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Veterinary Microbiology 237 (2019) 108419 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Prevalence of enteric pathogens in diarrheic and non-diarrheic samples from T pig farms with neonatal diarrhea in the North East of Spain Anna Vidala, , Gerard E. Martín-Vallsa, Montse Telloa, Enric Mateua,b, Marga Martína,b,1, ⁎ Laila Darwicha,b,1 a Departament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallès, Spain b UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallès, Spain ARTICLE INFO ABSTRACT Keywords: Diarrhea is one of the major causes of neonatal mortality in pigs. In the present study, 31 pig farms with Neonatal diarrhea outbreaks of neonatal diarrhea were investigated in Catalonia (NE Spain) from February 2017 until June 2018. Pigs Two hundred and fifteen diarrheic samples from 1 to 7 days old piglets were tested for a panel of enteric Rotavirus pathogens. In 19 of the studied farms additional fecal samples from apparently healthy pen-mates were collected Coronavirus and tested for the same panel of infectious agents. Samples were bacteriologically cultured and tested by PCR for Bacterial enteric pathogens E. coli virulence factors genes, C. perfringens types A and C toxins (Cpα, Cpβ, Cpβ2) and C. difficile toxins (TcdA, TcdB). Moreover, Rotavirus A (RVA), Rotavirus B (RVB), Rotavirus C (RVC), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were also determined by RT-qPCR. More than one pa- thogen could be detected in all of the outbreaks. Nevertheless, RVA was the only agent that could be statistically correlated with the outcome of diarrhea. For the other viruses and bacteria analyzed significant differences between the diseased pigs and the controls were not found. In spite of this, the individual analysis of each of the studied farms indicated that other agents such as RVB, RVC, toxigenic C. difficile or pathogenic E. coli could play a relevant role in the outbreak of diarrhea. In conclusion, the large diversity of agent combinations and disease situations detected in neonatal diarrhea outbreaks of this study stand for a more personalized diagnosis and management advice at a farm level. 1. Introduction have regained attention as agents of diarrhea in pigs. In regards to rotaviruses, although several genogroups (A, B, C, E and H) have been Neonatal diarrhea is one of the most frequently disease in modern associated with porcine diarrhea, rotavirus A (RVA) is the most fre- swine production, which can be associated with high mortality, de- quent (Marthaler et al., 2014). Other species such as rotavirus B (RVB) creased growth rates and increase of treatment costs (Sjölund et al., and C (RVC) have been identified less commonly in diarrheal outbreaks 2014). Infectious and non-infectious factors can be involved in diarrhea (Morin et al., 1990; Martella et al., 2007; Amimo et al., 2013b). RVB outbreaks in suckling piglets. Among non-infectious factors stress, poor has been reported in several Asian countries, North America, South husbandry and nutrition can contribute to an animal's susceptibility to Africa and Brazil, but rarely in Europe (Smitalova et al., 2009; Otto disease. Moreover, enteric outbreaks are usually associated to the pre- et al., 2015). However, there are still few studies conducted on RVB, sence of infectious agents, such as viruses, bacteria or coccidian, al- RVC and other genogroups to determine their importance in porcine though the presence of pathogens in piglets alone does not determine diarrhea outbreaks. the occurrence of diarrhea episodes (Ruiz et al., 2016). All those pa- Regarding porcine coronaviruses, transmissible gastroenteritis virus thogens can act as primary and sole agents of scours in piglets although (TGEV) and porcine epidemic diarrhea virus (PEDV) can also cause co-infections are commonly reported (Kongsted et al., 2018; Mesonero- diarrheal outbreaks with high morbidity and mortality in neonatal pigs. Escuredo et al., 2018). However, since 2014 the most recent outbreaks in Europe have been In recent years, viruses -particularly coronaviruses and rotaviruses- related with PEDV (Carvajal et al., 2015; Laranjo et al., 2015). Corresponding author at: Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Autonoma de Barcelona, Travessera dels Turons s/n, ⁎ CP 08193, Cerdanyola del Vallès, Spain. E-mail address: anna.vidal@uab.cat (A. Vidal). 1 Both authors supervised this work equally. https://doi.org/10.1016/j.vetmic.2019.108419 Received 4 June 2019; Received in revised form 6 September 2019; Accepted 6 September 2019 0378-1135/ © 2019 Elsevier B.V. All rights reserved. A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 As regards bacterial infections, Escherichia coli (E. coli) has histori- cally been considered one of the main agents causing neonatal diarrhea in pigs (Chan et al., 2013). Different E. coli pathotypes have been identified based on toxin production and other virulence factors. The most common are the enterotoxigenic E. coli (ETEC) strains, char- acterized by the production of enterotoxins (STa, STb and LT). Other pathotypes of E.coli have been detected in piglets, such as en- teropathogenic E. coli (EPEC) strains, producing intimin (eae gene), although less frequently (Toledo et al., 2012). Anaerobic bacterial pa- thogens such as enterotoxigenic strains of Clostridium perfringens (C. perfringens) type A (producing Cpα toxin), C. perfringens type C (pro- ducing Cpα and Cpβ toxins) and Clostridium difficile (C. difficile) pro- ducing enterotoxin A (TcdA) and/or cytotoxin B (TcdB) have also been reported in diseased piglets (Uzal and Songer, 2019). Fig. 1. Proportion of positive samples for each analyzed farms (n = 31) and Ideally, diagnosis of outbreaks of neonatal diarrhea should consider enteric pathogens by Boxplot. RVA/B/C, rotavirus A/B/C; PCoV, porcine cor- onaviruses (PEDV and TGEV); TcdA/B, C. difficile toxins; Cpα/β2, C. perfringens the clinical findings and lesions, the epidemiological pattern and the toxins; ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. coli; VTEC, detection of the infectious agents potentially involved. However, most verotoxigenic E. coli. The distribution of data is displayed as follows: the box is often the diagnosis of enteric diseases is mainly focused on some pre- determined by the Interquartile Range (IQR: 25th and 75th percentiles) and the dominant infectious agents. Yet, in most cases, several agents with the median line shows the middle value of the dataset; the whiskers are determined potential for producing diarrhea in piglets are found in the same out- by the 5th and 95th percentiles; minimum and maximum values are shown at break. the ends of the bars and outliers as gray dots. The objective of this study was to determine the prevalence of several pathogens related with neonatal diarrhea and to compare their Table 1 frequencies with that of healthy penmates in a framework of diagnostic Prevalence of viral agents and clostridial toxin genes detected by PCR in sam- analysis. ples of diarrheic piglets (n = 215) from 31 tested farms. Pathogen Piglets (N = 215) Farms (N = 31) 2. Materials and methods Number % Number % 2.1. Sampling Viral agents Thirty-one conventional farrow-to-finish farms presenting neonatal RVA 111 51.6 25 80.6 RVB 20 9.3 7 22.6 diarrhea in piglets aged between 1 and 7 days were included in the RVC 84 39.1 22 71 study. In each farm, 10 samples from diarrheic animals and 5 samples PCoV 11 5.1 7 22.6 from apparently healthy penmates were asked to be collected. Fecal C. perfringens samples were submitted for diagnostic to the Laboratori Veterinari de Cpα 152 70.7 31 100 Cpβ 7 3.3 2 6.4 Diagnosi de Malalties Infeccioses, of the Universitat Autònoma de Cpβ2 132 61.4 30 96.8 Barcelona (Spain), between February 2017 and June 2018. Farms were C. difficile located in Catalonia (NE of Spain), one of the regions of Europe with a TcdA 62 28.9 25 80.6 higher pig density (242 pigs/km2). Finally, a total of 215 diarrheic TcdB 73 34 25 80.6 samples were taken from the 31 tested farms (5–10 animals/farm). Additionally, from 19 of these studied farms, 88 fecal samples (3–5 animals/farm) were obtained from apparently healthy pen-mates that Table 2 Prevalence of E. coli pathotypes, virulence factors and toxins at animal and farm did not present diarrhea at the moment of sampling. One gram of fecal level. sample was obtained directly from the animals using rectal swabs. A farm was considered to be positive for a specific pathogen when at least Pathotype Adhesins Toxins Pigs Farms one sample of the tested animals was found positive for that pathogen. n % n % 2.2. Microbiological testing ETEC F4 STa, STb 2 1 1 3.2 ND LT 2 1 1 3.2 Stool samples were directly analyzed upon arrival for micro- LT, STb 1 0.5 1 3.2 STa, STb 1 0.5 1 3.2 biological identification of E. coli, C. perfringens and C. difficile and an STa 1 0.5 1 3.2 aliquot of each sample was stored at −80 °C. STb 12 5.6 6 19.4 For E. coli isolation, samples were aerobically cultured on Columbia EPEC F18, eae ND 1 0.5 1 3.2 blood agar (BD GmBh, Germany) and MacConkey agar plates (Oxoid, F41, eae 2 1 2 6.5 eae 14 6.5 8 25.8 UK), and were incubated during 24 h at 37 °C. VTEC ND VT1 3 1.5 3 9.7 To recover C. perfringens and C. difficile from faeces, samples were ETEC/EPEC F41, eae STb 1 0.5 1 3.2 firstly treated with ethanol (96%) 35 min to eliminate the vegetative ETEC/VTEC F4 STa, STb, VT2 1 0.5 1 3.2 cells and then centrifuged (x 8000g) as described by Koransky et al. ND VT2, STb 5 2.3 3 9.7 (1978). The pellet was then cultured on a selective medium Clostridium EPEC/VTEC eae VT1 3 1.5 1 3.2 difficile agar base (Conda Laboratorios, Spain), and incubated anaero- ND: not detected. bically for 48 h at 37 °C. sterile distilled water before centrifuging. The Nucleospin RNA ex- 2.3. Molecular diagnosis of viral agents traction kit (Macherey-Nagel, Germany) was used following the man- ufacturer’s instructions. The final extracted RNA was suspended in Faecal samples were centrifuged (6000g, 5 min) before the RNA 50 μL of RNAse-free water (Macherey-Nagel, Germany). Detection of extraction. Non-diarrheic samples were initially diluted in 500 μL of 2 A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 Table 3 Distribution of farms positive to the different panel of enteric pathogens. RVA/B/C, Rotavirus A/B/C; C. difficile, toxigenic strains (TcdA, TcdB); E. coli, pathogenic E.coli; PCoV, porcine coronaviruses; Cp A/C, C. perfringens A/C. Farms N RVA RVB RVC PCoV C.difficile Cp A Cp C E.coli Number of positive farms: 6 + – + – + + – + enteric pathogen 4 + – + – + + – – associations 3 + – – – + + – + 3 – + + – + + – + 2 + – + + + + – + 1 + + + + + + – + 1 + – – + + + – + 1 + – – + + + – – 1 + – + – + + + + 1 + – – – + + + + 1 + + + – + + – – 1 + + + + – + – – 1 + – + – – + – + 1 + – – + – + – – 1 + – – – – + – – 1 – + + – + + – – 1 – – + – + + – + 1 – – – – + + – – TOTAL FARMS 31 25 7 22 7 27 31 2 20 Table 4 each tube. Tubes were boiled in a water bath for 10 min, and then Proportion and statistical values of enteric agents between diarrheic (n = 140) centrifuged at 13,000 rpm for 5 min. After centrifugation, the super- and healthy (n = 88) animals. natant was recovered and stored at −80 °C until processed. Agent Proportion Proportion Pearson p-value Fisher’s The presence of E. coli adhesins (F4, F5, F6, F18, F41 and eae) and Cases % (n) Controls % (n) Chi-square exact test toxins (LT, Sta, STb, EAST1) was analysed using conventional PCR. VT1 (p) and VT2 toxins were included as a routine basis in this general diag- nostic panel of E. coli. C. perfringens (α, β and β2) and C. difficile (TcdA Viral agents RVA 61.4 (86) 31.8 (28) 18.95 0. 00013 – and TcdB) toxins were also evaluated by PCR. RVB 12.1 (17) 4.9 (6) 1.69 0.19 – For all PCR, the master mix consisted of: 1x PCR Buffer, 0.2 mM of RVC 33.6 (47) 36.4 (32) 0.18 0.67 – each dNTP (Bioline, France), 3 mM of MgCl2, 1 mM of each primer and PCoV 4.3 (6) 2.3 (2) 0.65 – 0.72 1 U of Taq DNA Polymerase (Bioline, France). A final volume of 2.5 μL C. perfringens of DNA was used in the PCR. In each reaction, positive and negative Cpα 73.5 (103) 79.5 (70) 1.05 0.30 – Cpβ 2.8 (4) 1.1 (1) 0.75 – 0.65 controls were included. Cpβ2 60.7 (85) 61.4 (54) 0.01 0.922 – The characterisation of E.coli, as regards to the presence of adhesins C. difficile and eae) and toxins was done using the primers described by Toledo TcdA 25.7 (36) 19.3 (17) 1.24 0.27 – et al. (2012). The PCR program consisted of 5 min at 94 °C, followed by TcdB 27.1 (38) 29.5 (26) 0.15 0.69 – E. coli adhesins 30 cycles of 1 min at 94 °C, 1 min of annealing at 63 °C and 1 min of F4 0 1.1 (1) – – – extension at 72°, and a final extension step of 7 min at 72 °C. F5 0.7 (1) 0 – – – The detection of toxigenic C. difficile strains was done by a stan- F6 0 0 – – – dardized PCR protocol for TcdA and TcdB previously described by F18 0.7 (1) 0 – – – Persson et al. (2008). The PCR program consisted of 10 min at 94 °C, F41 3.6 (5) 1.1 (1) 1.25 – 0.41 eae 13.6 (19) 14.8 (13) 0.065 0.8 – followed by 25 cycles of 50 s at 94 °C, 40 s of annealing at 53 °C and 50 s E. coli toxins of extension at 72 °C, and a final extension step of 3 min at 72 °C. For LT 0 0 – – – the detection of C. perfringens type A and C, specific PCR were carried Sta 1.4 (2) 0 – – – out using the primers described by van Asten et al. (2009), and the Stb 5.7 (8) 3.4 (3) 0.63 – 0.54 EAST1 57.1 (80) 67 (59) 2.23 0.14 – program consisted of 5 min at 95 °C, followed by 30 cycles of 1 min at VT1 2.9 (4) 0 – – – 94 °C, 1 min annealing at 53 °C and 1 min of extension at 72 °C, and a VT2 2.9 (4) 0 – – – final extension step of 10 min at 72 °C. Reference E. coli and C. perfringens strains used as positive controls were kindly donated by Dr. Blanco (E. coli Reference Laboratory, viral agents was done using the AgPath-ID™ One-Step RT-PCR kit Santiago de Compostela, Spain). Positive C. difficile strain was kindly (Applied Biosystems, ThermoFisher, USA). For RVA, PEDV and TGEV, provided by Dr. Sanfeliu (UDIAT Diagnostic Centre, Sabadell, Spain). the protocol designed by Masuda et al. (2016) was followed, and RVB PCR products were resolved in a 1.5% agarose gel by electrophor- and RVC were detected using a previously described RT-PCR by esis. Reference positive strains and a 100 bp ladder (Biotools B&M labs, Marthaler et al. (2014). Spain) were used to identify the positive samples. Amplified PCR pro- ducts were visualized using ethidium bromide staining under UV light. 2.4. Molecular diagnosis of bacterial agents 3. Results DNA was extracted from bacterial cultures by boiling. Briefly, all bacterial growth from MacConkey plates and Clostridium spp. selective 3.1. Overall prevalence of enteric pathogens in the diarrheic outbreaks medium plates from all samples were diluted in 600 μL of sterile dis- tilled water and 200 μL of the dilution were then transferred to a new The prevalence of enteric pathogens from the diarrheic cases eval- tube. Two-hundred microliters of sterile distilled water were added to uated in the study showed a high diversity in the proportion of positive 3 A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 samples per each of the 31 farm tested (Fig. 1). C. perfringens A, C. 3.2. Prevalence and combination of enteric pathogens at farm level difficile toxigenic strains, and RVA and RVC were the most frequently agents diagnosed at farm level (Fig. 1). Rotaviruses were detected in 30 out of 31 farms (Table 3). RVA Regarding the number of diarrheic animals in the overall population (80.6%) and RVC (71%) were isolated from most of the farms (25 and analyzed, viruses presented the following prevalence (Table 1): 51.6% 22, respectively), and were detected concomitantly in 17 of them (111/215) of samples were positive for RVA, 9.3% (20/215) for RVB (54.8%). By contrast, only 7 farms were positive to RVB, always found and 39.1% (84/215) for RVC. The percentage of samples positive to in co-infection with RVC. Finally, PEDV and TGEV were detected in 4 coronaviruses was low (11/215, 5.1%), 6 of them being positive to and 3 farms respectively. TGEV and 5 to PEDV. Regarding bacterial agents, C. perfringens Cpα As regards the bacterial agents, C. perfringens A was found in 100% toxin was found in 71% (152/215) of the samples while only 7 samples of farms, followed by C. difficile toxigenic strains (87.1% farms). (3.3%) from two different farms were found positive for Cpβ toxin Pathogenic E.coli, mainly ETEC and EPEC strains, was found in 64.5% (Table 1). Moreover, Cpβ2 toxin was detected in 87% of Cpα toxin of farms. Finally, 58% of farms were positive to RVA, RVC, C.difficile positive samples (132/152). TcdA and TcdB C. difficile isolates were and CpA co-infection (Table 3). found in 28.9% (62/215) and 34% (73/215) of the samples, respec- tively, 22.3% of the samples being TcdA/TcdB double positive. E. coli was isolated in pure culture in 44% (94/215) of the tested 3.3. Comparison of results between diarrheic and healthy piglets samples from diarrheic animals. The virulence factor characterization of these 94 isolates showed a low prevalence (< 5%) of E. coli toxins Diarrheic animals (n = 140) and non-diarrheic (n = 88) penmates and fimbriae, except for STb (10.7%), eae (9.8%) and EAST1 (56%) were sampled in 19 farms. RVA was the only pathogen statistically genes (Table 2). E. coli strains that could be classified into a pathotype associated with the cases of diarrhea [61.4% vs 31.8%, p < 0.001] were isolated from 21/31 (67.7%) farms but with a low proportion of (Table 4). Regarding bacterial pathogens no statistical differences were positive samples. The highest prevalence corresponded to the ETEC found when comparing diseased versus non-diseased pen-mates al- pathotype (12.1%), harboring STa, STb and/or LT genes, followed by though prevalence of C. difficile TcdA and E. coli F41 or STa toxigenic the EPEC pathotype (9.8%) with the eae gene, and lasting with an oc- strains were slightly higher in the diseased animals (Table 4). casional VTEC (5.1%) strains, none of them harboring neither VT1 and There were 6/19 farms in which RVA could not have a prominent VT2 genes. role in the diarrhea outbreak, either because of the absence of RVA positive animals in the farm or because the RVA prevalence was higher Fig. 2. Comparison of prevalence of positive samples between diarrheic (D, black bar) and non-diarrheic groups (ND, light bar) distributed by farms (Fn) and enteric pathogens. RVA/B/C, rotavirus A/B/C; PCoV, porcine coronaviruses (PEDV and TGEV); TcdA/B, C. difficile toxins; Cpα/β2, C. perfringens toxins; ETEC, en- terotoxigenic E. coli; EPEC, enteropathogenic E. coli; VTEC, verotoxigenic E. coli. 4 A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 Fig. 2. (continued) in healthy animals than in diseased cases. In those particular cases, along with other non-infectious causes, stands for the multifactorial other bacterial agents such as toxigenic C. difficile (TcdA / TcdB) or origin of the neonatal diarrhea in pigs and shows the actual complexity pathogenic E. coli (mainly ETEC or VTEC), or other viruses, such as of this condition. RVB, could be identified in a larger proportion of diseased pigs com- One of the main results of this study is the evidence of RVA as the pared to the healthy pen-mates (Fig. 2). most frequent agent involved in neonatal diarrhea of the studied cases. This result agrees with a recent case-control study conducted in pig 4. Discussion farms in Denmark, in which the authors concluded that RVA was the only agent that could be statistically associated to neonatal diarrhea The present study reports data of the prevalence of the main pa- (Kongsted et al., 2018). Nevertheless in some cases their role as a thogens associated with neonatal diarrhea in Spain. In general, C. per- causative agent of disease in pigs have been controversial. While some fringens type A, toxigenic C. difficile and rotavirus could be isolated from studies did not find a clear association between RVA infection and most of the analyzed farms. Furthermore, most of the analyzed samples neonatal disease (Ruiz et al., 2016; Amimo et al., 2013a), others did of this study, regardless the health status of the piglet, were positive for find a statistical relationship between neonatal diarrhea and RVA single multiple combinations of pathogens, involving principally RVA, RVC, E. infection (Linares et al., 2009; Kongsted et al., 2018; Mesonero- coli, and toxigenic strains of C. difficile and C. perfringens type A. Escuredo et al., 2018), or RVA combined with other factors such as co- Moreover, the large number of combinations of pathogens, possibly infections or management conditions (Ruiz et al., 2016). 5 A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 Fig. 2. (continued) As regards to other rotaviruses, most of the RVC positive farms the present study it must be mentioned that the PCR that we used could found in this study were also positive to RVA, and had similar fre- not distinguish TGEV from porcine respiratory coronavirus (PRCV) quencies of RVC positive animals in piglets suffering diarrhea and in the since the target gene was the viral nucleocapside. Besides this, the healthy penmates. A recent study made in Danish pigs affected by the spread of PRCV across Europe since its emergence in the 1980 decade new neonatal porcine diarrhea syndrome (NNPDS) found that re- reduced the prevalence of TGEV because of the crossed immunity be- garding rotaviruses only 9% of pigs were positive to RVA and none to tween the two viruses. Nowadays diarrhea caused by TGEV is un- RVC by RT-qPCR (Goecke et al., 2017). By contrast, for RVB, differ- common in Europe (Saif et al., 2012). ences in the frequency of this pathogen in diseased and healthy piglets The prevalence of C. perfringens type A, as well as the Cpβ2 positive were clearer although the global RVB prevalence was low. Thus, in the strains, was very high and similar between diarrheic and healthy pigs. context of our study, both RVA and RVB could be considered as pro- The role of the cpβ2 toxin in the pathogenesis of neonatal diarrhea is minent causing agents of diarrhea in some outbreaks. controversial and while some studies have associated it with diarrhea The number of papers on the prevalence of RVB and RVC in pigs is outbreaks (Garmory et al., 2000; Bueschel et al., 2003), others found no relatively scarce. In a study conducted in the United States, the authors differences between diseased and healthy pigs (Jäggi et al., 2009; reported similar rotaviruses prevalence with 62% RVA, followed by Farzan et al., 2013; Lee et al., 2014). Since C. perfringens type A is a 53% RVC and 33% RVB (Marthaler et al., 2014). Rotavirus B has also common gut microorganism, the detection of this agent cannot be in- been detected in several Asian countries, South Africa, and Brazil terpreted unambiguously, given that it is not possible to distinguish (Alekseev et al., 2015). In Europe, limited reports of RVB have been commensal from pathogenic strains. Thus, although it has been con- described in Germany (Otto et al., 2015) and the Czech Republic sidered as a main pathogen involved in persistent neonatal diarrhea (Smitalova et al., 2009) so far, with prevalence of 1.6% and 0.6% re- (Mesonero-Escuredo et al., 2018), a direct pathogen-toxin-disease as- spectively. The differences in the obtained prevalence between those sociation has not been yet determined (Kongsted et al., 2013, Kongsted studies and the present work could be explained by the study design, et al., 2018). As regards C. perfringens type C, the prevalence detected in the age of the animals or the geographical area of sampling. the present study was low. This could be the result of the routine Regarding the analyzed coronaviruses, PEDV and TGEV, only 13 vaccination plan implemented in sows (Salvarani et al., 2013). animals from 8 different farms were positive. Similar results were found It has been suggested that C. difficile could be one of the most im- recently in Spain by Mesonero-Escuredo et al. (2018), who reported a portant uncontrolled cause of neonatal diarrhea in pigs in some sce- 3.7% prevalence for PEDV. Regarding the positive samples to TGEV in narios (Songer and Anderson, 2006) with significantly higher 6 A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 prevalence in diarrheic piglets (Kim et al., 2018). However, in our Carvajal, A., Argüello, H., Martínez-Lobo, F.J., Costillas, S., Miranda, R., de Nova, P.J.G., study, the general prevalence of toxigenic C. difficile was similar in both Rubio, P., 2015. Porcine epidemic diarrhoea: new insights into an old disease. Porcine Health Manag. 1, 12. healthy and diarrheic animals. Other studies have reported a high Chan, G., Farzan, A., DeLay, J., McEwen, B., Prescott, J.F., Friendship, R.M., 2013. A prevalence of C. difficile toxins in healthy animals concluding no clear retrospective study on the etiological diagnoses of diarrhea in neonatal piglets in relationship between diarrheal outbreaks and the detection of toxigenic Ontario, Canada, between 2001 and 2010. Can. J. Vet. Res. 77, 254–260. Dubreuil, J.D., Isaacson, R.E., Schifferli, D.M., 2016. Animal enterotoxigenic Escherichia C. difficile in pigs (Yaeger et al., 2007; Álvarez-Pérez et al., 2009). coli. EcoSal Plus 7. https://doi.org/10.1128/ecosalplus.ESP-0006-2016. ETEC has been and still is considered the main agent responsible for Farzan, A., Kircanski, J., DeLay, J., Soltes, G., Songer, J.G., Friendship, R., Prescott, J.F., intestinal disorders in neonatal piglets being F4, F5, F6 and F41, the 2013. An investigation into the association between cpb2-encoding Clostridium perfringens type A and diarrhea in neonatal piglets. Can. J. Vet. Res. 77, 45–53. main fimbriae associated with diarrhea, (Dubreuil et al., 2016; Luppi Garmory, H.S., Chanter, N., French, N.P., Bueschel, D., Songer, J.G., Titball, R.W., 2000. et al., 2016). In the present study, ETEC strains were infrequently iso- Occurrence of Clostridium perfringens beta2-toxin amongst animals, determined lated from both diarrheic and non-diarrheic piglets, similar to the re- using genotyping and subtyping PCR assays. Epidemiol. Infect. 124, 61–67. Jäggi, M., Wollschläger, N., Abril, C., Albini, S., Brachelente, C., Wyder, M., Posthaus, H., sults reported previously by others (Kongsted et al., 2013; Kongsted 2009. Retrospective study on necrotizing enteritis in piglets in Switzerland. Schweiz. et al., 2018; Larsson et al., 2015; Mesonero-Escuredo et al., 2018). This Arch. Tierheilkd. 151, 369–375. low prevalence of E. coli pathotypes and virulent factors is probably Kim, H.Y., Cho, A., Kim, J.W., Kim, H., Kim, B., 2018. High prevalence of Clostridium related to the E. coli vaccination programs implemented in sows in the difficile PCR ribotype 078 in pigs in Korea. Anaerobe 51, 42–46. Kongsted, H., Jonach, B., Haugegaard, S., Angen, O., Jorsal, S.E., Kokotovic, B., Larsen, Spanish farms. Most of these vaccines available on the market contain L.E., Jensen, T.K., Nielsen, J.P., 2013. Microbiological, pathological and histological E. coli fimbriae (mostly F4, F5, F6 and F41) and toxoids (such as LT), findings in four Danish pig herds affected by a new neonatal diarrhoea syndrome. and therefore, prevent the infection caused by pathogenic strains of E. BMC Vet. Res. 9, 206. Kongsted, H., Pedersen, K., Hjulsager, C.K., Larsen, L.E., Pedersen, K.S., Jorsal, S.E., coli. By contrast, EAST1 positive E. coli were very common. The pa- Bækbo, P., 2018. Diarrhoea in neonatal piglets: a case control study on micro- thogenic role of the EAST1 toxin is not clear, given that it has also been biological findings. Porcine Health Manag. 4, 17. found in a high prevalence in strains from healthy animals of our study Koransky, J.R., Allen, S.D., Dowell, V.R., 1978. Use of ethanol for selective isolation of sporeforming microorganisms. Appl. Environ. Microbiol. 35, 762–765. in agreement with the results published by Zajacova et al. Laranjo, M., Allepuz, A., Pleguezuelos, P., López, S., Casal, J., Segalés, J., Martín, M., (2012).Nevertheless, in some farms in which RVA was not considered to Pujols, J., 2015. Estudio epidemiológico prospectivo en granjas con brotes recientes be the main causative agent, the diarrheic process of the piglets could de diarrea epidémica porcina. Unpublished report. Larsson, J., Aspán, A., Lindberg, R., Grandon, R., Båverud, V., Fall, N., Jacobson, M., be associated to pathogenic E. coli or toxigenic C. difficile strains. 2015. Pathological and bacteriological characterization of neonatal porcine diar- The high frequency of multiple infections detected in diarrheic and rhoea of uncertain aetiology. J. Med. Microbiol. 64, 916–926. healthy piglets makes the setting up of a final diagnosis a very difficult Lee, K.E., Lim, S.I., Shin, S.H., Kwon, Y.K., Kim, H.Y., Song, J.Y., An, D.J., 2014. Distribution of Clostridium perfringens isolates from piglets in South Korea. J. Vet. task. Moreover, presence of pathogenic agents in the healthy group Med. Sci. 76, 745–749. should be interpreted with caution since some of these animals could be Linares, R.C., Fernandes Barry, A., Fernandes Alfieri, A., Médici, K.C., Feronato, C., sampled during the incubation period of the infection. Additional in- Grieder, W., Alfieri, A.A., 2009. Frequency of group A rotavirus in piglet stool sam- formation supplied by complementary techniques or studies, may help ples from non-vaccinated brazilian pig herds. Braz. Arch. Biol. Technol. 52, 63–68. Luppi, A., Gibellini, M., Gin, T., Vangroenweghe, F., Vandenbroucke, V., Bauerfeind, R., to achieve a definitive diagnosis. Bonilauri, P., Labarque, G., Hidalgo, A., 2016. Prevalence of virulence factors in In conclusion, the large diversity of agent combinations and disease enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhea in situations detected in the different pig farms confirms the multifactorial Europe. Porcine Health Manag. 2, 20. Martella, V., Bányai, K., Lorusso, E., Bellacicco, A.L., Decaro, N., Camero, M., Bozzo, G., origin of the neonatal diarrhea in pigs and stand for a more persona- Moschidou, P., Arista, S., Pezzotti, G., Lavazza, A., Buonavoglia, C., 2007. Prevalence lized diagnosis and management advice at a farm level, including also of group C rotaviruses in weaning and post-weaning pigs with enteritis. Vet. non-infectious factors that can trigger neonatal diarrhea. Microbiol. 123, 26–33. Marthaler, D., Homwong, N., Rossow, K., Culhane, M., Goyal, S., Collins, J., Matthijnssens, J., Ciarlet, M., 2014. Rapid detection and high occurrence of porcine Declaration of Competing Interest rotavirus A, B and C by RT-qPCR in diagnostic samples. J. Virol. Methods 209, 30–34. Masuda, T., Tsuchiaka, S., Ashiba, T., Yamasato, H., Fukunari, K., Omatsu, T., Furuya, T., Shirai, J., Mizutani, T., Nagai, M., 2016. Development of one-step real-time reverse The authors do not have any conflict of interest. transcriptase-PCR-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea. Jpn. J. Vet. Res. 64, 5–14. Acknowledgements Mesonero-Escuredo, S., Strutzberg-Minder, K., Casanovas, C., Segalés, J., 2018. Viral and bacterial investigations on the aetiology of recurrent pig neonatal diarrhea cases in Spain. Porcine Health Manag. 5, 4–5. Heartfelt thanks to all the collaborating veterinarians. A. Vidal was Morin, M., Magar, R., Robinson, Y., 1990. Porcine group C rotavirus as a cause of neo- supported by a PIF fellowship from the Universitat Autònoma de natal diarrea in a Quebec swine herd. Can. J. Vet. Res. 54, 385–389. Barcelona. This work was partly funded by INIAE-RTA2015-00003- Otto, P.H., Rosenhain, S., Elschner, M.C., Hotzel, H., Machnowska, P., Trojnar, E., Hoffmann, K., Johne, R., 2015. Detection of rotavirus species A, B and C in domestic C02-01 grant. mammalian animals with diarrhea and genotyping of bovine species A rotavirus strains. Vet. Microbiol. 179, 168–176. References Persson, S., Torpdahl, M., Olsen, K.E., 2008. New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/ cdtB) genes applied to a Danish strain collection. Clin. Microbiol. Infect. 14, Alekseev, K.P., Penin, A.A., Mukhin, A.N., Khametova, K.M., Grebennikova, T.V., 1057–1064. Yuzhakov, A.G., Moskvina, A.S., Musienko, M.I., Raev, S.A., Mishin, A.M., Ruiz, V.L., Bersano, J.G., Carvalho, A.F., Catroxo, M.H., Chiebao, D.P., Gregori, F., Kotelnikov, A.P., Verkhovsky, O.A., Aliper, T.I., Nepoklonov, E.A., Herrera-Ibata, Miyashiro, S., Nassar, A.F., Oliveira, T.M., Ogata, R.A., Scarcelli, E.P., Tonietti, P.O., D.M., Shepherd, F.K., Marthaler, D.G., 2015. Genome characterization of a patho- 2016. Case-control study of pathogens involved in piglet diarrhea. BMC Res. Notes genic porcine Rotavirus B strain identified in Buryat Republic, Russia in 2015. 9, 22. Pathogens 7, 46. Saif, L.J., Pensaert, M.B., Sestak, K., Yeo, S.G., Jung, K., 2012. Coronaviruses. In: Álvarez-Pérez, S., Blanco, J.L., Bouza, E., Alba, P., Gibert, X., Maldonado, J., Garcia, M.E., Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), 2009. Prevalence of Clostridium difficile in diarrhoeic and non-diarrhoeic piglets. Diseases of Swine. Wiley-Blackwell, Hoboken, USA, pp. 501–524. Vet. Microbiol. 137, 302–305. Salvarani, F.M., Conceição, F.R., Cunha, C.E., Moreira, G.M., Pires, P.S., Silva, R.O., Alves, Amimo, J.O., Vlasova, A.N., Saif, L.J., 2013a. Detection and genetic diversity of porcine G.G., Lobato, F.C., 2013. Vaccination with recombinant Clostridium perfringens group A rotaviruses in historic (2004) and recent (2011 and 2012) swine fecal toxoids α and β promotes elevated antepartum and passive humoral immunity in samples in Ohio: predominance of the G9P[13] genotype in nursing piglets. J. Clin. swine. Vaccine 31, 4152–4155. Microbiol. 51, 1142–1151. Sjölund, M., Zoric, M., Wallgren, P., 2014. Financial impact of disease on pig production. Amimo, J.O., Vlasova, A.N., Saif, L.J., 2013b. Prevalence and heterogeneity of porcine In: Part III. Gastrointestinal Disorders. Proceedings of 6th European Symposium of group C rotaviruses in nursing and weaned piglets in Ohio, USA and identification of Porcine Health Management. Italy, Sorrento. p. 189. a potential new VP4 genotype. Vet. Microbiol. 164, 27–38. Smitalova, R., Rodak, L., Smid, B., Psikal, I., 2009. Detection of nongroup A rotaviruses in Bueschel, D.M., Jost, B.H., Billington, S.J., Trinh, H.T., Songer, J.G., 2003. Prevalence of faecal samples of pigs in the Czech Republic. Vet. Med. (Praha) 54, 12–18. cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of Songer, J.G., Anderson, M.A., 2006. Clostridium difficile: an important pathogen of food genotype with phenotype. Vet. Microbiol. 94, 121–129. animals. Anaerobe 12, 1–4. 7 A. Vidal, et al. Veterinary Microbiology 237 (2019) 108419 Toledo, A., Gómez, D., Cruz, C., Carreón, R., López, J., Giono, S., Castro, A.M., 2012. 136, 411–412. Prevalence of virulence genes in Escherichia coli strains isolated from piglets in the Yaeger, M.J., Kinyon, J.M., Songer, J.G., 2007. A prospective, case control study evalu- suckling and weaning period in Mexico. J. Med. Virol. 61, 148–156. ating the association between Clostridium difficile toxins in the colon of neonatal Uzal, F.A., Songer, J.G., 2019. Clostridial diseases. In: Zimmerman, J.J., Karriker, L.A., swine and gross and microscopic lesions. J. Vet. Diagn. Invest. 19, 52–59. Ramirez, A., Schwartz, K.J., Stevenson, G.W., Zhang, J. (Eds.), Diseases of Swine, Zajacova, Z.S., Konstantinova, L., Alexa, P., 2012. Detection of virulence factors of Eleventh Edition. John Wiley & Sons, Inc., Hoboken (NJ) USA, pp. 792–806. Escherichia coli focused on prevalence of EAST1 toxin in stool of diarrheic and non- Van Asten, A.J., van der Wiel, C.W., Nikolaou, G., Houwers, D.J., Gröne, A., 2009. A diarrheic piglets and presence of adhesion involving virulence factors in astA positive multiplex PCR for toxin typing of Clostridium perfringens isolates. Vet. Microbiol. strains. Vet. Microbiol. 154, 369–375. 8