Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes.

RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

In order to achieve host cell entry, the apicomplexan parasite Neospora caninum relies on the contents of distinct organelles, named micronemes, rhoptries and dense granules, which are secreted at defined timepoints during and after host cell entry. It was shown previously that a vaccine composed of a mixture of three recombinant antigens, corresponding to the two microneme antigens NcMIC1 and NcMIC3 and the rhoptry protein NcROP2, prevented disease and limited cerebral infection and transplacental transmission in mice. In this study, we selected predicted immunogenic domains of each of these proteins and created four different chimeric antigens, with the respective domains incorporated into these chimers in different orders. Following vaccination, mice were challenged intraperitoneally with 2 × 10(6)N. caninum tachzyoites and were then carefully monitored for clinical symptoms during 4 weeks post-infection. Of the four chimeric antigens, only recNcMIC3-1-R provided complete protection against disease with 100% survivors, compared to 40-80% of survivors in the other groups. Serology did not show any clear differences in total IgG, IgG1 and IgG2a levels between the different treatment groups. Vaccination with all four chimeric variants generated an IL-4 biased cytokine expression, which then shifted to an IFN-γ-dominated response following experimental infection. Sera of recNcMIC3-1-R vaccinated mice reacted with each individual recombinant antigen, as well as with three distinct bands in Neospora extracts with similar Mr as NcMIC1, NcMIC3 and NcROP2, and exhibited distinct apical labeling in tachyzoites. These results suggest that recNcMIC3-1-R is an interesting chimeric vaccine candidate and should be followed up in subsequent studies in a fetal infection model.

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.

[Acceptance of killing of animals: survey among veterinarians and other professions]

Professional veterinarians are one of the most affected professions when it comes to killing animals. However, in some situations the opinion about the acceptance of killing of animals differs between people, which can cause a dilemma for the executing person. In a pilot study based on questionnaires, veterinarians from different working fields and students of different branches stated their acceptance of killing of animals in diverse concrete situations. The result clearly demonstrates a higher acceptance of killing of animals among veterinarians with longtime experience in contrast to other groups and the almost same acceptance among agricultural students. The acceptance increased with age, however, we could not find a gender specific difference except of within a narrow age interval. The variability of acceptance within the same profession group differs between the situations. Veterinarians should be aware of their different thinking about killing of animals in some situations compared to other people and should know the reason of such differences. This is important not least to protect themselves and their opinion and to contribute to their societal responsibility by their veterinarian activity.

Cloning of farm animals: impact on animal health and welfare and implications in trade

The commercial use of animal cloning for breeding food producing animals has been limited so far by biological and technical constraints such as adverse effects on the health and welfare of animals, especially high perinatal and postnatal disease and mortality of clones. However, the improvement of the technique may overcome those problems in future and contribute to the spread of cloning in agricultural production, which raises concern not only on health and welfare aspects but also on food safety and ethics. This may cause conflict in international trade. The present article reviews these topics on the basis of up-to-date scientific opinions.

[Killing and dignity of animals: a problem for veterinarians?]

Killing of animals is an important task to be performed by veterinarians. Killing decisions and their implementation often raise ethical questions. As a result of an interdisciplinary workshop targeting the subject "killing of animals" with veterinarians and ethicists, a three-dimensional dimension scheme was developed. Whereas the first two dimensions are focused on the animal's past and future life and are discussed with regard to life quality and life accomplishment (the "telos"), the third dimension incorporates the reason to kill and may integrate the concept of dignity. This form of dignity and the weighing of interests are applied to example scenarios and the resulting responsibilities of veterinarians and society are discussed.

Mutations in the bovine ABCG2 and the ovine MSTN gene added to the few quantitative trait nucleotides identified in farm animals: a mini-review

The progress in molecular genetics in animal breeding is moderately effective as compared to traditional animal breeding using quantitative genetic approaches. There is an extensive disparity between the number of reported quantitative trait loci (QTLs) and their linked genetic variations in cattle, pig, and chicken. The identification of causative mutations affecting quantitative traits is still very challenging and hampered by the cloudy relationship between genotype and phenotype. There are relatively few reports in which a successful identification of a causative mutation for an animal production trait was demonstrated. The examples that have attracted considerable attention from the animal breeding community are briefly summarized and presented in a table. In this mini-review, the recent progress in mapping quantitative trait nucleotides (QTNs) are reviewed, including the ABCG2 gene mutation that underlies a QTL for fat and protein content and the ovine MSTN gene mutation that causes muscular hypertrophy in Texel sheep. It is concluded that the progress in molecular genetics might facilitate the elucidation of the genetic architecture of QTLs, so that also the high-hanging fruits can be harvested in order to contribute to efficient and sustainable animal production.

Brain tumors in children and adolescents and exposure to animals and farm life: a multicenter case-control study (CEFALO)

The etiology of brain tumors in children and adolescents is largely unknown, and very few environmental risk factors have been identified. The aim of this study was to examine the relationship between pre- or postnatal animal contacts or farm exposures and the risk of childhood brain tumors (CBTs), since infectious agents may pose a risk factor and a proposed mechanism is transferral of infectious agents from animals to humans.

Slow release antibiotics for treatment of septic arthritis in large animals

The search for an effective treatment for septic arthritis is ongoing. Current therapies are expensive since they require repeated joint lavage and long term antibiotic treatment. Local application of antimicrobial drugs is advantageous because high concentrations can be attained at the infection site, although repeated injections increase the risk of superinfection of the joint. Thus, slow release formulations, which have the advantage of local treatment yet single application of the drug, are appealing. Antibiotics used in slow release formulations are selected for tissue compatibility, an appropriate antibacterial spectrum, and stability both during the mixing procedure and within the carrier during the release period. Ideally the carriers should be bioresorbable. Promising reports on the clinical use of poly(methyl methacrylate) (PMMA) mixed with several different antibiotics, and of collagen sponges impregnated with gentamicin, should encourage the search for formulations optimally adapted to veterinary medical requirements.

Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection

The major route of transmission of Neospora caninum in cattle is transplacentally from an infected cow to its progeny. Therefore, a vaccine should be able to prevent both the horizontal transmission from contaminated food or water and the vertical transmission. We have previously shown that a chimeric vaccine composed of predicted immunogenic epitopes of NcMIC3, NcMIC1 and NcROP2 (recNcMIC3-1-R) significantly reduced the cerebral infection in BALB/c mice. In this study, mice were first vaccinated, then mated and pregnant mice were challenged with 2×10(6)N. caninum tachyzoites at day 7-9 of pregnancy. Partial protection was only observed in the mice vaccinated with a tachyzoite crude protein extract but no protection against vertical transmission or cerebral infection in the dams was observed in the group vaccinated with recNcMIC3-1-R. Serological and cytokine analysis showed an overall lower cytokine level in sera associated with a dominant IL-4 expression and high IgG1 titers. Thus, the Th2-type immune response observed in the pregnant mice was not protective against experimental neosporosis, in contrary to the mixed Th1-/Th2-type immune response observed in the non-pregnant mouse model. These results demonstrate that the immunomodulation that occurs during pregnancy was not favorable for the protection against N. caninum infection conferred by vaccination with recNcMIC3-1-R.

An Analysis of Capacities, Moral Hierarchies, and the Moral Status of Non-Human Animals in Contemporary Animal Rights Work

The primary objective of this thesis is to demonstrate the pernicious impact that moral hierarchies have on our perception and subsequent treatment of non-human animals. Moral hierarchies in general are characterized by a dynamic in which one group is considered to be fundamentally superior to a lesser group. This thesis focuses specifically on the moral hierarchies that arise when humans are assumed to be superior to non-human animals in virtue of their advanced mental capabilities. The operative hypothesis of this thesis is essentially that moral hierarchies thwart the provision of justice to non-human animals in that they function as a justification for otherwise impermissible actions. When humans are assumed to be fundamentally superior to non-human animals then it becomes morally permissible for humans to kill non-human animals and utilize them as mere instrumentalities. This thesis is driven primarily by an in-depth analysis of the approaches to animal rights that are provided by Peter Singer, Tom Regan, and Gary Francione. Each of these thinkers claim that they overcome anthropocentrism and provide approaches that preclude the establishment of a moral hierarchy. One of the major findings of this thesis, however, is that Singer and Regan offer approaches that remain highly anthropocentric despite the fact that each thinker claims that they have overcome anthropocentrism. The anthropocentrism persists in these respective approaches in that each thinkers gives humans Regan and Singer have different conceptions of the criteria that are required to afford a being moral worth, but they both give preference to beings that have the cognitive ability to form desires regarding the future.. As a result, a moral hierarchy emerges in which humans are regarded to be fundamentally superior. Francione, however, provides an approach that does not foster a moral hierarchy. Francione creates such an approach by applying the principle of equal consideration of interests in a consistent manner. Moreover, Francione argues that mere sentience is both a necessary and sufficient condition for being eligible and subsequently receiving moral consideration. The upshot of this thesis is essentially that the moral treatment of animals is not compatible with the presence of a moral hierarchy. As a result, this thesis demonstrates that future approaches to animal rights must avoid the establishment of moral hierarchies. The research and analysis within this thesis demonstrates that this is not a possibility, however, unless all theories of justice that are to accommodate animals abandon the notion that cognition matters morally.