A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.

Toll-like receptors in domestic animals

Toll-like receptors are pattern recognition receptors with which hosts recognize pathogen-associated molecular patterns (PAMP). This recognition process is translated rapidly into a meaningful defense reaction. This form of innate host defense is preserved in the animal kingdom: invertebrates heavily depend on it; higher vertebrates also have an adaptive immune system. Both adaptive and innate immune systems are intertwined in that the former also depends on an intact innate recognition and response system. Members of the TLR system cover recognition of parasitic, bacterial or viral germs. Due to the constraints imposed by the necessity to recognize PAMP and to interact with downstream signaling molecules, the TLR system is relatively conserved in evolution. Nevertheless, subtle species differences have been reported for several mammalian TLR members. Examples of this will be given. In all mammalian species investigated, part of the coding sequence is available for the most important TLR members, thus allowing study of expression of these TLR members in various tissues by reverse-transcription polymerase chain reaction in its classical (RT-PCR) and quantitative real time RT-PCR (qRT-PCR) form. In some species, the whole coding sequences of the most important or even all TLR members are known. This allows construction of cDNA and transfection of common host cells, thus permitting functional studies. Extensive investigations were devoted to the study of non-synonymous single nucleotide polymorphisms. In a few cases, expression of a given amino acid in the extracellular (ligand-binding) portion of TLR members could be associated with infectious diseases. This will be discussed below.

Alpine ibex (Capra i. ibex) is not a reservoir for chlamydial infections of domestic ruminants and humans

Chlamydophila (C.) abortus is the most common infectious abortigenic agent in small domestic ruminants in Switzerland. In contrast, the knowledge about chlamydiae in wild ruminants is scarce. As interactions between livestock and Alpine ibex (Capra i. ibex) occur on alpine pastures, the question raises if wild ruminants could play a role as carriers of chlamydiae. Thus, we investigated the prevalence of chlamydiae in Alpine ibex in Switzerland. In total, 624 sera, 676 eye swabs, 84 organ samples and 51 faecal samples from 664 ibex were investigated. Serum samples were tested by two commercial ELISA kits specific for C. abortus. Eye swabs, organs and faecal samples were examined by a Chlamydiaceae-specific real-time polymerase chain reaction (PCR). Positive cases were further investigated by the ArrayTube (AT) microarray method for chlamydial species determination. Of 624 serum samples investigated, 612 animals were negative, whereas nine sera (1.5%) reacted positively in one of the two tests and three sera showed an inconclusive result. Eye swabs of seven out of 412 ibex (1.7%) were tested positive for Chlamydiaceae by real-time PCR. By AT microarray, Chlamydophila (C.) pecorum was identified in two animals, Chlamydophila (C.) pneumoniae was detected in one animal and a mixed infection with C. abortus and C. pecorum was found in four animals. Organs and faecal samples were all negative by real-time PCR analysis. In summary, we conclude that C. abortus is not a common infectious agent in the Swiss ibex population. To our knowledge, this is the first description of C. pneumoniae in ibex. Further studies are necessary to elucidate the situation in other species of wild ruminants as chamois (Rupicapra r. rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus c. capreolus) in Switzerland.

Free-Ranging Wild Boar: A Disease Threat to Domestic Pigs in Switzerland?

The risk of transmission of pathogens from free-ranging wild boars (Sus scrofa scrofa) to outdoor domestic pigs (S. scrofa domesticus) is of increasing concern in many European countries. We assess this risk, using Switzerland as an example. We estimated 1) the prevalence of important pathogens in wild boars and 2) the risk of interactions between wild boars and outdoor pigs. First, we tested 252 wild boars from selected areas between 2008 and 2010 for infection with Brucella spp. Bacterial prevalence was estimated to 28.8% (confidence interval [CI] 23.0-34.0) when using bacterial culture (B. suis Biovar 2) and real-time polymerase chain reaction. Antibody prevalence was 35.8% (CI 30.0-42.0), which was significantly higher than in previous studies in Switzerland. We also tested 233 wild boars for porcine reproductive and respiratory syndrome virus (PRRSV). Antibody prevalence was 0.43% (CI 0.01-2.4) for EU-PRRSV and real-time reverse transcription polymerase chain reaction results were negative. These findings suggest that B. suis is increasingly widespread in wild boars and PRRSV is currently not of concern. Second, we documented the spatial overlap between free-ranging wild boars and outdoor piggeries by mapping data on their respective occurrence. Wild boars are most widespread in the mountain range along the western and northern Swiss borders, while most piggeries are located in central lowlands. A risk of interaction is mainly expected at the junction between these two bioregions. This risk may increase if wild boars expand eastward and southward beyond anthropogenic barriers believed to limit their range. Therefore, we evaluated the potential of expansion of the wild boar population. Population trends suggest a continuous increase of wild boars for the past 15 yr. Surveillance of selected wildlife passages using cameras on highways and main roads indicates that these barriers are permeable (average of up to 13 wild boar crossings per 100 days). Thus an increase of wild boar range should be considered. There may be a risk of B. suis spillover from wild boars in Switzerland, which could increase in the future. Data on the occurrence of interactions between pigs and wild boars are needed to assess this risk.

A study to demonstrate freedom of Trichinella spp. in domestic pigs in Switzerland

Trichinellosis is a food-borne zoonotic disease caused by the nematode Trichinella spp. Many omnivorous and carnivorous animal species can act as host for this parasite, including domestic pigs. To protect public health, it should be ensured that pork should not contain infective Trichinella larvae. Surveillance for Trichinella spp. can be done using direct (larval detection) and indirect (antibody detection) diagnostic techniques. The aim of this study was to demonstrate the absence of infection in Swiss domestic pigs. An ELISA was used as the initial screening test, and sera reacting in ELISA were further investigated using both a Western blot for serology and an artificial digestion test with 20 g of diaphragm tissue for larval detection. A total of 7412 adult pigs, 9973 finishing pigs and 2779 free-ranging pigs were tested. Samples from 17 (0.23%) adult pigs, 16 (0.16%) finishing pigs and nine (0.32%) free-ranging pigs were ELISA-positive, but all of these sera were subsequently negative by Western blot and by the artificial digestion method. Based on these findings, an absence of Trichinella infections in adult pigs (target prevalence 0.04%) and finishing pigs (target prevalence 0.03%) can be concluded. The results also demonstrated that the prevalence of Trichinella infections does not exceed 0.11% in free-ranging pigs, the group with the highest risk of exposure.

Comparing the demonstration of freedom from Trichinella infection of domestic pigs by traditional and risk-based surveillance

Traditionally, the routine artificial digestion test is applied to assess the presence of Trichinella larvae in pigs. However, this diagnostic method has a low sensitivity compared to serological tests. The results from artificial digestion tests in Switzerland were evaluated over a time period of 15 years to determine by when freedom from infection based on these data could be confirmed. Freedom was defined as a 95% probability that the prevalence of infection was below 0.0001%. Freedom was demonstrated after 12 years at the latest. A new risk-based surveillance approach was then developed based on serology. Risk-based surveillance was also assessed over 15 years, starting in 2010. It was shown that by using this design, the sample size could be reduced by at least a factor of 4 when compared with the traditional testing regimen, without lowering the level of confidence in the Trichinella-free status of the pig population.

Effect Of Yolk Testosterone On Gonadal Testosterone And Aromatase Activity In The Pre-Optic Area Of The Brain In Male Domestic C

Maternal effects are a mother¿s non-genetic contributions to development that alter phenotypic traits in offspring. Maternal effects can take the form of prenatal allocation of resources, such as the deposition of androgens into egg yolks. For example, elevated yolk testosterone increases male sexual behaviors such as copulation solicitation and courtship displays in some avian species, in addition to aggressive behaviors like pecks and intimidating postures towards same-sex competitors. However, the mechanism connecting in ovo testosterone exposure with changes in sexual and aggressive behaviors has yet to be elucidated. While testosterone released by the gonads is important in the activation of sexual behaviors, it must undergo conversion to estrogen by the enzyme aromatase in the pre-optic area (POA) of the avian brain for full expression of sexual activity. POA aromatase is also necessary for the activation of aggressive behaviors in male birds. This experiment tested the hypothesis that elevated yolk testosterone leads to changes in POA aromatase activity and levels of gonadal testosterone, as these two endocrine parameters may mediate the effect of yolk testosterone on the frequency of sexual and aggressive behaviors. The effect of elevated yolk testosterone on gonadal testosterone levels and aromatase activity in the POA of 3-day-old domestic chickens Gallus gallus domesticus was investigated. Unincubated eggs were injected with either 10 ng testosterone in 50 ¿L sesame oil (¿T chicks¿) or 50 ¿L sesame oil (¿C chicks¿). At 3 days post-hatch, gonadal testosterone content was measured after steroid extraction using an EIA, and aromatase activity in the POA was quantified by measuring the production of tritiated water from [1ß-3H]-androstenedione. I predicted that gonadal testosterone levels and brain aromatase activity would be higher in T chicks, however found no difference between treatments. Though juvenile T production peaks at 3 days post-hatch, it is possible that the reproductive systems, including the testes and POA, are not fully developed at this time.

Securities: Its Conception and Protection of Interests of its Holders

Theoretical studies of the problems of the securities markets in the Russian Federation incline to one or other of the two traditional approaches. The first consists of comparing the definition of "valuable paper" set forth in the current legislation of the Russian Federation, with the theoretical model of "Wertpapiere" elaborated by German scholars more than 90 years ago. The problem with this approach is, in Mr. Pentsov's opinion, that any new features of the definition of "security" that do not coincide with the theoretical model of "Wertpapiere" (such as valuable papers existing in non-material, electronic form) are claimed to be incorrect and removed from the current legislation of the Russian Federation. The second approach works on the basis of the differentiation between the Common Law concept of "security" and the Civil Law concept of "valuable paper". Mr. Pentsov's research, presented in an article written in English, uses both methodological tools and involves, firstly, a historical study of the origin and development of certain legal phenomena (securities) as they evolved in different countries, and secondly, a comparative, synchronic study of equivalent legal phenomena as they exist in different countries today. Employing the first method, Mr. Pentsov divided the historical development of the conception of "valuable paper" in Russia into five major stages. He found that, despite the existence of a relatively wide circulation of valuable papers, especially in the second half of the 19th century, Russian legislation before 1917 (the first stage) did not have a unified definition of valuable paper. The term was used, in both theoretical studies and legislation, but it covered a broad range of financial instruments such as stocks, bonds, government bonds, promissory notes, bills of exchange, etc. During the second stage, also, the legislation of the USSR did not have a unified definition of "valuable paper". After the end of the "new economic policy" (1922 - 1930) the stock exchanges and the securities markets in the USSR, with a very few exceptions, were abolished. And thus during the third stage (up to 1985), the use of valuable papers in practice was reduced to foreign economic relations (bills of exchange, stocks in enterprises outside the USSR) and to state bonds. Not surprisingly, there was still no unified definition of "valuable paper". After the beginning of Gorbachev's perestroika, a securities market began to re-appear in the USSR. However, the successful development of securities markets in the USSR was retarded by the absence of an appropriate regulatory framework. The first effort to improve the situation was the adoption of the Regulations on Valuable Papers, approved by resolution No. 590 of the Council of Ministers of the USSR, dated June 19, 1990. Section 1 of the Regulation contained the first statutory definition of "valuable paper" in the history of Russia. At the very beginning of the period of transition to a market economy, a number of acts contained different definitions of "valuable paper". This diversity clearly undermined the stability of the Russian securities market and did not achieve the goal of protecting the investor. The lack of unified criteria for the consideration of such non-standard financial instruments as "valuable papers" significantly contributed to the appearance of numerous fraudulent "pyramid" schemes that were outside of the regulatory scheme of Russia legislation. The situation was substantially improved by the adoption of the new Civil Code of the Russian Federation. According to Section 1 of Article 142 of the Civil Code, a valuable paper is a document that confirms, in compliance with an established form and mandatory requisites, certain material rights whose realisation or transfer are possible only in the process of its presentation. Finally, the recent Federal law No. 39 - FZ "On the Valuable Papers Market", dated April 22 1996, has also introduced the term "emission valuable papers". According to Article 2 of this Law, an "emission valuable paper" is any valuable paper, including non-documentary, that simultaneously has the following features: it fixes the composition of material and non-material rights that are subject to confirmation, cession and unconditional realisation in compliance with the form and procedure established by this federal law; it is placed by issues; and it has equal amount and time of realisation of rights within the same issue regardless of when the valuable paper was purchased. Thus the introduction of the conception of "emission valuable paper" became the starting point in the Russian federation's legislation for the differentiation between the legal regimes of "commercial papers" and "investment papers" similar to the Common Law approach. Moving now to the synchronic, comparative method of research, Mr. Pentsov notes that there are currently three major conceptions of "security" and, correspondingly, three approaches to its legal definition: the Common Law concept, the continental law concept, and the concept employed by Japanese Law. Mr. Pentsov proceeds to analyse the differences and similarities of all three, concluding that though the concept of "security" in the Common Law system substantially differs from that of "valuable paper" in the Continental Law system, nevertheless the two concepts are developing in similar directions. He predicts that in the foreseeable future the existing differences between these two concepts will become less and less significant. On the basis of his research, Mr. Pentsov arrived at the conclusion that the concept of "security" (and its equivalents) is not a static one. On the contrary, it is in the process of permanent evolution that reflects the introduction of new financial instruments onto the capital markets. He believes that the scope of the statutory definition of "security" plays an extremely important role in the protection of investors. While passing the Securities Act of 1933, the United States Congress determined that the best way to achieve the goal of protecting investors was to define the term "security" in sufficiently broad and general terms so as to include within the definition the many types of instruments that in the commercial world fall within the ordinary concept of "security' and to cover the countless and various devices used by those who seek to use the money of others on the promise of profits. On the other hand, the very limited scope of the current definition of "emission valuable paper" in the Federal Law of the Russian Federation entitled "On the Valuable Papers Market" does not allow the anti-fraud provisions of this law to be implemented in an efficient way. Consequently, there is no basis for the protection of investors. Mr. Pentsov proposes amendments which he believes would enable the Russian markets to become more efficient and attractive for both foreign and domestic investors.