Introduction

Canine Exocrine Pancreatic Insufficiency (EPI) is an alimentary tract disorder causing maldigestion, malabsorption and debilitations in affected individuals (German, 2012) and is characterized by a decrease or absence of trypsin, chymotrypsin, amylase, and lipase enzymes because of which proteins, starches, and fats found in their diet cannot be broken down and are passed out in the faeces undigested. Pancreatic acinar atrophy is the most common cause for the maldigestion signs of canine EPI ⁽Westermarck and Wiberg, 2012). Fortunately, the pancreas has a high reserve capacity, so signs of maldigestion do not occur until 90% of the exocrine pancreatic function is lost (Westermarck et al., 2005). In the veterinary clinical practice, it is difficult to diagnose the infections of pancreas and most of them go unnoticed. The symptoms exhibited by the affected ones are ambiguous and clueless in general. Not much information is available on the diagnostic aspects of EPI. Therefore, the present study has been planned with the objective to assess and suggest suitable diagnostic tests for EPI.

Materials and Methods

The investigation was conducted to study the diagnostic aspects of Exocrine Pancreatic Insufficiency (EPI) in canines and was accomplished on the clinical cases presented at the Teaching Veterinary Clinical Complex, Bhoiguda, Secunderabad; Campus Veterinary Hospital, College of Veterinary Science, Rajendranagar, Hyderabad and Veterinary Hospital, Department of Animal Husbandry, Shantinagar, Hyderabad for a period of 8 months. Among the cases that were treated for diarrhoea during the study period, 76 dogs were presented with the history of passing voluminous, loose, semi formed, grey colored glistening faeces, having caprophagia with marked reduction in body weight in spite of normal rather increased appetite and with the clinical signs of occasional vomiting, flatulence and poor hair coat were selected for the study and screened for EPI. Out of 76 cases selected, 18 cases were found positive for EPI when subjected to faecal examination and detailed haemato-biochemical investigation.

For faecal examination, approximately 2 grams of faecal sample was collected from each dog and screened for the presence of parasitic ova. The samples which are negative for parasitic ova were further screened macroscopically for trypsin by radiographic film test/ x-ray film digestion test and was done as per the standard procedure of Seybold et al. (1981) and further tested microscopically for the presence of muscle fibres by staining the faecal smear with aqueous methylene blue stain. Presence of fat in the faeces i.e. steatorrhoea was done as per the standard procedure of Drummey et al. (1961). Presence of starch in faeces was detected by staining the faecal smear with 2% Lugol’s Iodine solution and presence of numerous blue starch granules under low power of microscope was considered as positive for amylorrhoea (Seybold et al., 1981). Detection of faecal protein was done by staining direct smear of fresh faeces with Lugol’s Iodine for 5 minutes and viewed under low power of microscope. Presence of deep brown colour stained undigested muscle fibres with obvious striations and occasional nuclei was considered as positive for creatorrhoea (Simpson and Doxey, 1988).

Haematological investigations were conducted by collecting blood samples on day 0, 5 and 7 and estimated total erythrocyte count (x 10⁶ / µL), haemoglobin (g/dL), packed cell volume (%) and total leukocyte count (x 10³ / µL) by using animal blood count analyzer. Biochemical investigations were carried out with Star 21 Plus Semi-Automatic analyzer supplied by M/s Rapid Diagnostics Pvt. Ltd. Delhi by using diagnostic kits supplied by M/s Aspen Laboratories Pvt. Ltd., Delhi. The serum glucose levels (mg/dL) were estimated by GOD-POD method and serum Gamma Glutamyl Transferase (GGT) (IU/L) by kinetic method (Szacz, 1969). The estimation of serum Amylase (IU/L) was done by kinetic method as per the modified procedure (Wallenfels, 1978). Serum Lipase (U/L) concentration was done by kinetic method (Neely and Kalpan, 1984) using the diagnostic kits supplied by Lab Kits Diagnostics, Canovelles – Barcelona.

Results and Discussion

Diagnosis of exocrine pancreatic dysfunction is based on typical findings in clinical histories and clinical signs and is confirmed with pancreatic function tests (Watson, 2003). The present investigation was undertaken to assess suitable diagnostic tests for exocrine pancreatic insufficiency (EPI) in canines. Among the 76 dogs presented to the selected hospitals during the period of study with the history of passing voluminous, loose grey greasy coloured faeces were selected and screened by faecal examination and preliminary biochemical tests. Dogs passing voluminous and loose faeces; passing greasy and grey colored glistening faeces; and marked reduction in body weight in spite of normal appetite was the common clinical history reported by the owners which accounted for 100, 100 and 71.05 percent respectively (Table 1). These findings were in agreement with previous findings of Krishna Kumar et al. (1995) and  Gamet and Jergens (1998). The reason for changes in the colour and consistency of the faeces could be attributed to the infection and certain changes in the secretions of pancreatic juices (Williams et al, 1987).

Table 1: Clinical history of EPI in dogs

* Non significant at 5 percent (p < 0.05) level; ** Significant at 5 percent (p < 0.05) level.

On perusal of Table 2, it was evident that, out of 76 dogs, 37dogs (48.68%) had caprophagia, 43 (56.58%) had poor hair coat, 19 (25.0%) had occasional vomiting and 21 (27.63%) showed flatulence, similar signs were observed by Westermarck et al. (1993) and Whitney (1993).

Table 2: Clinical signs observed in EPI dogs

Screening of faecal sample revealed the positive results for x-ray film digestion test (Fig. 1 & 2), meat / muscle fibre digestion test, steatorrhoea, amylorrhoea and creatorrhoea as 41 (53.95%), 34 (44.74%), 21 (27.63), 31 (40.79) and 25 (32.89%) respectively as depicted in Table 3.

Fig. 1: Unprocessed X-ray film incubated in the faecal emulsion of EPI positive dog made with 5% sodium bicarbonate. Lack of clearance indicating the absence of trypsin.

Fig. 2: Unprocessed X-ray film incubated in the faecal emulsion of EPI negative dog made with 5% sodium bicarbonate. Clearing of X-ray film indicating the presence of trypsin in faeces.

Table3: Faecal examination

* Non significant at 5 percent (p < 0.05) level; ** Significant at 5 percent (p < 0.05) level.

Similar findings of steatorrhoea in pancreatic insufficiency were reported by Gamet and Jergens (1998) and Boari et al. (1994). Amylorrhoea in pancreatic insufficiency could be due to the inability of the pancreas to deliver adequate quantities of amylase to the intestinal lumen resulting in inadequate digestion of starch (Drazner, 1983). Creatorrhoea in pancreatic insufficiency was reported earlier by Krishna Kumar et al. (1991). The reason for the presence of undigested muscle fibres in the stool could be due to Trypsin and chymotrypsin deficiency Brobst, (1972).

In the present study, 18 EPI positive dogs have been divided into three (3) groups randomly and given different therapeutic regimens. The mean TEC levels recorded before and after treatment in Group I, II and III were 6.08 ± 0.47 and 5.91 ± 0.47; 5.20 ± 0.48 and 5.25 ± 0.42; and 5.28 ± 0.34 and 5.18 ± 0.31 ×10 ⁶ /µ.l of blood, respectively. There is no significant difference in the mean values of TEC before and after treatment in all three groups. These values are in accordance with the earlier findings of erythrocyte count as 5.4 to 7.4 ×10 ⁶ /µ.l in 20 EPI positive dogs (Hill 1972)  and  also normal TEC levels in EPI affected dogs has been recorded by Strombeck (1978). The mean Hb estimated before and after treatment in Group I, II and III were 9.60 ± 0.84 and 9.53 ± 0.79; 10.53 ± 1.96 and 10.38 ± 1.97; and 11.11 ± 1.16 and 11.06 ± 1.09 g/dl of blood respectively. All three groups showed non significant difference (p< 0.05) in mean Hb levels, following therapy. Similar observations are made by Hill (1972) and Remson (1964) who recorded Hb levels as 8.2 g/dl and 12.9 to 17.2 gram percent respectively. The mean PCV recorded before and after treatment in Group I, II and III were 38.33 ± 3.01 and 38.66 ± 2.69; 44.00 ± 2.71 and 44.50 ±2.40; and 44.66 ± 3.05 and 43.83 ± 2.97 percent, respectively. There is no significant difference in the mean values of PCV before and after treatment in all three groups. The present findings are in accordance with earlier reports of Pidgeon and Strombeek (1982) and Oskoui et al. (2008) who recorded the PCV level as 26-31 and 27% respectively. Whereas few authors observed the increased levels of PCV (54.48±2.82%) (Satish Kumar and Choudhuri, 2002) whereas, Pidgeon and Strombeek (1982) recorded decreased levels of PCV (26% and 31%) in EPI positive dog with mild anaemia. The mean TLC recorded before and after treatment in Group I, II and III were 9.60 ± 0.84 and 9.53 ± 0.79; 10.53 ± 1.96 and 10.38 ± 1.97; and 11.11 ± 1.16 and 11.06 ± 1.09 x 10³/µl of blood, respectively. All three groups showed non significant difference (p< 0.05) in mean TLC levels, following therapy. These values are in accordance with the previous findings of Satish Kumar and Choudhuri, (2002) who recorded the mean TLC values as 6.2 to 18.1 and 11.36 ± 1.46 × 10³ / µl respectively. However, the mean TLC values reported by Oskoui et al (2008) are on higher side and TLC levels will go up to 47,500/ µL in EPI with infectious origin.

The mean serum glucose levels recorded before and after treatment in Group I, II and III were 79.92 ± 7.66 and 84.50 ± 6.84; 76.60 ± 9.77 and 82.44 ± 7.40; and 78.74 ± 6.91 and 82.38 ± 8.41 mg/dl, respectively. However, there was no significant difference in the mean values of serum glucose before and after treatment in all three groups. Koutinas et al. (1994) made similar observations of normal serum glucose concentration in four Mongrel dogs. However, slight increase in serum glucose level by Kovacevic (2006) and significant increased serum glucose levels (182.24±2.16 mg/dl) were recorded by Satish Kumar and Choudhuri (2002) in the EPI positive cases complicated with diabetes mellitus. The mean serum Gamma Glutamyl Transferase (GGT) values before and after treatment in Group I, II and III were 4.77 ± 1.12 and 4.93 ± 1.22; 4.13 ± 0.72 and 4.41 ± 0.79; and 6.30 ± 1.51 and 6.36 ± 1.51 IU/L, respectively. All three groups showed no change and the difference was not significant in mean GGT levels, following therapy and is matching with the previous observation of Strombeck et al. (1984). The levels of mean serum Amylase before and after treatment in Group I, II and III were 215.09 ± 22.94 and 621.09 ± 79.22; 196.93 ± 26.31 and 625.79 ± 58.21; and 171.42 ± 21.74 and 700.79 ± 52.93 IU/L, respectively. In the present study serum Amylase levels were higher after the therapy and the increase in values were significant (P<0.05) in all the three groups. On perusal of table 4, it was found that the above enzyme increased gradually from day 5 onwards till day 7 which suggested the therapeutic agent used augmented the necessary secretion of enzymes. The above findings were in association with the observation of Oskoui et al. (2008) who recorded 735 IU/L of serum amylase in EPI positive case. Decreased levels of serum amylase in EPI affected dogs were also noticed by Jacobs et al. (1982) and Simpson and Doxey (1990). However, Kovacevic (2006) reported the increased serum amylase levels in confirmed cases of EPI. In contrast, opposite findings of neither serum isoamylase determination nor total amylase activity had adequate sensitivity to support their use in diagnosis of EPI has been reported by Jacobs et al. (1988). The mean serum lipase levels recorded before and after treatment in group I, II and III were 19.19 ± 1.93 and 203.66 ± 25.75; 14.53 ± 1.49 and 174.16 ± 36.06; and 20.35 ± 2.23 and 266.59 ± 16.30 U/L, respectively. The increase in serum Lipase levels following treatment was significant (P<0.05) in all the three groups. On scrutiny of the Table 4, it was evident that the levels of mean serum lipase Increase is almost three folds which suggest that the reason for increased levels was due to correction of secretary activity of the pancreas by the gut. It is evident that the dogs which were treated had sufficient quantity of pancreatic extract. Similar observations of decreased serum lipase levels 3-37 (IU/L) in EPI affected dogs has been made by Whitney et al. (1986). Impaired digestion could imply a pancreatic disease with a deficiency of enzymes necessary for hydrolysis of fat (Brobst, 1972) and deficient secretion of pancreatic lipase can be demonstrated by severe steatorrhoea Batt (1979).

Table 4: Mean haematological and serological findings in Group I, II & III on day 0 and 7.

* Non significant at 5 percent (p < 0.05) level; ** Significant at 5 percent (p < 0.05) level.

Conclusion

Based on the above findings, from the present investigation, it is recommended that the x-ray film digestion test, microscopic examination of faeces for the presence of fat, starch and proteins and serological estimation of pancreatic enzymes i.e. amylase and lipase are playing a vital role in diagnosing the EPI. Among all, decreased concentration of serum amylase and lipase were shown to be effective for the confirmatory diagnosis of Exocrine Pancreatic Insufficiency in canines.

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