Pathology of female mice experimentally infected with an in vitro cultured strain of Trypanosoma equiperdum

Dourine, caused by infection with Trypanosoma equiperdum, is one of the trypanosomiasis in equids. The clinical course of dourine is long-term, ranging from 1–2 months to several years. Since the pathogenesis of dourine has not yet been elucidated, experimental studies using mouse infection models are needed. Although mice are not susceptible to most T. equiperdum strains, some strains can infect mice. Even in such strains, infected mice develop rapidly transient parasitemia and die within 2–8 days. Therefore, mice experimentally infected with these T. equiperdum strains are not suitable for mouse infection models to analysis the pathogenesis of dourine. A sequential method of isolating parasites from dourine-affected horses and adapting them to in vitro cultures using soft agarose media was recently developed. Various T. equiperdum strains adapted to in vitro conditions have been established using this technique. We used one of these strains, the T. equiperdum IVM-t2 strain. In the present study, T. equiperdum IVM-t2 strain inoculated mice developed periodic parasitemia during the experimental period of 60 days. Histopathologically, vaginitis and dermatitis were observed. These findings were comparable to those of dourine-affected horses. Therefore, mice infected with T. equiperdum IVM-t2 strain may be a valuable tool for pathological, immunological, and parasitological in vivo research, and will contribute to investigations on the mechanisms underlying the disease process and the host-parasite relationship.     

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS–based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.     

Bovine immune response to papillomatous digital dermatitis (PDD)-associated spirochetes is skewed in isolate reactivity and subclass elicitation

Papillomatous digital dermatitis (PDD) is a growing cause of lameness of dairy cattle worldwide. Farms with PDD-afflicted cows experience economic loss due to treatment costs, decreased milk production, lower reproductive efficiency and premature culling. Cows exhibit both humoral and cellular immune responses to PDD-associated spirochetes. This study was undertaken to further characterize the bovine humoral response to PDD-associated spirochetes. Forty-seven sera samples collected from cattle (Field cattle) on three different dairy operations in Iowa were analyzed. In addition, sera were obtained from six young steers (Test cattle) that received a mixed inoculum of four previously isolated Treponema phagedenis-like spirochetes (1A, 3A, 4A and 5B) on two separate occasions. Relative levels of total IgG, IgG1, IgG2 and IgM reactive to each individual spirochete were determined. Field cattle had a higher mean antibody response to 5B compared to the other isolates and T. phagedenis. Test cattle reacted most strongly with 4A following initial exposure, shifting to a greater reactivity with 5B and a reactivity profile similar to field cattle following secondary exposure. No measurable IgM was detected. IgG1 was produced predominately in all cattle. Low to moderate levels of total IgG reactivity to T. phagedenis occurred with sera from all cattle.     

Induction of seroconversion and persistence of Salmonella Typhimurium in pigs are strain dependent

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Salmonella Typhimurium pathogenesis is host species specific. In addition, differences in in vitro behaviour of Salmonella Typhimurium strains have also been described, which may be reflected by a different course of infection within a host species. We compared the course of a Salmonella Typhimurium infection in pigs, using two Salmonella Typhimurium strains that were able to interfere with MHC II expression on porcine macrophages to a different extent in vitro. After experimental inoculation, blood and faecal samples from all pigs were collected at regular time points. At 40 days post inoculation (pi), animals were euthanized and tissue samples were bacteriologically analysed. The proportion of serologically positive piglets at 33 days pi was significantly higher in pigs that were inoculated with the strain that did not downregulate MHC II expression in vitro. Furthermore, this strain was less frequently shed and isolated in lower numbers from tonsils and ileocaecal lymph nodes than the strain that was able to markedly downregulate MHC II expression in vitro. We thus found that the delayed onset of seroconversion after oral inoculation of piglets with a particular Salmonella Typhimurium strain coincided with higher faecal shedding and increased persistence. Strain specific differences in Salmonella pathogenesis might thus have repercussions on the serological detection of Salmonella Typhimurium infections in pigs.     

Isolation of avian bornaviruses from psittacine birds using QT6 quail cells in Japan

Avian bornaviruses (ABVs) were recently discovered as the causative agents of proventricular dilatation disease (PDD). Although molecular epidemiological studies revealed that ABVs exist in Japan, no Japanese isolate has been reported thus far. In this study, we isolated four strains of Psittaciform 1 bornavirus from psittacine birds affected by PDD using QT6 quail cells. To our knowledge, this is the first report to isolate ABVs in Japan and to show that QT6 cells are available for ABV isolation. These isolates and QT6 cells would be powerful tools for elucidating the fundamental biology and pathogenicity of ABVs.     

Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract

Melissococcus plutonius is an important pathogen that causes European foulbrood (EFB) in honeybee larvae. Recently, we discovered a group of M. plutonius strains that are phenotypically and genetically distinct from other strains. These strains belong to clonal complex (CC) 12, as determined by multilocus sequence typing analysis, and show atypical cultural and biochemical characteristics in vitro compared with strains of other CCs tested. Although EFB is considered to be a purely intestinal infection according to early studies, it is unknown whether the recently found CC12 strains cause EFB by the same pathomechanism. In this study, to obtain a better understanding of EFB, we infected European honeybee (Apis mellifera) larvae per os with a well-characterized CC12 strain, DAT561, and analyzed the larvae histopathologically. Ingested DAT561 was mainly localized in the midgut lumen surrounded by the peritrophic matrix (PM) in the larvae. In badly affected larvae, the PM and midgut epithelial cells degenerated, and some bacterial cells were detected outside of the midgut. However, they did not proliferate in the deep tissues actively. By immunohistochemical analysis, the PM was stained with anti-M. plutonius serum in most of the DAT561-infected larvae. In some larvae, luminal surfaces of the PM were more strongly stained than the inside. These results suggest that infection of CC12 strain in honeybee larvae is essentially confined to the intestine. Moreover, our results imply the presence of M. plutonius-derived substances diffusing into the larval tissues in the course of infection.     

Studies of Aspergillus fumigatus; casein precipitating and proteolytic effects of mycelial filtrate

Schäffer (1900) and Butkewitch (1903) seem to have been the first to focus attention on the proteolytic activity of micro-fungi. The occurrence and properties of proteolytic enzymes from various fungus species were subsequently studied by several authors. Literature in this field was reviewed by e.g. Ito (1950), Gorbach & Koch (1955), Koch & Dedic (1957), Hagihara (1960), Davies (1963) and Roper & Fennell (1965).In connection with investigation of the proteolytic activity of several fungus species, a gelatin hydrolyzing effect of Aspergillus fumigatus (AF) was reported to be exerted by living organisms in pure culture (Jensen 1931), extracted mycelial material (Maxwell 1950, Dingle & Solomons 1952), and by fluid culture medium in which AF was cultured (Dion 1950a, b, Dingle & Solomons). Ay res & Tobie (1943) demonstrated moderate casein hydrolyzing activity in extracted mycelial material from 4 AF strains, and Amatayakul (1955) observed low fibrinolytic activity in 1 strain.Proteolytic activity measured by breakdown of gelatin and casein was demonstrated by Jonsson & Martin (1964) in culture medium in which AF had been cultured. Three activity optima were observed at pH values around 3, 6.5 and 10. A subsequent study indicated that the activity in neutral and alkaline environment was caused by the same enzyme (Martin & Jönsson 1965).By means of electrophoretic separation combined with reactions for enzyme characterization, Tran Van Ky et al. (1966) demonstrated proteolytic activity in mycelial extracts of 21-day AF cultures. A casein precipitating enzyme (CP enzyme) was demonstrated by Sandvik (1967) in the fluid phase of frozen and thawed skimmilk agar cultures from e.g. AF.In addition to haemolysin and toxin (Rutqvist 1965, 1968, Rutqvist & Persson 1966), mycelial filtrates of AF have proved to contain a proteolytic enzyme. An account is given in the following of a study of this enzyme with respect to (1) casein precipitating ability, (2) casein and gelatin hydrolyzing effect, and (3) relation to toxin and haemolysin.     

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis

Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne’s disease.     

High Prevalence of BlaCTX-M Group Genes in Aeromonas dhakensis Isolated from Aquaculture Fish Species in South Korea

The prevalence of resistant genes against β-lactams in 119 Aeromonas strains was determined. A large number (99.2%) of the present fish strains were resistant to one or more β- lactams including ceftiofur, amoxicillin-clavulanic acid, ampicillin, piperacillin and cefpodoxime. Among antibiotic resistance phenotypes, the simultaneous resistance to all β-lactams occurred in 25.2% (n=30) of all strains, which consisted of 18 strains of A. dhakensis, 8 strains of A. caviae, 2 strains of A. hydrophila and only one strain of A. veronii. For exploring genetic background of the antibiotic resistances, multiple PCR assays were subjected to detect β-lactamase-encoding genes, bla_TEM, bla_OXA-B and bla_CTX-M. In the results, the bla_TEM-1 gene was harbored in all strains, whereas only 3 strains harbored bla_OXA gene. In the case of bla_CTX-M gene, the gene was detected in 21.0% (25 out of 119) of all strains, which countered with 80% (20 out of 25) of A. dhakensis, 8% (2 out of 25) of A. caviae and 12% (3 out of 25) of A. hydrophila. In addition, most of the bla_CTX-M positive strains showed simultaneous resistance to all β-lactams (18 out of 30 strains). In sequence analysis for bla_CTX-M genes detected, they were CTX-M group 1-encoding genes including bla_CTX-M-33 from 3 eel strains of A. dhakensis. Therefore, A. dhakensis obtained from cultured fish could represent a reservoir for spreading genes encoding CTX-M group 1 enzymes and hence should be carefully monitored, especially for its potential risk to public health.     

Aging-related Changes in In Vitro-matured Bovine Oocytes: Oxidative Stress,Mitochondrial Activity and ATP Content After Nuclear Maturation

The objective of this research was to clarify the aging-related changes in in vitro-matured bovine oocytes. Firstly, we examined the fertilization and embryonic development of bovine oocytes after 22 and 30–34 h of in vitro maturation (IVM). The oocytes after 30–34 h of IVM (penetrated by sperm at around 40 h after starting IVM) showed a lower developmental rate to blastocysts (P<0.01), although normal fertilization rates were similar regardless of IVM duration. In the next experiment, reactive oxygen species (ROS), mitochondrial activity and ATP content in oocytes after 20, 30 and 40 h of IVM were examined. The lowest level of ROS was found in the group subjected to 30 h of IVM. The mitochondrial activity and ATP content in the group subjected to 40 h of IVM were higher than in the group subjected to 20 h of IVM (P<0.01), and those in the group subjected to 30 h of IVM showed intermediate values. Thereafter, the mitochondrial activities at 3 days after in vitro fertilization in embryos derived from the oocytes subjected to 22 and 34 h of IVM were evaluated. In the group subjected to 34 h of IVM, high-polarized mitochondria were frequently observed at the periphery of blastomeres. The present results suggest that high mitochondrial activity observed in oocytes after prolonged IVM culture and localization of high-polarized mitochondria at the periphery of blastomeres during early embryonic development may be associated with the low developmental competence in aged bovine oocytes.     

In vitro susceptibility of Brachyspira hyodysenteriae to organic acids and essential oil components

The antibacterial potential of organic acids and essential oil components against Brachyspira hyodysenteriae, the causative pathogen of swine dysentery, was evaluated. Minimum inhibitory concentrations (MIC) of 15 compounds were determined at pH 7.2 and pH 6.0, using a broth microdilution assay. In addition, possible synergism was determined. MIC values for the three tested strains were similar. For organic acids, MIC values at pH 6.0 were lower than at pH 7.2. B. hyodysenteriae was most sensitive to cinnamaldehyde and lauric acid, with MIC values <1.5 mM. Most antibacterial effects of binary combinations were additive, however, for thymol and carvacrol, synergism could be observed. In vitro results demonstrate the antibacterial action of certain essential oil components and organic acids against B. hyodysenteriae.     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Effect of 7,8-Dihydroxyflavone as an Antioxidant on In Vitro Maturation of Oocytes and Development of Parthenogenetic Embryos in Pigs

One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 μM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 μM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 μM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 μM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.     

Developmental competence of oocytes grown in vitro: Has it peaked already?

In vitro growth of immature oocytes provides opportunities to increase gametic resources and to understand the mechanisms underlying oocyte development. Many studies on the in vitro growth of oocytes have been reported thus far; however, only a few cases have been reported, which demonstrated that oocytes can support full-term development after in vitro fertilization. Our research group recently found that culture of mouse neonatal primordial follicles increased the birthrate; however, the establishment of an in vitro system that can completely mimic follicle or oocyte growth in vivo and control oogenesis remains an ongoing challenge.     

Cytologic detection of Toxoplasma gondii in the cerebrospinal fluid of a dog and in vitro isolation of a unique mouse-virulent recombinant strain

Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.     

Characterization of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from pigs presenting with diarrhea in Korea

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans.     

Prevalence,O-genotype and Shiga toxin (Stx) 2 subtype of Stx-producing Escherichia coli strains isolated from Argentinean beef cattle

The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin (Stx) 2 gene-subtype of Stx-producing Escherichia coli (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. stx genes were detected in 90 (32%) out of the 283 rectal swabs by stx gene-specific PCR assay. The positive cases were 13 with stx1, 58 with stx2, and 19 with both stx1 and stx2. Among 90 stx gene-positive samples, 45 STEC strains were isolated, which included 3 stx1, 34 stx2, and eight stx1 and stx2 genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various stx2 gene-subtypes were identified in 42 STEC strains: 13 positive cases for stx2a, 11 for stx2c, 3 for stx2g, 10 for stx2a and stx2d, 4 for stx2a and stx2c, and 1 for stx2b, stx2c and stx2g. efaI gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that stx2a and stx2c were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.