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CORD-19:ffbdb12b3da27e9f59a07f385d22a98453bd3a7a JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-80 Sentence denotes Natural and experimental infection of neonatal calves with Clostridium difficile
T2 82-90 Sentence denotes Abstract
T3 91-256 Sentence denotes Clostridium difficile toxins were associated with calf diarrhea in a recent retrospective study; however, no causal relationship has been prospectively investigated.
T4 257-419 Sentence denotes This infection study tested whether the oral inoculation of neonatal calves with a toxigenic strain of C. difficile (PCR-ribotype 077) results in enteric disease.
T5 420-593 Sentence denotes Fourteen 6-24 h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4 Â 10 8 AE 3.5 Â 10 8 cfu) (n = 8) or sterile culture broth (n = 6).
T6 594-679 Sentence denotes Calves were euthanized on day 6 or after the onset of diarrhea, whichever came first.
T7 680-819 Sentence denotes Fecal and intestinal samples were blindly cultured for C. difficile, and tested for its toxin A/B (C. difficile TOX A/B II ELISA, Techlab).
T8 820-889 Sentence denotes PCR-ribotyping was used to compare inoculated and recovered isolates.
T9 890-1052 Sentence denotes Diarrhea was observed in all control calves and 3/8 of inoculated calves ( p = 0.03), but it did not occur in calves that tested positive for C. difficile toxins.
T10 1053-1105 Sentence denotes Fecal toxins were identified only from two controls.
T11 1106-1236 Sentence denotes PCR-ribotyping confirmed the presence of C. difficile PCR-ribotype 077 in samples of all inoculated calves, but not from controls.
T12 1237-1411 Sentence denotes The identification of five other PCRribotypes in 3/8 (37.5%) and 2/6 (33.3%) of inoculated and control calves, respectively, indicated early natural infection ( 24 h of age).
T13 1412-1466 Sentence denotes Five of 14 cecal samples had C. difficile ( p = 0.01).
T14 1467-1660 Sentence denotes In conclusion, the oral administration of C. difficile PCR-ribotype 077 to neonatal calves resulted in fecal/intestinal colonization but not in detection of toxins, or signs of enteric disease.
T15 1661-1754 Sentence denotes Further studies are required to investigate the clinical relevance of C. difficile in calves.
T16 1755-1760 Sentence denotes Crown
T17 1762-2238 Sentence denotes Clostridium difficile is a spore-forming bacterium associated with the development of serious enteric diseases and diarrhea in humans . www.elsevier.com/locate/vetmic Veterinary Microbiology 124 (2007) [166] [167] [168] [169] [170] [171] [172] Outbreaks of severe C. difficile-associated disease (CDAD) in humans have increased in frequency worldwide, due in part to the emergence of epidemic strains PCR-ribotypes 017 and 027 (van den Berg et al., 2004; Warny et al., 2005) .
T18 2239-2461 Sentence denotes Recently, the isolation of these and other strains from feces of dairy calves (Rodriguez-Palacios et al., 2006) , and from retail meat products in Canada (Rodriguez-Palacios et al., 2007) has raised epidemiologic interest.
T19 2462-2598 Sentence denotes C. difficile is also considered a pathogen for certain animals, including horses, dogs and pigs (Baverud, 2002; Keel and Songer, 2006) .
T20 2599-2798 Sentence denotes In cattle, a recent retrospective study performed in Canadian farms showed a strong association between the presence of fecal C. difficile toxins and calf diarrhea (Rodriguez-Palacios et al., 2006) .
T21 2799-2957 Sentence denotes However, C. difficile was more frequently isolated from controls (14.9%; 20/134) than from diarrheic calves (7.6%; 11/144) (Rodriguez-Palacios et al., 2006) .
T22 2958-3027 Sentence denotes This finding suggests that a disease-free carrier status might exist.
T23 3028-3169 Sentence denotes In humans, asymptomatic carriage of toxigenic strains of C. difficile is more common in neonates, whereas disease is more common in elderly .
T24 3170-3296 Sentence denotes In contrast, neonatal foals and piglets are reported to be susceptible to disease (Arroyo et al., 2004; Songer et al., 2000) .
T25 3297-3442 Sentence denotes Despite the recent association between C. difficile toxins and calf diarrhea, the role of C. difficile as a causal pathogen in calves is unknown.
T26 3443-3631 Sentence denotes In other species, the histological and functional lesions attributable to C. difficile are associated with the local effect of two exotoxins, toxins A and B (Schirmer and Aktories, 2004) .
T27 3632-3744 Sentence denotes A binary toxin (known as CDT) has also been hypothesized to be another virulence cofactor (Geric et al., 2006 ).
T28 3745-3925 Sentence denotes An experimental model of disease is desirable to characterize the pathogenicity of C. difficile in calves, and to facilitate studies evaluating therapeutic and preventive measures.
T29 3926-4137 Sentence denotes Oral infection with C. difficile may induce diarrhea in hamsters (Sambol et al., 2001) , foals (Arroyo et al., 2004) , and piglets , but it is unknown if experimental infection would induce disease in ruminants.
T30 4138-4430 Sentence denotes This study investigated whether the oral administration of a toxigenic strain of C. difficile to neonatal calves would result in fecal and intestinal colonization, development of CDAD, and production of detectable C. difficile toxins in feces and intestinal contents using a commercial ELISA.
T31 4431-4560 Sentence denotes Holstein-Friesian male calves ( 12 h of age), obtained from three farms in fall 2005, were assigned before arrival to two groups.
T32 4561-4679 Sentence denotes Animals were eligible if calving was normal, and if the calf stood up without assistance within the first 2 h of life.
T33 4680-4760 Sentence denotes Four litres of colostrum from their dams were fed to calves within 2 h of birth.
T34 4761-4872 Sentence denotes Calves were transported to an isolation unit and housed in individual pens until the end of the study on day 6.
T35 4873-4934 Sentence denotes Barrier precautions were used to prevent cross-contamination.
T36 4935-5184 Sentence denotes Calves were inoculated with C. difficile or placebo if they had no diarrhea at the end of a 6 h observation period and if they tested negative on a fecal ELISA test for C. difficile toxin A/ B on arrival (C. difficile TOX A/B II, Techlab, Virginia).
T37 5185-5307 Sentence denotes If diarrhea occurred before or within 6 h of inoculation, the calf was assigned to a third group for descriptive analysis.
T38 5308-5387 Sentence denotes Calves were only bottle-fed fresh bovine antibiotic-free whole milk (2 L/12 h).
T39 5388-5564 Sentence denotes A C. difficile strain (PCR-ribotype 077) which contained genes encoding production of toxin A (tcdA + ) and toxin B (tcdB + ), but not CDT (cdtB À ) or tcdC deletions was used.
T40 5565-5695 Sentence denotes This strain had been isolated from a diarrheic calf (Rodriguez-Palacios et al., 2006) and is a recognized cause of CDAD in humans.
T41 5696-5948 Sentence denotes Initially, pure colonies of this strain were incubated anaerobically (37 8C for 48 h) in 50 mL of C. difficile selective broth supplemented with cysteine hydrochloride, moxalactam and norfloxacin (CDMN, Oxoid, England) (Aspinall and Hutchinson, 1992) .
T42 5949-6065 Sentence denotes This broth was used to inoculate bottles with 400 mL of pre-reduced brain heart infusion (BHI, Oxoid, Canada) broth.
T43 6066-6262 Sentence denotes After 24-36 h of incubation, and centrifugation at 4000 Â g for 10 min, the sediment was re-suspended in 50 mL of sterile BHI broth, which was administered to the calves within 2 h of preparation.
T44 6263-6328 Sentence denotes Sterile BHI broth (50 mL) was used as placebo for control calves.
T45 6329-6505 Sentence denotes Purity and quantification of C. difficile, vegetative cells and spores, were determined for each batch of inoculum on 1 mL aliquots sampled before administration to the calves.
T46 6506-6647 Sentence denotes Spore quantification was performed after mixing 1 mL aliquots with 96% ethanol (v/v, 30 min), followed by quantitative culture on blood agar.
T47 6648-6778 Sentence denotes After initial physical examination, blood samples were obtained for total plasma protein quantification (hand-held refractometer).
T48 6779-6855 Sentence denotes Fecal samples were collected for C. difficile culture and toxin A/B testing.
T49 6856-6969 Sentence denotes After 6 h of observation, calves were inoculated every 12 h with three doses of placebo or C. difficile inoculum.
T50 6970-7033 Sentence denotes Inocula were fed to the calves with their last quarter of milk.
T51 7034-7118 Sentence denotes Observation and physical exams were performed every 6-12 h by two blinded observers.
T52 7119-7175 Sentence denotes Fecal samples were collected from the rectum once a day.
T53 7176-7299 Sentence denotes Diarrhea was defined as the presence of stools with a fecal score of 4 (watery) in a 4-grade system (Larson et al., 1977) .
T54 7300-7477 Sentence denotes Calves were euthanized at the end of the study period on day 6, or after 24 h of the onset of diarrhea or systemic disease (marked depression/dehydration), whichever came first.
T55 7478-7663 Sentence denotes Samples of intestinal content (5 mL) from duodenum (30 cm from pylorus), ileum (30 cm from cecum), and cecal apex were collected during post-mortem examination by transmural aspiration.
T56 7664-7711 Sentence denotes Samples were stored at À70 8C until processing.
T57 7712-7850 Sentence denotes Histological examination of specimens preserved in 10% formaldehyde was conducted using light microscopy and haematoxylin and eosin stain.
T58 7851-7962 Sentence denotes Lesions were blindly scored (0-3) by a pathologist; an overall additive score was given per intestinal segment.
T59 7963-8054 Sentence denotes The Animal Care Committee of the University of Guelph approved the protocols of this study.
T60 8055-8200 Sentence denotes Testing for C. difficile toxin A/B was performed on feces of days 1, 3, 5 and 6, on samples of intestinal contents using C. difficile TOX A/B II.
T61 8201-8326 Sentence denotes Readings were performed visually by agreement between two observers: positive and negative controls were used with every run.
T62 8327-8563 Sentence denotes The isolation of C. difficile from fecal samples (days 1-6) and from intestinal samples was performed using CDMN broth (with 0.1% sodium taurocholate; Oxoid, Canada) and CDMN agar (with 5% laked horse blood; Sigma-Aldrich Inc., Canada).
T63 8564-8730 Sentence denotes After incubation of 1 mL of samples in 9 mL of CDMN broth for 5-7 days, this broth was mixed with 96% ethanol (v/v, for 30 min) and centrifuged (4000 Â g for 10 min).
T64 8731-8799 Sentence denotes Sediments were plated onto CDMN agar and incubated for up to 5 days.
T65 8800-9035 Sentence denotes Confirmation of suspicious C. difficile colonies (swarming, non-hemolytic), extraction of DNA from pure colonies, and PCRribotyping analysis were conducted as previously described (Bidet et al., 1999; Rodriguez-Palacios et al., 2006) .
T66 9036-9205 Sentence denotes Cultures for Salmonella spp., Clostridium perfringens and Escherichia coli was performed on fecal samples of day 2 at the Animal Health Laboratory, University of Guelph.
T67 9206-9250 Sentence denotes C. perfringens toxins were not investigated.
T68 9251-9443 Sentence denotes Multiplex PCR genotyping of E. coli isolates was performed using primers for enterotoxigenic (Sta, K99, F41) and verotoxigenic (stx1, stx2, eaeA, hlyA, VETC) markers (Slavic and Gyles, 2005) .
T69 9444-9584 Sentence denotes Qualitative tests for rotavirus type A (RotaScreen Latex, England), and bovine coronavirus (BIO-X Diagnostics, Belgium) were also performed.
T70 9585-10007 Sentence denotes Sample size estimations (Intercooled Stata v9.0, TX,) based on an 88% success rate after oral C. difficile infection in foals (Arroyo et al., 2004) , and an estimated rate of natural calf diarrhea of 20% (Larson et al., 1998) , indicated that two groups !12 calves would provide adequate study power (73%) if natural diarrhea occurred in 25% (3/12) of controls, and if infection induced diarrhea in >83% (10/12) of calves.
T71 10008-10085 Sentence denotes This study would be terminated if partial results led to reduced study power.
T72 10086-10201 Sentence denotes Continuous characteristic variables were tested using non-parametric two-sample Wilcoxon rank tests (SAS v9.1, NC).
T73 10202-10292 Sentence denotes Differences in proportions of dichotomous variables were tested using Fisher's exact test.
T74 10293-10377 Sentence denotes Measure agreement for categorical variables was estimated via Cohen's k coefficient.
T75 10378-10424 Sentence denotes Statistical significance was held at p < 0.05.
T76 10425-10591 Sentence denotes This study was terminated after the inclusion of 14 calves as the first 8 inoculated animals did not develop CDAD (diarrhea and concurrent fecal C. difficile toxins).
T77 10592-10754 Sentence denotes On arrival, 13 of 14 calves were clinically normal and had no signs of diarrhea; the calf with diarrhea was used for descriptive analysis and euthanized on day 2.
T78 10755-10975 Sentence denotes One of eight inoculated calves developed diarrhea within 6 h of inoculation; it was used for descriptive analysis, received two more doses of C. difficile, and was monitored until day 6 as its diarrhea improved by day 2.
T79 10976-11069 Sentence denotes Comparative statistical analysis was conducted with five control and seven inoculated calves.
T80 11070-11406 Sentence denotes There was no age difference between the groups (mean AE 1S.D.; 17.6 AE 7.4 h, range = 6-28 h, and 12.6 AE 8.1 h, range = 5-24 h, respectively; p = 0.24), and all calves had adequate plasma protein concentrations (inoculated calves, 73.3 AE 7.1, range = 64-85 g/L; controls, 63.5 AE 3.7, range = 60-70 g/L; p = 0.07) (Rea et al., 1996) .
T81 11407-11673 Sentence denotes The inocula administered to the calves had a concentration of 1.4 Â 10 8 AE 3.5 Â 10 8 cfu of C. difficile PCR-ribotype 077 organisms (range = 1 Â 10 5 , 1 Â 10 9 ; n = 15), of which, 7.3 Â 10 4 AE 1.5 Â 10 5 cfu were spores (range = 1 Â 10 2 , 4.6 Â 10 5 ; n = 10).
T82 11674-11853 Sentence denotes By the end of the study, 5/5 control calves and 2/7 inoculated calves developed diarrhea ( p = 0.03), but the average length of study was not different between groups (inoculated:
T83 11854-11900 Sentence denotes 5.7 AE 0.3 days; range = 5.5-6 days; controls:
T84 11901-11943 Sentence denotes 4.4 AE 1.5 days; range 3-6 days; p = 0.3).
T85 11944-12002 Sentence denotes Three of five control calves were euthanized before day 6.
T86 12003-12131 Sentence denotes In total, 24 and 43 fecal samples were collected from 6 control and 8 inoculated calves, respectively, for C. difficile culture.
T87 12132-12214 Sentence denotes Of these, 18 and 27 samples, respectively, were processed for C. difficile toxins.
T88 12215-12285 Sentence denotes All 14 calves tested negative with the toxin ELISA the day of arrival.
T89 12286-12433 Sentence denotes Toxins were detected in one of the fecal samples tested (1/18 (5.5%) versus 0/27 (0%), p = 0.4); it was from a control calf with diarrhea on day 3.
T90 12434-12621 Sentence denotes The ELISA was also positive in one of the intestinal samples tested (1/12 (8.3%) versus 0/16 (0%), p = 0.43); the ileum of another control calf euthanized on day 3 due to severe diarrhea.
T91 12622-12776 Sentence denotes C. difficile was isolated from 26/43 (60.5%) fecal samples of the 8 inoculated calves but not from 24 samples of the 6 non-inoculated calves ( p < 0.001).
T92 12777-12943 Sentence denotes C. difficile was also cultured from 6/42 (14.3%) intestinal samples representing six calves; 2/6 ceca of controls, and 3/8 ceca and 1/8 duodenum of inoculated calves.
T93 12944-13100 Sentence denotes Isolation of C. difficile was significantly more frequent from cecum (5/14) than collectively from duodenum (1/14) and ileum (0/14) ( p = 0.01) ( Table 1) .
T94 13101-13248 Sentence denotes A very low measured agreement (k < 0.2) was observed when the last fecal culture and the intestinal cultures interpreted in parallel were compared.
T95 13249-13397 Sentence denotes PCR-ribotyping confirmed that 22/26 (84.6%) fecal and 4/6 (66.6%) intestinal C. difficile isolates were the inoculated PCR-ribotype 077 ( Table 1 ).
T96 13398-13565 Sentence denotes The remaining six isolates, representing five calves (feces of three inoculated, and ceca of two control calves), showed five different PCR-ribotypes (data not shown).
T97 13566-13727 Sentence denotes Both control calves harbouring C. difficile in the cecum were euthanized due to severe watery diarrhea on days 2 and 3, but tested negative with the toxin ELISA.
T98 13728-13858 Sentence denotes These calves were the only animals that tested positive for enterotoxigenic E.coli; and one additionally harboured Salmonella spp.
T99 13859-13941 Sentence denotes C. perfringens was present in both groups (3/6 control, 5/ 8 inoculated; p = 1.0).
T100 13942-14022 Sentence denotes No verotoxigenic E. coli, rotavirus or coronavirus were identified in the study.
T101 14023-14180 Sentence denotes Three C. difficile isolates, one PCR-ribotype 077 and two of other ribotypes, were cultured from feces of three calves with <24 h of age, before inoculation.
T102 14181-14366 Sentence denotes Similarly, two other PCR-ribotypes, cultured from cecum of two controls at 48 and 72 h of age, and from feces of two inoculated calves at 48 h of age, indicated early natural infection.
T103 14367-14611 Sentence denotes Post-mortem analysis revealed mild-to-moderate non-specific macroscopic changes (intestinal mucosa congestion, increased intestinal fluid content, mesenteric edema and edematous lymph nodes) in 4/6 control and 2/8 inoculated calves ( p = 0.28).
T104 14612-14771 Sentence denotes Microscopic inflammatory changes were overall mildmoderate, but they were more commonly seen in cecum and ileo-cecal lymph node of inoculated calves (Fig. 1) .
T105 14772-14870 Sentence denotes No intestinal ulceration, edema, or mucosal pseudomembranes were identified in inoculated animals.
T106 14871-15113 Sentence denotes In this study, oral inoculation of toxigenic C. difficile PCR-ribotype 077 to colostrum-fed neonatal calves resulted in fecal shedding but no detection of C. difficile toxins, or induction of enteric disease for up to 5 days post-inoculation.
T107 15114-15345 Sentence denotes These finding contrasts previous studies in hamsters, piglets and foals where diarrhea was induced in most animals within 48-72 h of oral infection (Arroyo et al., 2004; Jones et al., 1988; Post et al., 2000; Sambol et al., 2001) .
T108 15346-15439 Sentence denotes It is uncertain if diarrhea would have developed later, if calves had been monitored further.
T109 15440-15689 Sentence denotes As CDAD in humans occur a few days after discontinuation of antimicrobial therapy (Beaugerie et al., 2003) , some models of infection include the concurrent administration of antimicrobials to facilitate the induction of CDAD (Sambol et al., 2002) .
T110 15690-15828 Sentence denotes Antimicrobials were not used in this study as the main objective was to investigate the sole effect of an early ingestion of C. difficile.
T111 15829-15912 Sentence denotes It is unknown if diarrhea had occurred if these calves had received antimicrobials.
T112 15913-16155 Sentence denotes However, the failure to induce disease in the absence of antimicrobial therapy indicates a difference in susceptibility compared to neonatal foals (Arroyo et al., 2004) , and questions the role of C. difficile as a primary pathogen in calves.
T113 16156-16313 Sentence denotes It is important to consider, however, that failure to induce disease experimentally does not rule out the possibility that C. difficile is a bovine pathogen.
T114 16314-16404 Sentence denotes Rather it indicates that simple exposure to C. difficile is not adequate to cause disease.
T115 16405-16644 Sentence denotes Previous infection studies of adult horses and dogs with toxigenic C. difficile also resulted in fecal shedding but not in signs of enteric disease or fecal detection of C. difficile toxins (Clooten et al., 2003; Gustafsson et al., 2004) .
T116 16645-16967 Sentence denotes Potential explanations for the outcome in these studies may include natural agedependent resistance, lack of immune-compromising conditions, presence of an established and nondisrupted intestinal flora, and perhaps unknown yet factors inhibiting C. difficile colonization, toxin production, or binding to intestinal cells.
T117 16968-17110 Sentence denotes Differences in toxin production and virulence across C. difficile strains could also be considered (Warny et al., 2005; Sambol et al., 2001) .
T118 17111-17461 Sentence denotes Although a potential protective effect of milk (impaired adhesion of C. difficile to cells, and inhibition of toxin A mediated cytotoxicity) was suggested in previous in vitro studies (Dallas and Rolfe, 1998; Naaber et al., 1996) , the roles of passive immunity and bovine whole milk on the transfer of anti-C. difficile factors to calves is unknown.
T119 17462-17568 Sentence denotes Considering the wide distribution of C. difficile in the environment (Simango, 2006) , the isolation of C.
T120 17569-17577 Sentence denotes Fig. 1 .
T121 17578-17764 Sentence denotes Average microscopic inflammatory score (AMIS) of intestinal segments and ileo-cecal lymph node of calves naturally (n = 2) and experimentally (n = 8) infected with Clostridium difficile.
T122 17765-17843 Sentence denotes AMIS represents the average number of lesions/severity identified per segment.
T123 17844-17950 Sentence denotes Each segment was given cumulative scores (0-3; mild, moderate, severe) for six indicators of inflammation.
T124 17951-18021 Sentence denotes Inoculated calves did not have diarrhea at necropsy; but controls did.
T125 18022-18392 Sentence denotes Ab, abomasum; Du, duodenum; Je, jejunum, Il, ileum; Ce, cecum; Sc; spiral colon; Ln; ileo-cecal lymph node. difficile from mature cows (Rodriguez et al., unpublished data) , and the common presence of serum titers of anti-C. difficile toxin immunoglobulins in humans ($70%) (Johnson, 1997) , it is plausible that passive immunity might play a protective role for calves.
T126 18393-18494 Sentence denotes C. difficile was isolated from calves under 24 h of age indicating early natural ingestion of spores.
T127 18495-18819 Sentence denotes The isolation of PCR-ribotype 077, a strain associated with CDAD in humans, from healthy and diarrheic calves in 2004 (Rodriguez-Palacios et al., 2006) , and from the calf that arrived naturally infected to this study suggests this ribotype is circulating in the cattle population, and that calves might be healthy carriers.
T128 18820-18902 Sentence denotes C. difficile was more frequently isolated from the cecum of calves in both groups.
T129 18903-19192 Sentence denotes Although there are no comparable studies for C. difficile in calves, the severity of the histological lesions seen in the cecum of infected piglets and hamsters (Borriello and Barclay, 1985; Keel and Songer, 2006) suggest that cecum could be a site for residence of C. difficile in calves.
T130 19193-19419 Sentence denotes The frequent presence of mild-moderate microscopic inflammatory changes observed in the cecum of inoculated calves compared to the other intestinal segments and two controls might be consistent with such observation (Fig. 1) .
T131 19420-19547 Sentence denotes However, the clinical significance of this finding is uncertain due to limited sample size and the absence of signs of disease.
T132 19548-19680 Sentence denotes The toxin ELISA test used in this study has been adequately validated for diagnostic purposes in humans (Wilkins and Lyerly, 2003) .
T133 19681-19769 Sentence denotes In animals, despite its regular use in hospital settings, validation studies are scarce.
T134 19770-19941 Sentence denotes In piglets the ELISA performance approached human standards (Post et al., 2002) ; but in dogs, it was considered poorly sensitive and specific (Chouicha and Marks, 2006) .
T135 19942-20047 Sentence denotes Validation of available ELISAs is needed to better understand the significance of C. difficile in cattle.
T136 20048-20184 Sentence denotes This study documented fecal/intestinal colonization for up to 5 days following oral inoculation of calves with a strain of C. difficile.
T137 20185-20273 Sentence denotes No induction of enteric disease or detection of fecal C. difficile toxins, was observed.
T138 20274-20328 Sentence denotes Cecum commonly yielded positive C. difficile cultures.
T139 20329-20413 Sentence denotes PCR-ribotyping showed that calves may naturally harbour C. difficile by 24 h of age.
T140 20414-20483 Sentence denotes Validation studies for diagnostic immunoassays for toxins are needed.
T141 20484-20559 Sentence denotes C. difficile should not be ruled out as a potential cause of calf diarrhea.