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Pancreatic exocrine function and necrotising enterocolitis
Early Human Development 35 (1993) 145-149
Pancreatic exocrine function and necrotising enterocolitis C.M. Wood”, P. MaeKay”, K.D. Willisb, G.A. Browntb, J.W.L. Puntis*c ‘Academic
Unit of Paediatrics
and Child Health University of Leeds, Lee&
UK
‘The Institute of Child Health, University of Birmingham, Birmingham, UK ‘Peter Congdon Neonatal Unit. Clarendon Wing, The General Infirmary at Leeds. Belmont Grove, Leeds LS2 9NS. UK
(Received 20 May 1993; revision received 17 August 1993; accepted 22 August 1993)
Abstract Pancreatic protease deficiency may be an aetiological factor in enteritis necroticans, a disease sharing some features of necrotising enterocolitis (NEC). Using faecal chymotrypsin measurement we have prospectively studied pancreatic exocrine function in infants at risk of NEC. No significant difference was found comparing those infants who subsequently developed NEC and those who did not. Key words:
Necrotising; Enterocolitis;
Pancreatic; Exocrine function; Chymotrypsin;
1. Iotmduction
Although NEC represents a major cause of morbidity and mortality in the premature infant [l], the aetiology remains uncertain. Studies of stool alpha-l antitrypsin concentration [2], breath hydrogen excretion [3], vasculo-cutaneous potential [4], and faecal flora [5] all suggest that the pathophysiology of NEC is evolving some time before clinical signs and symptoms become apparent. We have previously reported two infants with NEC in whom evidence of decreased pancreatic protease secretion anteceded signs and symptoms of the condition [6]. There are other similarities between NEC and enteritis necroticans (pigbel), a disease caused by C. perfiingens type C in which deficiency of pancreatic proteases has been suggested as * Corresponding author. tWe regret the loss of Geoff Brown, who died on the 12th of February 1992. 0378-3782/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0378-3782(93)01467-P
146
CM.
Wood et al. /Early Hum. Dev. 35 (1993) 145-149
an important aetiological factor [7]. We have therefore conducted a prospective study in infants less than 32 weeks’ gestation of pancreatic exocrine function, assessed by faecal chymotrypsin, in order to explore further the possible relationship with NEC. Faecal chymotrypsin has been shown to correlate well with chymotrypsin secretion following intravenous pancreozymin-secretin pancreatic function testing
PI. 2. Subjects and methods 2. I. Subjects
Ninety premature infants of 32 weeks’ gestation or less admitted consecutively to a regional neonatal intensive care unit were recruited to the study over an 8-month period. The assessment of gestational age was based on routine antenatal ultrasound measurement of biparietal diameter at 18 weeks’ gestation, together with clinical assessment at birth. In the course of the investigation, nine infants developed NEC, with typical clinical features in addition to intrahepatic or intramural bowel gas on abdominal radiography (NEC group). Their median (range) gestational age was 28 weeks (24-30), and weight 830 g (560-1520). Six of this group were normally grown with weights appropriate for gestational age (AGA), and three were small for gestational age (SGA) with a weight below the 10 centile. Eleven of the ninety infants originally recruited had clinical signs and symptoms suggestive of NEC but no intramural or intrahepatic gas was seen on radiological examination. These were excluded from further study. Seventy infants had no signs or symptoms of NEC and from these, nine were matched for gestational age and weight with the NEC group to provide a control group. The median (range) gestational age of the control group was 27 weeks (24-30), and weight 780 g (610-1980). Six controls were AGA and three SGA. There was no significant difference in gestational age or weight between the two groups. One infant in the NEC group received mothers milk and seven a low birthweight formula (Cow and Gate). Three control infants were fed with mothers milk and five with a low birthweight formula (Cow and Gate). One infant from each group received only parenteral feeding during the study period. Enteral feeds were usually commenced within the first 48 h of life. 2.2. Stool collection and analytical methods
A stool specimen was collected by nursing staff approximately every three days over the first month of life, starting as soon after birth as possible. Approximately 0.2 g of faeces was inoculated into a phial containing 1.8 ml of prereduced glycerol citrate broth. The weight of stool was determined by subtracting the known weight of the phial from the weight after the addition of the sample. One-hundred microlitres of fluid were used for bacteriological analysis as previously reported [5], whilst the rest was frozen and stored at -70°C until a batch analysis was performed at the completion of the study. Chymotryptic activity was measured using a proprietary test kit, the Boehringer Mannheim C-system, involving a photometric assay [9]. In the NEC group, only stools collected before the onset of clinical symptoms were analysed.
C.M.
Wood et al. /Early
Hum.
Dev. 35 (1993)
145-149
147
2.3. Statistical evaluation The significance of the differences between the two groups was examined using the Mann-Whitney V-test. 3. Results A diagnosis of NEC was made in nine infants at a median (range) age of 15 days [5-411. In each case two or more stools were collected prior to clinical onset of illness. In all, 53 stools from the NEC group and 73 from the control group were obtained. Duplicate samples of each specimen were analysed and the mean used to determine chymotrypsin concentration. The coefficient of variation for the control stool analyses was 7.05%. For individual patients results showed a wide scatter over the 4-week period. In the NEC group, three (6%) specimens produced a result below the lower reference limit of 120 pg chymotrypsin/g stool previously determined for neonates greater than 36 weeks gestation [lo]. In the control group, eight (11%) specimens fell below this limit. Overall, there was no significant difference in faecal chymotrypsin concentrations between the two groups. Results were also grouped together week by week (Fig. 1) and although in each of the first 3 weeks median chymotrypsin concentration was lower in infants who subsequently developed NEC than in the controls, the difference between the two groups week by week did not reach a level of statistical significance. C. perfringens was isolated in stool from two infants with NEC and two controls.
3000 2500
Stool
chymotrypsin
the ‘first
4 weeks
2.
2
2
.
0
.
0
g
2000
ub
1500
Ql
-
0
1. .
8
-
8 0 1
_rf
i
Q
2
‘f 4
7 yz
Week development
..
‘19
3-T f
Fig. 1. Stool chymotrypsin
??o
.I%
0
‘t
0
’
500
Q3
8
. ??
~looo-
-
0
. t;
o = NEC 0 = controls -median and
.
‘;z E 2 0.
of life
-
._E
in
5 3
4
of life
concentrations during the first four weeks of life in nine infants prior to the of NEC and nine controls. The median, and interquartile ranges are indicated.
148
C.M.
Wood et al. /Early
Hum. Dev. 35 (1993) 145-149
4. Discussion The range of faecal chymotrypsin concentration in this prospective study was similar to that previously described in a group of preterm infants 161, although the median value was somewhat lower. On the basis of faecal chymotrypsin measurement we did not find any evidence of decreased pancreatic exocrine function in those patients who developed NEC compared with control patients. NEC is a common and serious problem in the preterm infant [l] and yet the aetiology remains obscure. Some clinical and pathological features are shared with enteritis necroticans (pigbel), an important cause of illness and death amongst children in the highlands of Papua New Guinea. Although pigbel is related to infection with Clostridiwn perfringens type C [ 1l] many carriers remain asymptomatic and it has been hypothesised that reduced pancreatic protease activity is one of the prerequisites for the development of the disease [7]. In one study, proteolytic activity was found to be absent from the stools of 20 Papua New Guinean children tested, compared with only one of 12 European control children [7]. In an animal model where cultures of C. perfringens were given intragastrically, only those guinea-pigs who ingested food containing natural trypsin inhibitors developed necrotising enteritis [7]. Subnormal values of faecal chymotrypsin (suggesting pancreatic exocrine hypofunction) have been described anteceding clinical NEC in two infants enroled in a prospective investigation of pancreatic exocrine function in the preterm [6]. Impaired pancreatic exocrine function as a risk factor for development of NEC would be consistent with the increased prevalence of NEC in the premature and the growth retarded infant, since in both groups there is decreased production of pancreatic proteases [6,12,13]. Despite the fact that clostridial toxins are found in stools no more frequently in patients with NEC than in healthy newborns [14], this does not preclude the possibility that local action of toxin is important in the pathogenesis even when toxin cannot be found in stool. Reduced secretion or inhibition of proteases might lead to impaired toxin degradation or, through resulting maldigestion, create an intraluminal environment that favoured proliferation of micro-organisms. The presence of protein in the gut does appear to be essential in an animal model of NEC [ 151. As in pigbel, Cfostridium sp. also appear to play a role in the aetiology of some cases of NEC which have shown clustering [ 161. Although faecal assay correlates well with apparent duodenal secretion rates of chymotrypsin 181,it is at best an indirect assessment of pancreatic function and may not be sensitive enough to reflect subtle variation in pancreatic protease activity within the gut lumen. Alternatively, our failure to demonstrate any significant difference in faecal chymotrypsin concentration in infants who did or did not develop NEC may indicate that deficiency of pancreatic proteases is not important in the aetiology of the disease, although our numbers are small. We plan further investigation of the full range of pancreatic proteases in small bowel juice. This may further illuminate the relationship between small bowel milieu and the risk of NEC in a larger group of patients.
C.M.
Wood et al. /Early
Hum.
Dev. 35 (1993)
145-149
149
5. Acknowledgements
We are grateful to Miss J. Patterson for her secretarial assistance. 6. References I Yu V.Y.H., Joseph R., Bajuk B., Orgill A. and Astbury J. (1984): Perinatal risk factors for necrotising enterocolitis. Arch. Dis. Child., 59, 430-434. 2 Shulman R.J., Buffone G. and Wise L. (1985): Enteric protein loss in necrotising enterocolitis as measured by fecal alpha-I-antitrypsin excretion. J. Pediatr., 107, 287-289. 3 Cheu H.W., Brown D.R. and Rowe MI. (1989): Breath hydrogen excretion as a screening test for the early diagnosis of necrotising enterocolitis. Am. J. Dis. Child., 143, 156-159. 4 Millar M.R., MacKay P., Dealler S., Catto A., Scanlon J. and CongdonP. (1988): Reversed potential of vasculocutaneous potential and neonatal necrotising enterocolitis. Lancet, 334, 1417. 5 Hoy C., Millar M.R., MacKay P., Godwin P.G.R., Langdale V. and Levene MI. (1990): Quantitative changes in faecal microflora preceding necrotising enterocolitis in premature neonates. Arch. Dis. Child., 65, 1057-1059. 6 Kolacek S., Puntis J.W.L., Lloyd D.R., Brown G.A. and Booth I.W. (1990): Ontogeny of pancreatic exocrine function. Arch. Dis. Child., 65, 178-181. 7 Lawrence Cl. and Walker P.D. (1976): Pathogenesis of enteritis necroticans in Papua New Guinea. Lancet, i, 125-126. 8 Brown G.A., Sule D., Williams J., Puntis J.W.L., Booth I.W. and McNeish A.S. (1988): Faecal chymotrypsin: a reliable index of exocrine pancreatic function. Arch. Dis. Child., 63, 785-789. 9 Kaspar P., Moller G. and Wahlfeld A. (1984): New photometric assay for chymotrypsin in stool. Clin. Chem., 30, 1753-1757. IO Brown G.A., Halliday R.B., Turner P.J. and Smalley C.A. (1988): Faecal chymotrypsin concentrations in neonates with cystic fibrosis and healthy controls. Arch. Dis. Child., 63, 1229-1233. I1 Lawrence G.W., Lehmann D., Anian G., Coakley C.A., Saleu G., Barker M.J. and Davis M.W. (1990): Impact of active immunisation against enteritis necroticans in Papua New Guinea. Lancet. 336, I l65- I 167. 12 Zoppi G., Andreotti G., Pajno-Ferrara F., Njai D.M. and Gaburro D. (1972): Exocrine pancreatic function in premature and full term neonates. Pediatr. Res., 6, 880-886. I3 Lebenthal E. and Lee P.C. (1988): The impact of intrauterine and postnatal malnutrition on the development of the exocrine pancreas and small intestine. J. Pediatr. Gastroenterol. Nutr., 7, l-3. I4 Thomas D.F.M., Fernie D.S., Bayston R. and Spitz L. (1984): Clostridial toxins in neonatal necrotising enterocolitis. Arch. Dis. Child., 59, 270-272. 15 Clark D.A., Thompson J.E., Weiner L.B., Schneider J.A. and Rofahr J.E. (1985): Necrotising enterocolitis: intraluminal biochemistry in human neonates and a rabbit model. Pediatr. Res., 19, 919-921. 16 Kosloske A.M. and Ulrich J.A. (1980): A bacteriologic basis for the clinical presentations of necrotising enterocolitis. J. Pediatr. Surg., 15, 558-564.
Pancreatic exocrine function and necrotising enterocolitis C.M. Wood”, P. MaeKay”, K.D. Willisb, G.A. Browntb, J.W.L. Puntis*c ‘Academic
Unit of Paediatrics
and Child Health University of Leeds, Lee&
UK
‘The Institute of Child Health, University of Birmingham, Birmingham, UK ‘Peter Congdon Neonatal Unit. Clarendon Wing, The General Infirmary at Leeds. Belmont Grove, Leeds LS2 9NS. UK
(Received 20 May 1993; revision received 17 August 1993; accepted 22 August 1993)
Abstract Pancreatic protease deficiency may be an aetiological factor in enteritis necroticans, a disease sharing some features of necrotising enterocolitis (NEC). Using faecal chymotrypsin measurement we have prospectively studied pancreatic exocrine function in infants at risk of NEC. No significant difference was found comparing those infants who subsequently developed NEC and those who did not. Key words:
Necrotising; Enterocolitis;
Pancreatic; Exocrine function; Chymotrypsin;
1. Iotmduction
Although NEC represents a major cause of morbidity and mortality in the premature infant [l], the aetiology remains uncertain. Studies of stool alpha-l antitrypsin concentration [2], breath hydrogen excretion [3], vasculo-cutaneous potential [4], and faecal flora [5] all suggest that the pathophysiology of NEC is evolving some time before clinical signs and symptoms become apparent. We have previously reported two infants with NEC in whom evidence of decreased pancreatic protease secretion anteceded signs and symptoms of the condition [6]. There are other similarities between NEC and enteritis necroticans (pigbel), a disease caused by C. perfiingens type C in which deficiency of pancreatic proteases has been suggested as * Corresponding author. tWe regret the loss of Geoff Brown, who died on the 12th of February 1992. 0378-3782/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0378-3782(93)01467-P
146
CM.
Wood et al. /Early Hum. Dev. 35 (1993) 145-149
an important aetiological factor [7]. We have therefore conducted a prospective study in infants less than 32 weeks’ gestation of pancreatic exocrine function, assessed by faecal chymotrypsin, in order to explore further the possible relationship with NEC. Faecal chymotrypsin has been shown to correlate well with chymotrypsin secretion following intravenous pancreozymin-secretin pancreatic function testing
PI. 2. Subjects and methods 2. I. Subjects
Ninety premature infants of 32 weeks’ gestation or less admitted consecutively to a regional neonatal intensive care unit were recruited to the study over an 8-month period. The assessment of gestational age was based on routine antenatal ultrasound measurement of biparietal diameter at 18 weeks’ gestation, together with clinical assessment at birth. In the course of the investigation, nine infants developed NEC, with typical clinical features in addition to intrahepatic or intramural bowel gas on abdominal radiography (NEC group). Their median (range) gestational age was 28 weeks (24-30), and weight 830 g (560-1520). Six of this group were normally grown with weights appropriate for gestational age (AGA), and three were small for gestational age (SGA) with a weight below the 10 centile. Eleven of the ninety infants originally recruited had clinical signs and symptoms suggestive of NEC but no intramural or intrahepatic gas was seen on radiological examination. These were excluded from further study. Seventy infants had no signs or symptoms of NEC and from these, nine were matched for gestational age and weight with the NEC group to provide a control group. The median (range) gestational age of the control group was 27 weeks (24-30), and weight 780 g (610-1980). Six controls were AGA and three SGA. There was no significant difference in gestational age or weight between the two groups. One infant in the NEC group received mothers milk and seven a low birthweight formula (Cow and Gate). Three control infants were fed with mothers milk and five with a low birthweight formula (Cow and Gate). One infant from each group received only parenteral feeding during the study period. Enteral feeds were usually commenced within the first 48 h of life. 2.2. Stool collection and analytical methods
A stool specimen was collected by nursing staff approximately every three days over the first month of life, starting as soon after birth as possible. Approximately 0.2 g of faeces was inoculated into a phial containing 1.8 ml of prereduced glycerol citrate broth. The weight of stool was determined by subtracting the known weight of the phial from the weight after the addition of the sample. One-hundred microlitres of fluid were used for bacteriological analysis as previously reported [5], whilst the rest was frozen and stored at -70°C until a batch analysis was performed at the completion of the study. Chymotryptic activity was measured using a proprietary test kit, the Boehringer Mannheim C-system, involving a photometric assay [9]. In the NEC group, only stools collected before the onset of clinical symptoms were analysed.
C.M.
Wood et al. /Early
Hum.
Dev. 35 (1993)
145-149
147
2.3. Statistical evaluation The significance of the differences between the two groups was examined using the Mann-Whitney V-test. 3. Results A diagnosis of NEC was made in nine infants at a median (range) age of 15 days [5-411. In each case two or more stools were collected prior to clinical onset of illness. In all, 53 stools from the NEC group and 73 from the control group were obtained. Duplicate samples of each specimen were analysed and the mean used to determine chymotrypsin concentration. The coefficient of variation for the control stool analyses was 7.05%. For individual patients results showed a wide scatter over the 4-week period. In the NEC group, three (6%) specimens produced a result below the lower reference limit of 120 pg chymotrypsin/g stool previously determined for neonates greater than 36 weeks gestation [lo]. In the control group, eight (11%) specimens fell below this limit. Overall, there was no significant difference in faecal chymotrypsin concentrations between the two groups. Results were also grouped together week by week (Fig. 1) and although in each of the first 3 weeks median chymotrypsin concentration was lower in infants who subsequently developed NEC than in the controls, the difference between the two groups week by week did not reach a level of statistical significance. C. perfringens was isolated in stool from two infants with NEC and two controls.
3000 2500
Stool
chymotrypsin
the ‘first
4 weeks
2.
2
2
.
0
.
0
g
2000
ub
1500
Ql
-
0
1. .
8
-
8 0 1
_rf
i
Q
2
‘f 4
7 yz
Week development
..
‘19
3-T f
Fig. 1. Stool chymotrypsin
??o
.I%
0
‘t
0
’
500
Q3
8
. ??
~looo-
-
0
. t;
o = NEC 0 = controls -median and
.
‘;z E 2 0.
of life
-
._E
in
5 3
4
of life
concentrations during the first four weeks of life in nine infants prior to the of NEC and nine controls. The median, and interquartile ranges are indicated.
148
C.M.
Wood et al. /Early
Hum. Dev. 35 (1993) 145-149
4. Discussion The range of faecal chymotrypsin concentration in this prospective study was similar to that previously described in a group of preterm infants 161, although the median value was somewhat lower. On the basis of faecal chymotrypsin measurement we did not find any evidence of decreased pancreatic exocrine function in those patients who developed NEC compared with control patients. NEC is a common and serious problem in the preterm infant [l] and yet the aetiology remains obscure. Some clinical and pathological features are shared with enteritis necroticans (pigbel), an important cause of illness and death amongst children in the highlands of Papua New Guinea. Although pigbel is related to infection with Clostridiwn perfringens type C [ 1l] many carriers remain asymptomatic and it has been hypothesised that reduced pancreatic protease activity is one of the prerequisites for the development of the disease [7]. In one study, proteolytic activity was found to be absent from the stools of 20 Papua New Guinean children tested, compared with only one of 12 European control children [7]. In an animal model where cultures of C. perfringens were given intragastrically, only those guinea-pigs who ingested food containing natural trypsin inhibitors developed necrotising enteritis [7]. Subnormal values of faecal chymotrypsin (suggesting pancreatic exocrine hypofunction) have been described anteceding clinical NEC in two infants enroled in a prospective investigation of pancreatic exocrine function in the preterm [6]. Impaired pancreatic exocrine function as a risk factor for development of NEC would be consistent with the increased prevalence of NEC in the premature and the growth retarded infant, since in both groups there is decreased production of pancreatic proteases [6,12,13]. Despite the fact that clostridial toxins are found in stools no more frequently in patients with NEC than in healthy newborns [14], this does not preclude the possibility that local action of toxin is important in the pathogenesis even when toxin cannot be found in stool. Reduced secretion or inhibition of proteases might lead to impaired toxin degradation or, through resulting maldigestion, create an intraluminal environment that favoured proliferation of micro-organisms. The presence of protein in the gut does appear to be essential in an animal model of NEC [ 151. As in pigbel, Cfostridium sp. also appear to play a role in the aetiology of some cases of NEC which have shown clustering [ 161. Although faecal assay correlates well with apparent duodenal secretion rates of chymotrypsin 181,it is at best an indirect assessment of pancreatic function and may not be sensitive enough to reflect subtle variation in pancreatic protease activity within the gut lumen. Alternatively, our failure to demonstrate any significant difference in faecal chymotrypsin concentration in infants who did or did not develop NEC may indicate that deficiency of pancreatic proteases is not important in the aetiology of the disease, although our numbers are small. We plan further investigation of the full range of pancreatic proteases in small bowel juice. This may further illuminate the relationship between small bowel milieu and the risk of NEC in a larger group of patients.
C.M.
Wood et al. /Early
Hum.
Dev. 35 (1993)
145-149
149
5. Acknowledgements
We are grateful to Miss J. Patterson for her secretarial assistance. 6. References I Yu V.Y.H., Joseph R., Bajuk B., Orgill A. and Astbury J. (1984): Perinatal risk factors for necrotising enterocolitis. Arch. Dis. Child., 59, 430-434. 2 Shulman R.J., Buffone G. and Wise L. (1985): Enteric protein loss in necrotising enterocolitis as measured by fecal alpha-I-antitrypsin excretion. J. Pediatr., 107, 287-289. 3 Cheu H.W., Brown D.R. and Rowe MI. (1989): Breath hydrogen excretion as a screening test for the early diagnosis of necrotising enterocolitis. Am. J. Dis. Child., 143, 156-159. 4 Millar M.R., MacKay P., Dealler S., Catto A., Scanlon J. and CongdonP. (1988): Reversed potential of vasculocutaneous potential and neonatal necrotising enterocolitis. Lancet, 334, 1417. 5 Hoy C., Millar M.R., MacKay P., Godwin P.G.R., Langdale V. and Levene MI. (1990): Quantitative changes in faecal microflora preceding necrotising enterocolitis in premature neonates. Arch. Dis. Child., 65, 1057-1059. 6 Kolacek S., Puntis J.W.L., Lloyd D.R., Brown G.A. and Booth I.W. (1990): Ontogeny of pancreatic exocrine function. Arch. Dis. Child., 65, 178-181. 7 Lawrence Cl. and Walker P.D. (1976): Pathogenesis of enteritis necroticans in Papua New Guinea. Lancet, i, 125-126. 8 Brown G.A., Sule D., Williams J., Puntis J.W.L., Booth I.W. and McNeish A.S. (1988): Faecal chymotrypsin: a reliable index of exocrine pancreatic function. Arch. Dis. Child., 63, 785-789. 9 Kaspar P., Moller G. and Wahlfeld A. (1984): New photometric assay for chymotrypsin in stool. Clin. Chem., 30, 1753-1757. IO Brown G.A., Halliday R.B., Turner P.J. and Smalley C.A. (1988): Faecal chymotrypsin concentrations in neonates with cystic fibrosis and healthy controls. Arch. Dis. Child., 63, 1229-1233. I1 Lawrence G.W., Lehmann D., Anian G., Coakley C.A., Saleu G., Barker M.J. and Davis M.W. (1990): Impact of active immunisation against enteritis necroticans in Papua New Guinea. Lancet. 336, I l65- I 167. 12 Zoppi G., Andreotti G., Pajno-Ferrara F., Njai D.M. and Gaburro D. (1972): Exocrine pancreatic function in premature and full term neonates. Pediatr. Res., 6, 880-886. I3 Lebenthal E. and Lee P.C. (1988): The impact of intrauterine and postnatal malnutrition on the development of the exocrine pancreas and small intestine. J. Pediatr. Gastroenterol. Nutr., 7, l-3. I4 Thomas D.F.M., Fernie D.S., Bayston R. and Spitz L. (1984): Clostridial toxins in neonatal necrotising enterocolitis. Arch. Dis. Child., 59, 270-272. 15 Clark D.A., Thompson J.E., Weiner L.B., Schneider J.A. and Rofahr J.E. (1985): Necrotising enterocolitis: intraluminal biochemistry in human neonates and a rabbit model. Pediatr. Res., 19, 919-921. 16 Kosloske A.M. and Ulrich J.A. (1980): A bacteriologic basis for the clinical presentations of necrotising enterocolitis. J. Pediatr. Surg., 15, 558-564.