Increased viral read counts and metagenomic full genome characterization of porcine astrovirus 4 and Posavirus 1 in sows in a swine farm with unexplained neonatal piglet diarrhea.

Neonatal diarrhea in piglets may cause major losses in affected pig herds. The present study used random high-throughput RNA sequencing (metagenomic next generation sequencing, mNGS) to investigate the virome of sows from a farm with persistent neonatal piglet diarrhea in comparison to two control farms without diarrhea problems. A variety of known swine gastrointestinal viruses was detected in the control farms as well as in the problem farm (Mamastrovirus, Enterovirus, Picobirnavirus, Posavirus 1, Kobuvirus, Proprismacovirus). A substantial increase in normalized viral read counts was observed in the affected farm compared to the control farms. The increase was attributable to a single viral species in each of the sampled sows (porcine astrovirus 4 and Posavirus 1). The complete genomes of a porcine astrovirus 4 and two co-infecting Posavirus 1 were de novo assembled and characterized. The 6734 nt single-stranded RNA genome of porcine astrovirus 4 (PoAstV-4) strain Belgium/2019 contains three overlapping open reading frames (nonstructural protein 1ab, nonstructural protein 1a, capsid protein). Posavirus 1 strains Belgium/01/2019 and Belgium/02/2019 have a 9814 nt single-stranded positive-sense RNA genome encoding a single open reading frame (polyprotein precursor) containing the five expected Picornavirales-conserved protein domains. The study highlights the potential of mNGS workflows to study unexplained neonatal diarrhea in piglets and contributes to the scarce availability of both PoAstV-4 and Posavirus-1 whole genome sequences from Western Europe.

Activation of HIF-1α by δ-Opioid Receptors Induces COX-2 Expression in Breast Cancer Cells and Leads to Paracrine Activation of Vascular Endothelial Cells.

Opioids promote tumor angiogenesis in mammary malignancies, but the underlying signaling mechanism is largely unknown. The current study investigated the hypothesis that stimulation of δ-opioid receptors (DOR) in breast cancer (BCa) cells activates the hypoxia-inducible factor 1α (HIF-1α), which triggers synthesis and release of diverse angiogenic factors. Immunoblotting revealed that incubation of human MCF-7 and T47D breast cancer cells with the DOR agonist d-Ala2,d-Leu5-enkephalin (DADLE) resulted in a transient accumulation and thus activation of HIF-1α DADLE-induced HIF-1α activation preceded PI3K/Akt stimulation and was blocked by the DOR antagonist naltrindole and naloxone, pertussis toxin, different phosphoinositide 3-kinase (PI3K) inhibitors, and the Akt inhibitor Akti-1/2. Whereas DADLE exposure had no effect on the expression and secretion of vascular endothelial growth factor (VEGF) in BCa cells, an increased abundance of cyclooxygenase-2 (COX-2) and release of prostaglandin E2 (PGE2) was detected. DADLE-induced COX-2 expression was also observed in three-dimensional cultured MCF-7 cells and impaired by PI3K/Akt inhibitors and the HIF-1α inhibitor echinomycin. Supernatant from DADLE-treated MCF-7 cells triggered sprouting of endothelial (END) cells, which was blocked when MCF-7 cells were pretreated with echinomycin or the COX-2 inhibitor celecoxib. Also no sprouting was observed when END cells were exposed to the PGE2 receptor antagonist PF-04418948. The findings together indicate that DOR stimulation in BCa cells leads to PI3K/Akt-dependent HIF-1α activation and COX-2 expression, which trigger END cell sprouting by paracrine activation of PGE2 receptors. These findings provide a potential mechanism of opioid-driven tumor angiogenesis and thus therapeutic targets to combat the tumor-angiogenic opioid effect. SIGNIFICANCE STATEMENT: Opioids are indispensable analgesics for treating cancer-related pain. However, opioids were found to promote tumor growth and metastasis, which questions the use of these potent pain-relieving drugs in cancer patients. Enhanced tumor vascularization after opioid treatment implies that tumor progression results from angiogenic opioid effects. Thus, understanding the signaling mechanism of opioid-driven tumor angiogenesis helps to identify therapeutic targets to combat these undesired tumor effects. The present study reveals that stimulation of δ-opioid receptors in breast cancer cells leads to an activation of HIF-1α and expression of COX-2 via PI3K/Akt stimulation, which results in a paracrine activation of vascular endothelial cells by prostaglandin E2 receptors.

Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae.

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDSD28-D56 -61.90%, macroscopic lung lesions -88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) -67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.

Prevalence and chemical composition of uroliths in fattening pigs in Belgium.

The present study investigated the prevalence of uroliths in fattening pigs and assessed the composition of these urinary tract concrements. In total, 2,432 urinary bladders were sampled in the slaughterhouse and checked for abnormal content. Urinary samples were analysed microscopically, and samples of the urinary bladder wall were tested for histological signs of inflammation. The composition of the concrements was examined by infrared spectrophotometry. Macroscopic and microscopic abnormalities were detected in 8.4% and 52.8% of the samples respectively. Magnesium ammonium phosphate (struvite), calcium oxalate dihydrate (COD), calcium carbonate (calcite), calcium oxalate monohydrate (COM) and amorphous crystals were detected. Analysis of stones showed COD in all samples in different proportions. The calcium content of examined stones was always considerable (up to 34%), in contrast to the magnesium content which represented max 1.9%. Struvite was found in one third of the samples, but was never part of stones and grit. COD crystals were the second most common microscopic crystal. These COD crystals and some COD stones had a rectangular shape, and therefore, they can be harmful to the bladder mucosa. In conclusion, uroliths are present in a large proportion of male fattening pigs, and consequently, urinary concrements pose a life-threatening risk for urethra obstruction in male pigs. Further research is warranted to identify potential risk factors for urolithiasis and microscopic crystals.

Environmental dynamics of Campylobacter jejuni genotypes circulating in Luxembourg: what is the role of wild birds?

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide, but, unlike other foodborne pathogens, is not commonly reported as causing outbreaks. The population structure of the species is characterized by a high degree of genetic diversity, but the presence of stable clonally derived genotypes persisting in space and time, and potentially leading to diffuse outbreaks, has recently been identified. The spread of these recurring genotypes could be enhanced by wild birds, suspected to act as vectors for a wide range of microorganisms that can be transmissible to other animals or humans. This study assessed the genetic diversity of C. jejuni carriage in wild birds and surface waters to explore a potential link between these environments and the persistence over years of recurring lineages infecting humans in Luxembourg. These lineages corresponded to over 40 % of clinical isolates over a 4 year period from 2018 to 2021. While mainly exotic genotypes were recovered from environmental samples, 4 % of C. jejuni from wild birds corresponded to human recurring genotypes. Among them, a human clinical endemic lineage, occurring for over a decade in Luxembourg, was detected in one bird species, suggesting a possible contribution to the persistence of this clone and its multi-host feature. Whereas 27 % of wild birds were carriers of C. jejuni, confirming their role as spreader or reservoir, only three out of 59 genotypes overlapped with recurring human strains. While direct transmission of C. jejuni infection through wild birds remains questionable, they may play a key role in the environmental spreading of stable clones to livestock, and this issue merits further investigation.

Relationship between piglets' survivability and farrowing kinetics in hyper-prolific sows.

BACKGROUND: Prolonged farrowing and more piglets born with low birth weight are undesirable consequences of genetic selection for increased litter size. The objective of the present observational study was to evaluate the relationship between piglets' survivability and farrowing kinetics in hyperprolific sows. A total of 58 sows of different parities and 1190 piglets were included. The entire farrowing process was monitored and the following parameters were recorded: inter-piglet birth interval, birth order, total born, live born, dead born, and mummified piglets, obstetric intervention, weight at birth and 24h, colostrum yield and intake. RESULTS: The sows included in this study had on average 20.6 ± 0.6 total piglets born, of which 16.4 ± 0.6 were live born, 3.3 ± 0.4 were stillborn and 0.9 ± 0.2 were mummified piglets. The average farrowing duration and average birth interval were 411.3 ± 31.6 and 20.6 ± 1.7 min, respectively. Farrowing duration was positively associated (p < 0.05) with parity, number of stillborn and mummified piglets. Piglet mortality 24h after birth was negatively affected (p < 0.01) by birth weight and positively affected (p < 0.01) by cumulative birth interval. The last tercile of piglets born (birth order ≥ 17) had the highest (p < 0.01) inter-piglet birth interval (IPBI) (43.4 ± 4.17 min) compared to piglets born in the first (birth order between 2 and 7) (26.5 ± 3.8 min) and second (birth order between 8 and 16) terciles (21.9 ± 3.8 min). Cumulative birth interval, birth weight, occurrence of stillborn piglets and manual intervention were positively associated (p < 0.05) with IPBI. Piglet birth weight was also positively associated (p < 0.01) to individual colostrum intake. Piglets ingesting more colostrum had lower (p < 0.01) mortality from 24h after birth until weaning. Sow's parity and cumulative birth interval were positively associated with the presence of stillborn piglets (p = 0.02 and p < 0.01, respectively). CONCLUSION: Reducing farrowing duration may be crucial to decrease stillbirth rate and neonatal mortality in hyperprolific sows. Moreover, special care must be provided to the lighter piglets within a litter to increase their colostrum intake and minimize piglet's mortality throughout lactation.

Porcine ear necrosis in weaned piglets: prevalence and impact on daily weight gain.

BACKGROUND: Porcine ear necrosis (PEN) in pigs is characterized by a blue to black discoloration of the tip or margin of the ear followed by necrosis. The present study investigated the prevalence of PEN in a Belgian pig farm with PEN problems in nursery pigs, the effect of a mycotoxin detoxifier added to the feed on PEN prevalence, and the impact of PEN on the piglets' growth. Six consecutive batches of weaned piglets [565-751 piglets per batch, (n = 3898)] were included. For each weaning batch, the presence and severity of PEN during the nursery period (3-10 weeks of age) were recorded weekly. Average daily gain (ADG) was calculated by weighing 597 individual piglets divided over the six batches. Additionally different mycotoxins were measured in the feed using LC-MS/MS analysis, and to three randomly selected batches, a mycotoxin detoxifier (Mycofix® Plus 5E, Biomin) was added to the feed. RESULTS: At the end of the nursery period, 11.0-32.0% of the piglets in each batch were affected. The prevalence increased with the number of weeks post-weaning, especially from week 4 after weaning onwards. Mild, moderate, severe and very severe lesions represented 84.6%, 14.0%, 1.3% and 0.1% of all lesions, respectively. Different mycotoxins were present in the feed, but all at low concentrations. The mean ADG (± SD) for pigs without (n = 243) and with (n = 158) lesions was 391 g (± 71 g) and 394 g (± 65 g), respectively (P > 0.05). The ADG for mildly affected (387 g ± 68 g) and moderately affected piglets (420 g ± 44 g) was not significantly different (P > 0.05). The PEN prevalence in the batches without or with the mycotoxin detoxifier was 25% and 22%, respectively (P > 0.05). CONCLUSIONS: Twenty-three percent of animals showed lesions at the end of the nursery. Affected pigs did not have a lower ADG compared to non-affected animals, which might be explained by the fact that most affected piglets only had mild lesions. The addition of a mycotoxin detoxifier did not influence the prevalence of PEN, possibly because of the low levels of mycotoxin contamination. Further research is warranted to assess the impact of more severe PEN lesions and the effect of control measures.

Prophylactic Use of Meloxicam and Paracetamol in Peripartal Sows Suffering From Postpartum Dysgalactia Syndrome.

Postpartum dysgalactia syndrome (PPDS) is a major economic problem in modern sow farms. General treatment of PPDS consists of the use of oxytocin to promote milk ejection and non-steroidal anti-inflammatory drugs (NSAIDs) to alleviate inflammatory processes. So far, studies investigated the use of a single administration of NSAIDs after parturition in healthy and non-healthy sows. The current study investigated whether administration of meloxicam or paracetamol in sows prior to parturition improves sow and piglet health as well as performance in a farm with PPDS problems in sows. Sixty sows and 978 piglets from a Belgian farrow-to-finish farm were enrolled. Sows were randomly divided into three groups: a non-treated control group, a meloxicam-treated group and a paracetamol-treated group. Treatment was administered orally for 7 days from gestation day 113 onwards. Performance and health parameters investigated in sows were gestation length, farrowing duration, litter characteristics, colostrum yield and quality (Immunoglobulin G), litter weight gain, weaning-to-estrus interval, pregnancy rate, rectal temperature, acute phase proteins and inflammatory markers serum amyloid A, haptoglobin, interferon γ, interleukin 1ß and 6 backfat, constipation and feed refusal. Performance and health parameters in piglets were birthweight, average daily weight gain, colostrum intake and mortality. Paracetamol-treated sows showed a significantly (P = 0.04) lower rectal temperature (mean ± SD: 38.09 ± 0.18°C) than the meloxicam-treated sows (38.24 ± 0.18°C), but not than the control group (38.22 ± 0.18°C). Sows of the paracetamol-treated group had a significantly (P = 0.001) longer gestation length (116.3 ± 0.9 days) than sows of the control group (115.3 ± 0.6 days), but not than meloxicam-treated sows (115.9 ± 0.9 days). No significant differences between the three groups were found for all the other parameters. In conclusion, the prophylactic oral administration of either meloxicam or paracetamol for 7 days starting 2 days prior to farrowing did not show beneficial effects on both health and performance parameters of sows and piglets.

Use of non-steroidal anti-inflammatory drugs in porcine health management.

OBJECTIVE: Treatment of inflammation and pain management is an important topic in the welfare of pigs. It is very difficult for veterinary practitioners to choose the most appropriate product for a certain problem. This review aims to summarise and discuss the characteristics of different non-steroidal anti-inflammatory drugs (NSAIDs), as well as paracetamol and metamizole, available for pigs in the European Union. METHODS: The databases Pubmed, Google Scholar, CliniPharm CliniTox and European Medicines Agency were searched. Relevant terms (eg,'meloxicam', 'fever', 'swine', 'pig', 'inflammation', 'castration', 'pain') were used to search for original articles, reviews and books. Only peer-reviewed articles were used. References from studies were also analysed in order to find additional relevant studies. CONCLUSION: Studies which have investigated the efficacy of NSAIDs for different conditions, using different treatment regimens, are scarce. Most studies focused on the efficacy of NSAID-related pain alleviation in piglet castration, as well as the anti-inflammatory potential of NSAIDs in experimental inflammation models. Little research has been carried out on the use of metamizole, tolfenamic acid, paracetamol and sodium salicylate and their effect in pigs.

In vitro study to assess the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) for treating canine mammary tumours.

Therapy of canine mammary tumours (CMTs) with classical antitumour drugs is problematic, so better therapeutic options are needed. Palbociclib (PD-0332991) is an innovative and effective anticancer drug for the treatment of breast cancer in women. Palbociclib is an inhibitor of cyclin-dependent kinase 4 (CDK4) and CDK6, which are key regulators of the cell cycle machinery and thus cell proliferation. In the present in vitro study, we investigated whether Palbociclib also represents a candidate drug to combat CMT. For this purpose, the effect of Palbociclib was analysed in P114 and CF41 cells, two CMT cell lines with an endogenous CDK4/6 co-expression. Incubation of P114 and CF41 cells with Palbociclib resulted in a dose- and time-dependent loss of phosphorylated retinoblastoma protein (pRb), a classical CDK4/6 substrate within the cell cycle machinery. Moreover, treatment of CMT cells with Palbociclib-induced cell cycle arrest affected cell viability, prevented colony formation and impaired cell migration activity. Palbociclib also inhibited the growth of P114 and CF41 cell spheroids. Immunohistochemical analysis of canine patient samples revealed a consistent expression of CDK6 in different canine mammary carcinoma types, but an individual and tumour-specific expression pattern of phosphorylated pRb independent of the tumour grade. Together, our findings let us suggest that Palbociclib has antitumour effects on CMT cells and that canine patients may represent potential candidates for treatment with this CDK4/6 inhibitor.