- Email: [email protected]
Ultrasonographic Imaging of Experimentally Induced Pancreatitis in Cattle
The Veterinary Journal 2003, 165, 314–324 doi:10.1016/S1090-0233(02)00179-X
Ultrasonographic Imaging of Experimentally Induced Pancreatitis in Cattle T. MOHAMED, H. SATO, T. KUROSAWA, S. OIKAWA and A. NITANAI Department of Veterinary Internal Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido 069-8501, Japan
SUMMARY This study was conducted to determine the ultrasonographic patterns of pancreatitis evoked in cattle, with reference to laboratory and pathological findings. Using ultrasonographic guidance, acute necrotizing pancreatitis was induced in six cows by injecting chloroform into the pancreatic tissue. Ultrasonographic examination was then performed once daily for nine days. Pancreatic lesions were visible 24 h after induction of pancreatitis, as represented by a uniform increase in echogenicity and by intralobular and subcapsular fluid accumulation. As the experiment progressed, patchy hypoechogenic foci appeared within the gland parenchyma. Amylase and lipase activities showed rapid increases. Post mortem examination revealed gross and microscopic necrotic and haemorrhagic lesions in the body and right lobe of the pancreas, accompanied by oedema and fibrosis. Ultrasonography was found to be extremely useful for the detection and characterization of experimentally induced pancreatitis and to monitor its progression in the cow. These findings are of potential value as a reference for the diagnostic workup of bovine pancreatitis, and ultrasonography is seen as a promising non-invasive technique for the diagnosis of suspected pancreatitis in cattle. Ó 2002 Elsevier Science Ltd. All rights reserved.
KEYWORDS: Bovine pancreatitis; histopathology; imaging patterns; pancreatitis modelling; ultrasonography.
INTRODUCTION Although pancreatitis is considered to be less common in cattle than in humans, dogs, and cats, the incidence may be higher than is apparent from the scattered reports of its primary occurrence. Doherty et al. (1998) reported a three-year-old Friesian cow in which diabetes mellitus was associated with lymphocytic pancreatitis and in 2001, Braun et al. reported a six-year-old Simmental bull with diabetes mellitus caused by pancreatitis. In both studies, the animals were admitted to the clinic suffering from ketosis and in poor bodily condition, and pancreatitis in each case was incidentally diagnosed at necropsy. Other studies have discussed pancreolithiasis in cattle (Velling, 1975; Groom, 1994). In the bovine pancreas, inflammatory and degenerative changes are detected post mortem but are Correspondence to: Tharwat Mohamed, Department of Veterinary Internal Medicine, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan. Tel.: +81-11-388-4819; fax: +81-11-3875890; E-mail: [email protected]
rarely diagnosed clinically because of a lack of pathognomonic clinical signs and definitive laboratory findings (Velling, 1975; Groom, 1994; Doherty et al., 1998; Braun et al., 2001). Because the diseased pancreas often eludes clinical evaluation in animals, the veterinary clinician must rely on other diagnostic tools that can be used to support the history, clinical signs, physical examination findings, and laboratory results. Computed axial tomography is the most useful imaging modality for pancreatic evaluation in humans, but the expense, need for general anaesthesia and limited availability make it less useful in veterinary practice (Miles et al., 1988; Saunders et al., 1992; Williams, 2000). As a result, ultrasonography (US) is currently the imaging modality of choice in the evaluation of pancreatic diseases in domestic species (Saunders, 1991; Saunders et al., 1992). An early report on six dogs with experimentally induced pancreatitis suggested that pancreatic US may be a valuable technique for evaluating dogs with spontaneously-occurring acute pancreatitis (Murtaugh et al., 1985). In a large study population
1090-0233/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
PANCREATITIS IMAGING IN CATTLE
of 70 dogs having fatal acute pancreatitis, Hess et al. (1998) reported that acute pancreatitis was more consistently diagnosed using abdominal US than using abdominal radiography. Furthermore, although pancreatitis has historically been diagnosed in the human and the dog more frequently than in the cat, studies have established that US is also an extremely valuable tool for identifying pancreatitis in cats (Simpson et al., 1994; Wall et al., 2001). It was believed that the frequency of pancreatitis was low in this species (Duffel, 1975; Strombeck, 1979) and the disease often went undiagnosed until after an exploratory laparotomy or necropsy (Rogers, 1983). However, with advancement in US, there is now growing evidence that pancreatitis occurs more frequently in the cat than was once believed (Simpson et al., 1994; Steiner & Williams, 1997). The prevalence of bovine cases of pancreatitis is not clear. From time to time in our laboratory, naturally-occurring pancreatitis has been revealed during necropsy of cows suffering from other diseases. For example, during 2001, 525 cows were necropsied and nine of these cows were shown to have pancreatitis at post mortem examination. The cows had different conditions including fat necrosis, endocarditis, lymphosarcoma, and caudal vena cava thrombosis. These finding led us to consider that pancreatitis may also occur more frequently in cattle. However, the ultrasonographic patterns of pancreatitis have not been documented in cows, the purpose of the present study was to define the ultrasonographic characteristics of pancreatitis induced in cattle, as a step toward providing a potential reference guide for the diagnostic workup of bovine pancreatitis. Understanding the ultrasonographic characteristics of the disease may help to identify early cases of clinical pancreatitis. MATERIALS AND METHODS Animals Nine non-pregnant non-lactating Holstein cows weighing 580 52 kg (mean SD) were included in the study and divided into three groups of three animals each: experimental (healthy), control (healthy), and preliminary (one with a minor foot problem and two with chronic mastitis). The cows in the preliminary group were used only in the ultrasonographic, physical, and post mortem examinations. In all nine cows, the serum total amylase and lipase activities were within normal range. All cows were free of alimentary tract disease, as shown by the
315
history and by physical and laboratory examinations, and there were no abnormalities in their appetites. The animals received a daily ration in two equal portions at 0700 and 1700 h. Each animal was given a basal concentrate diet (up to 4 kg/cow/day) containing 18% crude protein and 74% total digestible nutrient, a constant amount of grass hay (about 1% of BW) and water was available ad libitum. Their care and handling were in accordance with the Laboratory Animal Control Guidelines of Rakuno Gakuen University for life science research, in conformity with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in USA (NIH publication No. 86-23, revised 1985). Ultrasonographic examination of the pancreas Before injecting chloroform (CF) into the pancreas, and once daily for nine days after injection, the organ of each cow was examined ultrasonographically as described by Pusterla and Braun (1997). We used an ultrasound scanner (Model EUB-26, Hitachi Co.) equipped with a 3.5 MHz linear transducer. Briefly, the procedure was as follows: the hair was clipped and shaved over an area between the right ninth intercostal space to approximately 12 cm caudal to the costal arch. A generous amount of alcohol was applied to the skin as a transmission medium instead of ultrasonic coupling gel. With the transducer placed parallel to the ribs of the cow in a standing position, the entire shaved region was scanned in a systematic fashion caudal to cranial as far as the costal arch and the intercostal spaces nine to twelve were scanned dorsoventrally. The pancreatic body and right lobe of the organ could be seen easily; however, the left lobe could not be seen. Induction of acute pancreatitis Because the injection needle must penetrate the liver parenchyma, carrying a significant risk of haemorrhage (Mohamed et al., 2002), platelet count, prothrombin time, and activated partial thromboplastin time (APTT) were determined for all cows before injection of the pancreas. The region was aseptically prepared and infiltrated with 10 mL of 2% procaine hydrochloride. Each cow was sedated with xylazine (0.07 mg/kg body weight, intravenously), and a 17-gauge needle (Ebara) (17.5 cm in length) was inserted percutaneously and directed sonographically to the pancreatic tissue. The needle was maintained parallel to the plane of the ultrasound beam at about a 20–40° angle from the long axis of the transducer.
316
THE VETERINARY JOURNAL, 165, 3
spiratory rate, and intestinal and ruminal motility; and auscultation of the thorax and heart. From the cows in the experimental group and controls, blood (10 mL) was sampled before injection of the pancreas (day zero) and daily for the following nine days. It was allowed to clot and the sera collected. Blood samples for determination of lipid profiles were collected in heparinized tubes. Harvested sera and plasma were stored at )30 °C pending assay.
Fig. 1. Intrapancreatic injection of chloroform (labeled S INJ). ND: needle; PV: portal vein.
Chloroform (Wako Pure Chemicals) was selected to induce pancreatitis following earlier work in cattle (Hassan et al., 1980) and in dogs (Mia et al., 1978; Akuzawa et al., 1994). Using ultrasonographic guidance, 8 mL CF was given to each of six cows (experimental group and preliminary group) by injection into five sites of the pancreatic parenchyma (approximately 1.5 mL at each site) (Fig. 1). To avoid inadvertent leakage of CF into the peritoneal cavity, the injection was made only to the thicker parts of the pancreatic parenchyma. Control cows were injected with the same amount of physiological saline (PS). Pancreatic US was performed once daily until the cows were euthanased nine days later and necropsied. To minimize stress or possible discomfort, the experimental period was limited to nine days. Suspected pancreatic lesions appearing on the ultrasonograms were confirmed by comparison with the normal anatomical findings from the pre-experimental ultrasonographic examination and in conjunction with the gross pathological findings at necropsy. The laboratory and pathological features of the model (experimental group) were compared with those of control cows. Pancreatitis was confirmed histopathologically. Physical examination and blood sampling All cows underwent a physical examination prior to the onset of the experiment and twice daily for the next nine days. Clinical evaluation included assessment of general appearance, activity, and appetite; determination of rectal temperature, pulse rate, re-
Laboratory examination Haemostatic data (platelet count, prothrombin time, and APTT) were determined in the Kishimoto Clinical Laboratory, Sapporo, Japan. Haemograms were measured using a Particle counter (Model F-820, Sysmex Inc.). The serum lipase activity was determined with a commercially available kit (No. 800-B Sigma Diagnostics) based on the titrimetric method of Tietz and Fiereck (1966). Clinical chemistry kits (Wako Pure Chemicals) were used to analyse other parameters, including the concentration of amylase, glucose, albumin, calcium, ornithine carbamoyltransferase (OCT), c-glutamyltransferase (GGT), blood urea nitrogen (BUN), creatinine, total cholesterol (TC), non-esterified fatty-acids (NEFA), triglycerides (TG), and phospholipids (PL). Statistical analysis All values are expressed as mean SD. Measurements used in statistical analysis were taken only from the experimental group and controls. Pre- and post-induction mean values were analysed using Duncan’s multiple-range test, and the difference was considered significant if P < 0:05 and highly significant if P < 0:01. RESULTS Ultrasonographic examination before injection of the pancreas In all cows, the pancreatic body and right lobe of the organ were consistently identified as a triangular structure; however, the left lobe of the pancreas could not be visualized. The echogenic pattern of the pancreas was homogeneous throughout and could be clearly differentiated from that of adjacent structures. The pancreas was slightly hyperechogenic compared to the liver parenchyma. The accessory pancreatic duct, recognized in five cows, appeared as a tubular, anechoic structure with a well-defined, hyperechogenic wall. The pancreaticoduodenal vein, identified in seven cows, appeared
PANCREATITIS IMAGING IN CATTLE
317
appeared normal except for a decreased appetite and mild fever in two of the animals on the second and third days of the study. In PS-treated controls, no changes were observed. The platelet count, prothrombin time and APTT, determined before injection of the pancreas, were within the normal ranges for cattle. At day 5 in the experimental group, the leukocyte count was significantly ðP < 0:05Þ higher than the pre-experimental values; however, no changes were observed in the other haematological parameters. Haematological values of control cows did not differ significantly from base-line values.
Fig. 2. Normal pancreatic sonograms before injection of chloroform into the pancreas. (A) The body and right limb of the pancreas are visible as a triangular structure. (B) The pancreaticoduodenal vein (PDV) appears as a bandlike anechoic structure coursing from the duodenum along the right lobe of the pancreas to where it joins the portal vein (PV). In both sonograms, the margins of the uniformly echogenic pancreas are designated by arrows.
as a band-like structure coursing from the duodenum along the right lobe of the pancreas to where it joined the portal vein (Fig. 2). Ultrasonographic examination after injection of the pancreas A uniform increase in echogenicity with subcapsular and intralobular fluid accumulation was identified in the pancreatic region on ultrasound scans obtained 24 h after CF-injection. On day 3, poorly circumscribed hypoechogenic foci appeared within the pancreatic parenchyma. As the experiment progressed, well-defined anechoic areas appeared in the pancreatic body and right lobe. On the last day of the experiment, changes in the ultrasonographic appearance were evident exclusively in the pancreatic parenchyma, the anechoic or hypoechogenic areas becoming larger and more clearly defined (Fig. 3). In the PS-injected cows, no changes occurred in the echogenicity pattern of the pancreatic parenchyma throughout the experimental period. In all groups, the hepatic parenchyma appeared normal at the start of the experiment and remained unchanged. Physical and haematological examinations In the six CF-treated cows, no changes were noted on physical examination and all the animals
Biochemical examination In marked contrast to the control cows, CF-injected cows showed rapid increases in the mean values of amylase (5.4-fold) and lipase (7.9-fold) activities, with the values parallel and reaching a peak concurrently 48 h after induction of pancreatitis. The serum amylase value returned to base-line levels earlier than did the mean serum lipase concentrations (Fig. 4). In both the experimental group and controls, changes in the mean concentrations of glucose, albumin, and calcium were negligible. Moreover, BUN as well as creatinine tested normal (Tables I, II). The serum OCT activity increased significantly ðP < 0:05Þ in CF-treated cows from day 2 to day 3; however, in none of the cows did any changes occur in the GOT activity. Mean activities of lipase, amylase, OCT, and GGT in control cows did not differ significantly from base-line values. The cows injected with CF did not develop any consistent alteration in plasma lipid patterns (Table I). Pathological examination In each of the cows injected with CF and euthanased nine days later, the pancreas was grossly enlarged (1052 205 g) with adhesions to surrounding viscera. Necrotic areas and haemorrhage with multiple, fluid-filled cystic spaces were identified within the pancreas. The body and right lobe of the pancreas consistently exhibited most of the pathological changes. Multiple tissue sections revealed degeneration and necrosis of pancreatic acinar cells, in addition to fibrosis and haemorrhage in various degrees of severity. Interlobular septa were markedly oedematous with varying populations of inflammatory cells, mainly lymphocytes and neutrophils (Fig. 5). Many venous thrombi were scattered throughout the inflamed tissue. In control cows, the results of the pathological examination were normal.
318
THE VETERINARY JOURNAL, 165, 3
Fig. 3. Pancreatic imaging after induction of pancreatitis. (A) Ultrasonographic image made 24 h after chloroform injection. An overall increase in echogenicity is clear. The margins of the pancreas are designated by arrows. (B) Ultrasonographic image made 72 h after induction of pancreatitis. Hypoechogenic foci can be identified within the right lobe of the pancreas. Arrows show the pancreatic margins. (B) The echogenicity of landscape-like hypoechoic region in (C) seem to increase in echogenicity (D).
DISCUSSION Ultrasonography is routinely used to diagnose pancreatic diseases in humans (Bennett & Hann, 2001; Hirooka et al., 2001; Koito et al., 2001) and in dogs (Lamb et al., 1995; Hess et al., 1998; Van Enkevort et al., 1999) and has gained increasing importance for detecting pancreatitis in the cat (Simpson et al., 1994; Wall et al., 2001). Pancreatic US in cattle is in its infancy, but with a clear understanding of the physical principles of ultrasound, detailed knowledge of normal and abnormal pancreatic ultrasonic
anatomy, and with experience, ultrasonic imaging of the pancreatic diseases in cows should assume an important role in veterinary practice. In the present study, our pre-experimental images of the pancreatic region concur with the normal ultrasonic anatomy of the bovine pancreas as described by Pusterla and Braun (1997). However for US of acute pancreatitis, no bovine studies are available for comparison. Acute pancreatitis has been surgically produced in cattle by injection of CF in situ into the pancreatic
PANCREATITIS IMAGING IN CATTLE
319
Fig. 4. Serum total amylase and lipase activities (mean SD) vs time in cows with experimentally induced pancreatitis ðn ¼ 3Þ and in control cows ðn ¼ 3Þ.
tissue (Hassan et al., 1980) and in dogs by a wide variety of means, such as perfusion of oleic acid into the pancreatic duct (Attix et al., 1981; Nyland et al., 1983; Murtaugh et al., 1985; Murtaugh & Jacobs, 1985; Chikamune et al., 1998) and injection of CF in situ into the pancreatic tissue (Mia et al., 1978; Akuzawa et al., 1994). In the current study, induction of acute pancreatitis was successful in the preliminary group as shown by US and pathology findings and successful in the experimental group as evidenced by US, haematological and biochemical values and pathology findings. Necrosis found in the pancreas at post mortem examination, leukocytosis and dramatic increases in serum amylase and lipase activities during the first 48 h after CF-infusion, in conjunction with the histological abnormalities,
show that CF-injection into the pancreatic parenchyma provides an effective means of experimentally elicting pancreatitis in the cows. The increased echogenicity of the pancreatic parenchyma observed 24 h after CF-injection was suspected to indicate frequent, diffuse involvement of the gland and make it more echogenic, presumbly due to oedema. The hypoechogenicity of the pancreas detected at day three is believed to represent necrosis, haemorrhage, and inflammatory exudate, as confirmed by the post mortem examination. This finding is in line with reports of acute pancreatitis in humans (Cox et al., 1980) and dogs (Nyland et al., 1983; Murtaugh et al., 1985), in which decreased echogenecity in the pancreatic region has also been attributed to oedema, haemorrhage, and
320
Table I Haematological and biochemical readings from chloroform-treated experimental cows (n = 3) Parameter 0
1
2
3
4
5
6
7
8
9
67 13 3:8 1:2 42 2 2:3 0:08 24 6 65 3:4 0:7 70 7 1:9 0:7 0:2 0:08 0:16 0:04 1:13 0:2
67 32 3:4 0:7 41 1 2:3 0:17 30 14 25 18 3:5 0:9 68 4 1:8 0:6 0:1 0:09 0:19 0:03 1:09 0:2
68 26 3:2 0:5 42 3 2:4 0:18 30 12 20 13 2:9 1:4 62 3 1:9 0:6 0:2 0:06 0:16 0:08 1:11 0:1
76 8 2:8 0:5 38 2 2:3 0:15 27 10 10 6 3:3 1:2 65 3 1:9 0:5 0:2 0:04 0:18 0:05 1:08 0:1
78 15 2:9 0:7 37 3 2:2 0:09 23 7 73 3:2 1:1 69 5 1:9 0:6 0:2 0:05 0:19 0:08 1:04 0:2
86 10 3:4 0:7 38 3 2:3 0:12 27 12 63 3:3 0:8 60 3 1:8 0:4 0:3 0:11 0:18 0:05 1:02 0:1
69 8 3:2 0:9 39 5 2:3 0:05 26 11 53 3:8 1:6 72 4 1:9 0:5 0:3 0:08 0:19 0:07 1:03 0:2
60 16 3:3 0:7 38 4 2:3 0:13 31 15 14 9 3:4 0:8 69 6 2:1 0:8 0:3 0:15 0:18 0:08 1:05 0:1
61 15 3:4 0:4 39 4 2:4 0:11 32 15 93 4:2 2:8 70 9 2:2 0:7 0:3 0:19 0:23 0:09 1:09 0:3
55 28 3:1 0:1 37 5 2:4 0:51 33 17 71 13:7 1:6 72 5 2:1 0:6 0:3 0:13 0:21 0:07 0:99 0:1
WBC: white blood cells; GGT: c -glutamyltransferase; OCT: ornithine carbamoyltransferase; BUN: blood urea nitrogen; Cr: creatinine; TC: total cholesterol; NEFA: non-esterified fattyacids; TG: triglycerides; PL: phospholipids. *P < 0:05 compared to zero day values.
THE VETERINARY JOURNAL, 165, 3
WBC ð102 =lLÞ Glucose (mmol/L) Albumin (g/L) Calcium (mmol/L) GGT (IU/L) OCT (IU/L) BUN (mmol/L) Cr (lmol/L) TC (mmol/L) NEFA (mmol/L) TG (mmol/L) PL (mmol/L)
Time after injection (day)
Table II Haematological and biochemical readings from control cows (n = 3) Parameter 0 2
WBC ð10 =lLÞ Glucose (mmol/L) Albumin (g/L) Calcium (mmol/L) GGT (IU/L) OCT (IU/L) BUN (mmol/L) Cr ðlmol=LÞ TC (mmol/L) NEFA (mmol/L) TG (mmol/L) PL (mmol/L)
1
2
3
4
5
6
7
73 20 76 7 70 15 68 22 71 9 75 20 76 13 66 10 3:2 0:8 3:3 0:5 3:9 0:7 2:8 0:5 2:9 0:8 2:8 0:4 2:8 0:6 2:7 0:9 44 5 44 2 43 3 43 1 41 5 43 5 41 6 44 7 2:6 0:22 2:7 0:15 2:7 0:28 2:6 0:11 2:8 0:13 2:6 0:19 2:6 0:14 10:9 0:6 27 12 28 10 27 7 26 11 24 8 23 6 19 15 2:7 16 84 66 54 53 64 64 93 52 3:5 0:4 3:8 0:5 4:2 1:5 2:6 0:7 3:7 0:9 3:2 0:8 3:3 0:4 3:2 0:6 90 8 110 3 105 7 100 4 95 6 93 7 1078 85 7 2:2 0:5 2:4 0:6 1:7 0:5 1:9 0:3 1:9 0:7 2:4 0:3 1:8 0:5 1:7 0:4 0:3 0:09 0:2 0:11 0:3 0:08 0:2 0:10 0:3 0:14 0:3 0:06 0:3 0:18 0:2 0:09 0:15 0:08 0:18 0:04 0:14 0:05 0:12 0:09 0:14 0:08 0:18 0:07 0:11 0:03 0:12 0:04 1:02 0:1 1:05 0:2 1:01 0:1 0:91 0:1 0:92 0:3 0:62 0:18 0:92 0:2 1:01 0:3
8
9
74 14 3:1 0:3 45 4 10:9 0:4 2:7 0:9 66 3:8 0:9 99 8 2:1 0:6 0:2 0:10 0:19 0:06 1:01 0:14
78 25 3:4 0:5 45 2 11:7 0:6 2:9 0:15 73 3:6 0:5 102 9 2:3 0:7 0:2 0:11 0:25 0:07 0:89 0:1
PANCREATITIS IMAGING IN CATTLE
Time after injection (day)
See Table I for Key.
321
322
THE VETERINARY JOURNAL, 165, 3
Fig. 5. Photomicrographs of pancreatic necropsy specimens obtained nine days after chloroform injection. (A) Pancreatic acini are necrotic. Numerous lymphocytes, plasma cells and a few neutrophils and eosinophils infiltrate the proliferating fibrous tissue between normal and necrotic areas (white arrows). Interstitial oedema is also evident (solid black arrows). (B) Epithelial cells of excretory ducts are desquamated (arrowhead) and haemorrhage is present in the necrotic area. An intravascular thrombus is also evident (open black arrow). H and Estain. Bar ¼ 300 lm.
inflammatory exudate. Pancreatomegaly and hypoechogenicity in our cows are in agreement with the ultrasonographic findings described in pancreatitis induced experimentally in dogs (Nyland et al., 1983; Murtaugh et al., 1985) and cats (Kitchell et al., 1986). An enlarged hypoechogenic pancreas has also been described in dogs and cats having naturally-occurring pancreatitis (Saunders, 1991; Simpson et al., 1994; Wall et al., 2001). It was somewhat surprising that on the daily physical examinations, the cows with induced pancreatitis displayed no apparent abnormalities, other than a slightly decreased appetite and mild fever in
two animals on the second and third days after CFinjection. Although the islets of Langerhans were necrotic in some areas of the histological sections examined, it might be argued that sufficient endocrine function of the organ is maintained by the cells in areas of viable tissue, as suggested by the absence of overt diabetes mellitus in any of the cows. The normal blood glucose level might be because the injection of CF was made only to the body and right lobe of the pancreas, possibly leaving the left lobe disease-free and, therefore, functionally normal for the duration of the experiments. The normal blood calcium levels in the cows may be explained, at least in part, by the normal serum albumin concentrations. This view is in keeping with the rationale behind studies on dogs with acute pancreatitis, in which hypocalcaemia was attributed to decreased serum albumin concentrations resulting in attenuation of non-ionized calcium (Attix et al., 1981; Feldman et al., 1981). The high serum OCT values in the present study suggest that the disturbance of hepatocytes might occur after CFinfusion. Induction of pancreatitis in the present study did not change the lipid profile patterns in any of the cows, indicating that plasma lipid profiles would appear to have little value in the diagnosis of acute pancreatitis in cows. In a study of 70 dogs with fatal acute pancreatitis, Hess et al. (1998) concluded that the diagnostic value of amylase and lipase is limited. Similarly, in the cat, serum amylase and lipase concentrations have been reported to be of little use in diagnosing pancreatitis (Hill & Van Winkle, 1993; Hardy, 1994; Stewart, 1994; Simpson, 1997). In the present cows, although pancreatitis resulted in rapid increases in amylase and lipase activities, the values returned quickly to within reference ranges. This finding indicates that in cows with chronic pancreatitis the amylase and lipase values would remain normal. We concluded that serum amylase and lipase concentrations appear to be of little use in diagnosing bovine pancreatitis. Elevations may be used in support of a diagnosis, but evidence indicates they can not be used as a standard for a definitive diagnosis. In cows it is difficult to draw definitive conclusions from cases of naturally occurring pancreatitis because either recovery or extended therapy may alter the disease pattern before pathological confirmation can be obtained. Because it is not certain to what degree of reliability the model mimics spontaneous pancreatitis in cows and whether the ultrasonographic characteristics would differ, studies are in progress to further delineate the ultraso-
PANCREATITIS IMAGING IN CATTLE
nographic features of pancreatitis in cows. Results of the present experimental model are deemed useful as potential reference values for the ultrasonographic, laboratory and pathological characteristics in the early recognition of cows with naturally occurring pancreatitis. Further studies are warranted from veterinarians making use of these values for evaluating suspected pancreatitis in cows. ACKNOWLEDGMENTS The authors are deeply grateful to Professor N. Kennedy (Biomedical English) for her critical reading of the manuscript, Dr. T. Onaga (Physiology) for his technical assistance and Dr. H. Okada (Pathology) for his comments on the pathological slides. REFERENCES AKUZAWA, M., MORIZONO, M., NAGATA, K., HAYANO, S., SAKAMOTO, H., YASUDA, N., OKAMOTO, K., KAWASAKI, Y. & DEGUCHI, E. (1994). Changes of serum amylase, its isozyme fractions and amylase-creatinine clearance ratio in dogs with experimentally induced acute pancreatitis. Journal of Veterinary Medical Science 56, 269–73. ATTIX, E., STROMBECK, D. R. & WHEELDON, E. B. (1981). Effects of anticholinergic and a corticosteriod on acute pancreatitis in experimental dogs. American Journal of Veterinary Research 42, 1668–74. BENNETT, G. L. & HANN, L. E. (2001). Pancreatic ultrasonography. Surgical Clinics of North America 81, 259–81. BRAUN, U., DURR, M., DIENER, M., OSSENT, P., HAMMON, H. & BLUM, J. W. (2001). Diabetes mellitus caused by pancreatitis in a bull. Schweiz. Arch. Tierheilk. 143, 99–104. CHIKAMUNE, T., KATAMOTO, H., NOMURA, K. & OHASHI, F. (1998). Lipoprotein profile in canine pancreatitis induced with oleic acid. Journal of Veterinary Medical Science 60, 413–21. COX, K. L., AMENT, M. E., SAMPLE, W. F., SARTI, D. A., O’DONNELL, M. & BYRNE, W. J. (1980). The ultrasonic and biochemical diagnosis of pancreatitis in children. Journal of Pediatrics 96, 407–11. DOHERTY, M. L., HEALY, A. M. & DONNELLY, W. J. C. (1998). Diabetes mellitus associated with lymphocytic pancreatitis in a cow. Veterinary Record 142, 493. DUFFEL, S. J. (1975). Some aspects of pancreatic disease in the cat. Journal of Small Animal Practice 16, 356–74. FELDMAN, B. F., ATTIX, E. A., STROMBECK, D. R. & O’NEILL, S. (1981). Biochemical and coagulation changes in a canine model of acute necrotizing pancreatitis. American Journal of Veterinary Research 42, 805–9. GROOM, S. (1994). Pancreolithiasis in a Holstein-Friesian cow. Canadian Veterinary Journal 35, 244. HARDY, R. M. (1994). Disease of the exocrine pancreas. In: The Cat; Diseases and Clinical Management, ed. R. G. Sherding, pp. 1287–92. New York: Churchill Livingstone.
323
HASSAN, M. S., AMER, A. A., EL-AMROUSI, S. & BAYOUMI, A. H. (1980). Some studies on pancreatic function in cattle and buffaloes. II. Induced acute pancreatitis. Assiut Veterinary Medical Journal 7, 295–306. HESS, R. S., SAUNDERS, H. M., VAN WINKLE, T. J., SCHOFER, F. S. & WASHABAU, R. J. (1998). Clinical, clinicopathologic, radiographic and ultrasonographic abnormalities in dogs with fatal acute pancreatitis: 70 cases (1986–1995). Journal of the American Veterinary Medical Association 213, 665–70. HILL, R. C. & VAN WINKLE, T. J. (1993). Acute necrotizing pancreatitis and acute suppurative pancreatitis in the cat: a retrospective study of 40 cases (1976–1989). Journal of Veterinary Internal Medicine 7, 25–33. HIROOKA, Y., GOTO, H., HASHIMOTO, S., HIRAI, T., NIWA, K., TAKEDA, K. & HAYAKAWA, T. (2001). Recent advances in US diagnosis of pancreatic cancer. Hepatogastroenterology 48, 916–22. KITCHELL, B. E., STROMBECK, D. R., CULLER, J. & HARROLD, D. (1986). Clinical and pathologic changes in experimentally induced acute pancreatitis in cats. American Journal of Veterinary Research 47, 1170–3. KOITO, K., NAMIENO, T., NAGAKAWA, T., HIROKAWA, N., ICHIMURA, T., SYONAI, T., YAMA, N., SOMEYA, M., NAKATA, K., SAKATA, K. & HAREYAMA, M. (2001). Pancreas: imaging diagnosis with color/power Doppler ultrasonography, endoscopic ultrasonography, and intraductal ultrasonography. European Journal of Radiology 38, 94–101. LAMB, C. R., SIMPSON, K. W., BOSWOOD, A. & MATTHEWMAN, L. A. (1995). Ultrasonography of pancreatic neoplasia in the dog: a retrospective review of 16 cases. Veterinary Record 137, 65–8. MIA, A. S., KOGER, H. D. & TIERNEY, M. M. (1978). Serum values of amylase and pancreatic lipase in healthy mature dogs and dogs with experimental pancreatitis. American Journal of Veterinary Research 39, 965–9. MILES, K., LATTIMER, J. C., KRAUSE, G. F., KNAPP, D. W. & SAYLES, C. E. (1988). The use of intraperitoneal fluid as a simple technique for enhancing sonographic visualization of the canine pancreas. Veterinary Radiology 29, 258–63. MOHAMED, T., SATO, H., KUROSAWA, T. & OIKAWA, S. (2002). Echo-guided studies on portal and hepatic blood in cattle. Journal of Veterinary Medical Science 64, 23–8. MURTAUGH, R. J., HERRING, D. S., JACOBS, R. M. & DEHOFF, W. D. (1985). Pancreatic ultrasonography in dogs with experimentally induced acute pancreatitis. Veterinary Radiology 26, 27–32. MURTAUGH, R. J. & JACOBS, R. M. (1985). Serum antiprotease concentrations in dogs with spontaneous and experimentally induced acute pancreatitis. American Journal of Veterinary Research 46, 80–3. NYLAND, T. G., MULVANY, M. H. & STROMBEK, D. R. (1983). Ultrasonic features of experimentally induced acute pancreatitis in the dog. Veterinary Radiology 24, 260–6. PUSTERLA, N. & BRAUN, U. (1997). Ultrasonographic examination of the pancreas in healthy cows. Veterinary Radiology and Ultrasound 38, 63–7. ROGERS, W. A. (1983). Diseases of the exocrine pancreas. In: Veterinary Internal Medicine, ed. S. J. Ettinger, pp. 1435–55. Philadelphia, PA: WB Saunders.
324
THE VETERINARY JOURNAL, 165, 3
SAUNDERS, H. M. (1991). Ultrasonography of the pancreas. In: Problems in Veterinary Medicine, ed. P. M. Kaplan, pp. 583–603. Philadelphia, PA: WB Saunders. SAUNDERS, H. M., PUGH, C. R. & RHODES, W. H. (1992). Expanding applications of abdominal Ultrasonography. Journal of the American Animal Hospital Association 28, 369–74. SIMPSON, K. W., SHIROMA, J. T., BILLER, D. S., WICKS, J., JOHNSON, S. E., DIMSKI, D. & CHEW, D. (1994). Ante mortem diagnosis of pancreatitis in four cats. Journal of Small Animal Practice 35, 93–9. SIMPSON, K. W. (1997). Acute pancreatitis. In: Consultations in Feline Internal Medicine, ed. J. R. August, pp. 91–8. Philadelphia, PA: WB Saunders. STEINER, J. M. & WILLIAMS, D. A. (1997). Feline exocrine pancreatic disorders: insufficiency, neoplasia and uncommon conditions. Compendium on Continuing Education for the Practicing Veterinarian 19, 836–49. STEWART, A. F. (1994). Pancreatitis in dogs and cats: cause, pathogenesis, diagnosis and treatment. Compendium on Continuing Education for the Practicing Veterinarian 16, 1423–30.
STROMBECK, D. R. (1979). Small Animal Gastroenterology pp. 301–31. Davis,CA: Stonegate Publishing Co.. TIETZ, N. W. & FIERECK, E. A. (1966). A specific method for serum lipase determination. Clinica Chimica Acta 13, 352. VAN ENKEVORT, B. A., O’BRIEN, R. T. & YOUNG, K. M. (1999). Pancreatic pseudocysts in 4 dogs and 2 cats: ultrasonographic and clinicopathologic findings. Journal of Veterinary Internal Medicine 13, 309–13. VELLING, K. (1975). Bovine pancreolithiasis in Denmark. Acta Veterinaria Scandinavica 16, 327–40. WALL, M., BILLER, D. S., SCHONING, P., OLSEN, D. & MOORE, L. E. (2001). Pancreatitis in a cat demonstrating pancreatic duct dilatation ultrasonographically. Journal of the American Animal Hospital Association 37, 49–53. WILLIAMS, D. A. (2000). Exocrine pancreatic disease. In: Textbook of Veterinary Internal Medicinevol. 5, eds. S. J. Ettinger, & E. C. Feldman, (vol. 5) pp. 1345–67. Philadelphia, PA: WB Saunders. (Accepted for publication 29 May 2002)
Ultrasonographic Imaging of Experimentally Induced Pancreatitis in Cattle T. MOHAMED, H. SATO, T. KUROSAWA, S. OIKAWA and A. NITANAI Department of Veterinary Internal Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido 069-8501, Japan
SUMMARY This study was conducted to determine the ultrasonographic patterns of pancreatitis evoked in cattle, with reference to laboratory and pathological findings. Using ultrasonographic guidance, acute necrotizing pancreatitis was induced in six cows by injecting chloroform into the pancreatic tissue. Ultrasonographic examination was then performed once daily for nine days. Pancreatic lesions were visible 24 h after induction of pancreatitis, as represented by a uniform increase in echogenicity and by intralobular and subcapsular fluid accumulation. As the experiment progressed, patchy hypoechogenic foci appeared within the gland parenchyma. Amylase and lipase activities showed rapid increases. Post mortem examination revealed gross and microscopic necrotic and haemorrhagic lesions in the body and right lobe of the pancreas, accompanied by oedema and fibrosis. Ultrasonography was found to be extremely useful for the detection and characterization of experimentally induced pancreatitis and to monitor its progression in the cow. These findings are of potential value as a reference for the diagnostic workup of bovine pancreatitis, and ultrasonography is seen as a promising non-invasive technique for the diagnosis of suspected pancreatitis in cattle. Ó 2002 Elsevier Science Ltd. All rights reserved.
KEYWORDS: Bovine pancreatitis; histopathology; imaging patterns; pancreatitis modelling; ultrasonography.
INTRODUCTION Although pancreatitis is considered to be less common in cattle than in humans, dogs, and cats, the incidence may be higher than is apparent from the scattered reports of its primary occurrence. Doherty et al. (1998) reported a three-year-old Friesian cow in which diabetes mellitus was associated with lymphocytic pancreatitis and in 2001, Braun et al. reported a six-year-old Simmental bull with diabetes mellitus caused by pancreatitis. In both studies, the animals were admitted to the clinic suffering from ketosis and in poor bodily condition, and pancreatitis in each case was incidentally diagnosed at necropsy. Other studies have discussed pancreolithiasis in cattle (Velling, 1975; Groom, 1994). In the bovine pancreas, inflammatory and degenerative changes are detected post mortem but are Correspondence to: Tharwat Mohamed, Department of Veterinary Internal Medicine, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan. Tel.: +81-11-388-4819; fax: +81-11-3875890; E-mail: [email protected]
rarely diagnosed clinically because of a lack of pathognomonic clinical signs and definitive laboratory findings (Velling, 1975; Groom, 1994; Doherty et al., 1998; Braun et al., 2001). Because the diseased pancreas often eludes clinical evaluation in animals, the veterinary clinician must rely on other diagnostic tools that can be used to support the history, clinical signs, physical examination findings, and laboratory results. Computed axial tomography is the most useful imaging modality for pancreatic evaluation in humans, but the expense, need for general anaesthesia and limited availability make it less useful in veterinary practice (Miles et al., 1988; Saunders et al., 1992; Williams, 2000). As a result, ultrasonography (US) is currently the imaging modality of choice in the evaluation of pancreatic diseases in domestic species (Saunders, 1991; Saunders et al., 1992). An early report on six dogs with experimentally induced pancreatitis suggested that pancreatic US may be a valuable technique for evaluating dogs with spontaneously-occurring acute pancreatitis (Murtaugh et al., 1985). In a large study population
1090-0233/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
PANCREATITIS IMAGING IN CATTLE
of 70 dogs having fatal acute pancreatitis, Hess et al. (1998) reported that acute pancreatitis was more consistently diagnosed using abdominal US than using abdominal radiography. Furthermore, although pancreatitis has historically been diagnosed in the human and the dog more frequently than in the cat, studies have established that US is also an extremely valuable tool for identifying pancreatitis in cats (Simpson et al., 1994; Wall et al., 2001). It was believed that the frequency of pancreatitis was low in this species (Duffel, 1975; Strombeck, 1979) and the disease often went undiagnosed until after an exploratory laparotomy or necropsy (Rogers, 1983). However, with advancement in US, there is now growing evidence that pancreatitis occurs more frequently in the cat than was once believed (Simpson et al., 1994; Steiner & Williams, 1997). The prevalence of bovine cases of pancreatitis is not clear. From time to time in our laboratory, naturally-occurring pancreatitis has been revealed during necropsy of cows suffering from other diseases. For example, during 2001, 525 cows were necropsied and nine of these cows were shown to have pancreatitis at post mortem examination. The cows had different conditions including fat necrosis, endocarditis, lymphosarcoma, and caudal vena cava thrombosis. These finding led us to consider that pancreatitis may also occur more frequently in cattle. However, the ultrasonographic patterns of pancreatitis have not been documented in cows, the purpose of the present study was to define the ultrasonographic characteristics of pancreatitis induced in cattle, as a step toward providing a potential reference guide for the diagnostic workup of bovine pancreatitis. Understanding the ultrasonographic characteristics of the disease may help to identify early cases of clinical pancreatitis. MATERIALS AND METHODS Animals Nine non-pregnant non-lactating Holstein cows weighing 580 52 kg (mean SD) were included in the study and divided into three groups of three animals each: experimental (healthy), control (healthy), and preliminary (one with a minor foot problem and two with chronic mastitis). The cows in the preliminary group were used only in the ultrasonographic, physical, and post mortem examinations. In all nine cows, the serum total amylase and lipase activities were within normal range. All cows were free of alimentary tract disease, as shown by the
315
history and by physical and laboratory examinations, and there were no abnormalities in their appetites. The animals received a daily ration in two equal portions at 0700 and 1700 h. Each animal was given a basal concentrate diet (up to 4 kg/cow/day) containing 18% crude protein and 74% total digestible nutrient, a constant amount of grass hay (about 1% of BW) and water was available ad libitum. Their care and handling were in accordance with the Laboratory Animal Control Guidelines of Rakuno Gakuen University for life science research, in conformity with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in USA (NIH publication No. 86-23, revised 1985). Ultrasonographic examination of the pancreas Before injecting chloroform (CF) into the pancreas, and once daily for nine days after injection, the organ of each cow was examined ultrasonographically as described by Pusterla and Braun (1997). We used an ultrasound scanner (Model EUB-26, Hitachi Co.) equipped with a 3.5 MHz linear transducer. Briefly, the procedure was as follows: the hair was clipped and shaved over an area between the right ninth intercostal space to approximately 12 cm caudal to the costal arch. A generous amount of alcohol was applied to the skin as a transmission medium instead of ultrasonic coupling gel. With the transducer placed parallel to the ribs of the cow in a standing position, the entire shaved region was scanned in a systematic fashion caudal to cranial as far as the costal arch and the intercostal spaces nine to twelve were scanned dorsoventrally. The pancreatic body and right lobe of the organ could be seen easily; however, the left lobe could not be seen. Induction of acute pancreatitis Because the injection needle must penetrate the liver parenchyma, carrying a significant risk of haemorrhage (Mohamed et al., 2002), platelet count, prothrombin time, and activated partial thromboplastin time (APTT) were determined for all cows before injection of the pancreas. The region was aseptically prepared and infiltrated with 10 mL of 2% procaine hydrochloride. Each cow was sedated with xylazine (0.07 mg/kg body weight, intravenously), and a 17-gauge needle (Ebara) (17.5 cm in length) was inserted percutaneously and directed sonographically to the pancreatic tissue. The needle was maintained parallel to the plane of the ultrasound beam at about a 20–40° angle from the long axis of the transducer.
316
THE VETERINARY JOURNAL, 165, 3
spiratory rate, and intestinal and ruminal motility; and auscultation of the thorax and heart. From the cows in the experimental group and controls, blood (10 mL) was sampled before injection of the pancreas (day zero) and daily for the following nine days. It was allowed to clot and the sera collected. Blood samples for determination of lipid profiles were collected in heparinized tubes. Harvested sera and plasma were stored at )30 °C pending assay.
Fig. 1. Intrapancreatic injection of chloroform (labeled S INJ). ND: needle; PV: portal vein.
Chloroform (Wako Pure Chemicals) was selected to induce pancreatitis following earlier work in cattle (Hassan et al., 1980) and in dogs (Mia et al., 1978; Akuzawa et al., 1994). Using ultrasonographic guidance, 8 mL CF was given to each of six cows (experimental group and preliminary group) by injection into five sites of the pancreatic parenchyma (approximately 1.5 mL at each site) (Fig. 1). To avoid inadvertent leakage of CF into the peritoneal cavity, the injection was made only to the thicker parts of the pancreatic parenchyma. Control cows were injected with the same amount of physiological saline (PS). Pancreatic US was performed once daily until the cows were euthanased nine days later and necropsied. To minimize stress or possible discomfort, the experimental period was limited to nine days. Suspected pancreatic lesions appearing on the ultrasonograms were confirmed by comparison with the normal anatomical findings from the pre-experimental ultrasonographic examination and in conjunction with the gross pathological findings at necropsy. The laboratory and pathological features of the model (experimental group) were compared with those of control cows. Pancreatitis was confirmed histopathologically. Physical examination and blood sampling All cows underwent a physical examination prior to the onset of the experiment and twice daily for the next nine days. Clinical evaluation included assessment of general appearance, activity, and appetite; determination of rectal temperature, pulse rate, re-
Laboratory examination Haemostatic data (platelet count, prothrombin time, and APTT) were determined in the Kishimoto Clinical Laboratory, Sapporo, Japan. Haemograms were measured using a Particle counter (Model F-820, Sysmex Inc.). The serum lipase activity was determined with a commercially available kit (No. 800-B Sigma Diagnostics) based on the titrimetric method of Tietz and Fiereck (1966). Clinical chemistry kits (Wako Pure Chemicals) were used to analyse other parameters, including the concentration of amylase, glucose, albumin, calcium, ornithine carbamoyltransferase (OCT), c-glutamyltransferase (GGT), blood urea nitrogen (BUN), creatinine, total cholesterol (TC), non-esterified fatty-acids (NEFA), triglycerides (TG), and phospholipids (PL). Statistical analysis All values are expressed as mean SD. Measurements used in statistical analysis were taken only from the experimental group and controls. Pre- and post-induction mean values were analysed using Duncan’s multiple-range test, and the difference was considered significant if P < 0:05 and highly significant if P < 0:01. RESULTS Ultrasonographic examination before injection of the pancreas In all cows, the pancreatic body and right lobe of the organ were consistently identified as a triangular structure; however, the left lobe of the pancreas could not be visualized. The echogenic pattern of the pancreas was homogeneous throughout and could be clearly differentiated from that of adjacent structures. The pancreas was slightly hyperechogenic compared to the liver parenchyma. The accessory pancreatic duct, recognized in five cows, appeared as a tubular, anechoic structure with a well-defined, hyperechogenic wall. The pancreaticoduodenal vein, identified in seven cows, appeared
PANCREATITIS IMAGING IN CATTLE
317
appeared normal except for a decreased appetite and mild fever in two of the animals on the second and third days of the study. In PS-treated controls, no changes were observed. The platelet count, prothrombin time and APTT, determined before injection of the pancreas, were within the normal ranges for cattle. At day 5 in the experimental group, the leukocyte count was significantly ðP < 0:05Þ higher than the pre-experimental values; however, no changes were observed in the other haematological parameters. Haematological values of control cows did not differ significantly from base-line values.
Fig. 2. Normal pancreatic sonograms before injection of chloroform into the pancreas. (A) The body and right limb of the pancreas are visible as a triangular structure. (B) The pancreaticoduodenal vein (PDV) appears as a bandlike anechoic structure coursing from the duodenum along the right lobe of the pancreas to where it joins the portal vein (PV). In both sonograms, the margins of the uniformly echogenic pancreas are designated by arrows.
as a band-like structure coursing from the duodenum along the right lobe of the pancreas to where it joined the portal vein (Fig. 2). Ultrasonographic examination after injection of the pancreas A uniform increase in echogenicity with subcapsular and intralobular fluid accumulation was identified in the pancreatic region on ultrasound scans obtained 24 h after CF-injection. On day 3, poorly circumscribed hypoechogenic foci appeared within the pancreatic parenchyma. As the experiment progressed, well-defined anechoic areas appeared in the pancreatic body and right lobe. On the last day of the experiment, changes in the ultrasonographic appearance were evident exclusively in the pancreatic parenchyma, the anechoic or hypoechogenic areas becoming larger and more clearly defined (Fig. 3). In the PS-injected cows, no changes occurred in the echogenicity pattern of the pancreatic parenchyma throughout the experimental period. In all groups, the hepatic parenchyma appeared normal at the start of the experiment and remained unchanged. Physical and haematological examinations In the six CF-treated cows, no changes were noted on physical examination and all the animals
Biochemical examination In marked contrast to the control cows, CF-injected cows showed rapid increases in the mean values of amylase (5.4-fold) and lipase (7.9-fold) activities, with the values parallel and reaching a peak concurrently 48 h after induction of pancreatitis. The serum amylase value returned to base-line levels earlier than did the mean serum lipase concentrations (Fig. 4). In both the experimental group and controls, changes in the mean concentrations of glucose, albumin, and calcium were negligible. Moreover, BUN as well as creatinine tested normal (Tables I, II). The serum OCT activity increased significantly ðP < 0:05Þ in CF-treated cows from day 2 to day 3; however, in none of the cows did any changes occur in the GOT activity. Mean activities of lipase, amylase, OCT, and GGT in control cows did not differ significantly from base-line values. The cows injected with CF did not develop any consistent alteration in plasma lipid patterns (Table I). Pathological examination In each of the cows injected with CF and euthanased nine days later, the pancreas was grossly enlarged (1052 205 g) with adhesions to surrounding viscera. Necrotic areas and haemorrhage with multiple, fluid-filled cystic spaces were identified within the pancreas. The body and right lobe of the pancreas consistently exhibited most of the pathological changes. Multiple tissue sections revealed degeneration and necrosis of pancreatic acinar cells, in addition to fibrosis and haemorrhage in various degrees of severity. Interlobular septa were markedly oedematous with varying populations of inflammatory cells, mainly lymphocytes and neutrophils (Fig. 5). Many venous thrombi were scattered throughout the inflamed tissue. In control cows, the results of the pathological examination were normal.
318
THE VETERINARY JOURNAL, 165, 3
Fig. 3. Pancreatic imaging after induction of pancreatitis. (A) Ultrasonographic image made 24 h after chloroform injection. An overall increase in echogenicity is clear. The margins of the pancreas are designated by arrows. (B) Ultrasonographic image made 72 h after induction of pancreatitis. Hypoechogenic foci can be identified within the right lobe of the pancreas. Arrows show the pancreatic margins. (B) The echogenicity of landscape-like hypoechoic region in (C) seem to increase in echogenicity (D).
DISCUSSION Ultrasonography is routinely used to diagnose pancreatic diseases in humans (Bennett & Hann, 2001; Hirooka et al., 2001; Koito et al., 2001) and in dogs (Lamb et al., 1995; Hess et al., 1998; Van Enkevort et al., 1999) and has gained increasing importance for detecting pancreatitis in the cat (Simpson et al., 1994; Wall et al., 2001). Pancreatic US in cattle is in its infancy, but with a clear understanding of the physical principles of ultrasound, detailed knowledge of normal and abnormal pancreatic ultrasonic
anatomy, and with experience, ultrasonic imaging of the pancreatic diseases in cows should assume an important role in veterinary practice. In the present study, our pre-experimental images of the pancreatic region concur with the normal ultrasonic anatomy of the bovine pancreas as described by Pusterla and Braun (1997). However for US of acute pancreatitis, no bovine studies are available for comparison. Acute pancreatitis has been surgically produced in cattle by injection of CF in situ into the pancreatic
PANCREATITIS IMAGING IN CATTLE
319
Fig. 4. Serum total amylase and lipase activities (mean SD) vs time in cows with experimentally induced pancreatitis ðn ¼ 3Þ and in control cows ðn ¼ 3Þ.
tissue (Hassan et al., 1980) and in dogs by a wide variety of means, such as perfusion of oleic acid into the pancreatic duct (Attix et al., 1981; Nyland et al., 1983; Murtaugh et al., 1985; Murtaugh & Jacobs, 1985; Chikamune et al., 1998) and injection of CF in situ into the pancreatic tissue (Mia et al., 1978; Akuzawa et al., 1994). In the current study, induction of acute pancreatitis was successful in the preliminary group as shown by US and pathology findings and successful in the experimental group as evidenced by US, haematological and biochemical values and pathology findings. Necrosis found in the pancreas at post mortem examination, leukocytosis and dramatic increases in serum amylase and lipase activities during the first 48 h after CF-infusion, in conjunction with the histological abnormalities,
show that CF-injection into the pancreatic parenchyma provides an effective means of experimentally elicting pancreatitis in the cows. The increased echogenicity of the pancreatic parenchyma observed 24 h after CF-injection was suspected to indicate frequent, diffuse involvement of the gland and make it more echogenic, presumbly due to oedema. The hypoechogenicity of the pancreas detected at day three is believed to represent necrosis, haemorrhage, and inflammatory exudate, as confirmed by the post mortem examination. This finding is in line with reports of acute pancreatitis in humans (Cox et al., 1980) and dogs (Nyland et al., 1983; Murtaugh et al., 1985), in which decreased echogenecity in the pancreatic region has also been attributed to oedema, haemorrhage, and
320
Table I Haematological and biochemical readings from chloroform-treated experimental cows (n = 3) Parameter 0
1
2
3
4
5
6
7
8
9
67 13 3:8 1:2 42 2 2:3 0:08 24 6 65 3:4 0:7 70 7 1:9 0:7 0:2 0:08 0:16 0:04 1:13 0:2
67 32 3:4 0:7 41 1 2:3 0:17 30 14 25 18 3:5 0:9 68 4 1:8 0:6 0:1 0:09 0:19 0:03 1:09 0:2
68 26 3:2 0:5 42 3 2:4 0:18 30 12 20 13 2:9 1:4 62 3 1:9 0:6 0:2 0:06 0:16 0:08 1:11 0:1
76 8 2:8 0:5 38 2 2:3 0:15 27 10 10 6 3:3 1:2 65 3 1:9 0:5 0:2 0:04 0:18 0:05 1:08 0:1
78 15 2:9 0:7 37 3 2:2 0:09 23 7 73 3:2 1:1 69 5 1:9 0:6 0:2 0:05 0:19 0:08 1:04 0:2
86 10 3:4 0:7 38 3 2:3 0:12 27 12 63 3:3 0:8 60 3 1:8 0:4 0:3 0:11 0:18 0:05 1:02 0:1
69 8 3:2 0:9 39 5 2:3 0:05 26 11 53 3:8 1:6 72 4 1:9 0:5 0:3 0:08 0:19 0:07 1:03 0:2
60 16 3:3 0:7 38 4 2:3 0:13 31 15 14 9 3:4 0:8 69 6 2:1 0:8 0:3 0:15 0:18 0:08 1:05 0:1
61 15 3:4 0:4 39 4 2:4 0:11 32 15 93 4:2 2:8 70 9 2:2 0:7 0:3 0:19 0:23 0:09 1:09 0:3
55 28 3:1 0:1 37 5 2:4 0:51 33 17 71 13:7 1:6 72 5 2:1 0:6 0:3 0:13 0:21 0:07 0:99 0:1
WBC: white blood cells; GGT: c -glutamyltransferase; OCT: ornithine carbamoyltransferase; BUN: blood urea nitrogen; Cr: creatinine; TC: total cholesterol; NEFA: non-esterified fattyacids; TG: triglycerides; PL: phospholipids. *P < 0:05 compared to zero day values.
THE VETERINARY JOURNAL, 165, 3
WBC ð102 =lLÞ Glucose (mmol/L) Albumin (g/L) Calcium (mmol/L) GGT (IU/L) OCT (IU/L) BUN (mmol/L) Cr (lmol/L) TC (mmol/L) NEFA (mmol/L) TG (mmol/L) PL (mmol/L)
Time after injection (day)
Table II Haematological and biochemical readings from control cows (n = 3) Parameter 0 2
WBC ð10 =lLÞ Glucose (mmol/L) Albumin (g/L) Calcium (mmol/L) GGT (IU/L) OCT (IU/L) BUN (mmol/L) Cr ðlmol=LÞ TC (mmol/L) NEFA (mmol/L) TG (mmol/L) PL (mmol/L)
1
2
3
4
5
6
7
73 20 76 7 70 15 68 22 71 9 75 20 76 13 66 10 3:2 0:8 3:3 0:5 3:9 0:7 2:8 0:5 2:9 0:8 2:8 0:4 2:8 0:6 2:7 0:9 44 5 44 2 43 3 43 1 41 5 43 5 41 6 44 7 2:6 0:22 2:7 0:15 2:7 0:28 2:6 0:11 2:8 0:13 2:6 0:19 2:6 0:14 10:9 0:6 27 12 28 10 27 7 26 11 24 8 23 6 19 15 2:7 16 84 66 54 53 64 64 93 52 3:5 0:4 3:8 0:5 4:2 1:5 2:6 0:7 3:7 0:9 3:2 0:8 3:3 0:4 3:2 0:6 90 8 110 3 105 7 100 4 95 6 93 7 1078 85 7 2:2 0:5 2:4 0:6 1:7 0:5 1:9 0:3 1:9 0:7 2:4 0:3 1:8 0:5 1:7 0:4 0:3 0:09 0:2 0:11 0:3 0:08 0:2 0:10 0:3 0:14 0:3 0:06 0:3 0:18 0:2 0:09 0:15 0:08 0:18 0:04 0:14 0:05 0:12 0:09 0:14 0:08 0:18 0:07 0:11 0:03 0:12 0:04 1:02 0:1 1:05 0:2 1:01 0:1 0:91 0:1 0:92 0:3 0:62 0:18 0:92 0:2 1:01 0:3
8
9
74 14 3:1 0:3 45 4 10:9 0:4 2:7 0:9 66 3:8 0:9 99 8 2:1 0:6 0:2 0:10 0:19 0:06 1:01 0:14
78 25 3:4 0:5 45 2 11:7 0:6 2:9 0:15 73 3:6 0:5 102 9 2:3 0:7 0:2 0:11 0:25 0:07 0:89 0:1
PANCREATITIS IMAGING IN CATTLE
Time after injection (day)
See Table I for Key.
321
322
THE VETERINARY JOURNAL, 165, 3
Fig. 5. Photomicrographs of pancreatic necropsy specimens obtained nine days after chloroform injection. (A) Pancreatic acini are necrotic. Numerous lymphocytes, plasma cells and a few neutrophils and eosinophils infiltrate the proliferating fibrous tissue between normal and necrotic areas (white arrows). Interstitial oedema is also evident (solid black arrows). (B) Epithelial cells of excretory ducts are desquamated (arrowhead) and haemorrhage is present in the necrotic area. An intravascular thrombus is also evident (open black arrow). H and Estain. Bar ¼ 300 lm.
inflammatory exudate. Pancreatomegaly and hypoechogenicity in our cows are in agreement with the ultrasonographic findings described in pancreatitis induced experimentally in dogs (Nyland et al., 1983; Murtaugh et al., 1985) and cats (Kitchell et al., 1986). An enlarged hypoechogenic pancreas has also been described in dogs and cats having naturally-occurring pancreatitis (Saunders, 1991; Simpson et al., 1994; Wall et al., 2001). It was somewhat surprising that on the daily physical examinations, the cows with induced pancreatitis displayed no apparent abnormalities, other than a slightly decreased appetite and mild fever in
two animals on the second and third days after CFinjection. Although the islets of Langerhans were necrotic in some areas of the histological sections examined, it might be argued that sufficient endocrine function of the organ is maintained by the cells in areas of viable tissue, as suggested by the absence of overt diabetes mellitus in any of the cows. The normal blood glucose level might be because the injection of CF was made only to the body and right lobe of the pancreas, possibly leaving the left lobe disease-free and, therefore, functionally normal for the duration of the experiments. The normal blood calcium levels in the cows may be explained, at least in part, by the normal serum albumin concentrations. This view is in keeping with the rationale behind studies on dogs with acute pancreatitis, in which hypocalcaemia was attributed to decreased serum albumin concentrations resulting in attenuation of non-ionized calcium (Attix et al., 1981; Feldman et al., 1981). The high serum OCT values in the present study suggest that the disturbance of hepatocytes might occur after CFinfusion. Induction of pancreatitis in the present study did not change the lipid profile patterns in any of the cows, indicating that plasma lipid profiles would appear to have little value in the diagnosis of acute pancreatitis in cows. In a study of 70 dogs with fatal acute pancreatitis, Hess et al. (1998) concluded that the diagnostic value of amylase and lipase is limited. Similarly, in the cat, serum amylase and lipase concentrations have been reported to be of little use in diagnosing pancreatitis (Hill & Van Winkle, 1993; Hardy, 1994; Stewart, 1994; Simpson, 1997). In the present cows, although pancreatitis resulted in rapid increases in amylase and lipase activities, the values returned quickly to within reference ranges. This finding indicates that in cows with chronic pancreatitis the amylase and lipase values would remain normal. We concluded that serum amylase and lipase concentrations appear to be of little use in diagnosing bovine pancreatitis. Elevations may be used in support of a diagnosis, but evidence indicates they can not be used as a standard for a definitive diagnosis. In cows it is difficult to draw definitive conclusions from cases of naturally occurring pancreatitis because either recovery or extended therapy may alter the disease pattern before pathological confirmation can be obtained. Because it is not certain to what degree of reliability the model mimics spontaneous pancreatitis in cows and whether the ultrasonographic characteristics would differ, studies are in progress to further delineate the ultraso-
PANCREATITIS IMAGING IN CATTLE
nographic features of pancreatitis in cows. Results of the present experimental model are deemed useful as potential reference values for the ultrasonographic, laboratory and pathological characteristics in the early recognition of cows with naturally occurring pancreatitis. Further studies are warranted from veterinarians making use of these values for evaluating suspected pancreatitis in cows. ACKNOWLEDGMENTS The authors are deeply grateful to Professor N. Kennedy (Biomedical English) for her critical reading of the manuscript, Dr. T. Onaga (Physiology) for his technical assistance and Dr. H. Okada (Pathology) for his comments on the pathological slides. REFERENCES AKUZAWA, M., MORIZONO, M., NAGATA, K., HAYANO, S., SAKAMOTO, H., YASUDA, N., OKAMOTO, K., KAWASAKI, Y. & DEGUCHI, E. (1994). Changes of serum amylase, its isozyme fractions and amylase-creatinine clearance ratio in dogs with experimentally induced acute pancreatitis. Journal of Veterinary Medical Science 56, 269–73. ATTIX, E., STROMBECK, D. R. & WHEELDON, E. B. (1981). Effects of anticholinergic and a corticosteriod on acute pancreatitis in experimental dogs. American Journal of Veterinary Research 42, 1668–74. BENNETT, G. L. & HANN, L. E. (2001). Pancreatic ultrasonography. Surgical Clinics of North America 81, 259–81. BRAUN, U., DURR, M., DIENER, M., OSSENT, P., HAMMON, H. & BLUM, J. W. (2001). Diabetes mellitus caused by pancreatitis in a bull. Schweiz. Arch. Tierheilk. 143, 99–104. CHIKAMUNE, T., KATAMOTO, H., NOMURA, K. & OHASHI, F. (1998). Lipoprotein profile in canine pancreatitis induced with oleic acid. Journal of Veterinary Medical Science 60, 413–21. COX, K. L., AMENT, M. E., SAMPLE, W. F., SARTI, D. A., O’DONNELL, M. & BYRNE, W. J. (1980). The ultrasonic and biochemical diagnosis of pancreatitis in children. Journal of Pediatrics 96, 407–11. DOHERTY, M. L., HEALY, A. M. & DONNELLY, W. J. C. (1998). Diabetes mellitus associated with lymphocytic pancreatitis in a cow. Veterinary Record 142, 493. DUFFEL, S. J. (1975). Some aspects of pancreatic disease in the cat. Journal of Small Animal Practice 16, 356–74. FELDMAN, B. F., ATTIX, E. A., STROMBECK, D. R. & O’NEILL, S. (1981). Biochemical and coagulation changes in a canine model of acute necrotizing pancreatitis. American Journal of Veterinary Research 42, 805–9. GROOM, S. (1994). Pancreolithiasis in a Holstein-Friesian cow. Canadian Veterinary Journal 35, 244. HARDY, R. M. (1994). Disease of the exocrine pancreas. In: The Cat; Diseases and Clinical Management, ed. R. G. Sherding, pp. 1287–92. New York: Churchill Livingstone.
323
HASSAN, M. S., AMER, A. A., EL-AMROUSI, S. & BAYOUMI, A. H. (1980). Some studies on pancreatic function in cattle and buffaloes. II. Induced acute pancreatitis. Assiut Veterinary Medical Journal 7, 295–306. HESS, R. S., SAUNDERS, H. M., VAN WINKLE, T. J., SCHOFER, F. S. & WASHABAU, R. J. (1998). Clinical, clinicopathologic, radiographic and ultrasonographic abnormalities in dogs with fatal acute pancreatitis: 70 cases (1986–1995). Journal of the American Veterinary Medical Association 213, 665–70. HILL, R. C. & VAN WINKLE, T. J. (1993). Acute necrotizing pancreatitis and acute suppurative pancreatitis in the cat: a retrospective study of 40 cases (1976–1989). Journal of Veterinary Internal Medicine 7, 25–33. HIROOKA, Y., GOTO, H., HASHIMOTO, S., HIRAI, T., NIWA, K., TAKEDA, K. & HAYAKAWA, T. (2001). Recent advances in US diagnosis of pancreatic cancer. Hepatogastroenterology 48, 916–22. KITCHELL, B. E., STROMBECK, D. R., CULLER, J. & HARROLD, D. (1986). Clinical and pathologic changes in experimentally induced acute pancreatitis in cats. American Journal of Veterinary Research 47, 1170–3. KOITO, K., NAMIENO, T., NAGAKAWA, T., HIROKAWA, N., ICHIMURA, T., SYONAI, T., YAMA, N., SOMEYA, M., NAKATA, K., SAKATA, K. & HAREYAMA, M. (2001). Pancreas: imaging diagnosis with color/power Doppler ultrasonography, endoscopic ultrasonography, and intraductal ultrasonography. European Journal of Radiology 38, 94–101. LAMB, C. R., SIMPSON, K. W., BOSWOOD, A. & MATTHEWMAN, L. A. (1995). Ultrasonography of pancreatic neoplasia in the dog: a retrospective review of 16 cases. Veterinary Record 137, 65–8. MIA, A. S., KOGER, H. D. & TIERNEY, M. M. (1978). Serum values of amylase and pancreatic lipase in healthy mature dogs and dogs with experimental pancreatitis. American Journal of Veterinary Research 39, 965–9. MILES, K., LATTIMER, J. C., KRAUSE, G. F., KNAPP, D. W. & SAYLES, C. E. (1988). The use of intraperitoneal fluid as a simple technique for enhancing sonographic visualization of the canine pancreas. Veterinary Radiology 29, 258–63. MOHAMED, T., SATO, H., KUROSAWA, T. & OIKAWA, S. (2002). Echo-guided studies on portal and hepatic blood in cattle. Journal of Veterinary Medical Science 64, 23–8. MURTAUGH, R. J., HERRING, D. S., JACOBS, R. M. & DEHOFF, W. D. (1985). Pancreatic ultrasonography in dogs with experimentally induced acute pancreatitis. Veterinary Radiology 26, 27–32. MURTAUGH, R. J. & JACOBS, R. M. (1985). Serum antiprotease concentrations in dogs with spontaneous and experimentally induced acute pancreatitis. American Journal of Veterinary Research 46, 80–3. NYLAND, T. G., MULVANY, M. H. & STROMBEK, D. R. (1983). Ultrasonic features of experimentally induced acute pancreatitis in the dog. Veterinary Radiology 24, 260–6. PUSTERLA, N. & BRAUN, U. (1997). Ultrasonographic examination of the pancreas in healthy cows. Veterinary Radiology and Ultrasound 38, 63–7. ROGERS, W. A. (1983). Diseases of the exocrine pancreas. In: Veterinary Internal Medicine, ed. S. J. Ettinger, pp. 1435–55. Philadelphia, PA: WB Saunders.
324
THE VETERINARY JOURNAL, 165, 3
SAUNDERS, H. M. (1991). Ultrasonography of the pancreas. In: Problems in Veterinary Medicine, ed. P. M. Kaplan, pp. 583–603. Philadelphia, PA: WB Saunders. SAUNDERS, H. M., PUGH, C. R. & RHODES, W. H. (1992). Expanding applications of abdominal Ultrasonography. Journal of the American Animal Hospital Association 28, 369–74. SIMPSON, K. W., SHIROMA, J. T., BILLER, D. S., WICKS, J., JOHNSON, S. E., DIMSKI, D. & CHEW, D. (1994). Ante mortem diagnosis of pancreatitis in four cats. Journal of Small Animal Practice 35, 93–9. SIMPSON, K. W. (1997). Acute pancreatitis. In: Consultations in Feline Internal Medicine, ed. J. R. August, pp. 91–8. Philadelphia, PA: WB Saunders. STEINER, J. M. & WILLIAMS, D. A. (1997). Feline exocrine pancreatic disorders: insufficiency, neoplasia and uncommon conditions. Compendium on Continuing Education for the Practicing Veterinarian 19, 836–49. STEWART, A. F. (1994). Pancreatitis in dogs and cats: cause, pathogenesis, diagnosis and treatment. Compendium on Continuing Education for the Practicing Veterinarian 16, 1423–30.
STROMBECK, D. R. (1979). Small Animal Gastroenterology pp. 301–31. Davis,CA: Stonegate Publishing Co.. TIETZ, N. W. & FIERECK, E. A. (1966). A specific method for serum lipase determination. Clinica Chimica Acta 13, 352. VAN ENKEVORT, B. A., O’BRIEN, R. T. & YOUNG, K. M. (1999). Pancreatic pseudocysts in 4 dogs and 2 cats: ultrasonographic and clinicopathologic findings. Journal of Veterinary Internal Medicine 13, 309–13. VELLING, K. (1975). Bovine pancreolithiasis in Denmark. Acta Veterinaria Scandinavica 16, 327–40. WALL, M., BILLER, D. S., SCHONING, P., OLSEN, D. & MOORE, L. E. (2001). Pancreatitis in a cat demonstrating pancreatic duct dilatation ultrasonographically. Journal of the American Animal Hospital Association 37, 49–53. WILLIAMS, D. A. (2000). Exocrine pancreatic disease. In: Textbook of Veterinary Internal Medicinevol. 5, eds. S. J. Ettinger, & E. C. Feldman, (vol. 5) pp. 1345–67. Philadelphia, PA: WB Saunders. (Accepted for publication 29 May 2002)