Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.     

The transmission potential of Rift Valley fever virus among livestock in the Netherlands: a modelling study

Abstracts

Rift Valley fever virus (RVFV) is a zoonotic vector-borne infection and causes a potentially severe disease. Many mammals are susceptible to infection including important livestock species. Although currently confined to Africa and the near-East, this disease causes concern in countries in temperate climates where both hosts and potential vectors are present, such as the Netherlands. Currently, an assessment of the probability of an outbreak occurring in this country is missing. To evaluate the transmission potential of RVFV, a mathematical model was developed and used to determine the initial growth and the Floquet ratio, which are indicators of the probability of an outbreak and of persistence in a periodic changing environment caused by seasonality. We show that several areas of the Netherlands have a high transmission potential and risk of persistence of the infection. Counter-intuitively, these are the sparsely populated livestock areas, due to the high vector-host ratios in these areas. Culex pipiens s.l. is found to be the main driver of the spread and persistence, because it is by far the most abundant mosquito. Our investigation underscores the importance to determine the vector competence of this mosquito species for RVFV and its host preference.     

Effect of hCG and eCG Treatments on Prostaglandins Synthesis in the Porcine Oviduct

The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross‐bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p < 0.05, p < 0.001, respectively) and protein prostaglandin endoperoxide synthase 2 expression (in the isthmus; p < 0.001) in gilts treated with human chorionic gonadotrophin/equine chorionic gonadotrophin (hCG/eCG) compared with Group II that were inseminated only. In addition, hCG and eCG treatment decreased mPGES‐1 mRNA levels in Groups III and IV, in both the isthmus (p < 0.01 in III, p < 0.001 in IV) and ampulla (p < 0.001). The prostaglandin E₂ content of oviductal tissues was significantly lower in Groups III (p < 0.05) and IV (p < 0.01 in isthmus, p < 0.0001 in ampulla) compared with Group II. mRNA and protein levels of PGFS in Group IV in the oviductal isthmus were higher (p < 0.01) compared with the non‐treated Group II. In effect, the amount of prostaglandin F_2α in oviductal tissues of gilts treated with hCG/eCG was significantly elevated (p < 0.001 in isthmus of Groups III and IV; p < 0.05 in ampulla of Group IV). Differential expression of the factors analysed in gilts treated with exogenous gonadotrophins suggests that hCG/eCG stimulation affects prostaglandins synthesis pathway. These changes can alter oviduct functions and in turn affect gamete maturation and fertilization as well as development of embryos and their transport to the uterus.     

Effects of Vasectomy on Seminal Plasma Alkaline Phosphatase in Male Alpacas (Vicugña pacos)

Azoospermia is a common finding in male alpacas which present for infertility. The challenge is to differentiate azoospermia of testicular origin from non‐testicular origin. In several species, alkaline phosphatase (AP) concentrations in seminal plasma have been used as a diagnostic marker of contributions of the testis and epididymis to the ejaculate. The purpose of this study was to determine whether AP assay could differentiate testicular from non‐testicular azoospermia in male alpacas. An experimental model of bilateral outflow obstruction (pre‐scrotal vasectomy) was used in 22 male alpacas, aged 2–9 years. No reproductive history was available. Animals were submitted for electroejaculation (EE) under general anaesthesia and vasectomy performed. Five weeks later, animals were submitted for EE. Vasectomy was not successful in one animal, which was removed from analysis. AP levels were compared in seminal plasma in the pre‐ and post‐vasectomy samples. The mean ± SEM concentration of AP in pre‐vasectomy seminal plasma was 504.29 ± 166.45 U/l (range 10–2910); the post‐vasectomy levels were 252.48 ± 81.77 U/l (range 0–1640; p = 0.06). In 71.4% of animals, AP levels decreased, varying from 18% to 100% reduction. Results of this study suggest that AP is not produced exclusively by the testis and epididymis in alpacas and that AP assay is not a valid diagnostic test for determination of origin of azoospermia; the gold standard for diagnosis of origin of azoospermia remains testicular biopsy.     

Role of lipid oxidation, chelating agents, and antioxidants in metallic flavor development in the oral cavity

This study investigated the production of metallic flavor, which is a combination of taste and retronasal odor. Chemical reactions in the oral cavity and saliva of healthy subjects were investigated after ingesting iron and copper solutions above and near threshold levels. Significant increase in lipid oxidation (p < 0.001) occurred after metal ingestion, detected as TBARS values. Ferrous ion caused the greatest flavor sensation and lipid oxidation, followed by cupric and cuprous ions. Ferric ion did not cause metallic sensation. Occurrence of oxidation was supported by damage to salivary proteins, detected as protein-carbonyls, and by a significant increase of odorous lipid oxidation related aldehydes. Sensory evaluation demonstrated that antioxidants (vitamins E and C) minimally reduced metallic flavor but that chelating agents (EDTA and lactoferrin) removed the metallic flavor. The role of lipid oxidation is essential for the production of a metallic flavor from ingestion of ferrous, cupric, and cuprous ions.     

Identification and functional characterization of brainstem cannabinoid CB2 receptors

The presence and function of CB2 receptors in central nervous system (CNS) neurons are controversial. We report the expression of CB2 receptor messenger RNA and protein localization on brainstem neurons. These functional CB2 receptors in the brainstem were activated by a CB2 receptor agonist, 2-arachidonoylglycerol, and by elevated endogenous levels of endocannabinoids, which also act at CB1 receptors. CB2 receptors represent an alternative site of action of endocannabinoids that opens the possibility of nonpsychotropic therapeutic interventions using enhanced endocannabinoid levels in localized brain areas.     

A mantle plume initiation model for the wrangellia flood basalt and other oceanic plateaus

The vast Wrangellia terrane of Alaska and British Columbia is an accreted oceanic plateau with Triassic strata that contain a 3- to 6-kilometers thick flood basalt, bounded above and below by marine sedimentary rocks. This enormous outpouring of basalt was preceded by rapid uplift and was followed by gradual subsidence of the plateau. The uplift and basalt eruptions occurred in less than approximately 5 million years, and were not accompanied by significant extension or rifting of the lithosphere. This sequence of events is predicted by a mantle plume initiation, or plume head, model that has recently been developed to explain continental flood volcanism. Evidence suggests that other large oceanic basalt plateaus, such as the Ontong-Java, Kerguelen, and Caribbean, were formed as the initial outbursts of the Louisville Ridge, Kerguelen, and Galapagos hot spots, respectively. Such events may play an important role in the creation and development of both oceanic and continental crust.