Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.     

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.     

Production of Middle White Piglets after Transfer of Embryos Produced In Vitro

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing Middle White piglets. After collection from three retired Middle White sows, a total of 222 oocytes were matured, fertilized and cultured in vitro, and a total of 50 embryos from the 4-cell to blastocyst stage were produced by the 4th or 5th day. These embryos were transferred individually into three recipients along with 5 in vivo-derived Duroc blastocysts. All of the recipients became pregnant, and they farrowed a total of 9 Middle White and 9 Duroc piglets. These results suggest that in vitro embryo production using ovaries from retired sows is useful for reproduction of pigs of pure breeds including the Middle White for breeding activities and conservation/utilization of genetic resources.     

Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India

Present study was undertaken to study the prevalence of β-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.     

Salmonella serotypes in wild boars (Sus scrofa) hunted in northern Italy

Background

Salmonella species (spp.) are zoonotic enteric bacteria able to infect humans, livestock and wildlife.However, little is known about the prevalence and the presence of the different serovars in wildlife. Considering the wide distribution of wild boars and the feeding behaviour (omnivorous scavengers), wild boars may be a good indicator for environmental presence of Salmonella spp. The aims of this study were to determine the presence of Salmonella spp. in hunted wild boars and to determine the serotype the isolated strains.

Findings

Over three hunting seasons, the intestinal contents of 1,313 boars hunted in northern Italy were sampled and cultured. Salmonella spp. were isolated from 326 boars (24.82%). Thirty different serovars belonging to three different S. enterica spp. were found. Twenty-one serovars of S. enterica subsp. Enterica were found including the human pathogens S. Typhimurium and S. Enteritidis. In addition, nine serovars belonging to S.enterica subsp. diarizonae and S. enterica subsp. houtenae were detected.

Conclusions

Considering the widespread occurrence of wild boars in Europe, the epidemiological role of this species in relation to salmonellosis might be relevant and should be further investigated. Wild boars may act as healthy carriers of a wide range of Salmonella serotypes.     

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

Comparison of in vivo and in vitro survival and fecundity rates of the poultry red mite, Dermanyssus gallinae

To assist in the testing of possible antigens in developing a vaccine against the poultry red mite (Dermanyssus gallinae De Geer), a rapid and reliable in vitro screening method is critical. This short paper describes how D. gallinae survival and fecundity rates in an in vivo feeding device compared to that of mites fed using an in vitro method. Results showed that survival of fed D. gallinae females and mites overall was greater in vitro, although there was no difference between male survival and fecundity between in vivo and in vitro designs. The in vitro feeding device described therefore has the potential to provide reliable results, comparable to those obtained by in vivo testing, to allow for the rapid screening of D. gallinae antigens.     

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh_s) and a 33 kDa β-domain (Tsh_β). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh_s (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh_β (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37℃ or 42℃. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26℃, 37℃, or 42℃, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37℃ in mucin agar, and it was not detected when the bacteria were grown at 26℃ in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.     

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area,Japan

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.     

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.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10⁷ C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.     

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.     

Conditional knockout of brca1/2 and p53 in mouse ovarian surface epithelium: Do they play a role in ovarian carcinogenesis?

Alterations of genes are known to be critical for the induction of tumorigenesis, but the mechanism of ovarian carcinogenesis is little understood and remains to be elucidated. In this study, we investigated the roles of brca1, brca2 and p53 genes in the development of ovarian cancer using conditional knockout mice generated by a Cre-loxP recombinant system. Following the application of recombinant adenovirus expressing Cre in vitro, the proliferation of ovarian surface epithelium (OSE) was increased. For instance, a significant increase in cell growth was observed in OSE cells in vitro by conditional knockout isolated from the mice bearing concurrent floxed copies of brca1 and brca2/p53. However, the proliferative effect of the ovarian cells was not observed in concurrent brca1/brca2 or p53 knockout mice in vivo, indicating that we could not observe the direct evidence of the involvement of brca1, brca2, and p53 in ovarian carcinogenesis. Since morphological changes including tumor formation were not observed in mice bearing floxed copies of concurrent brca1/brca2 or p53, the inactivation of brca1/2 or p53 is not sufficient for the induction of tumor formation. Taken together, these results suggest that the deficiency of these genes may not be involved directly in the mechanism of ovarian carcinogenesis.     

Anthelmintic activity of Cocos nucifera L. against sheep gastrointestinal nematodes

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This work evaluated the efficacy of Cocos nucifera fruit on sheep gastrointestinal parasites. The ethyl acetate extract obtained from the liquid of green coconut husk fiber (LGCHF) was submitted to in vitro and in vivo tests. The in vitro assay was based on egg hatching (EHT) and larval development tests (LDT) with Haemonchus contortus. The concentrations tested in the EHT were 0.31, 0.62, 1.25, 2.5 and 5 mg ml⁻¹, while in the LDT they were 5, 10, 20, 40 and 80 mg ml⁻¹. The in vivo assay was a controlled test. In this experiment, 18 sheep infected with gastrointestinal nematodes were divided into three groups (n = 6), with the following doses administered: G1—400 mg kg⁻¹ LGCHF ethyl acetate extract, G2—0.2 mg kg⁻¹ moxidectin (Cydectin^®) and G3—3% DMSO. The worm burden was analyzed. The results of the in vitro and in vivo tests were submitted to ANOVA and analyzed by the Tukey and Kruskal–Wallis tests, respectively. The extract efficacy in the EHT and LDT, at the highest concentrations tested, was 100% on egg hatching and 99.77% on larval development. The parameters evaluated in the controlled test were not statistically different, showing that despite the significant results of the in vitro tests, the LGCHF ethyl acetate extract showed no activity against sheep gastrointestinal nematodes.     

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.     

Vitreous Cryopreservation of Human Preantral Follicles Encapsulated in Alginate Beads with Mini Mesh Cups

To completely avoid ice crystal formation and thus get a higher survival rate, vitrification methods have been commonly used for cryopreservation of oocytes and embryos. However, currently used vitrification methods for oocytes and embryos are not suitable for the cryopreservation of preantral follicles (PFs). In the present study, stainless steel mesh was fabricated into mini mesh cups to vitrify isolated PFs. Moreover, isolated follicles were encapsulated and then subjected to vitreous cryopreservation to facilitate in vitro culture/maturation of follicles after warming. The results showed that the percentages of viable follicles did not differ significantly between the vitrification group and fresh group soon after warming (81.25% vs. 85.29%, P>0.05) and after a 7-day culture period (77.78% vs. 83.33%, P>0.05). No difference in mean follicular diameter was observed between cryopreserved and fresh follicles when cultured in vitro. Transmission electron microscopic analysis revealed that vitreous cryopreservation could maintain the ultrastructure of follicles in alginate beads. In conclusion, the present vitrification method could efficiently cryopreserve isolated human ovarian follicles encapsulated by calcium alginate, which could be put into immediate use (in vitro culture/ maturation) after warming. However, more follicles and some detailed biochemical analyses are required to further investigate the effects of vitrification on the long-term growth of human encapsulated PFs.     

Pluripotent cell derivation from male germline cells by suppression of Dmrt1 and Trp53

Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.