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Prevalence and risk factors associated with Clostridium difficile shedding in veal calves in Italy
Anaerobe 33 (2015) 42e47
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Prevalence and risk factors associated with Clostridium difficile shedding in veal calves in Italy Chiara Francesca Magistrali a, *, Carmen Maresca a, Lucilla Cucco a, Luca Bano b, Ilenia Drigo b, Giovanni Filippini a, Annalisa Dettori a, Sayra Broccatelli a, Giovanni Pezzotti a a b
Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, via Salvemini 1, 06126 Perugia, Italy Istituto Zooprofilattico Sperimentale delle Venezie e Treviso, vicolo Mazzini n. 4, Villorba, 31020 Treviso, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 February 2014 Received in revised form 15 January 2015 Accepted 27 January 2015 Available online 29 January 2015
The aim of this study is to describe the prevalence and risk factors of Clostridium difficile shedding in six farms belonging to two companies in Northern Italy. Four hundred and twenty veal calves, randomly selected and individually identified, were sampled three times: at 0e16, 90e120, and 150 days after introduction. C. difficile was isolated at least once from 87 out of the 420 calves (20.7%). The prevalence of shedding was 20.24% at the first sampling and dropped to 0.72% at the second sampling. None of the samples obtained at 150 days tested positive. Sampling of cecal contents and carcass swabs at slaughter was stratified according to the herd of origin of the animals. C. difficile was never isolated at slaughter, excluding a prevalence higher than 3.5% on the basis of previous investigations. Therefore, in this work, the veal calf could not be confirmed as a potential source of C. difficile for the consumer. Eight different ribotypes (RT) have been described, but the vast majority of the isolates (87.8%) belonged to three ribotypes only: RT-078, RT-012 and RT-126, which are also among the most common of the ribotypes detected in humans in Europe. Most isolates, and all the RT-078 isolates, harbored genes coding for toxins A and B, the binary toxin, and showed a deletion in the gene encoding toxin C, suggesting that the veal calf was a reservoir for epidemic hyper-virulent strains. A correlation between age and shedding was found: the odds ratio (OR) ranged from 2.79 for 36e45 days of age to 4.57 for 13e28 days of age. The presence of diarrhea at first sampling was significantly associated with the recovery of C. difficile in feces (OR 3.26). A correlation was found between the administration of antimicrobials and shedding: an increased risk was shown when the number of antimicrobials used was higher than 4 (OR 4.02) or 5e6 (OR 5.83) or when polymyxin E or beta-lactams were administered. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Clostridium difficile Veal calf Prevalence Antimicrobial Diarrhea Young age
1. Introduction Clostridium difficile is a spore-forming, Gram-positive bacterium, considered one of the leading causes of diarrhea in the industrialized world [1]. The C. difficileeinfection in humans (CDI) has a variety of clinical presentations, ranging from no symptoms to fulminant colitis, with pseudomembranous colitis being the most severe form of the disease [2,3]. The health care costs of CDI are
* Corresponding author. E-mail addresses: [email protected] (C.F. Magistrali), [email protected] (C. Maresca), [email protected] (L. Cucco), [email protected] (L. Bano), idrigo@ izsvenezie.it (I. Drigo), g.fi[email protected] (G. Filippini), [email protected] (A. Dettori), [email protected] (S. Broccatelli), [email protected] (G. Pezzotti). http://dx.doi.org/10.1016/j.anaerobe.2015.01.010 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
considered significant in both the United States and Europe, where they are estimated at $1.1 billion and V3000 million per annum, respectively [3]. In the last decade, the incidence and severity of CDI have been rising in the U.S., Canada, and Europe. This increase is linked to the spread of hypervirulent strains, in particular RT-027, and to a change in the epidemiological features of the infection [3,4]. Traditionally, CDI is nosocomial, targeting elderly hospitalized patients receiving antimicrobial therapy. Nevertheless, in the last ten years, cases in communities that were not exposed to known risk factors have been reported with increased frequency [4,5]. This kind of infection has been designated as community acquiredC. difficile disease (CA-CDI). It is often associated with RT-078, an emerging ribotype, reported in 2011 as the third most common
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ribotype in European hospitals [5]. The sources of CA-CDI are still under investigation. However, the possibility of an animal reservoir has been postulated, since RT-078 has been described as the leading ribotype in several domestic animals, in particular pigs and calves. Moreover, men and animals harbored strains belonging to same ribotypes and toxinotypes and this finding has suggested the possibility of an interspecies, or even foodborne, transmission [4,6]. Although the zoonotic nature of CDI has never been demonstrated, the finding of toxigenic, hypervirulent strains in food animals, such as pigs and cattle, has raised concerns on the possible role of farm animals as reservoir of the infection. These isolates are often multieresistant to antimicrobials, a characteristic which could favor the persistence along the animal production chain and which is often found in C. difficile epidemic strains in humans [7,8]. C. difficile is also associated with disease in a number of animal species. The role played by this bacterium in causing enteritis in piglets, hares and horses has been demonstrated [5,9,10]. C. difficile and its toxins have also been repeatedly isolated from the diarrhea of calves in several investigations. However, reports of its association with diarrhea in field conditions are not consistent among different studies. In addition, attempts to experimentally reproduce the disease in calves have never been completely successful. The inoculation of Toxin A (TcdA) and Toxin B (TcdB) in bovine intestinal loops have produced fluid accumulation, mucosal lesions and neutrophil accumulation but calves inoculated with C. difficile did not show any signs of disease [5,11]. Despite the importance of C. difficile in both public and animal health, there are very few longitudinal studies investigating the prevalence and risk factors of this infection from farm to slaughter in the current literature, and no data are available on this topic in Italy so far. It has been shown that a calf can acquire the bacterium within one day after birth, with the highest shedding rate early in life, approximately within one month of age, followed by a progressive decrease of shedding until the time of slaughter [8,12e14]. However, a recent longitudinal study carried out in veal calf farms showed a different shedding pattern, since C. difficile was shed in the last period of the production cycle [15]. This is an important issue to address, since high colonization rates at the time of slaughter could increase the risk of meat contamination [16]. Apart from the age effect, other risk factors are poorly described in this animal species: most importantly, antibiotic therapy, which is the principal risk factor for CDI in humans, was suspected to influence C. difficile prevalence in calves as well [8]. However, a direct link between the assumption of antimicrobials and C. difficile shedding in calves has never been shown. The aim of this work is, therefore, to describe the prevalence, ribotypes, and virulence determinants of C. difficile shedding from the introduction of the animals to slaughter in two large integrated veal companies in Northern Italy. Risk factors for C. difficile shedding, such as the effect of age, the use of antimicrobials, and the association with diarrhea were also investigated. 2. Materials and methods
43
provided to the calves in both companies and was supplemented with roughage. The roughage sources consisted of maize silage and high moisture corn in company A, where they were provided from the day of introduction to the herd. Company B calves were provided straw from the 20th day of life. In both companies, routine veterinary care was provided, and antibiotics were prescribed at the discretion of the attending veterinarian to control the onset of diseases. Calves were housed individually in separate stalls, with slatted floors, until approximately 2 months of age. After this time, thereafter, 4e7 animals were grouped per pen. All in/all out procedures were adopted per room between different groups of calves. The two companies did not provide a protocol of cleaning and disinfection procedures. Calves were slaughtered at 180e190 days of age in three different slaughterhouses located within 200 km of the herds. 2.2. Sampling on farm This work was carried out from February to October 2012. Four hundred and twenty animals were included in the study: 240 from company A and 180 from company B. The animal sample was calculated to estimate prevalence with 95% confidence intervals (CI) and 5% desired absolute precision. It was representative of the population, with animals selected at random. The calves were individually ear-tagged and data concerning the presence of diseases and therapy were recorded on an individual basis. Information regarding age, sex, diet and antibiotic treatments was recorded. Faecal samples were individually collected from the rectum at three different times: 0e16 days, 90e120 days, and approximately 150 days after the arrival to the farm. At every sampling time, an evaluation by the same veterinarian for the presence of diarrhea was recorded. The diarrhea was described as watery/pasty feces or as presence of perineal soiling. The third sampling of 60 calves belonging to company A and 30 from company B could not be performed because the slaughter time was brought forward. Moreover, 17 calves died and they were not be sampled until the end of the study. The number of calves from each company at different sampling times is recorded in Table 1. 2.3. Sample collection at slaughter A total of 152 animals were sampled at slaughter. Sampling was stratified according to the herd of origin of the animals, with a 3.5% expected prevalence, 95% CI, and 3% precision, on the basis of previous investigations [12]. At slaughter, cecal contents were individually sampled after evisceration and carcasses from the same animals were swabbed after chilling. Four different points (neck, brisket, flank and rump) were swabbed using a single hydrated sponge (Solar-cult Premoistened Sponges; Solar Biologicals Inc, Ogdensburg, New York), following the ISO 17604:2003 procedure [17]. All samples were stored at 4 C until processing, which occurred within 24e48 h.
2.1. Veal calves Six veal calf farms belonging to two large veal production companies were included in this study. Four farms were from company A and two from company B. The farms were located in Northern Italy. Both companies raised 10,561 calves during the survey, with 6757 being raised in company A, and 3804 in company B. The calves, all Holstein, originated from different small-medium sized farms in Northern Italy. They were introduced into the farm at 25 days of age on average. A complete milk replacer diet was
Table 1 Results from fecal samples at different sampling time. Total
Company A
Sampled þve %
Sampled þve %
Sampled þve %
240 238 169 647
180 178 144 502
0e16 days 420 90e120 days 416 ~150 days 313 Total 1149
85 3 0 88
20.24 0.72 0 7.66
71 2 0 73
Company B
29.58 0.84 0 11.28
14 1 0 15
7.77 0.56 0 2.98
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2.4. Culture
2.6. Statistical analysis
One gram (g) of feces or cecal contents was cultured following a modified single alcohol shock method [18]. Briefly, the samples were anaerobically (H2eCO2eN2, 5/5/90%) incubated at 37 C in 9 mL of cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, TCCFB (C. difficile selective supplement, Oxoid, UK; Sodium hydrogen phosphate, dehydrate, Carlo Erba, Pomezia, RM, Italy; D-() Fructose, Neutral red, Sodium phosphate monobasic, Sodium thaurocholate hydrate, SigmaeAldrich, Milan, Italy) for 7e10 days. After incubation, the broths were gently vortexed and 2 ml of each was put into a sterile plastic tube and treated with 2 mL of 96% ethanol for 1 h at room temperature. The tubes were then centrifuged at 3800 g for 10 min. The supernatant was discarded and the pellet streaked on blood agar supplemented with 5% horse red blood cells and 0.1% of esculin or ASEC (Blood Agar base, Biolife Italiana Srl, Milan, Italy; Esculin, Biolife Italiana Srl, Milan, Italy). The plates were anaerobically incubated at 37 C for 24e48 h. C. difficile-suspect colonies were identified from culture plates by the presence of a characteristic horse manure odor and morphological criteria, such as a black color (312 nm wavelength) detected with UV transillumination (TCX-20M, Uvitec, Cambridge, UK). The colonies were isolated on blood agar supplemented with 5% sheep blood (Blood Agar base, Biolife Italiana Srl, Milan, Italy), incubated anaerobically for 24 h at 37 C, and finally confirmed using Gram stain, a miniaturized biochemical test (Rapid ID32 A, Biomerieux Italia, Bagno a Ripoli, Florence, Italy) and by polymerase chain reaction (PCR). This led to the detection of the triose phosphate isomerase gene, (tpi), which is a housekeeping gene, as previously described [19]. Carcass swabs were inoculated in 30 mL of TCCFB and anaerobically incubated at 37 C for 7e10 days. The procedure then followed the same steps adopted for fecal and cecal samples.
At the first sampling, the overall prevalence, prevalence in each company, and 95% confidence interval (95% CI) were calculated. The variables (age, antimicrobial therapy and presence of diarrhea) were examined as descriptive of time and company. In statistical analysis, the age of each calf at first sampling was categorized into four groups (Table 2) and then included as a categorical variable. With antimicrobial therapy, the number of antimicrobials used and the antibiotic treatment were evaluated. The presence of diarrhea and the belonging company were variables recorded as ‘yes’ or ‘no’ for diarrhea, and ‘A’ or ‘B’ for company. The presence of a statistically significant association between C. difficile infection and hypothesized causal factors was tested using univariate analysis. An odds ratio (OR) and 95% confidence interval (95% CI) for each potential risk factor were determined. Statistical significance was tested using chi-square analysis with a p value <0.05 considered significant. Statistical analysis was performed with Stata software, version 11.1 (Copyright 2009 Stata Corp LP Stata Corp).
2.5. Bio-molecular characterization Strains were characterized by two previously described PCR tests targeting genes coding for Toxin A (TcdA), Toxin B (TcdB) [19] and binary toxin genes [20]. Because six isolates were lost in subculture or after freeze storage, PCR-ribotyping (RT) was performed on 82 out of 88 isolates [21]. The resulting patterns of field strains were compared with those obtained for 21 epidemic European strains (RT-001, RT-002, RT-005, RT-010, RT-012, RT-014, RT-016, RT-017, RT-018, RT-020, RT-027, RT-033, RT-045, RT-070, RT-078, RT-087, RT-081, RT-103, RT-126, RT-127, RT-150). Finally, the possible deletion of tcdC, the gene encoding toxin C, was investigated by a PCR test, as already described [22].
3. Results 3.1. Prevalence and age effect The age of the animals' introduction into the herd ranged from 4 to 78 days. Ninety three percent of the animals were introduced at 10e43 days of age (25 days on average). Data on the number of positive samples for each company at every sampling are shown in Table 1. C. difficile was isolated at least once from 87 out of 420 calves included in the survey. The prevalence at first sampling was 20.24% (CI 95%, 16.79%e24.07%) in both companies. Nevertheless, this data differs between the two companies: it was 29.58% (CI 95%, 24.36%e35.25%) in company A and 7.78% (CI 95%; 4.81%e11.93%) in company B. A calf was five times more likely to be C. difficile positive at first sampling if it belonged to company A than if it was from company B (OR 4.98; CI 2.70e9.18. p value 0.000). An association between the age of the calf at first sampling and the presence of C. difficile in feces was found and listed in Table 2. The OR ranged from 2.79 at 36e45 days of age to 4.57 at 13e28 days of age. The prevalence of shedding dropped to 0.72% at the second sampling, being 0.84% and 0.56% in company A and company B, respectively. One of the three calves which shed C. difficile at 90e120 days, was also positive at 0e16 days. No sample taken at 150 days or at the slaughterhouse (cecal contents and carcass swabs) had a positive result. Table 3 Risk factors for shedding of C. difficile in feces: antibiotic treatments. Risk factors
Table 2 Risk factors for C. difficile shedding in feces. Risk factors Age at sampling
Number of antimicrobials
Diarrhea
46e90 days 13e28 days 29e35 days 36e45 days 0e1 2 3 4 5e6 Absence Presence
OR
Inferior limit 95%
Superior limit 95%
p-value
1 4.57 4.16 2.79 e 1.16 2.81 4.02 5.83 1 3.26
e 1.98 1.79 1.17 e 0.31 0.8 1.13 1.14 e 1.97
e 10.53 9.69 6.67 e 4.27 9.81 14.32 29.86 e 5.4
e 0.000 0.001 0.021 e 0.827 0.106 0.032 0.034 e 0.000
Aminoglicosydes Beta-lactams Quinolones Macrolides Polymyxin E Sulfonamides Tetracyclines
No Yes No Yes No Yes No Yes No Yes No Yes No Yes
OR
Inferior limit 95%
Superior limit 95%
p-value
1 0.82 1 4.58 1 0.82 1 1.71 1 3.80 4.15 1 1 1.81
e 0.45 e 2.52 e 0.51 e 0.43 e 2.23 1.46 e e 0.83
e 1.48 e 8.33 e 1.33 e 6.77 e 6.46 11.79 e e 3.97
e 0.519 e 0.000 e 0.424 e 0.442 e 0.000 0.008 e e 0.139
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3.2. Antimicrobial use and C. difficile shedding
Table 5 Distribution of the genes coding for the major toxins, according to serotype.
During this survey, calves were treated with antibiotics to control the onset of diseases. The main reason reported for antimicrobial therapy after introduction to the herd was pneumonia. Treatment with tetracyclines and sulfonamides was common in both companies. Beta-lactams and polymyxin E were used only in herds belonging to company A, while florfenicol was used exclusively in calves belonging to company B. An association was found between antibiotic treatments before the first sampling and C. difficile shedding. In particular, an increased risk was shown when the number of antimicrobials used was higher than 4 (Table 2) or when polymyxin E or beta-lactams were administered (Table 3).
3.3. Presence of diarrhea and C. difficile Among calves excreting C. difficile at the first sampling, (n ¼ 85), 51 (48.2%) had diarrhea, while 44 (51.8%) showed no sign of diarrhea at the clinical examination. Among C. difficile negative animals at 0e16 days after introduction, (n ¼ 335), 68 (20.3%) had diarrhea, while 238 (71%) did not. The clinical evaluation could not be done in 29 cases (8.7%). The presence of diarrhea, detected at first sampling, was significantly associated with the recovery of C. difficile in feces: OR 3.26 (CI 1.97e5.40, p-value 0.000) (Table 2).
3.4. Bio-molecular characterization Data on ribotypes are shown in Table 4. Eight different ribotypes are described, five of which were present in farms of both companies. Polymerase chain reaction ribotypes for which reference strains were available were named according to standard Cardiff nomenclature (RT and number). The others were named according to internal nomenclature (Treviso, TV, and number). Seventy eight out of 82 isolates belonged to four ribotypes only: RT-078 (N ¼ 43; 52.5%), RT-012 (N ¼ 16; 19.5%), RT-0126 (N ¼ 13; 15.9%) and RT-033 (N ¼ 6; 7.3%). Ribotype 078 was by far the most common, since 43 isolates belonged to this ribotype. The distribution of the most common ribotypes was similar in the two companies, except for RT033, isolated five times in company B and only once in company A. The two isolates shed by the same calf at 0e16 and 90e120 days of age belonged to the same ribotype, RT-012. In Table 5, the distribution of the genes coding for the major toxins is shown. tcdA and tcdB were present in 95% of the isolates, the binary toxin in 80.5% and the 39 bp deletion of tcdC in 76.8%. The profile shown by all the RT-078 isolates was tcdAþ/tcdBþ/binary toxinþ/39 base pair deletion of tcdCþ. Table 4 Distribution of the different ribotypes. Ribotype
RT-078 RT-012 RT-126 RT-033 TV40 TV41 TV42 TV43 Total
45
Company A
Company B
Total
N
%
N.
%
N.
%
37 15 11 1 1 1 e 1 67
55.2 22.4 16.4 1.5 1.5 1.5 e 1.5 100.0
6 1 2 5 e e 1 e 14
40 6.7 13.3 33.3 e e 6.7 e 100.0
43 16 13 6 1 1 1 1 82
52.5 19.5 15.9 7.3 1.2 1.2 1.2 1.2 100.0
Ribotype
tcdA
tcdB
CDT
tcdC deletion
Total
RT-078 RT-012 RT-126 RT-033 TV40 TV41 TV42 TV43 Total
43 16 13 6 e 1 1 1 81
43 16 13 e e 1 1 1 75
43 3 13 6 e 1 e e 66
43 e 12 6 1 1 e e 63
43 16 13 6 1 1 1 1 82
4. Discussion and conclusions This work provides original data on the epidemiology of C. difficile in veal calf herds. In literature, the prevalence of calves shedding C. difficile is quite variable, ranging from 7.6% [23] to 61% [24] in different surveys [8,14,15,24,25]. In a recent study, a prevalence of 28% was found in veal calves, with marked differences between farms [15]. It was suggested that different sampling time or testing methodology can be any one of the possible reasons to explain this variability [5,8,11]. In our investigation, the sampling scheme and the culture tests were the same in both companies, so the observed differences in C. difficile shedding prevalence between the two companies can reasonably be attributed to real differences of shedding in calves. However, other factors such as the hygienic procedures, feeding or antibiotic treatments adopted by the two companies could have influenced our data. Moreover, the calves originated from multiple sources, and calves belonging to the same group were introduced into the herd on different days. As sampling was performed after the last calf of a group was introduced, a variability in the age of the calves at each of the sampling points was recorded. In this study, the time of major risk for C. difficile shedding was immediately after introduction to the herd. Approximately 20% of veal calves were positive at first sampling, and this percentage declined sharply at 90e120 days. This decreasing trend has also been described by other authors [26] who found higher prevalence of infection in feedlot cattle shortly after the arrival. As mentioned above, a different pattern of shedding was found in a study carried out on veal calves by Houser et al. [15], where the peak of shedding was described just before slaughter, at 20e22 weeks of age. The authors attributed this discrepancy to farmerelated factors, which could have increased the environmental contamination or determined an increased susceptibility of the animals to the infection [8,14,15,24]. Even though we are not able to rule out the influences of these factors in our study, we have found that the pattern of shedding can also be explained by an age effect, as young age at first sampling was shown to be significantly associated with a higher probability of C. difficile in faeces. The peak time was recorded at 13e28 days of age. This is not surprising, since a higher susceptibility of younger animals to infection has already been observed in pigs [12] and poultry [27]. In a longitudinal study on calves carried out in Belgium [13], the trend of C. difficile shedding was similar to that observed in this investigation. The peak of shedding at 1e2 weeks of age was reported in other studies [8,13,23]. In our study, the age effect was shown to occur in a brief period, during the first 2e3 months of age, after which there was a progressive decrease. Remarkably, a calf of less than 35 days of age had four times more probability of being a C. difficileeshedder than a calf of 46e90 days of age. Since the discrepancy between the prevalence at different ages was high, the younger age effect was shown to be statistically and biologically significant and should be taken into account in designing and interpreting prevalence studies in cattle.
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In our investigation, shedding was limited to the first production stages. No C. difficileeshedder was detected in the last sampling before slaughter, at approximately 170 days of age. Correspondingly, no cecal contents or carcass sponges were positive at slaughter, meaning that the prevalence of carcass contamination cannot be equal to or higher than 3.5%. This expected prevalence was chosen at the beginning of the study to determine the number of carcasses to be sampled, and it was based on data available in literature [12]. It is therefore possible that a few positive carcasses went undetected in our study. Data on carcass contamination by C. difficile reported in literature vary from study to study. The C. difficile housekeeping gene, tpi, was found in four out of one hundred veal calf carcass swabs before decontamination [19]. In a transversal study carried out in Belgium, C. difficile was isolated from 9.9% intestinal and 7.9% carcass samples from slaughtered cattle [28]. Again, the geographic origin of the animals, the hygiene procedures adopted at the slaughterhouse or the prevalence of colonization before slaughter, could have influenced the C. difficile carcass contamination [15]. It should be noted that in the present survey, the intestinal contents were not found positive at slaughter. Therefore, a contamination of carcasses from the same groups of animals seems less probable. According to our data, foodborne transmission via veal calf cannot be suspected as a risk for the consumer. The four most common ribotypes identified in this study, accounting for approximately 95% of all our isolates, corresponded to a profile of the international numerical designation: RT-078, RT012, RT-033 and RT-126. Our data are in agreement with previous findings on C. difficile ribotypes in cattle. Ribotype 078, which was prevalent in our investigation, is considered the most frequent ribotype in this animal [5,13,28] and both RT-033 and RT-012 were repeatedly described in the same species [5,13]. Ribotype 126, whose profile is very similar or even indistinguishable from RT-078, has already been shown as one of the predominant ribotypes in a veal calf farm in Belgium [13]. On the other hand, the three most common ribotypes isolated from calves, RT-078, RT-012 and RT-126, were also among the most common ribotypes detected in humans in Europe [4]. Calves shed mainly RT-078, belonging to toxinotype V, and RT078 is considered one of the emerging ribotypes in Europe, where it is reported as the third most common ribotype isolated from CDI in humans [4]. Most of the isolates collected in this work showed the encoding genes for the major toxins. The RT-033 isolates harbored tcdA and they were negative to tcdB, a profile which has been recently described in cattle [29]. All the RT-078 isolates were positive for the virulence-associated gene: this finding supports the hypothesis that calves, among other animal species, could act as a reservoir for zoonoticehypervirulent strains [7,12,30]. However, to reduce the burden of contamination on farms, information on the epidemiology of this microorganism in cattle is necessary and this is not readily available in literature yet. This work reduces this gap, providing new insight on possible risk factors for this infection in veal calf primary production. In the present study, a difference in the use of antimicrobials in the two companies was found. In particular, only herds belonging to company A used beta-lactams for therapy. The use of betalactams before first sampling was associated with an OR of 4.58 for C. difficile shedding. In humans, betaelactams are considered particularly effective in determining CDI. In a recent survey, the higher incidence of C. difficile in hospitals was characterized by the use of beta-lactams in patients [4]. Therefore, even if several management practices could explain the difference in prevalence between the two companies, it can be argued that a major role could have been played by antimicrobials. The number of antibiotics was also associated with an increased
risk for shedding, as calves treated with more than four classes of antimicrobials before sampling were four times more likely to shed C. difficile. As far as we know, this is the first time that the use of antimicrobials have been linked to C. difficile shedding in calves. It cannot be excluded that the association observed was indirect, since antibiotics were prescribed to control a disease and the health status could have influenced the shedding of C. difficile. However, this link has been already shown in other animal species, such as the horse, dog and cat, although with contrasting results in different studies [5]. Antimicrobial therapy is the major risk factor for CDI in man [2] and the first report of this association dates back to 1978 [31]. Antimicrobials favor CDI by the disruption of the intestinal microbiota, which in physiological conditions, protects the gut against C. difficile colonization and expansion [32]. It would be reasonable to presume that this mechanism justifies the same association in veal calves. The extensive use of antimicrobials in both human and animal medicine raises concerns regarding the emergence and spreading of multi-resistant organisms. In Italy, veterinary prescription is mandatory for antimicrobial therapy in animals. Nevertheless, in a survey done in 2004, the prophylactic use of antibiotics was common among cattle veterinarians and the use of fluoquinolones as first or second choice for the treatment of calf scours was reported [33]. Better prevention measures and improved biosecurity, implemented to reduce the use of antimicrobials in the veal calf production chain, can have positive consequences on the C. difficile infection as well. Finally, a link between the presence of diarrhea and C. difficile shedding was shown in the animals at first sampling. An association between enteric disorders and CDI in calves has been shown in other studies [11,23], but the question of whether C. difficile could be an agent of calf diarrhea is still debated in the literature. The experimental challenge of calves with C. difficile ribotype 077 failed to induce C. difficile-associated disease (CDAD), questioning the role of C. difficile as a primary pathogen in cattle [34]. However, the pathogenesis of CDAD can be more complex, involving the presence of predisposing factors causing a disruption of the intestinal flora and an increased susceptibility to the infection, like in humans [5]. Therefore, the role of C. difficile as a cause of diarrhea in cattle cannot be definitely ruled out [34]. It is possible that the administration of antimicrobials in veal calves had induced an increased susceptibility to CDAD in our study. On the other hand, the association observed during the present study could be justified by several factors, and it should not necessarily be linked to a role of C. difficile in cattle enteritis. For example, an increase in shedding during diarrhea could be postulated. Moreover, diarrhea and C. difficile shedding can result from a common risk factor, for example change of diet and/or a change in the gut flora determined by antimicrobials. In conclusion, this work further documents that C. difficile shedding in the veal calf is linked to younger age and to the presence of diarrhea. For the first time, the use of antimicrobials has been directly linked to the bacterial shedding in cattle, suggesting new approaches to the control of C. difficile spreading on farms. Even though shedding was limited to the first phase of the production chain, the presence of emerging ribotypes suggests a possible role of the veal calf as a reservoir of C. difficile for humans. Further work is therefore needed to investigate common risk factors and possible control measures in veal calf primary production.
Funding This work was funded by the Italian Ministry of Health (Progetto di ricerca corrente) IZSUM 07/2009.
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[18] L.G. Arroyo, J. Rousseau, B.M. Willey, D.E. Low, H. Staempfli, A. McGeer, J.S. Weese, Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions, J. ClinMicrobiol. 43 (10) (2005 Oct) 5341e5343. [19] L. Lemee, A. Dhalluin, S. Testelin, M.A. Mattrat, K. Maillard, J.F. Lemeland, J.L. Pons, Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (toxin A), and tcdB (toxin B) genes for toxigenic culture of Clostridium difficile, J. Clin. Microbiol. 42 (2004) 5710e5714. [20] S. Stubbs, M. Rupnik, M. Gibert, J. Brezier, B. Duerden, M. Popoff, Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile, FEMS Microbiol. Lett. 186 (2000) 307e312. [21] P. Bidet, F. Barbut, V. Lalande, B. Burghoffer, J.C. Petit, Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing, FEMS Microbiol. Lett. 175 (1999) 261e266. [22] J. Antikainen, E. Tarkka, K. Haukka, A. Siitonen, M. Vaara, J. Kirveskari, Detection of virulence genes of Clostridium difficile by multiplex PCR, APMIS 117 (2009) 607e613. €mpfli, T. Duffield, A.S. Peregrine, L.A. Trotz[23] A. Rodriguez-Palacios, H.R. Sta Williams, L.G. Arroyo, J.S. Brazier, J.S. Weese, Clostridium difficile PCR ribotypes in calves, Canada, Emerg. Infect. Dis. 12 (11) (2006 Nov) 1730e1736. [24] M.C. Costa, R. Reid-Smith, S. Gow, S.J. Hannon, C. Booker, J. Rousseau, K.M. Benedict, P.S. Morley, J.S. Weese, Prevalence and molecular characterization of Clostridium difficile isolated from feedlot beef cattle upon arrival and mid-feeding period, BMC Vet. Res. 8 (2012 Mar 28) 38, http://dx.doi.org/ 10.1186/1746-6148-8-38. [25] V. Romano, F. Albanese, S. Dumontet, K. Krovacek, O. Petrini, V. Pasquale, Prevalence and genotypic characterization of Clostridium difficile from ruminants in Switzerland, Zoonoses Public Health 59 (8) (2012 Dec) 545e548. [26] A. Rodriguez-Palacios, M. Koohmaraie, J.T. LeJeune, Prevalence, enumeration, and antimicrobial agent resistance of Clostridium difficile in cattle at harvest in the United States, J. Food Prot. 74 (10) (2011 Oct) 1618e1624. [27] V. Zidaric, M. Zemljic, S. Janezic, A. Kocuvan, M. Rupnik, High diversity of Clostridium difficile genotypes isolated from a single poultry farm producing replacement laying hens, Anaerobe 14 (6) (2008 Dec) 325e327. e, G. Daube, [28] C. Rodriguez, V. Avesani, J. Van Broeck, B. Taminiau, M. Delme Presence of Clostridium difficile in pigs and cattle intestinal contents and carcass contamination at the slaughterhouse in Belgium, Int. J. Food Microbiol. 166 (2) (2013 Sep 2) 256e262, http://dx.doi.org/10.1016/ j.ijfoodmicro.2013.07.017. [29] S. Janezic, V. Zidaric, B. Pardon, A. Indra, B. Kokotovic, J.L. Blanco, C. Seyboldt, C.R. Diaz, I.R. Poxton, V. Perreten, I. Drigo, A. Jiraskova, M. Ocepek, J.S. Weese, J.G. Songer, M.H. Wilcox, M. Rupnik, International Clostridium difficile animal strain collection and large diversity of animal associated strains, BMC Microbiol. 14 (1) (2014 Jun 28) 173. [30] M.A. Jhung, A.D. Thompson, G.E. Killgore, W.E. Zukowski, G. Songer, M. Warny, S. Johnson, D.N. Gerding, L.C. McDonald, B.M. Limbago, ToxinotypeV Clostridium difficile in humans and food animals, Emerg. Infect. Dis. 14 (7) (2008 Jul) 1039e1045. [31] A.M. Jones, E.J. Kuijper, M.H. Wilcox, Clostridium difficile: a European perspective, J. Infect. 66 (2) (2013 Feb) 115e128, http://dx.doi.org/10.1016/ j.jinf.2012.10.019. [32] K.M. Ng, J.A. Ferreyra, S.K. Higginbottom, J.B. Lynch, P.C. Kashyap, S. Gopinath, N. Naidu, B. Choudhury, B.C. Weimer, D.M. Monack, J.L. Sonnenburg, Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens, Nature 502 (7469) (2013 Oct 3) 96e99, http://dx.doi.org/10.1038/ nature12503. [33] L. Busani, C. Graziani, A. Franco, A. Di Egidio, N. Binkin, A. Battisti, Survey of the knowledge, attitudes and practice of Italian beef and dairy cattle veterinarians concerning the use of antibiotics, Vet. Rec. 155 (23) (2004 Dec 4) 733e738. [34] A. Rodriguez-Palacios, H.R. St€ ampfli, M. Stalker, T. Duffield, J.S. Weese, Natural and experimental infection of neonatal calves with Clostridium difficile, Vet. Microbiol. 124 (1e2) (2007 Sep 20) 166e172.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Prevalence and risk factors associated with Clostridium difficile shedding in veal calves in Italy Chiara Francesca Magistrali a, *, Carmen Maresca a, Lucilla Cucco a, Luca Bano b, Ilenia Drigo b, Giovanni Filippini a, Annalisa Dettori a, Sayra Broccatelli a, Giovanni Pezzotti a a b
Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, via Salvemini 1, 06126 Perugia, Italy Istituto Zooprofilattico Sperimentale delle Venezie e Treviso, vicolo Mazzini n. 4, Villorba, 31020 Treviso, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 February 2014 Received in revised form 15 January 2015 Accepted 27 January 2015 Available online 29 January 2015
The aim of this study is to describe the prevalence and risk factors of Clostridium difficile shedding in six farms belonging to two companies in Northern Italy. Four hundred and twenty veal calves, randomly selected and individually identified, were sampled three times: at 0e16, 90e120, and 150 days after introduction. C. difficile was isolated at least once from 87 out of the 420 calves (20.7%). The prevalence of shedding was 20.24% at the first sampling and dropped to 0.72% at the second sampling. None of the samples obtained at 150 days tested positive. Sampling of cecal contents and carcass swabs at slaughter was stratified according to the herd of origin of the animals. C. difficile was never isolated at slaughter, excluding a prevalence higher than 3.5% on the basis of previous investigations. Therefore, in this work, the veal calf could not be confirmed as a potential source of C. difficile for the consumer. Eight different ribotypes (RT) have been described, but the vast majority of the isolates (87.8%) belonged to three ribotypes only: RT-078, RT-012 and RT-126, which are also among the most common of the ribotypes detected in humans in Europe. Most isolates, and all the RT-078 isolates, harbored genes coding for toxins A and B, the binary toxin, and showed a deletion in the gene encoding toxin C, suggesting that the veal calf was a reservoir for epidemic hyper-virulent strains. A correlation between age and shedding was found: the odds ratio (OR) ranged from 2.79 for 36e45 days of age to 4.57 for 13e28 days of age. The presence of diarrhea at first sampling was significantly associated with the recovery of C. difficile in feces (OR 3.26). A correlation was found between the administration of antimicrobials and shedding: an increased risk was shown when the number of antimicrobials used was higher than 4 (OR 4.02) or 5e6 (OR 5.83) or when polymyxin E or beta-lactams were administered. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Clostridium difficile Veal calf Prevalence Antimicrobial Diarrhea Young age
1. Introduction Clostridium difficile is a spore-forming, Gram-positive bacterium, considered one of the leading causes of diarrhea in the industrialized world [1]. The C. difficileeinfection in humans (CDI) has a variety of clinical presentations, ranging from no symptoms to fulminant colitis, with pseudomembranous colitis being the most severe form of the disease [2,3]. The health care costs of CDI are
* Corresponding author. E-mail addresses: [email protected] (C.F. Magistrali), [email protected] (C. Maresca), [email protected] (L. Cucco), [email protected] (L. Bano), idrigo@ izsvenezie.it (I. Drigo), g.fi[email protected] (G. Filippini), [email protected] (A. Dettori), [email protected] (S. Broccatelli), [email protected] (G. Pezzotti). http://dx.doi.org/10.1016/j.anaerobe.2015.01.010 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
considered significant in both the United States and Europe, where they are estimated at $1.1 billion and V3000 million per annum, respectively [3]. In the last decade, the incidence and severity of CDI have been rising in the U.S., Canada, and Europe. This increase is linked to the spread of hypervirulent strains, in particular RT-027, and to a change in the epidemiological features of the infection [3,4]. Traditionally, CDI is nosocomial, targeting elderly hospitalized patients receiving antimicrobial therapy. Nevertheless, in the last ten years, cases in communities that were not exposed to known risk factors have been reported with increased frequency [4,5]. This kind of infection has been designated as community acquiredC. difficile disease (CA-CDI). It is often associated with RT-078, an emerging ribotype, reported in 2011 as the third most common
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ribotype in European hospitals [5]. The sources of CA-CDI are still under investigation. However, the possibility of an animal reservoir has been postulated, since RT-078 has been described as the leading ribotype in several domestic animals, in particular pigs and calves. Moreover, men and animals harbored strains belonging to same ribotypes and toxinotypes and this finding has suggested the possibility of an interspecies, or even foodborne, transmission [4,6]. Although the zoonotic nature of CDI has never been demonstrated, the finding of toxigenic, hypervirulent strains in food animals, such as pigs and cattle, has raised concerns on the possible role of farm animals as reservoir of the infection. These isolates are often multieresistant to antimicrobials, a characteristic which could favor the persistence along the animal production chain and which is often found in C. difficile epidemic strains in humans [7,8]. C. difficile is also associated with disease in a number of animal species. The role played by this bacterium in causing enteritis in piglets, hares and horses has been demonstrated [5,9,10]. C. difficile and its toxins have also been repeatedly isolated from the diarrhea of calves in several investigations. However, reports of its association with diarrhea in field conditions are not consistent among different studies. In addition, attempts to experimentally reproduce the disease in calves have never been completely successful. The inoculation of Toxin A (TcdA) and Toxin B (TcdB) in bovine intestinal loops have produced fluid accumulation, mucosal lesions and neutrophil accumulation but calves inoculated with C. difficile did not show any signs of disease [5,11]. Despite the importance of C. difficile in both public and animal health, there are very few longitudinal studies investigating the prevalence and risk factors of this infection from farm to slaughter in the current literature, and no data are available on this topic in Italy so far. It has been shown that a calf can acquire the bacterium within one day after birth, with the highest shedding rate early in life, approximately within one month of age, followed by a progressive decrease of shedding until the time of slaughter [8,12e14]. However, a recent longitudinal study carried out in veal calf farms showed a different shedding pattern, since C. difficile was shed in the last period of the production cycle [15]. This is an important issue to address, since high colonization rates at the time of slaughter could increase the risk of meat contamination [16]. Apart from the age effect, other risk factors are poorly described in this animal species: most importantly, antibiotic therapy, which is the principal risk factor for CDI in humans, was suspected to influence C. difficile prevalence in calves as well [8]. However, a direct link between the assumption of antimicrobials and C. difficile shedding in calves has never been shown. The aim of this work is, therefore, to describe the prevalence, ribotypes, and virulence determinants of C. difficile shedding from the introduction of the animals to slaughter in two large integrated veal companies in Northern Italy. Risk factors for C. difficile shedding, such as the effect of age, the use of antimicrobials, and the association with diarrhea were also investigated. 2. Materials and methods
43
provided to the calves in both companies and was supplemented with roughage. The roughage sources consisted of maize silage and high moisture corn in company A, where they were provided from the day of introduction to the herd. Company B calves were provided straw from the 20th day of life. In both companies, routine veterinary care was provided, and antibiotics were prescribed at the discretion of the attending veterinarian to control the onset of diseases. Calves were housed individually in separate stalls, with slatted floors, until approximately 2 months of age. After this time, thereafter, 4e7 animals were grouped per pen. All in/all out procedures were adopted per room between different groups of calves. The two companies did not provide a protocol of cleaning and disinfection procedures. Calves were slaughtered at 180e190 days of age in three different slaughterhouses located within 200 km of the herds. 2.2. Sampling on farm This work was carried out from February to October 2012. Four hundred and twenty animals were included in the study: 240 from company A and 180 from company B. The animal sample was calculated to estimate prevalence with 95% confidence intervals (CI) and 5% desired absolute precision. It was representative of the population, with animals selected at random. The calves were individually ear-tagged and data concerning the presence of diseases and therapy were recorded on an individual basis. Information regarding age, sex, diet and antibiotic treatments was recorded. Faecal samples were individually collected from the rectum at three different times: 0e16 days, 90e120 days, and approximately 150 days after the arrival to the farm. At every sampling time, an evaluation by the same veterinarian for the presence of diarrhea was recorded. The diarrhea was described as watery/pasty feces or as presence of perineal soiling. The third sampling of 60 calves belonging to company A and 30 from company B could not be performed because the slaughter time was brought forward. Moreover, 17 calves died and they were not be sampled until the end of the study. The number of calves from each company at different sampling times is recorded in Table 1. 2.3. Sample collection at slaughter A total of 152 animals were sampled at slaughter. Sampling was stratified according to the herd of origin of the animals, with a 3.5% expected prevalence, 95% CI, and 3% precision, on the basis of previous investigations [12]. At slaughter, cecal contents were individually sampled after evisceration and carcasses from the same animals were swabbed after chilling. Four different points (neck, brisket, flank and rump) were swabbed using a single hydrated sponge (Solar-cult Premoistened Sponges; Solar Biologicals Inc, Ogdensburg, New York), following the ISO 17604:2003 procedure [17]. All samples were stored at 4 C until processing, which occurred within 24e48 h.
2.1. Veal calves Six veal calf farms belonging to two large veal production companies were included in this study. Four farms were from company A and two from company B. The farms were located in Northern Italy. Both companies raised 10,561 calves during the survey, with 6757 being raised in company A, and 3804 in company B. The calves, all Holstein, originated from different small-medium sized farms in Northern Italy. They were introduced into the farm at 25 days of age on average. A complete milk replacer diet was
Table 1 Results from fecal samples at different sampling time. Total
Company A
Sampled þve %
Sampled þve %
Sampled þve %
240 238 169 647
180 178 144 502
0e16 days 420 90e120 days 416 ~150 days 313 Total 1149
85 3 0 88
20.24 0.72 0 7.66
71 2 0 73
Company B
29.58 0.84 0 11.28
14 1 0 15
7.77 0.56 0 2.98
44
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2.4. Culture
2.6. Statistical analysis
One gram (g) of feces or cecal contents was cultured following a modified single alcohol shock method [18]. Briefly, the samples were anaerobically (H2eCO2eN2, 5/5/90%) incubated at 37 C in 9 mL of cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, TCCFB (C. difficile selective supplement, Oxoid, UK; Sodium hydrogen phosphate, dehydrate, Carlo Erba, Pomezia, RM, Italy; D-() Fructose, Neutral red, Sodium phosphate monobasic, Sodium thaurocholate hydrate, SigmaeAldrich, Milan, Italy) for 7e10 days. After incubation, the broths were gently vortexed and 2 ml of each was put into a sterile plastic tube and treated with 2 mL of 96% ethanol for 1 h at room temperature. The tubes were then centrifuged at 3800 g for 10 min. The supernatant was discarded and the pellet streaked on blood agar supplemented with 5% horse red blood cells and 0.1% of esculin or ASEC (Blood Agar base, Biolife Italiana Srl, Milan, Italy; Esculin, Biolife Italiana Srl, Milan, Italy). The plates were anaerobically incubated at 37 C for 24e48 h. C. difficile-suspect colonies were identified from culture plates by the presence of a characteristic horse manure odor and morphological criteria, such as a black color (312 nm wavelength) detected with UV transillumination (TCX-20M, Uvitec, Cambridge, UK). The colonies were isolated on blood agar supplemented with 5% sheep blood (Blood Agar base, Biolife Italiana Srl, Milan, Italy), incubated anaerobically for 24 h at 37 C, and finally confirmed using Gram stain, a miniaturized biochemical test (Rapid ID32 A, Biomerieux Italia, Bagno a Ripoli, Florence, Italy) and by polymerase chain reaction (PCR). This led to the detection of the triose phosphate isomerase gene, (tpi), which is a housekeeping gene, as previously described [19]. Carcass swabs were inoculated in 30 mL of TCCFB and anaerobically incubated at 37 C for 7e10 days. The procedure then followed the same steps adopted for fecal and cecal samples.
At the first sampling, the overall prevalence, prevalence in each company, and 95% confidence interval (95% CI) were calculated. The variables (age, antimicrobial therapy and presence of diarrhea) were examined as descriptive of time and company. In statistical analysis, the age of each calf at first sampling was categorized into four groups (Table 2) and then included as a categorical variable. With antimicrobial therapy, the number of antimicrobials used and the antibiotic treatment were evaluated. The presence of diarrhea and the belonging company were variables recorded as ‘yes’ or ‘no’ for diarrhea, and ‘A’ or ‘B’ for company. The presence of a statistically significant association between C. difficile infection and hypothesized causal factors was tested using univariate analysis. An odds ratio (OR) and 95% confidence interval (95% CI) for each potential risk factor were determined. Statistical significance was tested using chi-square analysis with a p value <0.05 considered significant. Statistical analysis was performed with Stata software, version 11.1 (Copyright 2009 Stata Corp LP Stata Corp).
2.5. Bio-molecular characterization Strains were characterized by two previously described PCR tests targeting genes coding for Toxin A (TcdA), Toxin B (TcdB) [19] and binary toxin genes [20]. Because six isolates were lost in subculture or after freeze storage, PCR-ribotyping (RT) was performed on 82 out of 88 isolates [21]. The resulting patterns of field strains were compared with those obtained for 21 epidemic European strains (RT-001, RT-002, RT-005, RT-010, RT-012, RT-014, RT-016, RT-017, RT-018, RT-020, RT-027, RT-033, RT-045, RT-070, RT-078, RT-087, RT-081, RT-103, RT-126, RT-127, RT-150). Finally, the possible deletion of tcdC, the gene encoding toxin C, was investigated by a PCR test, as already described [22].
3. Results 3.1. Prevalence and age effect The age of the animals' introduction into the herd ranged from 4 to 78 days. Ninety three percent of the animals were introduced at 10e43 days of age (25 days on average). Data on the number of positive samples for each company at every sampling are shown in Table 1. C. difficile was isolated at least once from 87 out of 420 calves included in the survey. The prevalence at first sampling was 20.24% (CI 95%, 16.79%e24.07%) in both companies. Nevertheless, this data differs between the two companies: it was 29.58% (CI 95%, 24.36%e35.25%) in company A and 7.78% (CI 95%; 4.81%e11.93%) in company B. A calf was five times more likely to be C. difficile positive at first sampling if it belonged to company A than if it was from company B (OR 4.98; CI 2.70e9.18. p value 0.000). An association between the age of the calf at first sampling and the presence of C. difficile in feces was found and listed in Table 2. The OR ranged from 2.79 at 36e45 days of age to 4.57 at 13e28 days of age. The prevalence of shedding dropped to 0.72% at the second sampling, being 0.84% and 0.56% in company A and company B, respectively. One of the three calves which shed C. difficile at 90e120 days, was also positive at 0e16 days. No sample taken at 150 days or at the slaughterhouse (cecal contents and carcass swabs) had a positive result. Table 3 Risk factors for shedding of C. difficile in feces: antibiotic treatments. Risk factors
Table 2 Risk factors for C. difficile shedding in feces. Risk factors Age at sampling
Number of antimicrobials
Diarrhea
46e90 days 13e28 days 29e35 days 36e45 days 0e1 2 3 4 5e6 Absence Presence
OR
Inferior limit 95%
Superior limit 95%
p-value
1 4.57 4.16 2.79 e 1.16 2.81 4.02 5.83 1 3.26
e 1.98 1.79 1.17 e 0.31 0.8 1.13 1.14 e 1.97
e 10.53 9.69 6.67 e 4.27 9.81 14.32 29.86 e 5.4
e 0.000 0.001 0.021 e 0.827 0.106 0.032 0.034 e 0.000
Aminoglicosydes Beta-lactams Quinolones Macrolides Polymyxin E Sulfonamides Tetracyclines
No Yes No Yes No Yes No Yes No Yes No Yes No Yes
OR
Inferior limit 95%
Superior limit 95%
p-value
1 0.82 1 4.58 1 0.82 1 1.71 1 3.80 4.15 1 1 1.81
e 0.45 e 2.52 e 0.51 e 0.43 e 2.23 1.46 e e 0.83
e 1.48 e 8.33 e 1.33 e 6.77 e 6.46 11.79 e e 3.97
e 0.519 e 0.000 e 0.424 e 0.442 e 0.000 0.008 e e 0.139
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3.2. Antimicrobial use and C. difficile shedding
Table 5 Distribution of the genes coding for the major toxins, according to serotype.
During this survey, calves were treated with antibiotics to control the onset of diseases. The main reason reported for antimicrobial therapy after introduction to the herd was pneumonia. Treatment with tetracyclines and sulfonamides was common in both companies. Beta-lactams and polymyxin E were used only in herds belonging to company A, while florfenicol was used exclusively in calves belonging to company B. An association was found between antibiotic treatments before the first sampling and C. difficile shedding. In particular, an increased risk was shown when the number of antimicrobials used was higher than 4 (Table 2) or when polymyxin E or beta-lactams were administered (Table 3).
3.3. Presence of diarrhea and C. difficile Among calves excreting C. difficile at the first sampling, (n ¼ 85), 51 (48.2%) had diarrhea, while 44 (51.8%) showed no sign of diarrhea at the clinical examination. Among C. difficile negative animals at 0e16 days after introduction, (n ¼ 335), 68 (20.3%) had diarrhea, while 238 (71%) did not. The clinical evaluation could not be done in 29 cases (8.7%). The presence of diarrhea, detected at first sampling, was significantly associated with the recovery of C. difficile in feces: OR 3.26 (CI 1.97e5.40, p-value 0.000) (Table 2).
3.4. Bio-molecular characterization Data on ribotypes are shown in Table 4. Eight different ribotypes are described, five of which were present in farms of both companies. Polymerase chain reaction ribotypes for which reference strains were available were named according to standard Cardiff nomenclature (RT and number). The others were named according to internal nomenclature (Treviso, TV, and number). Seventy eight out of 82 isolates belonged to four ribotypes only: RT-078 (N ¼ 43; 52.5%), RT-012 (N ¼ 16; 19.5%), RT-0126 (N ¼ 13; 15.9%) and RT-033 (N ¼ 6; 7.3%). Ribotype 078 was by far the most common, since 43 isolates belonged to this ribotype. The distribution of the most common ribotypes was similar in the two companies, except for RT033, isolated five times in company B and only once in company A. The two isolates shed by the same calf at 0e16 and 90e120 days of age belonged to the same ribotype, RT-012. In Table 5, the distribution of the genes coding for the major toxins is shown. tcdA and tcdB were present in 95% of the isolates, the binary toxin in 80.5% and the 39 bp deletion of tcdC in 76.8%. The profile shown by all the RT-078 isolates was tcdAþ/tcdBþ/binary toxinþ/39 base pair deletion of tcdCþ. Table 4 Distribution of the different ribotypes. Ribotype
RT-078 RT-012 RT-126 RT-033 TV40 TV41 TV42 TV43 Total
45
Company A
Company B
Total
N
%
N.
%
N.
%
37 15 11 1 1 1 e 1 67
55.2 22.4 16.4 1.5 1.5 1.5 e 1.5 100.0
6 1 2 5 e e 1 e 14
40 6.7 13.3 33.3 e e 6.7 e 100.0
43 16 13 6 1 1 1 1 82
52.5 19.5 15.9 7.3 1.2 1.2 1.2 1.2 100.0
Ribotype
tcdA
tcdB
CDT
tcdC deletion
Total
RT-078 RT-012 RT-126 RT-033 TV40 TV41 TV42 TV43 Total
43 16 13 6 e 1 1 1 81
43 16 13 e e 1 1 1 75
43 3 13 6 e 1 e e 66
43 e 12 6 1 1 e e 63
43 16 13 6 1 1 1 1 82
4. Discussion and conclusions This work provides original data on the epidemiology of C. difficile in veal calf herds. In literature, the prevalence of calves shedding C. difficile is quite variable, ranging from 7.6% [23] to 61% [24] in different surveys [8,14,15,24,25]. In a recent study, a prevalence of 28% was found in veal calves, with marked differences between farms [15]. It was suggested that different sampling time or testing methodology can be any one of the possible reasons to explain this variability [5,8,11]. In our investigation, the sampling scheme and the culture tests were the same in both companies, so the observed differences in C. difficile shedding prevalence between the two companies can reasonably be attributed to real differences of shedding in calves. However, other factors such as the hygienic procedures, feeding or antibiotic treatments adopted by the two companies could have influenced our data. Moreover, the calves originated from multiple sources, and calves belonging to the same group were introduced into the herd on different days. As sampling was performed after the last calf of a group was introduced, a variability in the age of the calves at each of the sampling points was recorded. In this study, the time of major risk for C. difficile shedding was immediately after introduction to the herd. Approximately 20% of veal calves were positive at first sampling, and this percentage declined sharply at 90e120 days. This decreasing trend has also been described by other authors [26] who found higher prevalence of infection in feedlot cattle shortly after the arrival. As mentioned above, a different pattern of shedding was found in a study carried out on veal calves by Houser et al. [15], where the peak of shedding was described just before slaughter, at 20e22 weeks of age. The authors attributed this discrepancy to farmerelated factors, which could have increased the environmental contamination or determined an increased susceptibility of the animals to the infection [8,14,15,24]. Even though we are not able to rule out the influences of these factors in our study, we have found that the pattern of shedding can also be explained by an age effect, as young age at first sampling was shown to be significantly associated with a higher probability of C. difficile in faeces. The peak time was recorded at 13e28 days of age. This is not surprising, since a higher susceptibility of younger animals to infection has already been observed in pigs [12] and poultry [27]. In a longitudinal study on calves carried out in Belgium [13], the trend of C. difficile shedding was similar to that observed in this investigation. The peak of shedding at 1e2 weeks of age was reported in other studies [8,13,23]. In our study, the age effect was shown to occur in a brief period, during the first 2e3 months of age, after which there was a progressive decrease. Remarkably, a calf of less than 35 days of age had four times more probability of being a C. difficileeshedder than a calf of 46e90 days of age. Since the discrepancy between the prevalence at different ages was high, the younger age effect was shown to be statistically and biologically significant and should be taken into account in designing and interpreting prevalence studies in cattle.
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C.F. Magistrali et al. / Anaerobe 33 (2015) 42e47
In our investigation, shedding was limited to the first production stages. No C. difficileeshedder was detected in the last sampling before slaughter, at approximately 170 days of age. Correspondingly, no cecal contents or carcass sponges were positive at slaughter, meaning that the prevalence of carcass contamination cannot be equal to or higher than 3.5%. This expected prevalence was chosen at the beginning of the study to determine the number of carcasses to be sampled, and it was based on data available in literature [12]. It is therefore possible that a few positive carcasses went undetected in our study. Data on carcass contamination by C. difficile reported in literature vary from study to study. The C. difficile housekeeping gene, tpi, was found in four out of one hundred veal calf carcass swabs before decontamination [19]. In a transversal study carried out in Belgium, C. difficile was isolated from 9.9% intestinal and 7.9% carcass samples from slaughtered cattle [28]. Again, the geographic origin of the animals, the hygiene procedures adopted at the slaughterhouse or the prevalence of colonization before slaughter, could have influenced the C. difficile carcass contamination [15]. It should be noted that in the present survey, the intestinal contents were not found positive at slaughter. Therefore, a contamination of carcasses from the same groups of animals seems less probable. According to our data, foodborne transmission via veal calf cannot be suspected as a risk for the consumer. The four most common ribotypes identified in this study, accounting for approximately 95% of all our isolates, corresponded to a profile of the international numerical designation: RT-078, RT012, RT-033 and RT-126. Our data are in agreement with previous findings on C. difficile ribotypes in cattle. Ribotype 078, which was prevalent in our investigation, is considered the most frequent ribotype in this animal [5,13,28] and both RT-033 and RT-012 were repeatedly described in the same species [5,13]. Ribotype 126, whose profile is very similar or even indistinguishable from RT-078, has already been shown as one of the predominant ribotypes in a veal calf farm in Belgium [13]. On the other hand, the three most common ribotypes isolated from calves, RT-078, RT-012 and RT-126, were also among the most common ribotypes detected in humans in Europe [4]. Calves shed mainly RT-078, belonging to toxinotype V, and RT078 is considered one of the emerging ribotypes in Europe, where it is reported as the third most common ribotype isolated from CDI in humans [4]. Most of the isolates collected in this work showed the encoding genes for the major toxins. The RT-033 isolates harbored tcdA and they were negative to tcdB, a profile which has been recently described in cattle [29]. All the RT-078 isolates were positive for the virulence-associated gene: this finding supports the hypothesis that calves, among other animal species, could act as a reservoir for zoonoticehypervirulent strains [7,12,30]. However, to reduce the burden of contamination on farms, information on the epidemiology of this microorganism in cattle is necessary and this is not readily available in literature yet. This work reduces this gap, providing new insight on possible risk factors for this infection in veal calf primary production. In the present study, a difference in the use of antimicrobials in the two companies was found. In particular, only herds belonging to company A used beta-lactams for therapy. The use of betalactams before first sampling was associated with an OR of 4.58 for C. difficile shedding. In humans, betaelactams are considered particularly effective in determining CDI. In a recent survey, the higher incidence of C. difficile in hospitals was characterized by the use of beta-lactams in patients [4]. Therefore, even if several management practices could explain the difference in prevalence between the two companies, it can be argued that a major role could have been played by antimicrobials. The number of antibiotics was also associated with an increased
risk for shedding, as calves treated with more than four classes of antimicrobials before sampling were four times more likely to shed C. difficile. As far as we know, this is the first time that the use of antimicrobials have been linked to C. difficile shedding in calves. It cannot be excluded that the association observed was indirect, since antibiotics were prescribed to control a disease and the health status could have influenced the shedding of C. difficile. However, this link has been already shown in other animal species, such as the horse, dog and cat, although with contrasting results in different studies [5]. Antimicrobial therapy is the major risk factor for CDI in man [2] and the first report of this association dates back to 1978 [31]. Antimicrobials favor CDI by the disruption of the intestinal microbiota, which in physiological conditions, protects the gut against C. difficile colonization and expansion [32]. It would be reasonable to presume that this mechanism justifies the same association in veal calves. The extensive use of antimicrobials in both human and animal medicine raises concerns regarding the emergence and spreading of multi-resistant organisms. In Italy, veterinary prescription is mandatory for antimicrobial therapy in animals. Nevertheless, in a survey done in 2004, the prophylactic use of antibiotics was common among cattle veterinarians and the use of fluoquinolones as first or second choice for the treatment of calf scours was reported [33]. Better prevention measures and improved biosecurity, implemented to reduce the use of antimicrobials in the veal calf production chain, can have positive consequences on the C. difficile infection as well. Finally, a link between the presence of diarrhea and C. difficile shedding was shown in the animals at first sampling. An association between enteric disorders and CDI in calves has been shown in other studies [11,23], but the question of whether C. difficile could be an agent of calf diarrhea is still debated in the literature. The experimental challenge of calves with C. difficile ribotype 077 failed to induce C. difficile-associated disease (CDAD), questioning the role of C. difficile as a primary pathogen in cattle [34]. However, the pathogenesis of CDAD can be more complex, involving the presence of predisposing factors causing a disruption of the intestinal flora and an increased susceptibility to the infection, like in humans [5]. Therefore, the role of C. difficile as a cause of diarrhea in cattle cannot be definitely ruled out [34]. It is possible that the administration of antimicrobials in veal calves had induced an increased susceptibility to CDAD in our study. On the other hand, the association observed during the present study could be justified by several factors, and it should not necessarily be linked to a role of C. difficile in cattle enteritis. For example, an increase in shedding during diarrhea could be postulated. Moreover, diarrhea and C. difficile shedding can result from a common risk factor, for example change of diet and/or a change in the gut flora determined by antimicrobials. In conclusion, this work further documents that C. difficile shedding in the veal calf is linked to younger age and to the presence of diarrhea. For the first time, the use of antimicrobials has been directly linked to the bacterial shedding in cattle, suggesting new approaches to the control of C. difficile spreading on farms. Even though shedding was limited to the first phase of the production chain, the presence of emerging ribotypes suggests a possible role of the veal calf as a reservoir of C. difficile for humans. Further work is therefore needed to investigate common risk factors and possible control measures in veal calf primary production.
Funding This work was funded by the Italian Ministry of Health (Progetto di ricerca corrente) IZSUM 07/2009.
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