2010), and in cattle and buffalo suffering from rumen acidosis (Mohan et al. 2015). The resulting hyperglycemia could be attributed to the stress or to the decrease peripheral utilization of glucose coupled with hepatic glycogenolysis under the influence of corticosteroids released because of stress occurred (Gokce et al. 2004). Nevertheless, its concentration was significantly decreased in buffaloes showing enteritis. This finding was similar to that observed in buffaloes with diarrhea (Ghanem et al. 2012). The resulting hypoglycemia could be attributed to the lack of glucose absorption from the damaged intestine. On the other side, buffaloes with TRP showed no significant variations of serum glucose levels compared with other groups (P = 0.176). This result was in agreement with previous reports in dairy cows suffered from TRP (Kirbas et al. 2015).

Serum Hp and CRP were significantly elevated in all diseased animals compared with controls (P ≤ 0.05). These findings were in harmony with those reported in cattle and buffalo showing TRP (Jafarzadeh et al. 2004; Bozukluhan and Gokce 2007; El-Ashker et al. 2013), and dairy cattle exhibiting sub-acute ruminal acidosis (Khafipour et al. 2009) or in calves demonstrating acute rumen drinking (El-Ashker et al. 2012), but the finding was contradictory to that reported in lactating dairy cows who stated that sub-acute rumen acidosis did not affect marker of inflammation (Gozho et al. (2007). Serum Hp levels have been reported to increase to the range of 0.86–2.16 g/L in cattle and buffaloes with TRP (Jafarzadeh et al. 2004; Bozukluhan and Gokce 2007; El-Ashker et al. 2013). In addition, a positive correlation between Hp, CRP levels and WBC count has been detected (Murata and Miyamoto 1993; Hirvonen and PyӦrӓl 1998; Petersen et al. 2004; Ceciliani et al. 2012). Therefore, it is seemingly important to estimate serum Hp concentration and WBC counts simultaneously to determine the severity of inflammation in buffaloes with digestive troubles.

Conclusion

The results herein clearly demonstrated that buffaloes with enteritis had marked hematological and elemental alterations with significant inflammatory reaction with particular emphasis to hyperkalemia, hypoferremia, hypoglycemia, and marked increase of serum Hp. However, buffaloes showing acute rumen impaction had a significant hyponatremia and distinct elevation of serum enzymatic activities, while buffaloes exhibiting stomatitis had marked hypoproteinemia, hypoalbuminemia, hypocalcaemia with significant high values of serum CRP. Further studies are needed to explore the ameliorative value of using supplementary agents to help mitigate the associated biochemical and inflammatory reactions in buffaloes exhibiting digestive disorders.