Feline cholecystitis and acute neutrophilic cholangitis: clinical findings, bacterial isolates and response to treatment in six cases

Clinicopathological findings from six cats with confirmed cholecystitis or acute neutrophilic cholangitis are presented. Historical findings included lethargy and anorexia or inappetence of up to five days duration. On physical examination all cats were pyrexic and four out of six were jaundiced and had cranial abdominal pain. Bile samples were obtained by cholecystocentesis at exploratory coeliotomy (two cases) or by percutaneous, ultrasound-guided cholecystocentesis (four cases). Gall bladder rupture and bile peritonitis occurred subsequent to ultrasound-guided cholecystocentesis in one case. The most common bacterial isolate was Escherichia coli (four cases); E coli was isolated alone in two cases, in combination with a Streptococcus species (one case) and in combination with a Clostridium species (one case). Streptococcus species alone was isolated from one case, as was Salmonella enterica serovar Typhimurium. The latter is the first reported case of Salmonella-associated cholecystitis in a cat. Concurrent pancreatic or intestinal disease was detected histologically in three cases. All cases were treated with antimicrobials based on in vitro susceptibility results. Treatment was successful in five cases. One cat with concurrent diffuse epitheliotropic intestinal lymphoma was euthanased. Percutaneous ultrasound-guided cholecystocentesis is an effective, minimally-invasive technique enabling identification of bacterial isolates in cats with inflammatory hepatobiliary disease.     

Percutaneous ultrasound-guided cholecystocentesis in dogs

Percutaneous ultrasound-guided cholecystocentesis was performed on 13 healthy beagle dogs to determine whether percutaneous ultrasound-guided cholecystocentesis in the dog was a feasible and safe procedure. Clinical, laboratory and ultrasonographic examinations were done at 0 and 10 minutes, in the 2nd and 16th hour, and on the 7th day. They included a detailed physical examination of the mucous membranes, cardiorespiratory system and abdominal organs. Laboratory examinations of the blood consisted of a complete blood count, determination of packed cell volume (PCV), haemoglobin (Hb), total plasma protein (TPP), parameters of haemostasis including prothrombin time (PT), activated partial thromboplastin time (APTT), and enzyme activities reflecting hepatobiliary function, i.e. aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT). Ultrasonographic findings of the gallbladder (size, shape, wall, content) and appearance of the biliary tract and the surrounding cranial intraabdominal organs were also evaluated. Percutaneous ultrasound-guided cholecystocentesis was performed easily during the study, and dogs tolerated well the procedure performed without anaesthesia. All laboratory parameters of the blood remained within normal limits throughout the study. However, some follow-up values, i.e. PCV, TPP, APTT and ALT, demonstrated statistically significant differences when compared to baseline measurements, which might reflect the effect of 24-hour fasting before the experiment, as well as day-to-day metabolic fluctuations due to feeding and water supply during the study. There were no visible signs of bleeding from the liver, bile leakage from the gallbladder or accumulation of free peritoneal fluid during repeated ultrasonographic examinations. Percutaneous ultrasound-guided cholecystocentesis seems to be an important diagnostic procedure in canine gallbladder diseases and can be used safely and easily to gain gallbladder bile for diagnosis of bacterial cholecystitis or for investigating hepatobiliary function in the dog.     

Evaluation of serum bile acid concentrations for the diagnosis of portosystemic venous anomalies in the dog and cat

The serum concentration of bile acids was measured in dogs and cats with portosystemic venous anomalies (PSVA). In 14 dogs, the mean serum bile acid concentration after 12 hours of fasting was 61.7 +/- 68.7 mumol/L (normal, 2.3 +/- 0.4 mumol/L (SEM) and when measured 2 hours after a meal in 15 dogs was 229.9 +/- 87.7 mumol/L (normal, 8.3 +/- 2.2 mumol/L). The fasting serum bile acid concentration was within the normal range in 5 of 14 dogs. The postprandial concentration was determined in 3 of the 5 and in each case increased more than tenfold above the fasting value. The mean fasting serum bile acid concentration in 4 cats was 24.4 +/- 10.1 mumol/L (normal, 1.7 +/- 0.3 mumol/L) and in 2 of the cats increased to a mean of 120.6 mumol/L (normal, 8.3 +/- 0.8 mumol/L) 2 hours after feeding. The bile acid values in patients with PSVA were correlated with values for blood ammonia content, sulfobromophthalein (BSP) retention, and results of conventional tests of hepatic function. Bile acid concentrations were more sensitive than abnormalities in serum enzyme activities or BSP retention and equal in sensitivity to the ammonia tolerance test in detecting hepatobiliary insufficiency. Bile acid measurements were accomplished with less inconvenience to the patient and clinician, than tests of BSP excretion or ammonia tolerance. Used in combination with conventional laboratory tests for hepatic disease, pre- and postprandial serum bile acid concentrations appear to be a sensitive and specific indicator of hepatobiliary dysfunction of value in the diagnosis of PSVA in the dog and cat.     

Isolation and purification of Fasciola hepatica eggs

The present egg isolation method is both a rapid and simple technique for recovering large numbers of Fasciola hepatica eggs (1 X 10(7) eggs/gradient) from bovine bile. Bile from infected cattle was first passed through a 45 micron screen sieve. The F. hepatica eggs were collected from the surface of the screen by backwashing with a jet of distilled water. The resultant egg suspension was layered on a 60% to 100% (v/v) linear Percoll gradient prepared in distilled water. Centrifugation at 450 g for 20 min resulted in the formation of 2 visible bands and a pellet. The top band (density of 1.075 g ml-1) contained viscous debris and crystallized bile pigments. The second visible band (density of 1.093-1.099 g ml-1) consisted of a relatively pure population of F. hepatica eggs (greater than 93%) while the pellet contained only F. hepatica egg shells.     

Effects of iatrogenic hypercortisolism on gallbladder sludge formation and biochemical bile constituents in dogs

An association between gallbladder mucoceles and hypercortisolism (HC) was recently described in dogs. Because the formation of a mucocele from clear bile without the transitional formation of microprecipitates appears unlikely, the aim of this study was to investigate the effects of iatrogenic HC on sludge formation and changes in the biochemical composition of bile. Bile samples from 6 dogs obtained by percutaneous ultrasound-guided cholecystocentesis before (day 0), during (days 28, 56, and 84), and after (days 28p, 56p, and 84p) oral administration of hydrocortisone (8 mg/kg every 12 h) were analysed for calcium, cholesterol and bilirubin concentrations and pH. In addition the gallbladder was examined ultrasonographically for sludge. Six dogs receiving a placebo served as controls. Although gallbladder sludge was observed in all treated dogs at day 56, it was also noted in 50% of control dogs, and no significant differences were seen between groups at any sampling time. Bilirubin and cholesterol concentrations decreased significantly and reversibly during treatment, and calcium concentration showed a similar trend. Bile pH was consistently slightly alkaline during iatrogenic HC, whereas it was slightly acidic in control animals. A 3-month period of iatrogenic HC does not lead to ultrasonographically detectable gallbladder sludge or to an increase in bile constituents that are commonly implicated in sludge formation in humans.     

Changes in the serum levels of primary bile acids in dairy cows

Serum cholic acid (SCA) and serum chenodeoxycholic acid (SCDCA) concentrations were determined in healthy dairy cows by radioimmunoassay (RIA). The levels of these two primary bile acids were correlated with the cows' reproductive status. The lowest concentrations were measured in dry cows (SCA: 7.8 +/- 3.3 mumol/l, SCDCA.: 1.5 +/- 1.0 mumol/l). In freshly calved cows SCA and SCDCA was 17.8 +/- 6.9 mumol/l and 2.3 +/- 1.0 mumol/l, respectively, while in milking cows SCA and SCDCA was 15.8 +/- 5.7 and 2.3 +/- 0.8 mumol/l, respectively. SCA concentration showed a characteristic change on the days immediately after calving: on calving day it was close to the mean SCA concentration found for dry cows, then it underwent a striking abrupt rise and reached the value typical of post-parturient cows by post-partum (PP) day 4-5. Cholic acid was found to be the major primary bile acid in the blood of dairy cows. In dry cows the SCA:SCDCA ratio is 5:1. If the serum bile acid concentration rises, the SCA:SCDCA ratio will increase further.     

Diurnal and individual variations in bile acids in the plasma of normal dairy cows

Blood samples were collected every 2 h during a 24 h period from 6 cows of one herd and 10 cows of another herd. In a third herd 9 cows were sampled every 2 h from 6 a.m. to 8 p.m. Concentrations of total bile acids, acetoacetate, glucose and free fatty acids were determined in blood plasma. A marked difference in individual bile acid concentrations and patterns of diurnal variation was found. For most cows the highest bile acid values were observed between 2 and 6 a.m. (overall mean (+/- SD) at 6 a.m.: 104 +/- 84 mumol/l, range: 20-307 mumol/l). Fourteen cows with a bile acid value greater than 90 mumol/l at 6 a.m. ("high BA") were characterized as a group by showing a pronounced decrease in the mean bile acid concentration after morning feeding. In the group of 11 cows with a 6 a.m. bile acid value less than 90 mumol/l ("low BA") the time of day did not contribute significantly to the bile acid variation. For the "high BA" group a nearly synchronous variation between the mean values of the 3 feeding dependent parameters (acetoacetate, glucose and free fatty acids) and the mean values of bile acids was found. The within animal coefficients of correlation between bile acids and the feeding dependent parameters were significantly higher in the "high BA" group than in the "low BA" group. No direct connection was found between bile acid levels and the quantity of concentrates fed or the individual milk yield.     

Liver flukes promote cholelithiasis in sheep

The main objective of this study was to investigate whether cholelithiasis in sheep is related to parasitism or other commonly observed disorders such as liver abscesses. Additionally, the features of the observed biliary calculi are described. The livers of 254 randomly selected clinically healthy adult dairy sheep were used. All visible concretions in the bile were considered as stones. Based on the macroscopical examination, 60 livers were normal, 40 were parasitized with Fasciola hepatica, 42 were parasitized with Dicrocoelium dendriticum, 28 were parasitized with both D. dendriticum and F. hepatica, 40 livers had abscesses and 44 had hydatid cysts. Biliary calculi were detected in 40 livers. Twenty livers had pigment stones and 20 livers had cholesterol stones. The percentage of cholelithiasis was significantly higher in livers parasitized with flukes compared with the others (P<0.05) and the parasitoses with F. hepatica and D. dendriticum were proven to be significant risk factors (P<0.05) for the presence of cholelithiasis in sheep.     

Ultrasound-guided catheterization of the portal vein in 11 cows using the Seldinger technique

Catheterization of the portal vein using the Seldinger technique [Acta Radiol. (1958) 38, 368] was performed in 11 cows. Ultrasound-guided percutaneous portocentesis, using a 25-cm, 14-gauge steel cannula, was performed from the 10th, 11th or 12th intercostal space on the right side. A stylet was placed through the cannula, which was then removed, and a polyurethane balloon-tipped catheter was advanced over the stylet into the portal vein and sutured to the skin (Seldinger, 1952). Blood samples were collected from the catheter at least once daily. The catheter was removed 9-15 days later when blood could no longer be aspirated. The cows were then slaughtered and a post-mortem examination was performed. During the study, appetite remained normal in nine of 11 cows. In three cows, the general behaviour and demeanour were mildly, but transiently, abnormal. Four cows had leucopoenia. The most frequently encountered problem was occlusion of the catheter, which usually was resolved by flushing with heparinized saline. The most common post-mortem lesion observed was an increase in fibrous connective tissue at the site of cannulation. In nine of 11cows, there was a thrombus in the portal vein at the site of catheterization. Generally, the severity of the lesions was mild. The results of this study demonstrated that ultrasound-guided percutaneous catheterization of the portal vein, using the Seldinger technique, is possible in cows. The catheter may be left in place for collection of blood samples for up to 15 days.     

Serum bile acids in captive bustards

Blood samples were collected from clinically normal male and female houbara (Chlamydotis undulata macqueenii), kori (Ardeotis kori), buff-crested (Eupodotis ruficrista gindiana) and white-bellied bustards (E. senegalensis) to determine serum bile acid concentrations. Bile acid concentrations were determined by analysis with an Ultrospec 3000 ultraviolet/visible spectrophotometer, using an enzymatic bile acid test. The results provided values of serum bile acid concentrations for the four species, with means +/- standard errors of 35.8 +/- 2.8 mumol; 51.1 +/- 5.0 mumol; 18.4 +/- 2.1 mumol and 20.8 +/- 5.4 mumol for the houbara, kori, buff-crested and white-bellied bustard, respectively. Although no gender or age differences were detected within species, the results demonstrated significant differences in concentrations in clinically normal individuals between the different species.     

Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. 3. The histochemistry of mast cells and globule leucocytes

Comparative studies were made on the structure and histochemistry of mast cells and globule leucocytes in dicrocoeliasis and fascioliasis of the goat. Investigations showed that D. dendriticum and F. hepatica infections in the goat produce an increase in the number of hepatic mast cells and frequent occurrence of globule leucocytes in the epithelium of the bile ducts. The only difference between these infections was that globule leucocytes were more frequent in small bile ducts in dicrocoeliasis than in fascioliasis.Histochemical methods showed the presence of sulphomucins in the granules of mast cells, while both neutral mucins and carboxymucins were revealed in the globules of globule leucocytes. The acidity of the mucosubstances could not be shown to result from hyaluronic acid, chondroitin-4 and -6-sulphates, or sialic acid. The globules of globule leucocytes also contained histochemically identifiable highly basic proteins.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts, mast cell, globule leucocyte     

Production responses to internal parasite control in dairy cattle

A significant increase in milk production, averaging 164 litres per cow per lactation (a 4.8% increase), was seen after cows infected with gastrointestinal nematodes, paramphistomes and Fasciola hepatica were treated with broad-spectrum anthelmintics. Three hundred and ninety pairs of cows from eight herds with year-round calving were studied. One cow in each matched pair was given 7.5 mg/kg fenbendazole, 7.5 mg/kg levamisole hydrochloride and 15 mg/kg oxyclozanide in March, May and August of one year; the other cow in the pair received no anthelmintic. The number of nematode and trematode eggs was significantly decreased in the faeces of treated cows.     

Fasciola in horses in the Republic of South Africa: a single natural case of Fasciola hepatica and the failure to infest ten horses either with F. hepatica or Fasciola gigantica

The faeces of 11 horses were examined for Fasciola spp. eggs. One of them was positive for Fasciola hepatica, a finding which was confirmed post-mortem. The 10 negative horses were subsequently infested with either F. hepatica or Fasciola gigantica, each animal receiving orally from 500-9,500 metacercariae. No clinical signs were observed and no fluke eggs were detected in the faeces, and neither immature nor adult Fasciola worms were recovered from the horses slaughtered 16-26 weeks post-infestation with F. hepatica and 28-34 weeks post-infestation with F. gigantica, respectively. The results indicate that horses have a high level of resistance to both South African Fasciola spp. Factors which can probably explain the conflicting reports for the prevalences of fascioliasis in horses throughout the world and the varying results obtained in experimental infestations are briefly considered.     

Liver function tests in dogs with portosystemic shunts: measurement of serum bile acid concentration

Sulfobromophthalein excretion and plasma ammonia and serum bile acid concentrations were measured in 11 dogs with portal vascular anomalies. The fasting serum bile acid concentration was increased in all 11 dogs (78.9 +/- 16.1 mumol/L; normal, 2.6 +/- 0.4 mumol/L). For values measured in 8 dogs, the 2-hour postprandial serum bile acid concentration was increased further (177.0 +/- 26.4 mumol/L; normal, 7.6 +/- 2.3 mumol/L). The fasting plasma ammonia concentration was markedly increased in all 11 dogs (246.9 +/- 40.3 micrograms/dl; normal, 27 to 15 micrograms/dl). Thirty minutes after the oral administration of ammonium chloride, the plasma ammonia concentration was increased further in the 7 dogs (510.7 +/- 45.5 micrograms/dl; normal, 57.5 to 20.5 micrograms/dl). Results of the sulfobromophthalein excretion test were abnormal in 10 of 11 dogs (12.3 +/- 1.4%; normal, less than 5% retention after 30 minutes).     

Quantification of naloxone binding sites in brains from suckled beef cows during postpartum anestrus and resumption of estrous cycles

Thirty beef cows, approximately 3 yr of age, were randomly assigned to be slaughtered on d 7, 14, 28, 42 or 56 postpartum. Each cow suckled one calf until slaughter. Data from cows slaughtered on d 42 and 56 were pooled and further classified as anestrous or cyclic based on the presence of a corpus luteum and elevated serum concentrations of progesterone at slaughter. Specific binding of [3H]naloxone (3H-NAL) to homogenates of tissue from hypothalamus (HYP), preoptic area (POA) and basal forebrain (BF) was assessed using multiple-point Scatchard analyses. Nonspecific binding was estimated in the presence of 10(-6) M naloxone. Separation of bound from free 3H-NAL was achieved by centrifugation at 20,000 X g. Concentration (fmol/mg original tissue wet wt) of 3H-NAL binding sites in POA tissue was higher (P less than .05) on d 28 postpartum in anestrous cows than in cyclic cows on d 42 + 56 postpartum (2.58 +/- .32 vs 1.58 +/- .10). When all anestrous cows were compared with cyclic cows, concentrations of 3H-NAL binding sites in POA tissues and in BF tissue were higher (P less than .05) in anestrous cows (anestrous POA, 2.12 +/- .17, cyclic POA, 1.58 +/- .10; anestrous BF, 2.94 +/- .41, cyclic BF, 2.19 +/- .16). Compared across brain regions for all cows, the concentration of specific binding sites for 3H-NAL was greater (P less than .01) in BF (2.5 +/- .2) than in POA (1.9 +/- .1) and greater (P less than .01) in POA than in HYP (1.5 +/- .1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum bile acid analysis in dogs with experimentally induced cholestatic jaundice

Serum bile acid (SBA) concentration was determined weekly for 4 weeks in dogs with experimentally induced hyperbilirubinemic liver disease. Obstructive jaundice was created in 6 dogs by surgical ligation of the common bile duct, and hepatocellular jaundice was created in 6 sham-operated dogs by administration of dimethylnitrosamine; 6 other sham-operated dogs served as controls. Serum bile acid concentration increased rapidly after bile duct ligation (from 0.6 +/- 0.1 to 69.2 +/- 15.3 mumol/L at 3 days), peaked at 14 days (247.8 +/- 54.1 mumol/L), and then gradually decreased (179.9 +/- 27.1 mumol/L at 28 days). Serum bile acid concentration in dimethylnitrosamine-treated dogs increased more gradually to 38.9 +/- 10.7 mumol/L at 28 days, at which time the serum bilirubin concentration was comparable with that of bile duct-ligated dogs. Mean total SBA values in bile duct-ligated dogs were significantly (P less than 0.01) higher than those in control and dimethylnitrosamine-treated dogs at days 3 through 28, with no overlap of individual values. Serum bile acid concentration at day 28 correlated positively (P less than 0.01) with cholestasis and bile duct proliferation observed in liver biopsy specimens, but did not correlate with necrosis or inflammation. Serum bile acid concentration also correlated positively (P less than 0.01) with serum bilirubin and cholesterol concentrations and with serum alkaline phosphatase and alanine transaminase activities. Results of the study reported here indicated a relationship between SBA concentration and cholestasis in dogs; extrahepatic bile duct obstruction resulted in the highest SBA values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Therapeutic percutaneous ultrasound-guided cholecystocentesis in three dogs with extrahepatic biliary obstruction and pancreatitis

Three dogs were examined because of acute pancreatitis. In all 3, distension of the gallbladder was seen ultrasonographically, and extrahepatic biliary tract obstruction (EHBO) was diagnosed on the basis of ultrasonographic findings and serum biochemical abnormalities (i.e., high serum bilirubin and cholesterol concentrations and increased hepatic enzyme activities). In all 3 dogs, percutaneous ultrasound-guided cholecystocentesis (PUCC) was used to decompress the gallbladder, with cholecystocentesis performed multiple times in 1 dog. Serum bilirubin concentration was substantially decreased following the procedure in all 3 dogs. Two of the 3 dogs did not require surgery to resolve the obstruction. In the third dog, an exploratory laparotomy was performed because of concerns about development of abdominal effusion following the procedure. Bile staining of the mesenteric fat was seen during the laparotomy, but no defect in the gallbladder wall could be identified. In most dogs with EHBO secondary to pancreatitis, the obstruction resolves spontaneously as the acute pancreatitis improves so that surgery is not required. In those few dogs in which EHBO does not resolve or in which EHBO results in complications, therapeutic PUCC may be useful in relieving gallbladder distension.     

Liver fluke infestation in New World camelids. Parasitology, pathology, clinical findings and therapy]

In Llamas and Alpacas infestation with Fasciola hepatica or Dicrocoelium dendriticum can cause liver damage, sometimes even with lethal outcome. Once infected South American Camelids (SACs) react more sensitively to these parasites than other domestic ruminants. We report here on the pathology, parasitology, clinics and therapy of this disease. Concerning Dicrocoelium dendriticum we describe own clinical results and therapeutic outcome in addition to the pathological investigation. According to anatomic corrosion casts, the bile ducts of SACs show more similarity with the equine bile system than with the bile system of domestic ruminants.     

Ultrasound-guided percutaneous portocentesis in 21 cows

An ultrasound-guided portocentesis was performed in 21 clinically healthy cows, and blood was collected for haematological, biochemical and blood-gas analyses. At the same time blood samples were collected from the left jugular vein of each cow for the same analyses. Immediately after the centesis, portal blood pressure was measured. The cows were examined daily thereafter for eight days and then slaughtered for postmortem examination of the internal organs. The mean (sd) portal blood pressure was 37.1 (5.47) mmHg, and ranged from 24 to 49 mmHg. There were small but significant differences between many of the haematological and biochemical parameters in jugular and portal blood, and the concentration of bile acids was on average 2.7 times higher, and the concentration of ammonia was 19.3 times higher in portal blood. The demeanour and behaviour of the cows, and their appetite and rectal temperature remained normal during the eight-day observation period. The haematocrit, total and differential leucocyte counts, and the concentrations of total solids and fibrinogen were not affected by portocentesis. On postmortem examination, no lesions were apparent in the peritoneum, liver and portal vein.     

Effects of charcoal-extracted follicular fluid on reproductive function in postpartum cows

In order to determine the role of follicle-stimulating hormone (FSH) on the resumption of ovarian function in cows early postpartum (PP), bovine follicular fluid (FF) was used to selectively suppress concentrations of FSH. Calves were removed from all cows within 24 hr of birth. Follicular fluid that was treated with charcoal to remove steroids (15 ml; n = 14) or serum (S) from an ovariectomized cow (15 ml, n = 14) was injected i.m. twice daily from days 1 to 10 PP. Blood samples were collected before each injection and frequent samples (every 15 min for 6 hr) taken on days 5 and 10 PP. Eight cows from each group (FF and S) were slaughtered on the morning of day 11 PP and pituitaries and ovaries collected. The remaining cows (n = 6) were observed for estrus. Treatment with FF delayed follicular growth (P less than 0.01), as evidenced by the largest follicle per cow observed at time of slaughter (3.6 +/- 0.42 vs 11.5 +/- 1.77 mm dia; FF vs S). The intervals from parturition to first estrus (P less than 0.11) and to first progesterone rise (25.3 +/- 1.97 vs 18.0 +/- 3.62 d; P less than 0.06) tended to be delayed by treatment with FF vs S. Many of the cows treated with S ovulated by day 10 PP, they were divided retrospectively into those that had ovulated by (n = 9) or after (n = 5) day 10 PP for analysis. Cows treated with FF had lower (P less than 0.05) and less variable (P less than 0.01) serum FSH concentrations while levels of luteinizing hormone (LH) tended (P less than 0.08) to be greater on days 5 and 10 PP. Follicular fluid decreased levels of FSH (P less than 0.001), but not LH (P less than 0.15), in the samples obtained twice daily compared to S-treated cows that did not ovulate by day 10 PP. Anterior pituitaries were dissociated, and cells from each cow were cultured in order to ascertain whether treatment with FF in vivo would affect gonadotropin secretion in vitro. Estradiol-17 beta (E) was incubated with pituitary cells to determine the effect of E on gonadotropin secretion from cells of PP cows, and to ascertain whether treatment with FF in vivo and with E in vitro would interact to affect secretion of FSH and LH in culture. After 2 d of incubation, cells were treated with 10(-9) M E or vehicle (1% ethanol).(ABSTRACT TRUNCATED AT 400 WORDS)