High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Is an intraoperative liver function assessment possible? Application of the 13C-methacetin-breath-test during major liver resections - a pilot study.

BACKGROUND: As prevention of posthepatectomy-liver-failure is crucial, there is need of dynamic assessment of liver function, even intraoperatively. 13C-methacetin-breath-test estimates the organ's microsomal functional capacity. This is its first intraoperative evaluation in major liver surgery. METHODS: 30 patients planed for resection of ≥3 liver segments, between March-November 2019, were prospectively enrolled in this "single-center", pilot study. Using the 13C-methacetin-breath-test, liver function was assessed four times: preoperatively, intraoperatively before and after resection and postoperatively. The resulted maximum-liver-function-capacity (LiMAx)-values and delta-over-baseline (DOB)-curves were compared, further analyzed and correlated to respective liver volumes. RESULTS: The intraoperative LiMAx-values before resection were mostly lower than the preoperative ones (-11.3% ± 28%). The intraoperative measurements after resection resulted to mostly higher values than the postoperative ones (42.35% ± 46.19%). Pharmacokinetically, an interference between the two intraoperative tests was observed. There was no strong correlation between residual liver volume and function with a percentual residual-LiMAx mostly lower than the percentual residual volume (-17.7% ± 4.1%). CONCLUSIONS: Intraoperative application of the 13C-methacetin-breath-test during major liver resections seems to deliver lower values than the standard preoperative test. As multiple intraoperative tests interfere significantly to each other, a single intraoperative measurement is suggested. Multicentric standardized measurements could define the "normal" range for intraoperative measurements and control their predictive value.

A dual role of lysophosphatidic acid type 2 receptor (LPAR2) in nonsteroidal anti-inflammatory drug-induced mouse enteropathy.

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2-/-) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2-/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1ß, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2-/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy.

The Production of Recombinant African Swine Fever Virus Lv17/WB/Rie1 Strains and Their In Vitro and In Vivo Characterizations.

Lv17/WB/Rie1-Δ24 was produced via illegitimate recombination mediated by low-dilution serial passage in the Cos7 cell line and isolated on PAM cell culture. The virus contains a huge ~26.4 Kb deletion in the left end of its genome. Lv17/WB/Rie1-ΔCD-ΔGL was generated via homologous recombination, crossing two ASFV strains (Lv17/WB/Rie1-ΔCD and Lv17/WB/Rie1-ΔGL containing eGFP and mCherry markers) during PAM co-infection. The presence of unique parental markers in the Lv17/WB/Rie1-ΔCD-ΔGL genome indicates at least two recombination events during the crossing, suggesting that homologous recombination is a relatively frequent event in the ASFV genome during replication in PAM. Pigs infected with Lv17/WB/Rie1-Δ24 and Lv17/WB/Rie1/ΔCD-ΔGL strains have shown mild clinical signs despite that ASFV could not be detected in their sera until a challenge infection with the Armenia/07 ASFV strain. The two viruses were not able to induce protective immunity in pigs against a virulent Armenia/07 challenge.

Identification of herpesvirus transcripts from genomic regions around the replication origins.

Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a 'super regulatory center' in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries.

Pregabalin-Tolperisone Combination to Treat Neuropathic Pain: Improved Analgesia and Reduced Side Effects in Rats.

The current treatment of neuropathic pain (NP) is unsatisfactory; therefore, effective novel agents or combination-based analgesic therapies are needed. Herein, oral tolperisone, pregabalin, and duloxetine were tested for their antinociceptive effect against rat partial sciatic nerve ligation (pSNL)-induced tactile allodynia described by a decrease in the paw withdrawal threshold (PWT) measured by a dynamic plantar aesthesiometer. On day 7 after the operation, PWTs were assessed at 60, 120, and 180 min post-treatment. Chronic treatment was continued for 2 weeks, and again, PWTs were measured on day 14 and 21. None of the test compounds produced an acute antiallodynic effect. In contrast, after chronic treatment, tolperisone and pregabalin alleviated allodynia. In other experiments, on day 14, the acute antiallodynic effect of the tolperisone/pregabalin or duloxetine combination was measured. As a novel finding, a single dose of the tolperisone/pregabalin combination could remarkably alleviate allodynia acutely. It also restored the neuropathy-induced elevated CSF glutamate content. Furthermore, the combination is devoid of adverse effects related to motor and gastrointestinal transit functions. Tolperisone and pregabalin target voltage-gated sodium and calcium channels, respectively. The dual blockade effect of the combination might explain its advantageous acute analgesic effect in the present work.

Evaluation of Haematological and Immunological Parameters of the ASFV Lv17/WB/Rie1 Strain and Its Derived Mutant Lv17/WB/Rie1/d110-11L against ASFV Challenge Infection in Domestic Pigs.

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune-pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig's immune system. A better understanding of these immune-pathological mechanisms would help to design suitable vaccines against this disease.

Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1.

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5'-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Involvement of the MGF 110-11L Gene in the African Swine Fever Replication and Virulence.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease that causes extensive economic and animal welfare losses in the Eurasian pig (Sus scrofa) population. To date, no effective and safe vaccines have been marketed against ASF. A starting point for vaccine development is using naturally occurring attenuated strains as a vaccine base. Here, we aimed to remove the multigene family (MGF) 110 gene of unknown function from the Lv17/WB/Rie1 genome to improve the usability of the virus as a live-attenuated vaccine, reducing unwanted side effects. The MGF 110-11L gene was deleted using the CRISPR/Cas9 method, and the safety and efficacy of the virus were tested in pigs after isolation. The vaccine candidates administered at high doses showed reduced pathogenicity compared to the parental strain and induced immunity in vaccinated animals, although several mild clinical signs were observed. Although Lv17/WB/Rie1/d110-11L cannot be used as a vaccine in its current form, it was encouraging that the undesirable side effects of Lv17/WB/Rie1 at high doses can be reduced by additional mutations without a significant reduction in its protective capacity.

Interactions between NSAIDs, opioids and the gut microbiota - Future perspectives in the management of inflammation and pain.

The composition of intestinal microbiota is influenced by a number of factors, including medications, which may have a substantial impact on host physiology. Nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics are among those widely used medications that have been shown to alter microbiota composition in both animals and humans. Although much effort has been devoted to identify microbiota signatures associated with these medications, much less is known about the underlying mechanisms. Mucosal inflammation, changes in intestinal motility, luminal pH and bile acid metabolism, or direct drug-induced inhibitory effect on bacterial growth are all potential contributors to NSAID- and opioid-induced dysbiosis, however, only a few studies have addressed directly these issues. In addition, there is a notable overlap between the microbiota signatures of these drugs and certain diseases in which they are used, such as spondyloarthritis (SpA), rheumatoid arthritis (RA) and neuropathic pain associated with type 2 diabetes (T2D). The aims of the present review are threefold. First, we aim to provide a comprehensive up-to-date summary on the bacterial alterations caused by NSAIDs and opioids. Second, we critically review the available data on the possible underlying mechanisms of dysbiosis. Third, we review the current knowledge on gut dysbiosis associated with SpA, RA and neuropathic pain in T2D, and highlight the similarities between them and those caused by NSAIDs and opioids. We posit that drug-induced dysbiosis may contribute to the persistence of these diseases, and may potentially limit the therapeutic effect of these medications by long-term use. In this context, we will review the available literature data on the effect of probiotic supplementation and fecal microbiota transplantation on the therapeutic efficacy of NSAIDs and opioids in these diseases.

Performance of the intracerebroventricularly injected streptozotocin Alzheimer's disease model in a translationally relevant, aged and experienced rat population.

The intracerebroventricularly (icv) injected streptozotocin (STZ) induced brain state is a widely used model of sporadic Alzheimer-disease (AD). However, data have been generated in young, naive albino rats. We postulate that the translationally most relevant animal population of an AD model should be that of aged rats with substantial learning history. The objective of the study was thus to probe the model in old rats with knowledge in various cognitive domains. Long-Evans rats of 23 and 10 months age with acquired knowledge in five-choice serial reaction time task (5-CSRTT), a cooperation task, Morris water-maze (MWM) and "pot-jumping" exercise were treated with 3 × 1.5 mg/kg icv. STZ and their performance were followed for 3 months in the above and additional behavioral assays. Both STZ-treated age groups showed significant impairment in the MWM (spatial learning) and novel object recognition test (recognition memory) but not in passive avoidance and fear conditioning paradigms (fear memory). In young STZ treated rats, significant differences were also found in the 5CSRTT (attention) and pot jumping test (procedural learning) while in old rats a significant increase in hippocampal phospho-tau/tau protein ratio was observed. No significant difference was found in the cooperation (social cognition) and pairwise discrimination (visual memory) assays and hippocampal ß-amyloid levels. STZ treated old animals showed impulsivity-like behavior in several tests. Our results partly coincide with partly deviate from those published on young, albino, unexperienced rats. Beside the age, strain and experience level of the animals differences can also be attributed to the increased dose of STZ, and the applied food restriction regime. The observed cognitive and non-cognitive activity pattern of icv. STZ in aged experienced rats call for more extensive studies with the STZ model to further strengthen and specify its translational validity.

Introduction of a pharmacological neurovascular uncoupling model in rats based on results of mice.

Our aim was to establish a pharmacologically induced neurovascular uncoupling (NVU) method in rats as a model of human cognitive decline. Pharmacologically induced NVU with subsequent neurological and cognitive defects was described in mice, but not in rats so far. We used 32 male Hannover Wistar rats. NVU was induced by intraperitoneal administration of a pharmacological "cocktail" consisting of N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MSPPOH, a specific inhibitor of epoxyeicosatrienoic acid-producing epoxidases, 5 mg kg-1), L-NG-nitroarginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 10 mg kg-1) and indomethacin (a nonselective inhibitor of cyclooxygenases, 1 mg kg-1) and injected twice daily for 8 consecutive days. Cognitive performance was tested in the Morris water-maze and fear-conditioning assays. We also monitored blood pressure. In a terminal operation a laser Doppler probe was used to detect changes in blood-flow (CBF) in the barrel cortex while the contralateral whisker pad was stimulated. Brain and small intestine tissue samples were collected post mortem and examined for prostaglandin E2 (PGE2) level. Animals treated with the "cocktail" showed no impairment in their performance in any of the cognitive tasks. They had higher blood pressure and showed cca. 50% decrease in CBF. Intestinal bleeding and ulcers were found in some animals with significantly decreased levels of PGE2 in the brain and small intestine. Although we could evoke NVU by the applied mixture of pharmacons, it also induced adverse side effects such as hypertension and intestinal malformations while the treatment did not cause cognitive impairment. Thus, further refinements are still required for the development of an applicable model.

The Acute Antiallodynic Effect of Tolperisone in Rat Neuropathic Pain and Evaluation of Its Mechanism of Action.

Current treatment approaches to manage neuropathic pain have a slow onset and their use is largely hampered by side-effects, thus there is a significant need for finding new medications. Tolperisone, a centrally acting muscle relaxant with a favorable side effect profile, has been reported to affect ion channels, which are targets for current first-line medications in neuropathic pain. Our aim was to explore its antinociceptive potency in rats developing neuropathic pain evoked by partial sciatic nerve ligation and the mechanisms involved. Acute oral tolperisone restores both the decreased paw pressure threshold and the elevated glutamate level in cerebrospinal fluid in neuropathic rats. These effects were comparable to those of pregabalin, a first-line medication in neuropathy. Tolperisone also inhibits release of glutamate from rat brain synaptosomes primarily by blockade of voltage-dependent sodium channels, although inhibition of calcium channels may also be involved at higher concentrations. However, pregabalin fails to affect glutamate release under our present conditions, indicating a different mechanism of action. These results lay the foundation of the avenue for repurposing tolperisone as an analgesic drug to relieve neuropathic pain.

Allele-Specific Dual PCRs to Identify Members of the 27a Cluster of PPV.

Porcine Parvovirus (PPV) is one of the most important infectious agents causing severe reproductive failure in pigs. In the last two decades a particular, a novel genotype emerged in Europe and PPV-27a was named as the prototype of this genetic cluster. It was suggested that members of the PPV-27a cluster may adversely influence effective vaccination against PPV. For a reliable updated 27a definition, we aligned 93 databank-deposited partial or full nucleotide and protein sequences of the VP2 of different PPV isolates. We confirmed that the 27a cluster could indeed be distinguished from other members of the species, however, some divergences were identified compared to earlier defined genetic markers. Based on genetic differences, we developed a dual allele-specific polymerase chain reaction for the easy and quick discrimination of members of the 27a cluster from other PPV strains. The detection limit of dual PCR was found <1.66 × 104 copies/reaction. To sensitize and make it more user friendly, the method was further developed for qPCR application with fluorescent probes. Regarding the detection limit of the two PCRs (<1.66 × 104 copies/reaction of the dual PCR versus <2.40 × 102 copy/reaction of the dual qPCR), approximately two log improvement was achieved in the sensitivity of the method.

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate-early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.

Hemangioma of the uterine cervix: report of four cases and review of the literature. / Haemangioma a méhnyakban: 4 eset ismertetése és irodalmi áttekintés.

Összefoglaló. A haemangioma a noi nemi szervekben viszonylag ritkán, a méhnyakban pedig még ritkábban fordul elo. Kis mérete és szegényes megjelenése miatt elkerülheti a figyelmet, elofordul azonban, hogy mutéti ellátást igénylo vérzést okoz. Az évek során 4 esetben (ebbol 2 esetben terhesség alatt) diagnosztizáltunk méhnyak-haemangiomát (2 esetben cervicalis intraepithelialis carcinomával társulva), melyeknek ismertetjük változatos tüneteit, kolposzkópos megjelenését és a diagnózist biztosító szövettani (immunhisztokémiai) illusztrációit, valamint a képlet terhesség alatti fejlodésének kolposzkópos monitorizálását. 2 esetben capillaris (cavernosus) haemangiomát, 2 esetben arteriovenosus malformatiót igazoltunk. Az általunk hozzáférheto szakirodalomban nem találtunk magyar szerzo(k)tol beszámolót errol a cervicalis lokalizációjú, ritka, jóindulatú, de gyakran veszélyes vascularis daganatról. Orv Hetil. 2022; 163(5): 187-194. Summary. Hemangioma is relatively rare in the female genital organs and even less common in the uterine cervix. Its small size and poor appearance often result in a missed diagnosis, but it may cause bleeding that requires surgery. Over the years, we have confirmed the diagnosis of cervical hemangioma in 4 cases including two in pregnancy. 2 cases were associated with cervical intraepithelial neoplasia. This case report describes the symptoms, colposcopic appearance, and histological characteristics including immunohistochemical findings, and the colposcopic monitoring of development of the condition during pregnancy. In 2 cases, a capillary (cavernous) hemangioma, in 2 cases an arteriovenous malformation was diagnosed. We did not find any report from Hungarian author(s) about this rare, benign, but often dangerous vascular tumor with cervical localization. Orv Hetil. 2022; 163(5): 187-194.

Corrigendum: Intracerebroventricularly Injected Streptozotocin Exerts Subtle Effects on the Cognitive Performance of Long-Evans Rats.

[This corrects the article DOI: 10.3389/fphar.2021.662173.].

Shedding Light on the Pharmacological Interactions between µ-Opioid Analgesics and Angiotensin Receptor Modulators: A New Option for Treating Chronic Pain.

The current protocols for neuropathic pain management include µ-opioid receptor (MOR) analgesics alongside other drugs; however, there is debate on the effectiveness of opioids. Nevertheless, dose escalation is required to maintain their analgesia, which, in turn, contributes to a further increase in opioid side effects. Finding novel approaches to effectively control chronic pain, particularly neuropathic pain, is a great challenge clinically. Literature data related to pain transmission reveal that angiotensin and its receptors (the AT1R, AT2R, and MAS receptors) could affect the nociception both in the periphery and CNS. The MOR and angiotensin receptors or drugs interacting with these receptors have been independently investigated in relation to analgesia. However, the interaction between the MOR and angiotensin receptors has not been excessively studied in chronic pain, particularly neuropathy. This review aims to shed light on existing literature information in relation to the analgesic action of AT1R and AT2R or MASR ligands in neuropathic pain conditions. Finally, based on literature data, we can hypothesize that combining MOR agonists with AT1R or AT2R antagonists might improve analgesia.

Genetic Variability of PRRSV Vaccine Strains Used in the National Eradication Programme, Hungary.

Porcine reproductive and respiratory syndrome (PRRS) is a globally spread, highly infectious viral disease. Live, attenuated vaccines against PRRS virus (PRRSV) decrease virus excretion and evoke protective immunity reducing the economic damage caused by the disease. In a longitudinal molecular epidemiological study accompanying ongoing national eradication programme we evaluated the suitability of PRRSV ORF5 and ORF7 sequences to identify possible field strains of vaccine-origin. In total, 2342 ORF5 sequences and 478 ORF7 sequences were analysed. Vaccine strains were identified by sequence identity values and phylogenetic network analysis. Strains that shared greater than 98% nucleotide identity within ORF5 and/or ORF7 were considered to have originated from vaccine. A total of 882 (37.6%) ORF5 and 88 (18.4%) ORF7 sequences met these criteria. In detail, 618, 179 and 35 ORF5 and 51, 29 and 8 ORF7 sequences were related to Porcilis PRRS vaccine, Unistrain PRRS vaccine, and ReproCyc PRRS EU vaccine, respectively. Data showed that the Porcilis vaccine was genetically more stable. Whereas, the variability of the Unistrain and the ReproCyc strains was significantly higher. Given that ORF7 shares, in some instances, complete identity between a particular vaccine strain and some historic variants of field PRRSV strains, care must be taken when evaluating vaccine relatedness of a field isolate based on the ORF7. On the contrary, ORF5 sequences were more suitable to predict the vaccine origin making a distinction more robustly between field and vaccine strains. We conclude that ORF5 based molecular epidemiological studies support more efficiently the ongoing PRRS eradication programmes. The conclusions presented in this large-scale PRRS molecular epidemiological study provides a framework for future eradication programmes planned in other countries.

The Nonsteroidal Anti-Inflammatory Drug Ketorolac Alters the Small Intestinal Microbiota and Bile Acids Without Inducing Intestinal Damage or Delaying Peristalsis in the Rat.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) induce significant damage to the small intestine, which is accompanied by changes in intestinal bacteria (dysbiosis) and bile acids. However, it is still a question of debate whether besides mucosal inflammation also other factors, such as direct antibacterial effects or delayed peristalsis, contribute to NSAID-induced dysbiosis. Here we aimed to assess whether ketorolac, an NSAID lacking direct effects on gut bacteria, has any significant impact on intestinal microbiota and bile acids in the absence of mucosal inflammation. We also addressed the possibility that ketorolac-induced bacterial and bile acid alterations are due to a delay in gastrointestinal (GI) transit. Methods: Vehicle or ketorolac (1, 3 and 10 mg/kg) were given to rats by oral gavage once daily for four weeks, and the severity of mucosal inflammation was evaluated macroscopically, histologically, and by measuring the levels of inflammatory proteins and claudin-1 in the distal jejunal tissue. The luminal amount of bile acids was measured by liquid chromatography-tandem mass spectrometry, whereas the composition of microbiota by sequencing of bacterial 16S rRNA. GI transit was assessed by the charcoal meal method. Results: Ketorolac up to 3 mg/kg did not cause any signs of mucosal damage to the small intestine. However, 3 mg/kg of ketorolac induced dysbiosis, which was characterized by a loss of families belonging to Firmicutes (Paenibacillaceae, Clostridiales Family XIII, Christensenellaceae) and bloom of Enterobacteriaceae. Ketorolac also changed the composition of small intestinal bile by decreasing the concentration of conjugated bile acids and by increasing the amount of hyodeoxycholic acid (HDCA). The level of conjugated bile acids correlated negatively with the abundance of Erysipelotrichaceae, Ruminococcaceae, Clostridiaceae 1, Muribaculaceae, Bacteroidaceae, Burkholderiaceae and Bifidobacteriaceae. Ketorolac, under the present experimental conditions, did not change the GI transit. Conclusion: This is the first demonstration that low-dose ketorolac disturbed the delicate balance between small intestinal bacteria and bile acids, despite having no significant effect on intestinal mucosal integrity and peristalsis. Other, yet unidentified, factors may contribute to ketorolac-induced dysbiosis and bile dysmetabolism.