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Background Iron deficiency (ID) is often a neglected condition in inflammatory bowel disease (IBD), even though it is one of the most common extraintestinal manifestation. Our questions were the follows: If iron deficiency is part of the T2T management as CRP or fecal calprotectin and requires optimisation of the current therapy, how does this affect the medium-term outcome of the disease and the effectiveness of IV iron replacement? If the disease is active, does optimisation of therapy is sufficient to manage iron deficiency or iv. iron supplementation is also necessary? Methods Patients with ID defined by the ECCO guideline (Figure 1.) were randomly included in our prospective one-center, four arms study. Patients were divided into an active (A) and a remission (R) group. Activity was defined by CRP ≥10 mg/l or clinical activity scores CDAI ≥150, pMayo ≥3. Therapy optimisation was performed in the active group, further randomisation was performed into two subgroups: therapy modification alone (A1) or modification with iv. ferric-carboxymaltose (A2). All patients received iron supplementation in the remission group with (R2 – iron T2T group) or without therapy optimisation (R2). Every two months laboratory parameters and clinical activities were assessed until 6 months. Results 65 patients were included (CD 83.4%). Average follow-up was 3.9 months. Number of patients per group: A1:17, A2:23, R1:16, R2:9. Based on our results, iv. iron supplementation with modification in treatment was more efficient, than therapy optimisation alone, as the difference in increase in haemoglobin (p Conclusion Our results suggest, that therapy optimisation in addition to iron replacement is more effective in patients with remission and therefore biomarkers of IDA should be involved in the thight monitoring algorhytm to react T2T. However, therapy optimisation has to be associated with iv iron replacement in IBD with IDA and activity without the risk of worsening disease activity. Based on our results, ferric-carboximaltose is an effective and safe therapeutic method in the treatment of IDA associated with active or inactive IBD.

Background Iron deficiency (ID) is one of the most common extraintestinal manifestation of inflammatory bowel disease, which often remains untreated, despite the fact, that ID significantly impairs patients’ quality of life. Since anaemia develops through several pathomechanisms (e.g., occult bleeding, chronic inflammation, medicines), treating anaemia requires to manage the underlying pathological changes as well. Methods In our prospective, two-centers study, we assessed the frequency of ID among our IBD patients. The diagnosis of iron deficiency (iron deficiency anaemia [IDA], anaemia of chronic inflammation [CDA], anaemia of mixed origin [AMO]) was based on the ECCO’s anaemia guideline. According to the guideline, haemoglobin, CRP, ferritin, transferrin saturation, serum iron values, total iron binding capacity, hematocrit, and platelet counts were collected from our patients on a one-off basis. We aimed to compare the frequency of ID in a biologic centre, and in a non-biologic centre hospital as well. In addition, we assessed the frequency of the need for oral and intravenous iron replacement, respectively, based on the guideline as well. Results 720 patients were included in our study (CD 58.8%, UC 41.2 %). Average age was 43.3 years. We found, that 26.6% of our patients had anemia. There was no significant difference in the prevalence of ID (p=0.65) between CD and UC patients. Based on the ECCO criteria, 61.8% of them have ID (33.0% IDA, 23.0% AMO, 5.8% CDA), and in the rest of the cases (38.2%) it was classified to be anaemia of other origin (AOO), however, in 68.5% of these cases ID can be suspected at some degree, as ferritin was < 100 mg/l and/or transferrin saturation ≤ 20%. In total, 133 patients (69.6%) had anaemia in remission, and 58 patients (30.4%) in case of activity (CRP ≥10 mg/l). In addition, we found an association between elevated CRP levels and the prevalence/severity of anemia (p120–130 g/l) already after 2 months in 76.5% of the cases. Conclusion Based on our results, anemia and iron deficiency are common complications of IBD and correlate well with disease activity. Consequently, we suggest, that iron deficiency should be a part of the treat to target approach, and in case of ID, iv. ferric-carboximaltose is efficient in activity as well.

Background Bowel preparation prior to colonoscopy is a routine procedure and supposed to be safe. However, the exacerbation of the complaints could be observed in numerous cases after colonoscopy in patients with inflammatory bowel disease (IBD, Crohn’s disease CD, ulcerative colitis UC). Therefore, our study aimed to investigate the short- and long-term changes of the fecal microbiota composition influenced by the bowel preparation with sodium-picosulphate and magnesium-oxide. Methods IBD patients (inactive or mild) preparing for colonoscopy with bowel preparation were enrolled in the study. Control group (HC) consisted of non-IBD patients who were recommended to undergo colonoscopy due to abdominal complaints. Clinical data, blood and stool samples were collected before colonoscopy (point A), 3 days later (point B) and 4 weeks later (point C) to assess disease activity and the changes of the gut microbiome. Fecal microbiota structure was determined by sequencing V4 region of the 16S rRNA gene. Microbiome structure was determined at family level. Statistical analysis included differential abundance analysis and Mann-Whitney test, values of p Results 41 patients (9 CD, 13 UC, 19 HC) were enrolled in our study. After the bowel preparation, alpha diversity in the CD group was lower compared to UC(p=0.01) and control(p=0.02) patients at point B. Alpha diversity of UC patients was significantly higher than CD (p‹0.01) and control (p=0.03) groups at point C. Alpha diversity of UC patients increased from point A to C (p=0.04). However, a decreasing tendency in alpha diversity was observed in patients with CD at A to C period (p=0.26). These changes couldn’t be observed in the control group. Beta diversity showed slight differences between the IBD groups and the control group. After bowel preparation, beta diversity of CD patients immediately (from A to B) changed p=0.03), while beta diversity of UC patients seems to continually change from point A to C(p=0.03). Based on the differential abundance analysis, Clostridiales and Pseudomonadaceae families increased, while Bifidobacteriaceae, Carnobacteriaceae, Veillonellaceae and Pasteurellaceae families decreased in CD patients compared to the HC at point B. The most growing families in HC group were Brucellaceae, Moraxellaceae and Alcaligenaceae at A to B period. Conclusion Our findings may suggest that bowel preparation can result in a significant change in faecal microbial composition in IBD. Microbial alterations recovering earlier in UC patients due to changed mucosa. Reduced alpha diversity and altered abundance in CD may have potential role in disease exacerbation. Further examinations are needed to clarify our pilot results.

Background Changes in microbiome of the gut and decreased diversity in inflammatory bowel disease(IBD) is the subject of intense researches. The exact pathophysiological connection is lacking, but the systemic inflammation due to the microbial alterations are assumed. This study aimed to perform analysis of faecal microbiota composition simultaneously with body composition by bioelectrical impedance and nutrition habits in Crohn’s disease(CD) patients to find specific microbiota profiles connected with obesity, mesenterial fat, signs of systemic inflammation, nutritional and body composition characteristics. Methods In our prospective cohort study, CD patients were enrolled. At the inclusion, demographic and clinical data, blood, and faecal samples were obtained. Clinical disease activity was assessed by CDAI, and SES-CD. Laboratory tests were made including C-reactive protein(CRP), albumin and lipids (triglyceride, cholesterol). Faecal bacterial composition was assessed using shotgun metagenomics sequencing technique. Each patient underwent a body composition analysis via bioelectrical impedance analysis. A nutritional questionnaire was filled by each subject. Results Data of 27 CD patients with different localisation was analysed in this study (mean age was 35±11 years). According to the gut inflammation, some Blautia species correlated positively with SES-CD (p=0,004), and correlated with faecal calprotectin negatively (p=0,02), while Roseburia hominis negatively with CDAI scores(p=0,01). High CDAI scores were also associated with dysbiosis. Adlercreutzia equolifaciens and Blautia hansenii associated with triglyceride(negatively p=0,04, positively p=0,05). Firmicutes has a lower, while Bacteroidetes has a higher abundance at high cholesterol level(p=0,001, p=0,023). Lower Adlercreutzia and Lactobacillus abundance were seen in patients with high visceral fat area(p=0,05, p=0,01), while Clostridia correlated with percent body fat(p=0,025).Bifidobacterium bifidum were associated in people with low body weight or low skeletal muscle mass(p=0,01, p=0,03). Beside Clostridia, Eubacteriales also correlated with waist-hip ratio(p=0,04, p=0,04). Conclusion To our knowledge this is the first study to analyse microbiota profiles by shotgun sequencing technique in CD patients depending on body composition via bioelectrical impedance and nutrition. Changes in Blautia, Lactobacillus, Adlercreutzia, Roseburia and Bifidobacterium abundances in patients with CD and obesity highlight the importance of correlations between lipid metabolism, adipose tissue mass, and chronic inflammation. Through this, it helps to develop new therapeutic approaches, such as personalized pre- or probiotics or faecal microbiota transplantation.

Background Anti-tumor necrosis factor alpha treatments are effective and safe as first line therapy, however, their long-term efficacy is limited by primary (PNR) and secondary non-response (LOR) resulting in treatment discontinuation in approximately, 40–50% of cases. Vedolizumab (VDZ) and ustekinumab (UST) therapy could be good alternative in patient with anti-TNF failure, however, no direct comparison has been made. This study aimed to assess treatment persistence and long-term efficacy of VDZ and UST as second- and third line biological therapies in Crohn’s disease (CD) patients refractory to anti-TNF therapy. Methods In this multicentre study, CD patients on VDZ or UST maintenance therapy were enrolled. We analysed data of medical history retrospectively in case of both agents. Demographic and clinical data at baseline and one-year were obtained. Clinical (CDAI Results 200 UST and, 96 VDZ-treated patients were included in the study. After exclusion of confounders, 162 UST and, 77 VDZ-treated patients were completed the follow-up. Mean age was, 41,1 (±16,5) and, 37,9 (±11,3) years at both agents at baseline. Steroid-free clinical and biochemical remission rates did not differ between VDZ and UST groups at one year (73% vs, 73% and, 67% vs., 51%). Kaplan-Meyer analysis revealed superiority of UST during follow up (86,5% vs., 62,9%, p Conclusion We confirmed that VDZ and UST are effective and safe alternatives to CD patients who have failed to anti-TNF therapy. UST has a superiority in drug persistence compared to VDZ. Second compared to third line of sequential therapy results in better outcome in drug persistence in case of both agents, however, head-to-head comparison of both agents are needed to verify our data.

Background Inflammatory bowel disease (IBD) is characterized by an unregulated immune response generating unbalance cytokine homeostasis. Analysis of patient-specific cytokine profiles may open new therapeutic targets or identify biomarkers that distinguish responders from non-responders before initiating therapy. Therefore, the aim of the present study was to determine cytokine profile of IBD patients and identify cytokines with predictive potential. Methods Blood and biopsy samples were obtained from 79 clinically active IBD patients and 28 controls without IBD. Inclusion criteria included change of therapy or initiation of new treatment for IBD patients. Organoid culture (OC) was generated from 5 clinically active IBD patients and 5 controls. Cytokine Array was used to analyse cytokine expression patterns. Total protein and mRNA were isolated from biopsies and from OCs. Protein levels were measured by ELISA and gene expression by qRT-PCR in serum, mucosa and in OCs. Results Pro-inflammatory cytokines were not detected in control samples, whereas in IBD biopsies the cytokine profiles enabled the discrimination between inflamed or non-inflamed areas. The mucosal expression of Serpin E1 was detected in all inflamed biopsy samples, whereas it was below the detection limit in healthy subjects. In responders Serpin E1 gene expressions were significantly (p Conclusion Our results suggest that serum and mucosal Serpin E1 expression reflects endoscopic activity in IBD, which could be used to follow-up disease activity and possibly therapeutic response. Organoids mimicked the expression of Serpin E1 in the original biopsies suggesting that OCs can be used to study the effect of Serpin E1 inhibition.

Background Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract which is associated with imbalanced pro- and anti-inflammatory cytokines. Anti-TNF-α therapy is widely used, but 10–30% of the patients are primarily not responding to the treatment or lose response over time. In vitro organoid cultures (OCs) generated from tissue specific adult stem cells can mimic cellular diversity and function of the organ of origin and might be used as a predictive tool for patient specific therapeutic response. However, the reductionist nature of the OCs and lack of knowledge about the proper representation of the disease features currently limit the utilization of the OCs as ex vivo IBD models. Methods Therefore our aims were to determine and compare cytokine profiles of colonic biopsies collected from IBD patients and OCs generated from the same biopsies, and to follow the changes of cytokine expression over time. In this study samples from 20 IBD and 8 non-IBD patients were used. Biopsies were taken during colonoscopy from inflamed part of the colon of IBD patients. Crypts were isolated from biopsy samples to establish colon OCs. Total protein and RNA were isolated from biopsies and OCs. Cytokine Array was used to determine cytokine patterns in our samples. Gene expressions of control and IBD OCs after first passage was compared by qPCR. TNF-α concentrations and cellular expression was determined by EILSA and immunostainings, respectively in control and IBD OCs. To determine therapeutic response OCs were treated with anti-TNF-α therapy. Results Cytokine patterns of colon biopsies and OCs were remarkably similar until first passage. The major pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were detected both in biopsies and OCs. After second passage the cytokine expression decreased or disappeared in OCs. TNF-α was also measurable in the OCs, however a similar decrease was observed, which was also confirmed by immunostaining. Gene expression analysis in IBD OCs showed an increased TNF-α, IL-1β and IL-6 levels compared to healthy control OCs after first passage. ELISA also showed higher TNF-α concentration in organoids from inflamed origin compared to controls. After anti-TNF-α treatment we detected a decrease in the IL-6 gene expression in treated OCs. Conclusion Our results suggest that colon OCs maintain the cytokine expression ex vivo until the 2nd passage and show inflammatory characteristics. Moreover, in vitro treatment induces changes in the cytokine expression. Based on these results the utility of patient-derived organoids to predict the therapeutic response can be investigated.

Background Inflammatory bowel disease potentially elevates the risk of infections, furthermore, disease activity and medical treatment(s) can increase the risk as well. However, both international data and recent studies do not confirm these preliminary conceptions regarding the SARS-CoV-2 infection. In addition, a number of studies have reported that less antibodies are produced against the virus in IBD patients. In January, 2021, the vaccination campaign has begun in Hungary as well, however, questions have been raised about the effectiveness and safety of the vaccine. Methods In this multicentre study, we assessed the prevalence and risk factors of COVID-19 infection, the willingness to receive COVID-19 vaccine and the efficacy of vaccination among IBD patients receiving biological therapy, based on a cross-sectional questionnaire-based study. To assess safety and antibody response to COVID-19 vaccines, we conducted a prospective study in the same Hungarian IBD centers. IgG antibody was quantified to SARS-CoV-2 spike protein and nucleocapsid, 1 week before and after the first vaccine and, 4 and, 8 weeks after the second vaccinane, respectively. Results 472 patients were enrolled in the first part of our study. SARS-CoV-2 infection was confirmed in, 16.9% of patients. Wearing gloves and masks were found to be effective in preventing infection (p=0.02; p=0.005), avoidance of communal areas had no effect on infection rates. Male sex increased the risk (p=0.008) of viral infection. Based on subjective complaints, UC patients had a worse disease course (p=0.002). Biological therapies did not increase the risk of infections. Patients vaccinated with mRNA vaccine had a significantly higher spike protein antibody titer one month after the second vaccination (p=0.004) compared to other vaccine types (Sinopharm©, Sputnik V©, Astra Zeneca©). Seropositivity was detected in, 98% of patients. Sinopharm© vaccination triggered the lowest number of side effect (p Conclusion Face mask was the most effective preventive tool. The risk of infection was not increased by biological therapy, therefore therapy discontinuation is not justified. Almost every vaccinated patient developed seropositivity two month after vaccination independently from the type of the vaccine, however, spike protein antibody was significantly higher following mRNA vaccinations.

Background Since almost its 1 year outbreak declared by the World Health Organization on 11th March 2020, COVID-19 pandemic still cannot be controlled successfully. Inflammatory bowel disease potentially elevates the risk of infections, independently from the age, while disease activity and medical treatment(s) can increase the risk as well. Based on international data, in total, 4% receives the infection. Furthermore, 1.8% of the patients on biologic therapy needs intensive therapeutic care and 1% passes away. Methods This was an observational, questionnaire based study, carried out in Hungary, between February and March 2021. Our questionnaire consisted of 45 questions, that surveyed the impact of the pandemic among IBD patients, and the severity and outcome of the infection. Participants were on biologic therapy. Our aim was to determine the frequency and outcome of SARS-CoV2 infection in patients with inflammatory bowel disease on different biological therapy. Results In total, 387 respondents completed the questionnaire, and 47 participants (12%) developed COVID-19 infection. 66.9% of them were receiving anti-TNF inhibitor, 16.8% vedolizumab, 12.1% ustekinumab, and 4.1% tofacitinib. Based on our cohort, different biologic therapies didn’t elevate the risk of infection (p=0.3486), nor the hospitalization rate (p=0.277). No one was in ICU or ventilator, and nobody passed away. Furthermore, 38.3% suspended the current biologic therapy, but it didn’t decrease the rate of hospitalization (p=0.533), however, it didn’t cause flare-ups either in the primary disease (p=0.415). Based on our cohort, neither vitamin supplementations meant protection against the infection (p=0.117), only regular mask wearing seems to protect patients with IBD (p=0.009). Conclusion Based on our cohort, more IBD patients develop the infection in Hungary, compared to international data, however, the outcome of the infection is more favourable. It seems, that the different biological treatments don’t affect the infection rate, and neither elevates the hospitalization rate. In generally, there is no need to suspend the current biologic therapy, however, it should be a matter of individual judgment. After all, we claim that mask-wearing still seems to be the most effective form of prevention.

Background Budesonide is an oral corticosteroid, which is widely used in moderate-to-severe ulcerative colitis (UC) to obtain and maintain remission in those cases, where 5-aminosalicylic-acid was ineffective. Unlike previous forms of budesonide absorbing from the ileal and ascending colon region, the new-generation budesonide-MMX contains a formula, that allows absorption throughout the whole colon, and therefore in the whole potentially inflamed area in UC. We aimed to evaluate the effects and safety of budesonide-MMX in our UC patients who meet the above mentioned therapeutic demands in a real-life study. Methods We enrolled 22 patients with mild to severe UC in this single-centre prospective study until August of 2019. Patients received 9 mg oral budesonide-MMX once daily until 8 weeks. Laboratory parameters (cholesterol, triglyceride, CRP) and serum hormone levels (parathormone [PTH], dehydroepiandrosterone [DHEA] and cortisol) were monitored before and after the 8-week therapy to follow metabolic and hormonal changes. During these visits, body composition analysis was also performed with InBody 770 machine to observe the adverse steroid effects of budesonide-MMX in respect of body fat mass, body mass index, protein content of the body and bone mineral content. Disease activity was followed by the partial Mayo (pMayo) score. Statistical analysis was performed by paired t-test and Wilcoxon signed-rank test with SigmaPlot 1.25. Results The total of 22 patients (age: 44.4 ± 15.1 years, 6 male and 16 female patients) had received the 2-month budesonide-MMX therapy (2.0 ± 0.3 months). Mean disease duration was 8.3 years. By the end of follow-up, 15 (68.2%) patients experienced remission and 7 patients (31.8%) were primary non-responders. The disease activity decreased significantly from the mean of 3.95 to 1.64 (p < 0.001). No significant changes were observable in case of any body composition analysis parameter. Regarding the laboratory parameters, serum cholesterin level showed a significant increase (p < 0.001), while triglyceride and CRP showed did not show significant changes. Serum cortisol levels were elevated (p < 0.001), while PTH and DHEA showed no significant decrease. Only two patients experienced side effects: one of them hypertonia, headache and acnes, while the other patient experienced mild diarrhoea. One patient had a relapse during the treatment. Conclusion In our study, budesonide-MMX proved to be safe by bringing up a low number of side effects, while more than two-thirds of the patients could reach remission with this short-term therapy. Hormonal changes were not mentionable, although the drug’s effects on serum lipid content have to be examined further.

Background Tofacitinib (TFC) is an oral, small-molecule Janus kinase inhibitor, which was recently approved for moderate to severe ulcerative colitis (UC). The aim of the current real-life study was to determine efficacy of TFC induction therapy regarding the clinical response and remission in patients with active UC. We evaluated short-term efficacy data in a Hungarian cohort with prior exposure to other biological agents such as anti-TNF drugs and vedolizumab. Methods In this single-centre retrospective study, patients with TFC introduction were included. Since January 2019, a total of 16 patients received an oral TFC induction therapy in a dose of 10 mg twice daily for 8 weeks. Endoscopic activity was evaluated by endoscopic Mayo (eMayo) score before the introduction of TFC and in case of an inadequate therapeutic response to the 5-mg-therapy to confirm therapeutic decision-making. Based on the evaluation of clinical symptoms and laboratory parameters, we either kept the dosage or reduced the dose to 5 mg according to local regulations. We also collected data from the 16. and 24. weeks of the therapy. Primary endpoints were a clinical response (as a reduction in partial Mayo Score [pMayo] by minimum 3 points) or remission (as a Mayo score of the maximum of 2 points and without blood in stool) at week 8. Results Sixteen patients had received the induction therapy (mean age: 36 years, 7 males and 9 females) in our centre. After 8 weeks, 12 (75%) patients responded to the TFC induction therapy and 6 (37.5%) of them were in remission. Four patients were primary non-responders (25%). Corticosteroid therapy (18 ± 7 mg) was required during the induction in 4 responder cases, which could be stepped down by week 8. As a continuous maintenance therapy, 4 patients have already reached the 16th week and 8 have completed the 24th week. By the end of the follow-up, 12 patients responded and 10 was in remission. During the observation period, 3 patients had to remain on 10 mg TFC dose, 6 patients required dose escalation from 5 mg to 10 mg and 5 mg was sufficient in case of only 3 patients after the introduction. Endoscopic activity showed a moderate decrease from 2.5 ± 0.5 eMayo score to 2 ± 1 (n = 7) until week 16. In respect the responder patients, CRP levels decreased from the mean of 7.23 to 5.02. No serious side-effects were observed during the follow-up. Conclusion After the 8-week TFC induction therapy, the response rate was high and only every fourth patients were non-responder. A low number of patients had adequate reactions to the 5 mg-therapy after the introduction, but TFC is effective with dose-escalation in respect of clinical response and remission in patients with UC, who have had an inadequate response to previous biological therapy.

Background Faecal drug concentration is not routinely measured as per therapeutic drug monitoring strategies in inflammatory bowel disease (IBD) patients receiving anti-TNF therapy. However, our previous research work suggested the importance of faecal drug monitoring of anti-TNF agents since active disease may be present in spite of normal serum anti-TNF levels. The aim of the present study was to examine the correlation between faecal calprotectin and faecal infliximab (IFX) concentration and to evaluate the cut-off value of faecal drug concentration in the respect of faecal calprotectin in patients treated with maintenance IFX therapy. Methods Consecutive patients with IBD receiving maintenance IFX therapy at 1st Department of Medicine, University of Szeged were enrolled in the present study. Faecal samples were obtained before the subsequent IFX infusion. Faecal calprotectin and IFX concentrations were determined with ELISA. The correlation between faecal calprotectin and faecal IFX concentration was statistically assessed. Results Sixty-seven IBD patients were enrolled. Female/male ratio was 49%–51%. Sixty-five point six% of the patients were diagnosed with Crohn’s disease and 34.3% with ulcerative colitis. Mean disease duration was 14.9 years (SD 9.7) at the time of analysis. Mean duration of IFX therapy was 42.3 month (SD 31.6). Twenty-eight point three% of the patients received escalated IFX therapy. Mean faecal calprotectin concentration was 545.7 µg/g (SD 379.4 µg/g). Mean faecal IFX concentration was 1 ng/ml (SD 1.3). Faecal calprotectin and faecal IFX concentration showed significant correlation (r=0.37, p = 0.002). A cut-off value of faecal IFX level of 0.6 ng/ml was determined at faecal calprotectin concentration of 500 µg/g. Conclusion According to our results IFX is detectable in the faeces at a calprotectin concentration of 500 µg/g. The cut-off value for faecal IFX concentration proved to be 0.6 ng/ml. Simultaneous determination of faecal anti-TNF and faecal calprotectin concentration is supposed to have a higher benefit in the evaluation of response to IFX therapy.

Porcine reproductive and respiratory syndrome virus 1 is a major cause of swine morbidity and mortality in various parts of the world, including Hungary. A national elimination programme to reduce the associated economic burden was initiated in Hungary in 2012. Using extensive laboratory surveillance, we identified and isolated an unusual PRRSV strain. The complete coding sequence of this isolate was determined and analyzed. The genome of this Hungarian PRRSV1 strain, HUN60077/16, is 15,081 nucleotides in length. Phylogenetic and recombination analysis showed a mosaic structure of the genome where a large fragment of ORF1b and the genomic region coding for ORF3 to ORF7 showed a very close genetic relationship to the vaccine virus Unistrain, while the ORF1a region, the 3’ end of ORF1b, and the whole ORF2 were only distantly related to this or any other PRRSV1 strain whose genome sequence is available in the GenBank database. Genomic characterization of PRRSV strains is crucial when possible vaccine-associated cases are identified. This approach not only helps to identify genetic interactions between vaccine and wild-type PRRSV1 strains but may also be needed to prevent trust in commercial vaccines from being undermined.

Background Unlike previous forms of budesonide absorbing from the ileal and ascending colon region, the new-generation budesonide-MMX contains a formula, that allows absorption throughout the whole colon. Budesonide is degraded in the liver by cytochrome P450 3A enzyme, but so far, there is no study examining the relationship between the budesonide’s effect and the enzyme activity. CYP3A5 is absent in 90% of the European/caucasian population due to a functional loss mutation (CYP3A5*3), whereas patients with the wild-type CYP3A5*1 allele may be expected to have increased metabolism. The most common genetic polymorphisms in CYP3A4 (CYP3A4*1B and CYP3A4*22) result in increased and decreased expression, respectively. Methods We enrolled 33 patients with UC in this prospective study until January of 2021. Patients received 9 mg oral budesonide-MMX once daily until 8 weeks. Laboratory parameters (cholesterol, triglyceride, CRP) and serum hormone levels (parathormone, dehydroepiandrosterone and cortisol) were monitored before and after the 8-week therapy to follow metabolic and hormonal changes. During these visits, body composition analysis was also performed with InBody 770 machine to observe the adverse effects of budesonide-MMX in respect of body fat mass, body mass index, protein content of the body and bone mineral content. We examined the CYP450 3A (CYP3A5 and CYP3A4) enzyme genotype of the patients, to see, whether the different alleles of this drug-degrading enzyme affect the efficacy and safety. Results 33 patients had received the 2-month therapy. By the end of follow-up, based on partial Mayo score, 26 (78.8%) patients experienced remission and 6 patients (18.2%) were primary non-responders. Mean pMayo score decreased from 4.18 to 1.63 (p Conclusion In our study, budesonide-MMX proved to be safe and effective in the therapy of UC, however, cholesterol was elevated in the serum. Based on our cohort, different genotypes of CYP3A don’t have an impact on the effect of the drug, however, CYP3A allele variants are rare, therefore, further examinations should be performed.

Background Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract which is associated with the imbalanced pro- and anti-inflammatory cytokines. Anti-TNF-α is widely used as a therapeutic agent, but 10–30% of the patients are not responding to the treatment or in time it become less affective. In vitro organoid cultures generated from stem cells can mimic the cellular diversity and function of the organ of origin and might be used as a predictive tool for patient specific response. However, the in vitro cytokine expression pattern of the IBD colon organoid cultures have never been determined or compared with the cytokine expression of the colon mucosa. Methods Our aim was to determine and compare the cytokine profiles of the colon biopsy samples in IBD patients and organoid cultures generated from these biopsies. In this study samples from 5 IBD patients were used. Biopsies were taken during colonoscopy from inflamed part of the colon. Crypts were isolated from the biopsy samples to start colon organoid culture. Total protein was isolated from the biopsies and the organoids. Cytokine Array was used to determine the cytokine patterns in both samples. ELISA was used to measure the TNF-α concentrations and immunofluorescence staining to localize it in the organoids. Results We determined the cytokine profile of the biopsy samples and the colon organoid cultures. The cytokine patterns were remarkably similar until the first passage. The major pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were detected in the organoids after the first passage. After the second passage the cytokine expression decreased or disappeared. ELISA revealed that TNF-α was also expressed in the organoid cultures, however a similar decrease in the expression was observed, which was also confirmed by immunofluorescence staining. TNF-α positive cells were present in the organoids, but it decreased after every passage. Conclusion Our results suggest that the colon organoid cultures maintain the cytokine profile of the tissue of origin until a limited time (until the first passage). Therefore, organoids might be used to determine patient specific therapy for drug tests.

Background Inflammatory Bowel Disease (IBD) occurs as a consequence of abnormal immune response generating unbalance between pro- and anti-inflammatory signalling. Analysis of cytokine profiles in view of different cytokine targeting or immunosuppressive therapy may open up new therapeutic targets and may reveal biological profiles that distinguish responders from non-responders before initiating therapy. The aim of present study was to determine cytokine profile of IBD patients and identify cytokines with predictive potential. Methods IBD patients with clinically active disease were enrolled in study. Blood and biopsy samples were obtained from 22 IBD patients and 5 healthy controls. Biopsies were taken from inflamed and non-inflamed part of colon of IBD patients. Total protein and mRNA were isolated from biopsy samples. Cytokine Array was used to analyse cytokine expression patterns. Serum and mucosal SerpinE1 levels were measured by ELISA and qRT-PCR. Results In samples from IBD patients, remarkable discrimination between inflamed, or non-inflamed areas was observed, whereas no pro-inflammatory cytokines were detected in control samples. SerpinE1 was presented in every inflamed biopsy samples, which was analyzed in more details. Mucosal expression of SerpinE1 differed significantly in healthy subjects compared to IBD patients with active disease (0 vs 24.06 pg/mg, p=0.02). After therapy induction a remarkable decrease was observed in the mucosal SerpinE1 concentration in responders (45.5 vs 9.7 pg/mg, p=0.02) versus non-responders (45 vs 61.2 pg/mg, p=0.3). Moreover, mean value of mucosal SerpinE1 did not differ significantly in healthy subjects compared to responders (5.7 vs. 0 pg/mg, p=0.12). In non-responders the fold changes of SerpinE1 gene expressions were significantly (p=0.001) higher than in responders. Lowest expression of SerpinE1 gene was measured in control samples, whereas the highest in untreated, inflamed biopsy samples. Serum and mucosal SerpinE1 concentrations were significantly higher in patients with active disease compared to inactive (tissue: 5 vs 47.4 pg/mg, p=0.00003; serum: 22.4 vs 25.94 mg/ml, p=0.022). Correlation analysis revealed that serum SerpinE1 correlates with disease activity (p0.1, sensitivity=72%, specificity=77.8%, accuracy=73.5%). Conclusion These results suggest that serum and mucosal SeprinE1 expression reflects endoscopic activity of IBD. Correlation of SerpinE1expression between the blood and the bowel mucosa would open up new possibilities in non-invasive disease monitoring of IBD.

Background The risk of relapse after anti-tumour necrosis factor [TNF] therapy cessation in Crohn’s disease [CD] patients with perianal fistulas is unclear. We aimed to assess the risk of relapse after anti-TNF cessation in a large cohort and to identify risk factors. Methods A systemic literature search was conducted to identify cohort studies reporting on the incidence of relapse after cessation of anti-TNF therapy in CD patients. Individual patient data [IPD] were requested from the original study cohorts. Inclusion criteria for IPD-meta-analysis (IPD-MA) included age ³ 18 years, perianal fistulizing CD as indication for start of anti-TNF therapy, minimal treatment duration ³3 doses, and remission of luminal and perianal CD at cessation of anti-TNF therapy. Primary outcome was CD relapse [either perianal or luminal]. Perianal fistula relapse was defined as recurrence of draining perianal fistula related to previous or new fistula tracks, or abscess. Luminal relapse was defined as a clinical, biochemical, endoscopic, or radiological relapse requiring treatment or dose optimization of IBD medication or surgery. In a secondary analysis, risk factors associated with relapse were assessed using multivariate logistic regression analysis. Results A total of 307 patients from 12 studies in 9 countries were included in this IPD-MA. The median duration of anti-TNF treatment prior to therapy cessation was 14 months [IQR 6.1 – 29.9]. In 272/307 patients [89%] anti-TNF therapy was started for active perianal fistula and in 34 [11%] for both active perianal fistula and luminal CD. 169 patients [55%] developed a relapse [either perianal or luminal] after a median follow-up after cessation of 25 months [IQR 12 – 54]. Overall cumulative incidence of relapse was 31% and 43% at 1 and 2 years after anti-TNF cessation. Risk factor for CD relapse include upper GI involvement (L4) [HR 1.9], whereas older age [A3 vs A1, HR 0.48] and continuation of concomitant immunomodulators [HR 0.62] were protective factors. For a subgroup of patients with active perianal fistula and in luminal remission at start of anti-TNF, the cumulative incidence relapse rates were 25% and 43% at 1 and 2 years. No considerable differences in risk factors were found within this subgroup regarding risk of recurrence. Of the 179 patients who relapsed, 104 were retreated with anti-TNF with a response rate of 85%. Conclusion According to this IPD-MA, approximately two-thirds of CD patients with perianal fistula remain in remission with regard to fistulizing and luminal disease during 2 years after cessation of anti-TNF therapy. Further risk stratification based on perianal fistula characteristics is required.

This study investigated the effects of three different commercial diets on the growth, feed utilization, and product quality of pike (Esox lucius). The first diet had low lipid (12%) and low protein (37%) (diet A) content, the second diet had low lipid (12%) and high protein (52%) content (diet B), the third diet had higher lipid concentration (15%) and high protein content (52%) (diet C). Two hundred and seven pike were stocked in an experimental RAS (Recirculating Aquaculture System) and were fed ad libitum for the duration of the nine week trial. Specific growth rates, feed conversion rates, protein utilization, as well as body composition and somatic indices were calculated. Fish fed with diets B and C had significantly better SGR (Specific Growth Rate) and FCR (Feed Conversion Rate) than fish fed with diet A. The higher dietary lipid concentration does not cause significantly better protein utilization. Moreover, it had a negative effect on the product quality by increasing the body lipid concentration and the visceral fat deposition.

Background Inflammatory bowel diseases (IBD) patients are at increased risk for mental health problems. Managing IBD patients requires cooperation between gastroenterologists and psychologists and psychiatrists. We would like to assess the frequency of depression and suicidal thoughts among Hungarian IBD patients. Methods Multicentre, questionnaire-based survey among inflammatory bowel diseases patients. Demographic data and patient’s medical history was obtained with self-filling questionnaires. Patients’ psychic status was measured with Patient’s Health Questionnaire (PHQ-9), Beck Hopelessness Scale (short version). The survey was conducted in 3 IBD centres in Hungary, First Department of Medicine, University of Szeged, First Department of Surgery, University of Semmelweis and First Department of Medicine, University of Pécs. Results Three hundred IBD patients filled the questionnaires from the 3 IBD centres. Female-male ratio was 57% - 47%. Mean age at the time of the study was 38.8 years (SD 12.5) and mean disease duration was 11.5 years (SD 8.2). Sixty-two point three% was diagnosed with Crohn’s disease, 37.7% with ulcerative colitis, one patients reported IBD unclassified and three patients did not know the type of IBD. Sixty-nine point three% of the patients self-reported the remission state of IBD, while 30.7% self-reported to have active IBD. Proportion of patients on each category of PHQ-9 scale were the followings: severe 2%, moderate to severe 5.3%, moderate 21.3%, mild 33.6% and 37.6% had normal score. Nine point three% of the patients had elevated suicidal risk. More severe PHQ-9 category was associated with elevated risk for suicide. Significant difference was shown in the Hopelessness Scale’s results among the two genders, female patients had higher risks for suicidal thoughts. Those IBD patients in remission have lower probability for moderate or severe depression. Conclusion We have to be aware of that every IBD patient has increased risk for depression and suicide. Among our patients the rate of moderate to severe and severe depression was relatively low. Every tenth of our patients had elevated risk of suicide.

Background Patients with IBD may have associated spondyloarthropathy. Vedolizumab is used for IBD with its actions considered to be gut specific but the impact of this treatment on existing arthritis or development of arthritis is uncertain. Integrins and adhesion molecules play a role in the interception of recirculating activated lymphocytes between the gut and the synovial membrane due to the inhibition of the alpha4beta7 integrin homing at the level of the gut. We aimed to describe characteristics and outcomes of spondyloarthropathy in IBD patients treated with vedolizumab. Methods We retrospectively collected data on a multicentre cohort of IBD patients with arthropathy which developed following use of Vedolizumab for IBD. This was a part of the European Crohn`s and Colitis Organisation (ECCO) initiated CONFER (Collaborative Network for Exceptionally Rare case reports) project. Data were recorded on a standardised case report form and analysed with descriptive statistics. Results We report data on 14 cases of new onset spondyloarthropathy and 1 case of worsening of pre-existing arthropathy from 7 contributing centres. Vedolizumab was used for 6 patients with CD and 8 patients with UC. IBD was in remission in all but 3 patients at the time of development of the joint features. Sacroiliitis was the manifestation in 10 patients, 3 of whom had both sacroiliitis and peripheral arthritis. Three patients had peripheral arthritis alone and one patient had peripheral arthritis with enthesitis. One patient had worsening of peripheral arthropathy following use of vedolizumab. Raised inflammatory markers (CRP, ESR or both) were recorded only in one-third of the patients. MRI was abnormal in 9 patients being the marrow oedema in SI joints the commonest feature. One patient had abnormal bone scintigraphy and in another patient plain X-ray and ultrasound was used in confirming diagnosis. Eight of the patients developed arthropathy within days after a single infusion of vedolizumab. five patients needed hospitalisation for treatment The treatments used included systemic steroids (11 patients), Intraarticular steroids (3) NSAID (2 patients), Cox inhibitor (1), methotrexate (2) Hydroxychloroquine (1). Vedolizumab was discontinued in 11 of the patients. In the majority of patients (11/15) it took many months to resolve the arthropathy. On re-challenge with vedolizumab, the arthropathy recurred in 2 out of the 3 patients. Conclusion This case series demonstrates the potential risk for development of spondyloarthropathy with vedlizumab in IBD patients which is unrelated to the activity of IBD and can have significant impact including need for use for systemic steroids and hospitalisation. Larger prospective cohorts are needed to confirm these results.

Background Anti-tumour necrosis factor (TNF) therapy has dramatically changed the treatment of inflammatory bowel disease (IBD). The relationship between clinical outcomes and serum anti-TNF levels is controversial. This study aimed to perform simultaneous analyses of the serum, mucosal and faecal infliximab and adalimumab levels to determine the mucosal expression of TNF-α and assess the relationship of the anti-TNF-α levels in these biological samples with the endoscopic and clinical activities and body composition in IBD patients. Methods Consecutive IBD patients who received maintenance anti-TNF-α therapy and underwent colonoscopy were enrolled. We assessed the clinical disease activity, collected the blood, faecal and biopsy samples. The number of TNF-α positive cells in the mucosa was detected using immunofluorescent labelling. Serum, mucosal and faecal anti-TNF-α levels, anti-drug antibody level in the serum and faecal calprotectin were determined using ELISA. Each patient underwent body composition analysis. Results Fifty consecutive patients have been analysed in the study. The number TNF-α positive cells was significantly higher in the inflamed than in the un-inflamed part of the colon (p = 0.01); however, such a difference was not detected in case of tissue drug levels. Tissue drug level did not correlate with the serum drug level. Nevertheless, tissue drug levels of the samples obtained from the un-inflamed part of the colon were significantly higher in patients who were in remission (p = 0.03467). The presence of the drug in the faeces was significantly different according to disease activity (p = 0.0022). Drug levels in the biological samples did not show any association with the presence of anti-drug antibodies. Body composition parameters had no significant impact on the serum and tissue drug levels. Faecal calprotectin showed significant correlation with faecal drug concentration (p = 0.0162) and clinical and endoscopic activity (p < 0.001). Conclusion To the best our knowledge, this is the first study to measure drug concentrations in all the relevant biological samples. Our findings highlight the importance of faecal drug monitoring because active disease may be present in spite of normal serum anti-TNF levels. Simultaneous determination of faecal drug concentration and faecal calprotectin may be more accurate for evaluating therapeutic responses in the future.

Background There is a lack of data regarding clinical and therapeutic characteristics in middle and late adolescent inflammatory bowel diseases (IBD) patients in real life. Our aim was to retrospectively evaluate patient’s characteristics and therapeutic features among the Hungarian adolescent inflammatory bowel diseases population. Also our aim was to assess whether these patients before the transition have more serious disease phenotype than those after transition. Methods Retrospective, epidemiological study using the National Health Insurance Fund social security databases. Adolescent Hungarian IBD patients who were observable in the database between 2008 and 2017 were enrolled. Patients were analysed according to two groups: patients between ages of 14–17 years were defined middle adolescent patients, and patients between 18–21 years were defined late adolescent patients. Results The incidence of IBD was 24.82/100 000 in the middle and 38.17/100 000 in the late adolescent patients. Prevalence rates showed an increasing tendency in both groups. Incidence and prevalence rates were higher in the late adolescent patients. Mesalazine was used in high proportion of patients (66.6% vs. 69.3% of the middle and late adolescent patients, respectively), also in UC and CD patients. While, the use of sulphasalazine (13.1% vs. 19.6%, respectively) and azathioprine (20.3% vs. 11%, respectively) was relatively low. The need of biologics was higher in the middle adolescence group (p < 0.001). Use of anti-TNF agents increased during the observational period in both groups (p < 0.001). Corticosteroid use was similar in both groups; a decreasing tendency was observed. Rate of hospital admissions to gastroenterology department was higher than to surgery department in both groups. The number of hospital admissions to gastroenterology (p < 0.001, p = 0.001) and surgery departments (p < 0.001 and p = 0.031) decreased significantly in both groups throughout the observational period. Conclusion We have analysed the data of huge number of adolescent patients diagnosed with IBD to get better overview on the clinical and treatment characteristics of patients in these sensitive age groups. Overall, the increasing use of anti-TNF therapy and decreasing tendency of corticosteroid use reflects significant changes and improvement in treatment paradigm not only in adult but paediatric care. The higher need of anti-TNF agents reflects that these adolescent patients before the transition have more severe disease phenotype.

Bile acids play important physiological role in the solubilisation and absorption of dietary lipids. However, under pathophysiological conditions, such as short bowel syndrome, they can reach the colon in high concentrations inducing diarrhoea. In this study, our aim was to characterise the cellular pathomechanism of bile-induced diarrhoea using human samples. Colonic crypts were isolated from biopsies of patients (controls with negative colonoscopic findings) and of cholecystectomised/ileum-resected patients with or without diarrhoea. In vitro measurement of the transporter activities revealed impaired Na⁺/H⁺ exchanger (NHE) and Cl⁻/HCO₃⁻ exchanger (CBE) activities in cholecystectomised/ileum-resected patients suffering from diarrhoea, compared to control patients. Acute treatment of colonic crypts with 0.3 mM chenodeoxycholate caused dose-dependent intracellular acidosis; moreover, the activities of acid/base transporters (NHE and CBE) were strongly impaired. This concentration of chenodeoxycholate did not cause morphological changes in colonic epithelial cells, although significantly reduced the intracellular ATP level, decreased mitochondrial transmembrane potential and caused sustained intracellular Ca²⁺ elevation. We also showed that chenodeoxycholate induced Ca²⁺ release from the endoplasmic reticulum and extracellular Ca²⁺ influx contributing to the Ca²⁺ elevation. Importantly, our results suggest that the chenodeoxycholate-induced inhibition of NHE activities was ATP-dependent, whereas the inhibition of CBE activity was mediated by the sustained Ca²⁺ elevation. We suggest that bile acids inhibit the function of ion transporters via cellular energy breakdown and Ca²⁺ overload in human colonic epithelial cells, which can reduce fluid and electrolyte absorption in the colon and promote the development of diarrhea.

Aliment Pharmacol Ther 2011; 34: 911–922 Summary Background Adalimumab is a fully human monoclonal antibody targeting tumour necrosis factor with proven efficacy in the treatment of Crohn’s disease (CD). Aim To investigate the predictors of medium-term clinical efficacy and mucosal healing during adalimumab therapy, in patients with CD, in specialised centres approved for biological therapy in Hungary. Methods Data capture of the 201 CD patients was standardised and prospective (male/female: 112/89, median age: 33.0 years, duration: 8 years). Previous infliximab therapy had been administered in 48% of patients, concomitant steroids in 41%, azathioprine in 69% and combined therapy in 27% of patients. Results Overall clinical response and remission rates at 24 weeks were 78% and 52%, respectively; at 52 weeks were 69% and 44%, respectively. Endoscopic improvement and healing were achieved in 43% and 24% of patients. In a logistic regression model, clinical efficacy and CRP at week 12, need for combined immunosuppression at induction, shorter disease duration and smoking were identified as independent predictors for 12-month clinical outcome, whereas CRP at week 12, clinical remission at week 24, inflammatory parameters and nonsmoking were associated to endoscopic improvement/healing. Intensification to weekly dosing was needed in 16% of patients. Parallel azathioprine therapy and clinical remission at week 12 were inversely associated with dose escalation. Conclusions Clinical efficacy and normalised CRP at week 12 (early deep clinical remission) are associated with medium-term clinical efficacy and mucosal healing during adalimumab therapy, whereas need for combined immunosuppression at induction and smoking status are predictors for non-response. Parallel azathioprine therapy may decrease the probability for dose escalation.

The high cost of infliximab inhibits the regular retreatment of all patients in Hungary with Crohn's disease (CD) after beneficial induction therapy. This study is set out to evaluate the medium-term efficacy of induction therapy with infliximab without retreatment in CD patients with chronic activity and/or fistulae refractory to conventional therapy.A retrospective 1-year review was undertaken of all CD patients with successfully induced remission or fistula closure with 3 infusions of infliximab. Infliximab was administered in a dose of 5 mg/kg 3 times, in weeks 0, 2 and 6. Clinical remission was defined as symptom resolution and an estimated Crohn's Disease Activity Index (CDAI)150 and complete fistula closure. We evaluated the clinical response, the estimated CDAI, the number of draining fistulae, the dosages of steroid and immunosuppressive drugs at 6 and 12 months after the last infusion, and the needs for hospitalization and surgical intervention during this period. Breslow (Generalized Wilcoxon) test was used as the statistical method.The data of the 50 patients (19 luminal, 31 fistulizing disease; average age 29. 3 [13-59] years, disease localization: 23 colon, 13 ileum, 13 ileocolon, 1 duodenum) were suitable for analysis. Infliximab induction therapy without retreatment resulted in a beneficial effect lasting for at least 1 year in 22 of the 50 patients (44%). 11 of the 19 patients (57.9%) with luminal disease remained in steroid-free complete remission, while the fistulae persisted closed in only 11 of the 31 patients (35.5%) (p0.05).Infliximab induction therapy alone may result in sustained remission mainly in patients with luminal disease. These results suggest the need for maintenance therapy with infliximab after successful therapy induction in patients with fistulae, while luminal CD patients could possibly participate in regular retreatment only if needed. If these data are confirmed, this modification of the therapeutic procedure could well increase the cost-effectiveness of infliximab.

BACKGROUND: A recent study reported that a non-synonymous single nucleotide polymorphism (rs11209026, p.Arg381Gln) located in the IL23R gene is a protective marker for inflammatory bowel disease. AIM: To analyse the frequency of p.Arg381Gln in three independent European inflammatory bowel disease cohorts and to evaluate how this variant influences disease behaviour. METHODS: We assessed a European cohort of 919 inflammatory bowel disease patients and compared the IL23R p.Arg381Gln genotype frequency with 845 healthy controls. Inflammatory bowel disease patients originated from Germany [Crohn's disease (CD): n = 318; ulcerative colitis (UC): n = 178], Hungary (CD: n = 148; UC: n = 118) and the Netherlands (CD: n = 157). Ethnically matched controls were included. We performed subtyping analysis in respect to CARD15 alterations and clinical characteristics. RESULTS: The frequency of the glutamine allele of p.Arg381Gln was significantly lower in inflammatory bowel disease patients compared with controls in a pooled analysis of all three cohorts (P < 0.000001) as well as in the individual cohorts (Germany: P = 0.001, Hungary: P = 0.02 and the Netherlands: P = 0.0002). The p.Arg381Gln genotype distribution was similar between CD and UC. We did not observe either statistical interactions between p.Arg381Gln and CARD15 variants or any significant associations between p.Arg381Gln genotype and subphenotypes. CONCLUSIONS: The p.Arg381Gln IL23R variant confers a protective effect against both CD and UC, but does not determine disease phenotype.

Rhodococcus equi was isolated from lung, liver, spleen, and stomach content of two aborted equine fetuses of 7 and 8 months gestation from two different farms. Lesions included diffuse pyogranulomatous pneumonia with numerous Gram-positive coccobacilli within the cytoplasm of macrophages, multinucleated Langhans giant cells and neutrophils, and enhanced extramedullary hematopoiesis with megakaryocytosis within the liver and spleen. Detection of R. equi was made by bacteriology and immunohistochemistry for R. equi and VapA, the virulence factor of R. equi. R. equi and VapA were identified within the lungs of both fetuses, and its distribution correlated with lesions. Fetal lesions were similar to those observed in foals. We speculate that the fetuses contracted infection from the placenta by normal breathing movements or by swallowing of the amniotic fluid contaminated with R. equi.