Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Histopathological lesions in reproductive organs, distal spinal cord and peripheral nerves of horses naturally infected with Trypanosoma equiperdum.

BACKGROUND: Dourine, a venereal transmitted trypanosomosis caused by Trypanosoma equiperdum, has different clinical signs related to the reproductive and nervous system. Pathologic tissue changes associated with the disease are poorly described. The present study describes the histopathological lesions in naturally T. equiperdum-infected horses in the chronical stage of dourine. RESULTS: Four chronically dourine diseased horses underwent a post-mortem examination. They were Woo test negative, but CATT/T. evansi positive, had a low packed cell volume (PCV) and exhibited obvious clinical signs of dourine. Post-mortem examination did not reveal gross lesions in the organs assumed to be responsible for the symptomatology. On histopathology, genital organs were affected, with mononuclear cell infiltration and erosions and degeneration of seminiferous tubules and perivascular lymphoplasmacytic cuffing in the uterus. In the nervous system, mononuclear cell infiltration was located in peripheral nerves, ganglia and in the spinal cord, leading to axonal degeneration. Real-time PCR using ITS primer revealed the presence of trypanosomes in these organs and conventional PCRs using maxicircle and RoTat1.2 primers further confirmed the involvement of T. equiperdum since the DNAs from the vagina, testicle, distal spinal cord, sciatic and obturator nerves found to be positive for maxicircle and negative for RoTat 1.2. CONCLUSIONS: The histopathological lesions in the spinal cord and peripheral nerves explain the incoordination of the hind legs in T. equiperdum-infected horses, whilst its presence in the genital tract exemplifies the venereal transmission.

Tissue (re)distribution of Trypanosoma equiperdum in venereal infected and blood transfused horses.

Dourine, caused by Trypanosoma equiperdum, is a life-threatening venereal disease in equidae. So far, there is no clear evidence on how and when stallions become infectious, nor which tissues are affected by the parasite in diseased animals. Post-infection, after a transient, temporary phase of parasitaemia, the parasite disperses to different tissues in an unknown distribution pattern. This study describes the distribution of the parasite after infection by artificial insemination (AI) or blood transfusion. Mares (N = 4) were artificially inseminated with T. equiperdum spiked semen whereas stallions (N = 4) were infected by blood transfusion. The course of the disease was monitored by parasitological (Woo) and molecular (PCR) tests and clinical signs and haematological parameters were recorded. At 120 days post infection, horses had a full necropsy, histopathology and PCR. A similar pattern of parasitaemia, disease progression and tissue distribution were seen in all horses. Ejaculated semen in the preclinical stage and epididymal semen in the chronic stage of the disease was positive on PCR and caused infection in mice. Cymelarsan® treatment in the chronic stage did not result in a clinico-haematological or histopathological improvement. At necropsy, lesions were observed in the nervous and reproductive system. Histopathological lesions were most severe in the peripheral nerves and associated ganglia, the testicles and genital mucosae with multifocal infiltration of lymphocytes, plasma cells and histocytes. The parasites disseminated to several tissues including the nervous system, testicles and semen. The results indicate that transmission of T. equiperdum is possible through semen even from symptomless stallions post-treatment.

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

Safety assessment of the butyrate-producing Butyricicoccus pullicaecorum strain 25-3(T), a potential probiotic for patients with inflammatory bowel disease, based on oral toxicity tests and whole genome sequencing.

Inflammatory bowel disease (IBD) is a chronic inflammation of the digestive tract, characterized by dysbiosis of the intestinal microbiota. Probiotics have been suggested as a strategy to reduce active disease or extend remission. We isolated and characterized the butyrate-producing strain Butyricicoccus pullicaecorum 25-3(T) and identified it as a potential probiotic for patients with IBD. To evaluate the safety of 25-3(T) for use in humans, we conducted a standard acute oral toxicity test and a 28-day repeated oral dose toxicity test. The complete genome of B. pullicaecorum 25-3(T) was sequenced to search for virulence factors and antibiotic resistance determinants. The minimum inhibitory concentration (MIC) of 21 antimicrobials was determined. Results showed no adverse effects in the oral toxicity tests. B. pullicaecorum 25-3(T) is resistant against aminoglycosides and trimethoprim. The genome of 25-3(T) contains no virulence factors, one gene related to harmful metabolites and 52 sequences with high similarity to antimicrobial and toxic compound resistance genes, that did not correspond with a resistant phenotype. This first report of a safety assessment of a butyrate-producing strain from Clostridium cluster IV shows that B. pullicaecorum 25-3(T) is a non-pathogenic strain, but carries antibiotic resistance genes with the risk of transfer, that need further investigation.

Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus's egg?

BACKGROUND: The establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated. RESULTS: Florfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 µg/ml for florfenicol and 8.0 µg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 µg/ml florfenicol or 16 µg/ml trimethoprim combined with 80 µg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 µg/ml but not to 10 µg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 µg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection. CONCLUSIONS: We thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Tensile strength testing for resorbable mesh fixation systems in laparoscopic ventral hernia repair.

BACKGROUND: In an attempt to improve patient outcome and quality of life after laparoscopic ventral hernia repair, resorbable fixation devices have been developed to allow adequate mesh fixation while minimizing accompanying side-effects as tack erosion and adhesion formation. MATERIALS AND METHODS: In experimental set-up, 24 pigs were treated by laparoscopic mesh placement. Two different meshes (PP/ORC and PP/ePTFE) and four fixation devices were evaluated: a 6.4 mm poly(D,L: )-lactide pushpin (tack I), a 6.8 mm poly(D,L: )-lactide with blunt tip (tack II), a 4.1 mm poly(glycolide-co-L-lactide) (tack III) and one titanium tack (control tack). A first group of animals (n = 12) was euthanized after 2 weeks survival and a second group (n = 12) after 6 months. At euthanasia, a relaparoscopy was performed to assess adhesion formation followed by laparotomy with excision of the entire abdominal wall. Tensile strength of the individual fixation systems was tested with the use of a tensiometer by measuring the force to pull the tack out of the mesh. Additionally, the foreign body reaction to the fixation systems was evaluated histologically as was their potential degradation. RESULTS: At 2 weeks the tensile strength was significantly higher for the control tack (31.98 N/cm²) compared to the resorbable devices. Except for tack II, the tensile strength was higher when the devices were fixed in a PP/ePTFE mesh compared to the PP/ORC mesh. After 6 months only tack III was completely resorbed, while tack I (9.292 N/cm²) had the lowest tensile strength. At this time-point similar tensile strength was observed for both tack II (29.56 N/cm²) and the control tack (27.77 N/cm²). Adhesions seem to be more depending on the type of mesh, in favor of PP/ePTFE. CONCLUSION: At long term, the 4.1 mm poly(glycolide-co-L-lactide) tack was the only tack completely resorbed while the 6.8 mm poly(D,L: )-lactide tack with blunt tip reached equal strengths to the permanent tack.