Bovine intestinal obstruction: blood gas analysis, serum C-reactive protein and clinical, haematological and biochemical alterations

Abstract

The present study was conducted on six cattle and two buffaloes with intestinal obstruction. Four cases had intestinal volvulus, three had intussusception and one case could have been volvulus or torsion. The haemato-biochemical parameters of these eight animals were studied, together with 10 healthy cows and 10 buffaloes as control. Rectal examination was helpful in subjective assessment of volvulus and intussusception but could not help in definitive diagnosis. Multiple dilated intestinal loops in pelvic cavity could be a diagnostic feature of intestinal volvulus. Complete anorexia, colic, loss of defecation, rumen atony, dehydration, tachycardia and tachypenia were the most pronounced clinical symptoms. The animals with intestinal obstruction had significantly higher packed cell volume, neutrophil count and significantly lower lymphocytic count. Reversal of neutrophil: lymphocyte ratio and moderate to marked left shift along with moderate to severe toxic changes in the neutrophils was a consistent finding. Serum biochemical analysis showed significant increase of aspartate aminotrasferase, fibrinogen, lactate and C-reactive protein levels and significant reduction in albumin, fibrinogen ratio, potassium, chloride, calcium and phosphorus levels. However, fibrinogen and fibrinogen ratio may be misleading and need to be interpreted cautiously.The rumen chloride level was increased. Peritoneal fluid changes were consistent with septic peritonitis, increased specific gravity, total protein, total cell count and number of neutrophils. Blood gas analysis revealed hypochloremic hypokalemic metabolic alkalosis with compensatory respiratory acidosis. So these biochemical changes should be taken into consideration while dealing with intestinal obstruction in cattle and buffaloes.

Keywords:

• intestinal obstruction
• bovine
• blood gas analysis
• CRP
• rectal examination
• biochemistry

1. Introduction

The complex pathophysiological changes of intestinal obstruction in bovines are remarkably different from those in monogastric animals (Makhdoomi & Singh 1995) because abomasal reflux following intestinal obstruction brings about changes in rumen contents and blood (Braun et al. 1988). In cattle and buffaloes, there are many causes of obstruction of the intestine such as intussusception and volvulus, feed boluses, blood clots or hair balls (Radostits et al. 2007; Pravettoni et al. 2009). The clinical signs are abdominal pain (restlessness, kicking at the abdomen, lying down and getting up frequently, abnormal posture), scant or absence of faeces, blood stained faeces, increased heart rate, abdominal distension, progressive dehydration and toxaemia leading to shock and recumbency (Anderson & Ewoldt 2005; Fubini & Divers 2007; Radostits et al. 2007). Most of the previous studies on biochemical changes in intestinal obstruction have been carried out on few parameters (Makhdoomi & Singh 1995; Mohan et al. 2006; Tharwat 2011). So the present study was undertaken to investigate several aspects of blood biochemical profile in cattle and buffaloes showing intestinal obstruction.

2. Material and methods

2.1. Selection of animals and criteria for diagnosis of intestinal obstruction

The study included six Holstein cross-bred cows and two Murrah buffaloes, out of 268 animals (95 cattle and 173 buffaloes) clinical cases of gastrointestinal disorders, presented at Large Animal Clinics of Veterinary Teaching Hospital, Department of Teaching Veterinary Clinical Complex, GADVASU, Ludhiana, during the study period from May 2009 to April 2010. Tentative diagnosis of intestinal obstruction was based on clinical findings especially rectal examination, and was confirmed on post-mortem (PM) examination in seven animals and on exploratory laparotomy in one animal. Detailed signalment and history were noted in every case. Routine clinical examinations were carried out and body temperature, pulse and respiratory rate were recorded for each animal. All animals were subjected to a thorough clinical examination, as described by Rosenberger (1979). Also rectal examination of all the eight animals was carried out.

2.2. Control group

The control group for haematological and biochemical variables consisted of 10 healthy Holstein Friesian cross-bred cows and 10 buffaloes (Murrah and Niliravi breeds) from the university dairy farm. The selected animals were in different stages of lactation. They had not history of any disease for the current lactation period and were clinically healthy at the time of sampling.

2.3. Haematological parameters

Blood samples were aseptically collected from jugular vein in K₃ EDTA coated vials (Accuvete-PLUS, Quantum Biologicals Pvt. Ltd). Haemoglobin (Hb; g/dl), white blood count (WBC; /µl) and packed cell volume (PCV; %) were estimated by ADVIVA 2120 Haematology System (Siemens). Differential leukocyte count (DLC; %) was performed manually under oil immersion of light microscope in blood smear stained by Wright Giemsa stain or Leishman stain (Jain 1986). Further a thorough examination of stained blood smear was also done to determine blood cell changes such as differentiation of mature and immature neutrophil, left shift and toxic changes in neutrophils, if any.

2.4. Blood biochemical analysis

Blood samples were collected in acid free vials without any anticoagulant. Serum was separated and transferred to a dry clean vial for storage at –20°C till further evaluation. For glucose estimation blood was collected in vials containing sodium fluoride. Blood samples (2 mL) were collected in sodium citrate coated vials (Accuvete Disposables) for fibrinogen estimation. Fibrinogen was estimated by heat precipitation method using hand held refractometer (Feldman et al. 2000). Fibrinogen ratio was calculated by subtracting the fibrinogen value from total protein concentration and then dividing that value by fibrinogen concentration. VITROS DT-II Chemistry system (Ortho-Clinical Diagnostics, Johnson and Johnson Company) was used for estimation of total bilirubin, aspartate aminotrasferase (AST), alkaline phosphatase (ALP), gamma glutamyltrasferase (GGT), glucose, lactate, total protein, albumin, blood urea nitrogen (BUN), creatinine, sodium, potassium, chloride, calcium, phosphorus and magnesium. Globulin was estimated by subtracting albumin from total protein value. Cholesterol and C-reactive protein (CRP) were estimated by Bayer's diagnostic kits with the help of Microlab Autoanlyser (Merck). Blood samples were collected in heparinized syringes (1:1000) for blood gas analysis. Blood gas analysis was performed by RADIOMETER ABL 77 SERIES (ABL 77 v1.41, Analyser), within 5 minutes of collection. Further, it was assured that no atmospheric air entered the blood samples used for blood gas analysis.

2.5. Peritoneal fluid sampling

Abdominocentesis, as per the method of Radostits et al. (2007), was tried in all the eight animals, but peritoneal fluid could be collected from three animals only. Peritoneal fluid samples were analyzed for specific gravity (by refractometer), total protein (g/dl), total cell count (/μl), DLC (%), by methods as described for blood samples.

2.6. Rumen liquor sampling

Rumen liquor samples (10–15 ml) were collected using 16-gauge, 4 or 6 inch long needle inserted perpendicularly into the left paralumbar fossa (Hussain et al. 2013, 2014). Rumen liquor was filtered through a double layer muslin cloth and then centrifuged. Rumen chloride was estimated with the help of Bayer's diagnostic kits (colorimetric method) by using Microlab Autoanalyser (Merck).

2.7. Statistical analyses

The obtained data are presented as median and/or mean ± S.E. The data were analyzed using t-test to test for significant differences between control and animals with intestinal obstruction. The differences in means were considered statistically significant at P < 0.05 and P < 0.01.

3. Results and discussion

The prevalence of intestinal obstruction was 2.98% (8/268). The prevalence may be actually higher than in the general population of cows and buffaloes, owing to inclusion of only those cows and buffaloes that were confirmed to have gastrointestinal dysfunction and that too at a referral hospital. Also many intestinal obstruction cases usually die before being referred or noticed. From the results of this study, it was not possible to establish the aetiology. The diagnosis was made on the basis of PM examination or laparotomy. All the animals were presented with a primary complaint of colic and/or constipation. The animals were 2.5–8 years old females (median = 6 years, mean ± S.E = 5.31 ± 0.77 years) and had been ill for 2–10 days (median = 4.5 days, mean ± S.E = 5.31 ± 0.77 days). One cow was 2.5 month pregnant and one buffalo was 6 month pregnant, other six animals were non-pregnant. Seven animals were completely anorectic while one cow was taking little fodder. All animals had reduced water intake and sudden reduction in milk yield in milking animals (6/8). Faeces were absent (6/8) or scant (2/8). Two animals had history of regurgitation. On physical examination the animals were depressed and dehydrated, and had congested mucous membranes, tachycardia (95.50 ± 6.25 beats/min), tachypnea (44.35 ± 3.98 breaths/min) and normal temperature (101.53 ± 0.61°F). Rumen was atonic in seven and hypomotile in one animal. Six animals showed severe and persistent abdominal pain characterized by kicking at belly, restlessness, frequently lying down and getting up and sometimes rolling. There was no history of abdominal pain in two animals but on clinical examination these two animals showed signs of pain like arched back, frequently lying down and frequent treading on hindlimbs and occasional flick of hindlimbs. Persistent tympany was observed in four animals. Auscultation of heart and lung, revealed no abnormality. Excessive fluid was present in intestines on simultaneous auscultation and percussion (succession) of lower right abdomen. Rectal examination revealed absent or negligible faeces, sticky rectal mucosa and restricted hand movements in all the eight cases. In five animals (four cows and one buffalo) multiple and severely dilated intestinal loops were palpable in pelvic cavity and right abdominal quadrant. In two cases (one cow and one buffalo) sausage-shaped structure suggestive of intussusception, was palpable in right abdominal quadrant. One cow had mild to moderately dilated intestines. Seven animals died on the day of presentation while laparotomy was done in one animal suffering from intussusception.

These present clinical findings were similar to that of Radostits et al. (2007) and Tharwat (2011). For intestinal volvulus similar signs have been reported by Anderson et al. (1993). However, in present study, multiple intestinal loops palpable in pelvic cavity could be a diagnostic feature for advanced intestinal volvulus, as four out of five animals with this feature were confirmed as volvulus on PM while the fifth was volvulus or torsion on PM. The abdominal pain is considered as the characteristic feature of intussusception or volvulus (Radostits et al. 2007). But in two animals of present study there was no history of abdominal pain. It was assumed that in these two animals signs of pain were overlooked by the owners because the animals showed signs of pain on clinical examination. The inflamed peritoneum may have resulted in paresis of abdominal organs due to pain and toxaemia leading to ruminal atony/hypomotility. The regurgitation could be attributed to inability of ingesta to pass further down the digestive tract due to reticular/omasal malfunction secondary to generalized peritonitis as observed on PM examination. Persistent tympany which was attributed to generalized ileus of gastrointestinal tract (GIT) due to peritonitis.

3.1. haematological findings

The mean PCV and neutrophil count (P < 0.05) were significantly higher than their respective control values (Table 1). The lymphocyte count was significantly (P < 0.01) lower than respective control value. The mean WBC (P < 0.05) was higher than the normal reference range but did not differ significantly from control value (Table 1). Seven out of eight animals showed moderate to marked left shift along with moderate to severe toxic changes in the neutrophils. Reversal of neutrophil: lymphocyte ratio and moderate to marked left shift along with moderate to severe toxic changes in the neutrophils was a consistent finding. The inflammatory leukogram with increased number of immature neutrophils may be due ischemic necrosis of the intestine (Anderson & Ewoldt 2005). Anderson et al. (1993) also reported leukocytosis with mild left shift in intestinal volvulus of cattle. The rise in PCV value may be due dehydration as a result of hypovolemia due to obstruction.

Table 1. Haematological and biochemical parameters of control group and those with intestinal obstruction (Mean ± SE).

Measurement	Control (n = 20)	Intestinal obstruction (n = 8)	Reference range
Hb (g/dl)	10.65 ± 0.32	10.88 ± 0.84	8–15
PCV (%)	32.72 ± 1.03	40.25 ± 2.71*	24–46
TLC (/µl)	9723 ± 387.57	12,500 ± 2377	4000–12,000
Neutrophils (/µl)	3561 ± 152.8	9857 ± 2261.6*	600–4000
Lymphocytes (/µl)	5860 ± 252.5	2583 ± 447.1**	2500–7500
Total bilirubin (mg/dl)	0.09 ± 0.004	1.64 ± 0.67	0.01–0.5
AST (U/l)	101.05 ± 7.86	369.12 ± 99.88*	78–132
ALP (U/l)	89.55 ± 7.82	142.00 ± 37.33	27–107
GGT (U/l)	39.15 ± 2.09	60.75 ± 29.92	15–39
Total protein (g/dl)	7.47 ± 0.10	7.06 ± 0.36	5.7–8.1
Albumin (g/dl)	3.41 ± 0.05	2.65 ± 0.13**	2.1–3.6
Globulin (g/dl)	4.06 ± 0.06	4.41 ± 0.27	2.9–4.9
Fibrinogen (g/dl)	0.33 ± 0.03	0.64 ±0.09*	2–7
Fibrinogen ratio	25.35 ± 2.17	11.08 ± 1.04**	>15
BUN (mg/dl)	17.20 ± 1.06	35.75 ± 8.16	6–27
Creatinine (mg/dl)	1.19 ± 0.11	2.52 ± 0.67	1–2
Glucose (mg/dl)	60.10 ± 2.0	71.12 ± 13.74	45–75
Cholesterol (mg/dl)	84.40 ± 6.91	85.3 ± 13.17	62–193
Lactate (mmol/l)	1.51 ± 0.18	11.91 ± 1.65**	0.6–2.2
Sodium (mmol/l)	139.00 ± 1.29	129.38 ± 4.2	132–152
Potassium (mmol/l)	4.15 ± 0.09	3.10 ± 0.33*	3.9–5.8
Chloride (mg/dl)	101.70 ± 1.43	77.38 ± 4.97**	97–111
Calcium (mmol/l)	9.30 ± 0.24	7.80 ± 0.46*	9.7–12.4
Phosphorus (mg/dl)	5.88 ± 0.12	4.45 ± 0.52*	5.6–6.5
Magnesium (mg/dl)	2.36 ± 0.13	3.3 ± 0.5	1.7–3
CRP (mg/dl)	0.23 ± 0.09	4.16 ± 1.1*	0–1

* Significant difference at P < 0.05; **Significant difference at P < 0.01; TLC, total leukocyte count.

3.2. Biochemical findings

The mean values of estimated biochemical parameters of diseased animals and control group are presented in Table 1. The mean values of AST fibrinogen and CRP were significantly (P < 0.05) higher than respective control values. The mean lactate was also significantly (P < 0.01) higher than respective control value. Albumin, fibrinogen ratio and chloride levels were significantly (P < 0.01) lower than the respective control levels. The mean values of potassium, calcium and phosphorus were significantly (P < 0.05) lower than the respective control levels.

Absorption of toxic products from the rumen or alimentary tracts, starvation and constipation leading to cellular disturbances of liver parenchyma could have resulted in increased levels of total bilirubin, AST and ALP (Garry 2002; Kaneko et al. 2008). The increased AST activity may also be due to degenerative lesions in intestines (Kaneko et al. 2008). Significantly increased CRP and neutrophilia along with left shift indicated an inflammatory response in these cases. So the expected change in total protein would have been increased in its concentration. But on the contrary, the total protein concentration was within the normal reference range of 5.7–8.1 g/dl. Although albumin level was within the normal reference range, it was towards the lower side.The lower albumin and normal total protein concentration may be attributed to third space loses. The noted hemoconcentration (clinical dehydration and higher PCV), yet a tendency for normal total protein suggested significant third space loses. Due to intestinal obstruction and subsequent necrosis, there could have been probable peritoneal cell injury leading to release of vasoactive amines, further leading to increased vascular permeability and plasma exudation. This plasma exudation may be the probable cause for lower albumin and lower total protein. In other words it may be stated that leaky vessels caused a third space loss into the intestinal lumen or peritoneum and resulted in lower albumin and total protein concentrations. Smith (2002) has also stated that one of the adverse effects of peritoneal contamination is rapid influx of protein rich fluid, leading to hypoproteinemia. It is documented that the normal fibrinogen ratio in bovines gives more realistic picture of increased fibrinogen in inflammatory conditions and rules out any alterations due to dehydration (Jain 1986). So, in the present study the lower fibrinogen ratio (11.07 ± 4.07) could have indicated increased fibrinogen. However, it is unexpected to have significant hyperfibrinogenemia in such cases of intestinal obstruction with 4.5 days as median duration of illness. In this study, lower albumin concentration may be a potential cause for lower fibrinogen ratio. So in intestinal obstruction with expected third space loses, the fibrinogen ratio needs may be misleading and needs to be interpreted cautiously.

The mild to moderate degree of azotemia could also be attributed abomasal reflux (Avery et al. 1986; Kuiper & Breukink 1986). The increase in glucose corroborated well with the findings of Anderson et al. (1993) and may be attributed to stress caused by obstruction and peripheral resistance to glucose. It has been proposed that serum lactic acid levels may be an accurate biochemical parameter in advanced ischemic events. Lange and Jackel (1994) reported that plasma lactate concentrations were the best marker, reflecting the degree of ischemia in patients with mesenteric ischemia. Similarly, Kocdor et al. (2003) reported that higher lactate levels may be expected in intestinal obstruction cases with strangulation, in advanced periods of obstruction or when general ischemia is more prominent. They concluded that lactate could be a suitable biochemical parameter for predicting intestinal obstruction or in the follow-up of intestinal obstruction. In present study, dehydration and intestinal ischemia could be the causes for hypoperfusion and increased lactate level (Allen & Holm 2008).

3.3. Acid base and electrolyte changes

Acid-base balance is a highly integrated process in which certain variables chiefly pH, partial pressure of carbon dioxide (pCO₂) and actual bicarbonate (HCO₃) are controlled. The changes in acid-base balance are the early manifestations of many diseases and minor alterations in blood pH can alter the cellular functions of the body, so acid-base disorders should be interpreted cautiously. Hence the acid-base imbalances were interpreted on the basis of blood pH, pCO₂ and standard base excess (SBE) as per the guidelines of Haskens (1977). Further the normal intervals for interpretation of different parameters were taken as per Kaneko et al. (2008). The blood pH (7.552 ± 0.033) was higher than reference interval (7.32–7.44) indicating alkalosis. The higher or upper value of pCO₂ (45.67 ± 3.38 mmHg, reference interval, 35–45 mmHg), indicated respiratory acidosis. The SBE was 9.77 ± 3.71 mmol/l (reference interval, –4 to +4 mmol/l), indicating metabolic alkalosis. So the overall imbalance was primary metabolic alkalosis and compensatory respiratory acidosis. Abomasal reflux (indicated by increased rumen chloride, 41.75 ± 8.32 mEq/l) could be the cause for dehydration, hypochloremia, hypokalemia and azotemia (Avery et al. 1986; Kuiper & Breukink 1986; Braun et al. 1990; Behl et al. 1997). Hypochloremic, hypokalemic metabolic alkalosis with a compensatory respiratory acidosis has been reported earlier in cows with jejunal haemorrhage syndrome (Abutarbush et al. 2004), duodenal obstruction (Lejeune & Lorenz 2008), functional pyloric stenosis (Braun et al. 1990), intussusception and toxaemias (Radostits et al. 2007). Although lower albumin may have caused alterations in the ratios of chloride, potassium and bicarbonate, it seems more probable that in intestinal obstruction, abomasal reflux precedes the third space losses. So abomasal reflux may be the more likely cause for hypokalaemia, hypochloremia and high bicarbonate levels. Third space loses may have resulted in low sodium level but the compensating renal responses causing water retention, may have resulted in about normal sodium concentration (Smith 2002). The lower calcium and phosphorus concentration were ascribed to intestinal ischemia (leading decreased absorption) and anorexia in these animals (Sethuraman & Rathor 1979; Smith 2002; Radostits et al. 2007). Anorexia causes poor absorption of nutrients from intestines (Kaneko et al. 2008). Large increase or decrease in serum calcium concentration is generally the result of failure in the normal mechanisms of calcium homeostasis rather than a reflection of absolute calcium deficit (Smith 2002). In addition to anorexia lower albumin level may be the cause for lower calcium levels. Metabolic alkalosis reducing the parathyroid function may be another explanation for hypocalcaemia.

3.4. Serum CRP concentration

CRP is one of the most abundant acute phase proteins in animal serum. The liver rapidly synthesizes CRP when animals are sick or under severe stress (Sarikaputi et al. 1991; Godson et al. 1996). The concentration of CRP in the blood of healthy human beings ranges from 0 to 1 mg/dl, but during acute inflammation, the CRP levels may increase 1000 fold (Pepys & Baltz 1983). The CRP concentration for experimental sheep, irrespective of breed, has been reported to be 6.280 ± 0.429 mg/L (Vojtic & Krajnc 2000). In healthy dogs the serum CRP concentration has been reported to be <10 µg/ml (Holm et al. 2004). The markedly higher CRP level (4.16 ± 0.69 mg/dl) in present study may be attributed to inflammatory response due to ischemic necrosis of the obstructed part of intestine. It has been reported that diseases accelerate the synthesis and release of CRP from liver to the blood stream (Caspi et al. 1987). The results of present study are in agreement with that of Lee et al. (2003) who observed threefold increase in CRP level in cattle with mastitis as compared to healthy lactating cattle. According to Gewurz et al. (1982) CRP has no specificity for disease, but increases rapidly in presence of inflammation or tissue destruction.

3.5. Peritoneal fluid changes

Peritoneal fluid could be collected in three animals. In all the three cases, total cell count was more than 10,000/µl with a mean of 14,200 ± 1977/µl. In all the three cases, there was massive neutrophilia (>90%) along with presence of markedly degenerated neutrophils. The specific gravity (1.035 ± 0.005) and total protein (6.2 ± 0.76 g/dl) concentration were increased. Bacteria (cocci) were present in two and pyoperitonium (severely degenerated neutrophils with engulfed bacteria) was diagnosed in one case. Leakage of contents was observed in two cases. The peritoneal fluid changes were consistent with septic peritonitis and could be attributed to ischemic necrosis of the obstructed intestinal part leading to inflammatory response in peritoneum.

3.6. PM findings

Out of eight animals, seven were confirmed on PM examination. All these cases were suspected for intestinal obstruction by per rectal and physical examination and jejunojejunal intussusception had been diagnosed in one animal on laparotomy. On PM five cases were having intestinal volvulus (four had jejunal volvulus and one case had volvulus of proximal ileum) and two intussusception (jejunojejunal intussusception). Increased peritoneal fluid was observed in two cases, being red tinged in one and yellowish in other. In one case there was omental bursitis along with reticular adhesions. The omental bursitis was assumed to be secondary to peritonitis. Omasum was dilated and impacted in one case and normal in six cases. Reticular foreign bodies were absent in all cases. The intestinal serosa was having necrotic changes at the site of obstruction, in all cases. In majority (6/7) of cases intestines were gas filled and distended proximal to the site of obstruction. The length of devitalized portion of intestine in volvulus and intussusception was 15–25 cm and 7–15 cm (approximately), respectively. Abomasal mucosa was slightly hyperemic in one case. There were no gross abnormalities in other organs except epicardial haemorrhages in two cases and congestion of lungs in one case.

4. Conclusion

Rectal examination was helpful in subjective assessment of volvulus and intussusception but could not help in definitive diagnosis. Multiple dilated intestinal loops in pelvic cavity could be a diagnostic feature of intestinal volvulus. Reversal of neutrophil: lymphocyte ratio and moderate to marked left shift along with moderate to severe toxic changes in the neutrophils was a consistent finding. Liver and kidney function were deranged along with decreased levels of electrolytes and minerals. Fibrinogen and fibrinogen ratio may be misleading and need to be interpreted cautiously. Blood gas analysis revealed hypochloremic hypokalemic metabolic alkalosis with compensatory respiratory acidosis. The peritoneal fluid changes were consistent with septic peritonitis. These biochemical changes should be taken into consideration while dealing with intestinal obstruction in cattle and buffaloes.

Acknowledgements

The authors would like to thank Dr Sushil Prabhakar, Professor cum Head, Department of Teaching Veterinary Clinical Complex, and Dr Kirti Dua, Senior Scientist cum Head, Department of Clinical Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India, for providing the research facilities.