A Reliable, Practical, and Economical Protocol for Inducing Diarrhea and Severe Dehydration in the Neonatal Calf

Pamela G. Walker, Peter D. Constable, Dawn E. Morin, James K. Drackley, Jonathan H. Foreman, and John C. Thurmon

ABSTRACT metabolic acidosis, absence of phosphore, la creatinine, I'albu- hyponatremia, renal instead of mine et les proteines totales furent Fifteen healthy, colostrum-fed, enteric loss of chloride, renal as tous augmentes. Ce protocole pro- male dairy calves, aged 2 to 7 d well as enteric loss of free water, duit des effets clinicopathologiques were used in a study to develop a absence of profound clinical depres- comparables a ceux d'une diarrhee diarrhea protocol for neonatal sion and suspected differences in associee a un syndrome de mal- calves that is reliable, practical, and the morphologic and functional digestion avec augmentation de la economical. After instrumentation effect on intestinal epithelium. pression osmotique telle que ren- and recording baseline data, diar- Despite these differences, the contree lors de diarrhee aux virus rhea and dehydration were induced sucrose/diuretic protocol should be rota, corona ou a la cryptospori- by administering milk replacer useful in the initial screening of new diose, dans 100 % des inductions [16.5 mL/kg of body weight (BW), treatment modalities for calf diar- realisees. Ce modele ne peut induire PO], sucrose (2 g/kg in a 20% aque- rhea. To confirm their efficacy, the une acidose severe, une hypona- ous solution, PO), spironolactone most effective treatment methods tremie, une perte renale de chlore and hydrochlorothiazide (1 mg/kg, should then be examined in calves au lieu d'intestinale, une perte PO) every 8 h, and furosemide with naturally-acquired diarrhea. d'eau renale et intestinale, et pro- (2 mg/kg, IM, q6h). Calves were voque une depression profonde et administered sucrose and diuretic des differences possibles dans la agents for 48 h to induce diarrhea RESUME morphologie et le fonctionnement and severe dehydration. Clinical de 1'e'pithe'lium intestinal. Malgre' changes after 48 h were severe Quinze veaux males en sante ages ces differences, ce protocole peut watery diarrhea, severe depression, de 2 a 7 jours et ayant requ leur etre utile, dans un premier temps, and marked dehydration (mean, colostrum, ont ete utilises dans un pour verifier l'efficacite de nou- 14% BW loss). Cardiac output, protocole servant a simuler la diar- veaux traitements de la diarrhee stroke volume, mean central venous rhee netonatale. La diarrhee et la des veaux avant de les appliquer a pressure, plasma volume, thio- deshydratation furent induites a des veaux infectes. cyanate space, blood pH and bicar- l'aide du melange suivant: lait (Traduitpar docteur Pascal Dubreuil) bonate concentration, base excess, de remplacement (16,5 mL/kg), serum chloride concentration, sucrose (2 g/kg en solution aqueuse and fetlock temperature were 20 %), spironolactone et hydro- INTRODUCTION decreased. Plasma lactate concen- chlorothiazide (1 mg/kg) donne a tration, hematocrit, and serum toutes les huit heures, et du furo- Diarrhea in neonatal calves is a potassium, creatinine, phosphorus, semide (2 mg/kg, im) administre a major source of economic loss to the total protein and albumin concen- toutes les 6 heures. Les veaux ont cattle industry. Diarrhea is the leading trations were increased. This non- recu cette combinaison pendant cause of death in dairy heifer and beef infectious calf diarrhea protocol has 48 heures, 'a la fin desquelles une calves aged less than 4 mo old, with a 100% response rate, while provid- diarrhe'e severe, une depression annual mortality rates of 6.6% and ing a consistent and predictable marquee et une perte de poids 5.6% for dairy heifer and beef calves, hypovolemic state with diarrhea (14 %) furent notees. Le volume respectively (1,2). Financial losses that reflects most of the clinico- d'ejection cardiaque, la pression occur not only from calf mortality, pathologic changes observed in veineuse centrale, le volume plas- but also from the cost of medication osmotic/maldigestive diarrhea caused matique, 1'espace determine' par le and labor needed to treat sick calves. by infection with rotavirus, coron- thiocyanate, le pH, les bicarbonates, Considering the economic importance avirus or cryptosporidia. Limita- les chlorures ainsi que la tempera- of calf diarrhea, a consistent protocol tions of the protocol, when com- ture des extremites furent tous for inducing diarrhea would facilitate pared to infectious diarrhea models, reduits. Par contre, I'hematocrite, comparison of treatment modalities include failure to induce a severe les lactates, les ions potassium et and increase our understanding of the

College of Veterinary Medicine, Department of Veterinary Clinical Medicine (Walker, Constable, Morin, Foreman, Thurmon), College of Agricul- tural, Consumer and Environmental Sciences, Department of Animal Sciences (Drackley), University of Illinois, Urbana, Illinois 61802 USA. Received May 27, 1997.

Can J Vet Res 1998; 62: 205-213 205 pathophysiology of neonatal diarrhea. MATERIALS AND METHODS catheter was connected to calibrated Development of a reliable, practical fluid-filled transducers (P23XL trans- and economical protocol for inducing ANIMALS ducer, Viggo Spectramed, Oxnard diarrhea in calves would provide an The experimental protocol was California, USA) and correct catheter important research tool for the initial approved by our institutional animal position assessed by monitoring the screening of new treatment modalities care and use committee. Fifteen pressure signals on a strip chart under controlled conditions. healthy male dairy calves, colostrum- recorder (5/6 Recorder, Gilson Medical Numerous methods have been used fed and aged between 2 and 6 d, were Electronics Inc, Middleton, Wisconsin, to experimentally induce diarrhea in obtained from a local source. All USA). The catheter introducer was neonatal calves. These methods can calves had an adequate passive trans- then withdrawn from the jugular vein, be grouped into 2 categories, infec- fer of colostral immunoglobulins as and the Swan-Ganz catheter fastened tious and non-infectious. The ideal evaluated by the serum total protein to the neck. This Swan-Ganz catheter model would be reliable, economical, concentration; a reliable predictor of was used to determine cardiac output, and practical, while accurately reflect- serum immunoglobulin concentration mean central venous pressure, and ing the pathophysiologic changes in normally hydrated calves (34). The blood temperature. associated with naturally-occurring calves were housed at an ambient Fetlock temperature was measured diarrhea in calves. It would also mini- temperature of 20 to 22°C in individ- by utilizing a thermistor on a second mize suffering and decrease the risk ual calf stalls and fed a high quality Swan-Ganz catheter. The thermistor of pathogen transmission among (all milk-based protein) milk replacer was placed on the medial surface of calves, and decrease the risk of (Caf-gro Super Supreme, Farm & the rear fetlock with a small amount zoonotic disease in research workers. Fleet Stores Inc, Janesville, Wisconsin, of surgical lubricant (Johnson & Infectious diarrhea models used in USA) (crude protein > 22%, crude Johnson Medical Inc, Arlington, neonatal calves include enterotoxi- fat > 20%, crude fiber < 0.15%) at Texas, USA) to facilitate heat conduc- genic Escherichia coli (3-9), Cryp- 10% of their BW/d, divided into tion, and the entire Swan-Ganz tosporidium parvum (10,11) coron- 3 feedings at 7 AM, 3 PM, and catheter completely covered with tape avirus (12,13), rotavirus, or a 11 PM. The milk replacer was recon- (Johnson & Johnson Medical Inc) to combination of infectious agents stituted according to manufacturers' insulate from room air. Xylazine (5,9,12,14-16). Non-infectious agents directions at the rate of 227 g of dry sedation was reversed after instru- that have been administered orally to powder per 1.892 mL of warm tap mentation by administering tolazoline experimentally induce diarrhea in water (35-40'C). (1 mg/kg, IV) (Sigma Chemical Com- pany, St. Louis, Missouri, USA) and calves are sucrose (17-24), lactose INSTRUMENTATION (17,18,22,25-27), sorbitol (28), man- the calf was placed in its movable nitol (29), castor oil (29), chloram- Calves were acclimatized to their stall. The proximal and distal ports of phenicol, neomycin, ampicillin, and surroundings and diet until they were the intravascular Swan-Ganz catheter tetracycline (30,31), and purified STa between 3 and 7 d of age. All calves were flushed with heparinized saline toxin from E. coli (32). Subcutaneous had a normal appetite for the milk (100 IU/mL) (Abbott Laboratories, injection of reserpine has also been replacer and normal appearing and North Chicago, Illinois, USA) every used (33). well formed feces before instrumenta- 8 h for the duration of the study. tion. At this time, each calf was A urine collection device was fitted Although each of these infectious sedated with and xylazine (Xylazine to the calf after complete recovery non-infectious models can cause 20 mg/mL, Miles Inc, Animal Health from xylazine sedation. The urine col- diarrhea, most do not fulfill the crite- Products, Shawnee Mission, Kansas, lection device consisted of an empty ria for an ideal calf diarrhea model. USA) (0.20 mg/kg, IM), and the right 1 L intravenous fluid bag (Abbott Problems include a large variation in jugular furrow was clipped and surgi- Laboratories) with an IV drip set response, ranging from no effect, to cally scrubbed for aseptic placement (Abbott Laboratories) attached and mortality despite treatment (3,6,8,9, of an intravascular catheter. The secured with tape (Johnson & Johnson 12,15,16), and an unpredictable time catheter site was anesthetized with Medical Inc). The opposite end of the to the onset of diarrhea and dehydra- 1 mL of lidocaine hydrochloride bag was cut off and the bag placed tion (10-12,14-16). Such variability (Lidocaine 2%, The Butler Company, around the prepuce of the calf and increases the number of calves needed Columbus, Ohio, USA) and an 8-F glued with a commercial adhesive to compare treatment modalities. The catheter introducer (Argon Medical, (Goop, Eclectic Products, USA). The purpose of the study reported here Edward Weck Inc, Athens, Texas, end of the IV drip set was placed was therefore to develop a reliable, USA) placed using the Seldinger tech- through the bottom slats of the mov- economical, and practical diarrhea nique (35). A 90-cm 7F thermodilu- able calf stall, and placed in a 1 L glass protocol that reflects the pathophysi- tion catheter (Model 131H-7F container to store the voided urine. ology of naturally-occurring diarrhea. Edwards, Swan-Ganz catheter, Baxter Sucrose was chosen to induce diar- Healthcare Corporation, Edwards BASELINE MEASUREMENTS rhea because of predictable results in Critical Care Division, Irvine, Following instrumentation, calves other studies (17-24) in inducing California, USA) was advanced until were kept in their movable stalls for diarrhea. Diuretic agents were used in the distal port was in the pulmonary at least 8 h before recording baseline addition to sucrose to facilitate induc- artery and the proximal port was in or values and obtaining baseline blood tion of dehydration. near the right atrium. The Swan-Ganz samples. During the period between

206 instrumentation and recording base- the proximal port of the Swan-Ganz then releasing the skin. Thoracic skin line values all calves had a normal catheter, with the scapulohumeral tent duration was defined as the time appetite for milk replacer and normal joint taken as the zero pressure refer- taken for the skin to return to its ini- appearing and well formed feces. ence point. Measurements were taken tial, non-tented, position. The extent Calves did not have access to water or with the calf facing forward, standing of eyeball recession into the orbit was feed. or in sternal recumbency, with the determined by gently rolling out the Baseline values consisted of: heart head in a normal alert, non-feeding lower eyelid to its normal position rate; respiratory rate; rectal, fetlock position. Mean central venous pres- and estimating the distance between and blood temperatures; cardiac out- sure was defined as the height of the the globe and palpebral conjunctiva. put; mean central venous pressure; saline column (in cm H20) relative to Hatch marks in 5 mm increments were fecal consistency and pH; clinical the reference level. made on the edge of the thumb used to hydration; depression status; urine Fecal consistency was determined pull down the eyelid to assist in esti- volume; and BW. Blood samples were and fecal samples for analysis were mating eyeball recession. obtained from the distal port of the collected by digital rectal stimulation Body weight was measured by Swan-Ganz catheter for measurement with a gloved hand. Fecal pH was weighing the calf and the movable of: mixed venous blood gas tension measured immediately and the fecal stall, and subtracting the weight of the and blood pH; hematocrit; plasma lac- sample retained for subsequent deter- stall. Calves were weighed after all tate, glucose and protein concentra- mination of fecal dry matter content. measurements were taken and before tion; serum biochemical analysis; The amount of urine produced was administration of milk replacer; the plasma volume (Evans blue dye measured in a graduated cylinder for BW therefore represented fasted BW. method); and extracellular volume each 8 h period, and urine production Each calf was stimulated to urinate (thiocyanate space). (in mL/h) at the end of the collection and defecate by rubbing the perineal Heart rate (beats per minute) was period calculated (collected urine area before being weighed. measured by auscultating the thorax volume/8 h). with a stethoscope for a minimum of Fecal consistency, the degree of EXPERIMENTAL PROTOCOL 30 s. Respiratory rate (breaths per clinical dehydration, and the degree Osmotic/maldigestive diarrhea and minute) was measured by counting of clinical depression were given dehydration were induced by admin- thoracic excursions for a minimum of numerical scores (24) as follows: istering milk replacer solution 15 s; this was repeated if the calf was Fecal consistency: 0 = normal, (16.5 mL/kg, q8h; equivalent to a sniffing or vocalizing. Respiratory manure is normal and well formed; milk replacer feeding rate of 5% rate was obtained before any other 1 = abnormal feces but not diarrhea, BW/d) and twice isotonic sucrose measurements were taken and fre- manure is pasty (softer than normal); solution [2 g/kg sucrose (Flavorite quently with the calves apparently 2 = mild diarrhea (semi-liquid, but Sugar, Chaska, Minnesota, USA) in a unaware of any human presence. Rec- still has a solid component); 3 = pure 20% aqueous solution; calculated tal temperature was measured (B-D liquid feces. Diarrhea was defined as 600 mOsm/L, PO, q8h], furosemide thermometer, Becton Dickinson Con- the voiding of feces that splashed (Furosemide 50 mg/mL, Hoechst sumer Products, Westminster, South when hitting the ground, with failure Roussel, Somerville, New Jersey, Carolina, USA) before the calf was to form a firm cow pat. Degree of USA) (2 mg/kg, IM, q6h), spironolac- stimulated to defecate. A thermodilu- clinical depression: 0 = normal; 1 = tone (Aldactazide, 50 mg tablets, tion cardiac output computer (COM-1 mild depression, calf suckles but not G.D. Searle and Co, Chicago, Illinois, Cardiac output computer, American vigorously; 2 = moderate depression, USA) (1 mg/kg, PO, q8h) and Edwards Laboratories) was used to calf able to stand, suckling is weak or hydrochlorothiazide (Aldactazide, measure blood temperature at the time disorganized; 3 = severe depression, 50 mg tablets, G.D. Searle and Co) of cardiac output determination, fol- calf unable to stand or suckle. Degree (1 mg/kg, PO, q8h). Calves suckled lowed by fetlock temperature mea- of clinical dehydration: 0 = normal, the milk replacer-sucrose solution surement utilizing the thermistor con- eyes are bright and skin feels pliable; readily. Calves were administered nector on the second Swan-Ganz 1 = mild dehydration, slight loss of sucrose and diuretic agents for 48 h to catheter taped to the rear fetlock. Car- skin elasticity, skin tent < 3 s, eyes induce diarrhea and severe dehydra- diac output was measured by injecting not recessed into orbit; 2 = moderate tion. Calves were examined every 8 h 5 mL of cold (0°C) 5% dextrose dehydration, skin tent > 3 s but < 10 s, at feeding time and the rectal temper- through the proximal port and moni- eyes slightly recessed into orbit; 3 = ature, heart rate, respiratory rate and toring the change in pulmonary artery severe dehydration, skin tent > 10 s, urine volume recorded. At 24 and temperature by a thermodilution car- eyes markedly recessed into orbit. A 48 h post-induction of diarrhea, mixed diac output computer. The mean of total clinical score (TCS) was calcu- venous blood gas tension and blood 3 cardiac output determinations was lated by adding the 3 scores for the pH, hematocrit, plasma protein con- used for the cardiac output at each clinical evaluations described above, centration, cardiac output, blood tem- time interval. Mean central venous ranging in value from normal (0) to perature, fetlock temperature and BW pressure was determined by connect- markedly abnormal (9). were measured. Serum biochemical ing a graduated pipette (Medex Water Skin pliability was measured at the analysis, plasma lactate and glucose manometer set, American Pharmaseal lateral thorax over the 6th to 9th rib, concentrations, fecal dry matter content, Company, Medex Inc, Hilliard, Ohio, midway down the thorax by tenting fecal pH, plasma volume, thiocyanate USA) filled with heparinized saline to and twisting the skin 900 for 1 s and space, and hydration measurements

207 were determined at 48 h post-induc- green), and serum total protein con- supernatant was mixed 1:4 with 16% tion of diarrhea. centration (biuret) (Hitachi 704 Auto- ferric nitrate (Mallinckrodt Inc., Paris, Plasma volume was determined by matic Analyzer, Tokyo, Japan). Kentucky, USA) solution, covered rapidly injecting 20 to 25 mg of Evans Plasma glucose (glucose oxidase) and with a dark cloth for 15 min, and the blue dye (Sigma Chemical Company), lactate (oxidase) concentrations were absorbance immediately read at as a 0.5% solution, into the proximal also determined (YSI model 2300 Stat 460 nm against a reagent blank (2 mL port of the intravascular Swan-Ganz Plus Glucose and L-Lactate Analyzer, of TCA and 1.5 mL ferric nitrate catheter, obtaining pulmonary artery Yellow Springs, Ohio, USA). Blood reagent) (42). The pre-injection blood from the distal port at 15, 20, for serum and plasma analysis was plasma absorbance was subtracted 25, and 30 min after injection, and kept at 4°C for 2-4 h before being from the thiocyanate-containing extrapolating the Evans blue dye con- centrifuged, the serum and plasma plasma samples and the concentration centration back to zero time as previ- was then separated and frozen at of sodium thiocyanate determined ously reported (36,37). Plasma vol- -20°C for less than 2 mo before from the standard curve as previously ume determined by the Evans blue being analyzed. Hematocrit was described (36). dye technique in calves is similar to determined by microcentrifugation A Jenco portable pH meter (Jenco that obtained using radiolabeled and plasma and serum protein concen- Model 6009, Micro Computer based albumin (38). Blood volume was cal- tration by refractometry. Samples for portable pH meter, San Diego, Cali- culated from the plasma volume and blood pH, blood gas tension, hemo- fornia, USA) was used to measure hematocrit, after correcting for globin concentration, and plasma ion- fecal pH levels (immediately after trapped plasma, whereby blood ized calcium concentration were ana- collection) by total immersion of the volume in L = [plasma volume (L) X lyzed by a blood gas analyzer probe in the feces; the average of 2 100]/[100 - (hematocrit % x 0.94)] (238 pH/blood gas analyzer, Ciba such measurements was taken as the (39). Corning, Halstead, England) and the fecal pH value. Fecal dry matter con- Thiocyanate space (correlated with blood pH and blood gas tension val- tent was determined by placing a extracellular fluid volume) (40) was ues corrected for rectal temperature. known amount of feces in a ceramic determined by slowly injecting 1.0 to Samples for blood pH and blood gas crucible and drying at 60°C. Each 1.1 g of sodium thiocyanate (Sigma tension analysis were kept at 4°C sample was weighed every 3 d until Chemical Company), made up as a before being analyzed within 4 h. the weight remained constant; the 3.3% solution, into the proximal port Evans blue dye concentration in fecal dry matter content was then cal- of the intravascular Swan-Ganz plasma was determined spectrophoto- culated as: [(fecal dry weight)/(fecal catheter over 1 min. Pulmonary artery metrically (Coleman 55 Spectropho- wet weight)] X 100. blood samples were obtained from the tometer, Coleman Instruments Divi- distal port of the intravascular Swan- sion, Oak Brook, Illinois, USA) at STATISTICAL ANALYSIS Ganz catheter at 60, 80, 100 and 620 nm by interpolation from a stan- Data are presented as means ± sx. 120 min after injection. Thiocyanate dard curve. To prepare a standard curve Paired t-tests were used to compare space was determined by extrapolat- for each calf, 1 mL of pre-injection normally distributed continuous vari- ing the thiocyanate concentration plasma was added to 1 mL of a known ables and Wilcoxon signed rank tests back to zero time as previously concentration of Evans Blue Dye in were used to compare ordinal and reported (36). Thiocyanate space in isotonic saline and the absorbance non-normally distributed variables. calves is similar to that obtained by measured against a plasma blank. The normalcy of the distribution was radiolabeled sodium (38). Preparation of the samples consisted evaluated by examining the skewness Forty-eight hours after induction of of taking 1-mL aliquots each of the and kurtosis of the data and calculat- watery diarrhea and severe dehydra- post-injection plasma and isotonic ing the Shapiro-Wilk statistic. Vari- tion, the 15 calves were randomly saline and measuring absorbance at ables with non-normal distributions or assigned to 3 groups (2 treatment and 620 nm against the pre-injection unequal variances were log trans- 1 control), and the results are reported plasma blank. All plasma volume formed before being compared by a elsewhere (41). All 15 calves (includ- samples were analyzed within 24 h of paired t-tests. Paired comparisons ing 5 control calves) survived the 24 h collection, after storage at 4°C. were between baseline and time = treatment phase, and at the end of the Plasma thiocyanate concentration 48 h values only. The time = 24 h val- 24 h treatment phase, diarrhea was determined spectrophotometri- ues were not statistically compared to stopped within 8 h of cessation of cally (Coleman 55 Spectrophotome- the other values, but were included sucrose administration. ter, Coleman Instruments Division, for descriptive purposes. A P value Oak Brook, Illinois, USA) after < 0.05 was considered significant. SAMPLE ANALYSIS deproteinization of plasma to remove Automated methods were used to Evans Blue Dye. This process measure serum sodium, potassium, involved adding 1 mL of plasma to RESULTS and chloride concentrations (Ion- 10 mL of cold 10% trichloroacetic Selective electrode), serum phosphate acid (TCA) (EK Industries, Addison, The administration of sucrose and 3 concentration (ammonium phospho- Illinois, USA), centrifuging for different diuretic agents resulted in molybdate), serum creatinine concen- 10 min and removing the supernatant. profuse watery diarrhea, moderate tration (Jaffe picric acid), serum albu- The supernatant was stored at room dehydration and an average weight min concentration (bromcresol temperature for up to 3 wk. The loss of 3.9 kg, equivalent to 10% of

208 initial BW, in 24 h without evidence Table I. Physical examination findings, hemodynamic values, and fecal characteristics in of profound depression. Severe neonatal dairy calves (n = 15) with experimentally-induced diarrhea watery diarrhea (fecal score 3) was Baseline evident in all calves within 8 24 h 48 h Probability h of Physical examination findings starting sucrose administration, and Body weight (kg) 39.6 ± 2.6 35.7 ± 2.3 34.2 ± 2.3 < 0.0001 was maintained the entire time that Rectal temperature (°C) 39.1 ± 0.1 39.6 ± 0.1 39.6 ± 0.1 0.0082 sucrose was fed. This clinical obser- Blood temperature (°C) 39.2 ± 0.1 39.6 ± 0.1 39.6 ± 0.1 0.011 vation was consistent with the signifi- Fetlock temperature (°C) 34.3 ± 0.7 27.9 ± 1.2 26.5 ± 0.9 < 0.0001 cant decrease in fecal Rectal-fetlock temperature difference 4.8 ± 0.7 11.7 ± 1.1 13.1 ± 3.7 0.0001 dry matter con- Heart rate (beats/min) 126 ± 4 137 ± 4 131 ± 7 NS tent (Table I). Fecal pH decreased Respiratory rate (breaths/min) 38 ± 2 35 ± 2 37 ± 2 NS significantly from 5.89 to 5.10 follow- Thoracic skin tent duration (s) 2.1 ± 0.1 ND 8.9 ± 1.7 < 0.0001 ing diarrhea induction with sucrose. Eyeball recession into orbit (mm) 0 ± 0 ND 7.7 ± 0.4 < 0.0001 Watery diarrhea was maintained until Hydration score (0 to 3) 0 ± 0 1.9 ± 0.2 2.9 ± 0.1 < 0.0001 Clinical depression score (0 to 3) 0 ± 0 0.3 ± 0.1 1.2 ± 0.2 < 0.0001 48 h, at which time the average BW Total clinical score (0 to 9) 0 ± 0 5.2 ± 0.2 7.1 ± 0.2 < 0.0001 loss was 5.4 kg, corresponding to 14% dehydration (Table I). Calves had Hemodynamic values Thiocyanate space (L) 17.4 ± 1.3 ND 11.6 ± 0.8 < 0.0001 clinical evidence of severe dehydra- Plasma volume (L) 3.4 ± 0.3 ND 2.5 ± 0.2 < 0.0001 tion at that time, as measured by eye- Estimated interstitial fluid volume (L) 14.0 ± 1.3 ND 9.1 ± 0.7 < 0.0001 ball recession into orbit (7.7 mm), Blood volume (L) 4.6 ± 0.4 ND 3.9 ± 0.3 0.0130 prolonged thoracic skin tent duration Plasma volume/Thiocyanate space(%) 20.6 ± 1.6 ND 22.2 ± 1.5 NS (> 6 s for all calves), increased Cardiac output (L/min) 8.80 ± 0.42 4.47 ± 0.26 3.89 ± 0.26 < 0.0001 hydra- Stroke volume (mL) 71 ± 4 33 ± 2 32 ± 3 < 0.0001 tion score, rectal-fetlock temperature Mean central venous pressure (cm H20) 0.6 ± 0.8 ND -1.9 ± 1.0 0.0029 difference, and decreased urine pro- Urine production (mL/h) 81 ± 15 61 ± 10 12 ± 6 < 0.0001 duction (Table I). All calves were Fecal characteristics examined every 8 h and monitored Fecal consistency score (0 to 3) 0 ± 0 3.0 ± 0 3.0 ± 0 < 0.0001 periodically between examination/ Fecal dry matter content (%) 28 ± 2 ND 9 ± 1 < 0.0001 treatment periods. Calves preferred to Fecal pH 5.89 ± 0.10 ND 5.10 ± 0.09 < 0.0001 stay in sternal recumbency between Values are mean ± s. examination periods, but would stand ND = not determined and suckle at feeding time. If at any NS = not significant time throughout the study a calf Table II. Laboratory findings in neonatal dairy calves (n = 15) with experimentally-induced received a clinical depression score of diarrhea 3 (unable to stand or suckle) the calf was immediately euthanized. All Baseline 24 h 48 h Probability calves survived the 48 h diarrhea/ Mixed venous pH 7.36 ± 0.01 7.35 ± 0.01 7.31 ± 0.02 0.0029 Mixed venous PCO2 (mmHg) 60 ± 1 64 ± 1 60 ± 2 NS dehydration induction phase (includ- Mixed venous P02 (mmHg) 31 ± 1 27 ± 1 32 ± 1 NS ing all 5 calves in the control group), Mixed venous HCO3- (mEq/L) 34.3 ± 0.5 35.9 ± 1.1 31.0 ± 1.6 0.0172 and were able to stand and suckle Mixed venous Base excess (mEq/L) +8.6 ± 0.6 +9.9 ± 1.2 +4.7 ± 1.8 0.015 throughout this period. Plasma lactate (mM/L) 1.1 ± 0.1 ND 2.8 ± 0.5 0.0008 Rectal and blood temperatures were Hemoglobin (g/dL) 7.5 ± 0.2 9.6 ± 0.3 9.8 ± 0.3 < 0.0001 Hematocrit (%) 28.2 ± 1.2 32.5 ± 1.0 36.9 ± 1.3 <0.0001 significantly increased by 48 h, Plasma protein (g/dL) 6.4 ± 0.2 7.5 ± 0.3 8.1 ± 0.2 < 0.0001 whereas fetlock temperature decreased Serum sodium (mEq/L) 136 ± 1 ND 139 ± 2 NS by 6.4°C in 24 h and by 7.8°C in 48 h Serum potassium (mEq/L) 4.8 ± 0.1 ND 6.4 ± 0.5 0.017 (Table I). The rectal-fetlock tempera- Serum chloride (mEq/L) 94 ± 1 ND 90 ± 2 0.033 Plasma calcium (mEq/L) 5.2 ± 0.3 ND 4.5 ± 0.1 < 0.0001 ture difference also increased from Serum phosphorus (mg/dL) 7.0 ± 0.3 ND 11.0 ± 0.3 < 0.0001 4.80C to 13.1°C by 48 h. The large Serum total protein (g/dL) 5.6 ± 0.2 ND 7.4 ± 0.4 0.0005 reduction in fetlock temperature was Serum albumin (g/dL) 2.6 ± 0.1 ND 3.4 ± 0.1 < 0.0001 consistent with the clinical observa- Serum creatinine (mg/dL) 0.8 ± 0.1 ND 5.1 ± 0.6 < 0.0001 tion that all extremities were cool to Plasma glucose (mg/dL) 68 ±4 ND 66± 5 NS the touch. Heart rate and respiratory Values are mean ± SX ND = not determined rate were not significantly changed by NS = not significant 48 h; however, it appeared that depth of respiration increased toward the and blood volume (Table I). The by significant increases in hematocrit, end of the 48 h time period. reduction in thiocyanate space (5.8 L) hemoglobin concentration, plasma Plasma volume decreased by 26% was greater than the reduction in BW protein concentration, and serum con- and thiocyanate space decreased by (5.4 kg), indicating that fluid and centrations of albumin, total protein, 33% after 48 h of diarrhea and dehy- weight loss was primarily from the creatinine, and phosphate (Table II). dration (Table I). The reduction in extracellular space, and that there Serum sodium concentration was plasma volume and thiocyanate space may have been some expansion of the unchanged, whereas serum potassium was accompanied by a significant intracellular space. concentration increased and serum decrease in cardiac output, stroke vol- Hypovolemia and decreased glo- chloride concentration decreased. ume, mean central venous pressure, merular filtration rate were reflected Hyperphosphatemia was accompanied

209 by a decrease in plasma ionized cal- diarrhea in calves (decrease in fecal sodium, and potassium similar to cium concentration. pH from 6.8 to 6.0) (25). In contrast, those in calves with naturally- Despite the severe dehydration fecal pH increased from 5.5 to 7.1 in acquired diarrhea (19,20), but results induced by sucrose and diuretic calves with experimentally-induced in a smaller fecal chloride loss (19,20). administration, calves developed only enterotoxigenic E. coli diarrhea, a Accordingly, 3 diuretic agents with a mild metabolic acidosis, as indi- classical secretory diarrhea agent different sites of action were used in cated by a small decrease in blood pH, (47). the present study to produce elec- bicarbonate concentration, and base The mean dry matter content of trolyte losses in calves administered excess, and a corresponding small normal calf feces is 24% (48), sucrose similar to those occurring in increase in plasma lactate concentra- whereas the dry matter content of calf calves with naturally-acquired diar- tion (Table II). feces classified as loose or watery was rhea. Furosemide is a loop diuretic 13%, and 9%, respectively (25). In the which inhibits the reabsorption of study reported here, we observed a sodium in the proximal and distal DISCUSSION decrease in fecal dry matter content tubules and in the ascending limb of from 28% to 9%, consistent with the loop of Henle. Furosemide is a The oral administration of sucrose, sucrose-induced diarrhea. Reduction potent diuretic, natriuretic, and spironolactone, and hydrochloroth- in fecal dry matter content induced by chloruretic agent in the presence of iazide, and IM administration of sucrose is thought to facilitate bacte- acidosis or alkalosis, and has no furosemide, provided a reliable, prac- rial multiplication in the large intes- inhibitory effect on carbonic anhy- tical, and economical protocol for tine and increase bacterial concentra- drase or aldosterone activity in the inducing diarrhea and severe dehydra- tions in large intestinal fluid and feces distal tubule (51,52). Furosemide is tion in neonatal calves. Watery diar- (23), potentially exacerbating elec- widely used in cattle to induce diure- rhea was induced in all calves within trolyte and acid base derangements sis and treat udder edema (53,54), but 8 h, and maintained for the entire time associated with diarrhea. tends to induce hypokalemia in most that sucrose was administered. A pre- Whitten et al conducted a study to species (52,54). Hydrochlorothiazide dictable and severe dehydrated state measure intestinal exchanges of elec- promotes the excretion of sodium and was also induced in all calves, with trolytes in healthy calves and calves water primarily by inhibiting their the severity of the dehydration with diarrhea. Two calves contracted reabsorption in the cortical diluting increasing with time. diarrhea spontaneously and 2 calves segment of the distal renal tubule. Its Calves do not possess intestinal had sucrose-induced diarrhea when principal action is enhancement of the sucrase and are therefore unable to fed as an isotonic formulation (2 g excretion of approximately equal utilize dietary sucrose (18,43). This sucrose/kg, 10% aqueous solution, amounts of sodium and chloride makes sucrose an unsuitable energy q8h). Histologic changes in the intes- (51,55,56). Hypokalemia may develop source for feeding calves, but an ideal tine were similar in calves with natu- as a result of profound diuresis, par- agent for inducing diarrhea. As such, rally-acquired and sucrose-induced ticularly when used concomitantly sucrose has been used in numerous diarrhea, and included shortening and with loop diuretics such as furo- studies to reliably and rapidly induce blunting of the intestinal villi and semide (51,55). Like furosemide, watery diarrhea in neonatal calves damage to the lamina propria (21). In hydrochlorothiazide is used in cattle (17-24). Sucrose-induced diarrhea is another study to determine the effect to induce diuresis and treat udder associated with decreased intestinal of sucrose administration on suscepti- edema (53,56). Spironolactone acts as transit time and increased fecal bacte- bility to Cryptosporidium parvum a specific pharmacologic aldosterone rial concentration (17,18,23); both infection, infant mice were adminis- antagonist, primarily by competitive changes are identical to those tered oral hypertonic sucrose solution binding of receptors at the aldos- observed in calves with naturally- to evaluate the interaction of an terone-dependent sodium-potassium acquired diarrhea (25,44). Sucrose is intestinal lectin-like receptor with exchange site in the distal convoluted fermented in the small intestine by carbohydrates. Histologic examina- renal tubule (51). Spironolactone will enteric bacteria, with metabolism of tion showed marked vacuolation of therefore antagonize the hypokalemic 38% and 41% of an ingested sucrose jejunal and ileal epithelium after 1 d effect of furosemide and hydrochlor- load in the small intestine of 4-6 mo of hypertonic sucrose solution admin- othiazide, but exert a weaker diuretic, old steers and 4 wk old calves, respec- istration with return to normal tissue natriuretic, and chloruretic effect than tively (45,46). Sucrose fermentation 3 d after cessation of treatment (49). furosemide (51). In 4-week-old calves, to volatile fatty acids also occurs in Although unsubstantiated; similar his- spironolactone (1 mg/kg, PO, q24h) the large intestine, thereby lowering tologic lesions may have been present increased the renal excretion of fecal pH (46), as observed in the pre- in the calves in this study, as we fed sodium and chloride and inhibited sent study. Sucrose can therefore be sucrose in a hypertonic formulation aldosterone activity, resulting in regarded as a classical osmotic/ (2 g sucrose/kg, 20% aqueous solu- decreased plasma volume, decreased maldigestive diarrhea agent in calves. tion, q8h) and hypertonic solutions plasma sodium and chloride concen- Interestingly, the change in fecal pH can directly damage intestinal epithe- trations, and increased plasma potas- induced by feeding sucrose (decrease lium, particularly at the villus tip (50). sium concentration (57). from 5.9 to 5.1) was similar in magni- Sucrose administration (without The severe dehydration (14% BW) tude and direction to that observed in concurrent administration of diuretic produced in this study was not accom- naturally-acquired undifferentiated agents) induces fecal losses of water, panied by mortality or severe

210 acidemia. All calves were able to and therefore contributes to the devel- perature alone. A rectal-fetlock tem- stand and suckle at feeding time with opment of hypovolemic shock in perature difference of 13°C was this degree of dehydration but pre- affected calves. In the study reported reported in one comatose and near ferred to stay in sternal recumbency here, the decrease in thiocyanate death calf with experimentally- between examination periods. Previ- space, which is correlated with extra- induced coronavirus diarrhea (67). In ous calf studies have indicated that cellular volume (36,38), was slightly the study reported here, we found the calves with diarrhea die when they greater than the decrease in BW, 5.8 L normal rectal-fetlock temperature dif- have lost 12.7-13.4% of their BW and 5.4 kg respectively, indicating ference to be 4-5°C in calves kept at (19,58), and guidelines have been that fluid and weight loss in this calf an ambient temperature of 20-22°C, developed that suggest a 12-14% diarrhea model was primarily from and the temperature difference to decrease in BW due to dehydration is the extracellular space, and that there increase to 13°C in severely dehy- fatal (59,60). The results of the study may have been some expansion of the drated calves. Other studies have reported here clearly suggest that intracellular space. The ratio of reported that the normal rectal-fetlock death from diarrhea is not solely due plasma volume to thiocyanate space temperature difference in neonatal to dehydration, but more likely due to remained constant before (0.21) and calves was 2-3°C, and that the tem- acid-base and electrolyte derange- after (0.22) diarrhea induction, indi- perature difference increased to ments, specifically severe acidemia cating that water was not lost prefer- 8-15°C in calves with experimen- and hyperkalemia (19,61-66). Results entially from the intravascular or tally-induced dehydration (29-35% of previous calf diarrhea studies indi- interstitial compartments. Taken reduction in plasma volume) (7,75). cate that the primary cause for together, these findings indicate that Further evaluation of the rectal-fet- acidemia is excessive loss of bicar- the sucrose/diuretic calf diarrhea pro- lock temperature difference in dehy- bonate in the diarrheic feces (15,19, tocol produced similar alterations in drated calves with diarrhea is indi- 21,61,65), rather than severe dehydra- body fluid compartments as seen in cated in order to determine its clinical tion and accompanying lactic acido- experimentally-induced viral diarrhea utility. sis. Blood or plasma lactate concen- (14) and naturally-acquired diarrhea The protocol reported here, com- trations do not usually increase above (69). However, because diuretic bining sucrose and diuretic agents, 3 mM/L in calves with diarrhea, but agents were administrated to facilitate can be classified as an osmotic/ can rapidly increase above 3 mM/L extracellular fluid loss, the route of maldigestive diarrhea, and is there- during the final hours before death free water loss in the sucrose/diuretic fore functionally similar to diarrhea (67,68). Lactate concentration can protocol (intestinal and renal) differs caused by rotavirus, coronavirus, and vary with the age of the calf, with from that seen in naturally-acquired cryptosporidia (28). A similar proto- calves less than 1 wk old having a diarrhea (intestinal only). col using only one diuretic agent greater tendency to develop lactic aci- Measurement of fetlock tempera- (furosemide) and a shorter duration of dosis (70). ture may be clinically valuable in treatment (24 h) produced severe Dehydration in calves with diarrhea calves with diarrhea, as an indicator watery diarrhea and moderate dehy- is accompanied by a large decrease in of peripheral blood flow and response dration (24), but was not used for the extracellular fluid volume and a to treatment. Most calves with severe present study because pilot studies in smaller increase in intracellular fluid dehydration and diarrhea have cold 8 calves indicated that it was inca- volume (14). During diarrhea there is extremities due to poor peripheral pable of producing severe dehydration increased intestinal loss of sodium, perfusion. Phillips and Lewis and (> 10% decrease in BW), within 48 h. potassium, and chloride, with a con- Jones et al measured fetlock (dorsal Biochemical changes commonly current decrease in plasma sodium aspect) and hock (dorsal aspect) tem- observed in calf diarrhea include prer- concentration, resulting in hypo- peratures in calves with experimen- enal azotemia, metabolic acidosis, osmotic plasma and extracellular fluid tally-induced or naturally-acquired mild hyponatremia and hyperlac- (14,20,69-72). The diarrhea-induced diarrhea (14,74), and found that fet- tatemia, a variable degree of hyper- extracellular hypo-osmolality causes lock temperature appeared to be more kalemia and hyperphosphatemia, and free water to move from the extracel- sensitive to changes in hydration sta- hypoglycemia (13,76,77). Serum lular to intracellular fluid space, tus. Normal fetlock temperature was chloride concentration usually is thereby increasing the latter. Intracel- 34 to 37°C, and decreased to 26°C unchanged (58). The sucrose/diuretic lular fluid volume may also increase after the onset of diarrhea (14). The diarrhea protocol used in this study independently of extracellular hypo- changes in fetlock temperature produced marked prerenal azotemia osmolality changes in severely dehy- observed in the study reported here and hyperkalemia, mild metabolic drated diarrheic calves, as hypov- (decrease from 34 to 27°C over 48 h) acidosis, hyperphosphatemia, hypo- olemic shock results in cellular closely followed those in calves with calcemia, hypochloremia, and hyper- hypoxia, partial depolarization of the experimentally-induced and naturally- lactatemia, with no change in serum resting membrane potential, and acquired viral diarrhea (14,74). sodium concentration. This suggests increased intracellular sodium con- Because diarrheic calves may be that the protocol causes slightly dif- centration, resulting in cellular hypothermic or pyrexic, the differ- ferent electrolyte losses than that swelling (61,73). Whatever the mech- ence between rectal temperature and associated with naturally-acquired anism, the increase in intracellular fetlock temperature is likely to pro- diarrhea. fluid volume in calves with diarrhea is vide a more accurate guide to periph- The sucrose/diuretic protocol was at the expense of extracellular fluid eral blood flow than the fetlock tem- economical, with an average cost of

211 $9 for a 50 kg calf, excluding the cost acquired diarrhea), a different route diarrheic calves supplemented with oral of housing and feed. The protocol had for chloride loss (renal instead of electrolytes. Can J Vet Res 1989; 53: a 477-485. 100% response rate within 12 h with enteric), 2 routes for free water loss 10. TZIPORI S, SMITH M, HALPIN C, no mortality within 48 h, in contrast (enteric and renal) instead of one ANGUS KW, SHERWOOD D, CAMP- to E. coli diarrhea models that had a route in naturally-acquired diarrhea, BELL I. Experimental cryptosporidiosis 53% to 100% response rate in I to 4 d, failure to induce hyponatremia, lack in calves: Clinical manifestations and with up to 85% mortality (3,5,7,8). A of pathologic findings. Vet Rec 1983; 112: profound clinical depression, and 116-120. similar trend is seen in models that possible differences in the morpho- 11. HARP JA, WOODMANSEE DB, use coronavirus/rotavirus, with a 50% logic and functional alterations in MOON HW. Effects of colostral antibody to 100% response rate in 1 to 6 d, and intestinal epithelium, when compared on susceptibility of calves to Cryp- 33 to 86% mortality (12,14-16,61, to viral or E. coli diarrhea. We there- tosporidium parvum infection. Am J Vet Res 1989; 50: 2117-2119. 71,73), and Cryptosporidium, with a fore suggest that the sucrose/diuretic 12. SAIF LJ, REDMAN DR, MOORHEAD 86 to 100% response rate in 3 to 6 d protocol is useful for the initial PD, THEIL KW. Experimentally induced (10,1 1). screening of fluid resuscitation meth- coronavirus infections in calves: Viral Initial studies using sucrose investi- ods in treating moderately to severely replication in the respiratory and intestinal gated the capacity of the neonatal calf dehydrated calves with diarrhea that tracts. Am J Vet Res 1986; 47: 1426-1432. 13. LEWIS LD, PHILLIPS RW. Pathophysi- to utilize various carbohydrates as an are not severely acidemic. To confirm ologic changes due to coronavirus-induced energy source in milk replacer formu- their efficacy, the most effective treat- diarrhea in the calf. J Am Vet Med Assoc lations. To that end it was discovered ment methods should then be exam- 1978; 173: 636-642. that neonatal calves were unable to ined in calves with naturally-acquired 14. PHILLIPS RW, LEWIS LD. Viral induced changes in intestinal transport and hydrolyze sucrose and therefore diarrhea. resultant body fluid alterations in neonatal unable to utilize sucrose as an energy calves. Ann Rech vdter 1973; 4: 87-98. source (17,22). In addition it was 15. BOOTH AJ, NAYLOR JM. Correction of noted that diarrhea resulted within REFERENCES metabolic acidosis in diarrheal calves by several h of feeding sucrose, but feces oral administration of electrolyte solutions with or without bicarbonate. J Am Vet always returned to normal consistency 1. USDA. 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