Detection of Mycobacterium avium subsp. paratuberculosis in Slovakian wildlife.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of infectious enteritis called paratuberculosis that has a high economic impact on the worldwide livestock production. A central important question arises: Can wildlife animals serve as a reservoir for transmission of MAP to domestic ruminants? With this in mind, we devised a study to detect MAP in various Slovakian wildlife species found in the areas where paratuberculosis had been documented in domestic ruminants. The samples of parenchymatous organs (intestines, ileocecal valve and mesenteric lymphatic nodes) from 83 wildlife animals representing 13 species, inclu- ding 7 herbivorous, 5 carnivorous and 1 omnivorous species were collected during a four-year period. The clinical and pathological examinations failed to demonstrate any manifestations of paratuberculosis in any of the wildlife samples. The detection of MAP was done by widely used tests, i.e. cultivation and the PCR analysis. The bacterial cultures revealed the growth of Mycobacterium spp. colonies in 58 (70%) of all of the wild animals, but the PCR testing demonstrated paratuberculosis only in one (7.69%) of the roe deer population.

How viruses infiltrate the central nervous system.

Central nervous system is protected by the blood-brain barrier, which represents a physical, metabolic and transport barrier and is considered to be a part of a highly dynamic system termed neurovascular unit. Several pathogens, among them viruses, are able to invade the brain. Traversal of viruses across the blood-brain barrier is an essential step for the invasion of the central nervous system and can occur by different mechanisms - by paracellular, transcellular and/or by "Trojan horse" pathway. Penetration of viruses to brain can lead to the blood-brain barrier dysfunction, including increased permeability, pleocytosis and encephalopathy. Viruses causing the central nervous system infections include human immunodeficiency virus type 1, rhabdovirus, different flaviviruses, mouse adenovirus type 1, herpes simplex virus, influenza virus, parainfluenza virus, reovirus, lymphocytic choriomeningitis virus, arbovirus, cytomegalovirus, mumps virus, parvovirus B19, measles virus, human T-cell leukemia virus, enterovirus, morbillivirus, bunyaviruses, togaviruses and others. In this review we summarized what is known about the routes of how some viruses enter the brain and how neurons and glial cells react to infection.

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and ß-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine.

Omics approaches to probe markers of disease resistance in animal sciences.

Omics technologies have been developed in recent decades and used in different thematics. More advancements were done in human and plant thematics. Omics is the conjugation of different techniques, studying all biological molecules (DNA, RNA, proteins, metabolites, etc.). Omics is then able to study entire pathways, elucidating phenotypes and their control. Thus, thanks to Omics, it is possible to have a broad overview of the linkage between genotypes and phenotypes. Disease phenotypes (tolerance or resistance) are important to understand in both production and health. Nowadays a plethora of research articles are presenting results in the field of natural disease resistance of animals using Omics technologies. Moreover, thanks to advanced high throughput technologies novel modes of infections (infection pathways) are coming to the surface. Such pathways are complex (hundreds to thousands of molecules implied, with complicated control mechanisms), and Omics can generate useful knowledge to understand those pathways. Here we aim to review several angles of Omics used to probe markers of disease resistance with recent publications and data on the field, and present perspectives and its utilization for a better understanding of diseases.

Animal board invited review: advances in proteomics for animal and food sciences.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002--Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

EPR-TL correlation studies on Bi co-doped CaSO4:Dy phosphor.

CaSO(4):Dy, CaSO(4):(Dy, Bi) and CaSO(4):Bi phosphors were prepared through re-crystallization method. Thermoluminescence (TL) characteristics of these phosphor samples were investigated. The radiation induced radical ions formed in these phosphors were investigated using electron paramagnetic resonance (EPR) spectroscopy. The main signals observed in both CaSO(4):(Dy, Bi) and CaSO(4):Bi were identified as SO(4)(-) (II), SO(4)(-) (⊥) and SO(3)(-) (isotropic) with "g" values 2.023, 2.0089 and 2.004, respectively. In order to understand the TL mechanism, CaSO(4):(Dy, Bi) phosphor samples were annealed between 100 and 250 °C and their EPR spectra were studied. It was observed that EPR signal intensities reduce drastically in 250 °C annealed phosphor confirming the role of SO(4)(-) and SO(3)(-) types of defect centers in the dosimetric peak. The reduction in the TL sensitivity with increase in Bi(3+) co-dopant in the phosphor samples was correlated with quenching of TL by Bi(3+) ions rather than the reduction in the concentration of the above defect centers. An effort was also made to use the Bi(3+) co-doped CaSO(4):Dy phosphor for dosimetry of chilled or frozen food irradiation.

Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle.

Toll like receptors (TLRs) are a class of pattern recognition receptors belonging to the innate immune system. Mutations in the protein coding region of TLRs are associated with altered responsiveness to pathogen-associated molecular patterns (PAMPs). A search was performed for novel mutations in bovine TLR1, TLR2 and TLR4 genes associated with the Mycobacterium avium subsp. paratuberculosis (MAP) infection. The work was also focused on the assessment of linkage between well known mutations in TLR genes (TLR2: Arg677Trp, Pro681His and Arg753Gln; TLR4: Asp299Gly and Thr399Ile), and the susceptibility of cattle to MAP infection. Detection of MAP infection in cattle population (n=711) was based on IS900 PCR, which revealed 22.50% (n=160) MAP positivity. Known mutations in TLR2 and TLR4 genes were not found in cattle population. A novel mutation Val220Met was associated (Odd's ratio, OR-3.459) with increased susceptibility to MAP infection. Toll/interleukin-1 receptor (TIR) domain of TLR2 was screened for the presence of mutations, wherein a novel Ile680Val mutation was linked with MAP infection. In silico analysis of the bovine TLR4 ectodomain (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR motifs. LRR11 of the TLR4 showed five missense mutations possibly linked with the increased susceptibility to MAP infection. The most critical position that may alter the pathogen recognition of TLR molecule was 4th residue downstream to LRR domain. Two such missense mutations in TLR4 (Asp299Asn downstream to LRR11, and Gly389Ser downstream to LRR15) were associated with MAP infection. Briefly, the work describes novel mutations in the bovine TLRs and presents their association with the MAP infection.

PrP gene polymorphism in sheep breeds in Slovakia and susceptibility to scrapie.

We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.

Monitoring of humans and animals for the presence of various Rickettsiae and Coxiella burnetii by serological methods.

Serological examination of humans in Slovakia suspected of having rickettsial infections revealed the presence of antibodies to spotted fever group rickettsiae (R. conorii, R. slovaca, and R. typhi). Of interest is the finding of serological positivity to the newly recognized "IRS" agent. Antibodies to these rickettsiae and to C. burnetii were demonstrated also in domestic and hunting dogs and pet animals. These results confirm the occurrence and possible circulation of these rickettsiae and C. burnetii in the Slovak Republic.

Toll-like receptor gene polymorphism and its relationship with somatic cell concentration and natural bacterial infections of the mammary gland in sheep.

Possible correlation between Toll-like receptor (TLR)-gene mutations and the susceptibility of the mammary gland to bacterial infections and also the associate breed-dependent aspects of somatic cell concentration (SCC), bacterial infection and TLR-gene mutations in sheep are described. In Polish Lowland Sheep (PLS), milk samples exceeding the level of 500/microL (i.e. 5 x 10(5) per mL) of SCC were recorded almost twice more frequently than in Polish Heath Sheep (PHS) (40 and 22.3%, respectively). The frequency of bacterial infections was also found in a similar ratio (20 and 12.7%, respectively). During detection of the TLR-gene mutation we recorded 2 alleles of TLR1, 6 alleles of TLR2 and 10 alleles of TLR4 genes in PHS sheep, while PLS sheep possessed 2, 4 and 6 alleles, respectively. Statistical analyses revealed a relationship between the specified TLR alleles, SCC and the frequency of incidence of bacterial inflammations of mammary gland. The data may serve as a benchmark for further study of TLR-gene mutation-dependent predisposition of mammary gland defensive cells to recognize the pathogen properly and initiate the immunological response, and may help in identifying one of the markers of natural resistance against sheep mastitis.

Reactions of superoxide radicals with curcumin: probable mechanisms by optical spectroscopy and EPR.

Reactions of superoxide-crown ether complex with curcumin have been studied in acetonitrile. Optical absorption spectra showed that curcumin on reaction with superoxide forms a blue color intermediate absorbing at 560 nm, which subsequently decayed in a few hours with the development of the absorption band corresponding to the parent curcumin. The regeneration was 100% at low superoxide concentrations (1:1, or 1:2 or 1:3 of curcumin:superoxide) but reduced to 60% at high superoxide concentration (>1:5). The regeneration of curcumin is confirmed by HPLC analysis. Stopped-flow studies in acetonitrile following either the decay of parent curcumin at 420 nm or formation of 560 nm absorption have been used to determine the rate constant for the reaction of superoxide with curcumin. EPR studies confirmed the disappearance of characteristic superoxide signal in presence of curcumin with the formation of new featureless signal with g = 2.0067. Based on these studies it is concluded that at low superoxide concentrations curcumin effectively causes superoxide dismutation without itself undergoing any chemical change. At higher concentrations of superoxide, curcumin inhibits superoxide activity by reacting with it.

Protein A/G dependent ELISA a promising diagnostic tool in Lyme disease seroprevalence in game animals and hunting dogs.

One of the major problems in serodiagnosis in wild animals is unavailability of specific antiglobulin conjugate. Our study focuses on validation of Protein A/G dependent ELISA in game animals like deer and mouflons as well as in hunting dogs. Binding ability of Protein A/G-conjugate to antibodies was the highest in dogs followed by fallow deer and mouflons. Three different whole cell Borrelia antigens were used to evaluate antigen dependent variation. In new Protein A/G-ELISA the highest sensitivities (90.50%, deer; 85.37%, mouflon & 94.29%, dog) were obtained by B. garinii antigen, with no statistically significant variation (chi(2), P>0.05) among all other antigens used. Average seroprevalences observed in deer, mouflons and dogs were 44.90%, 29.41% and 30.43%, respectively. Marked influence of age on seroprevalence was noticed. Protein A/G-ELISA proved to be sensitive and promising diagnostic tool in serodiagnosis of Lyme disease in game ungulates and it can be used effectively for serosurvey in different wild mammals.

Immunoglobulin G antibodies to Borrelia burgdorferi in game animals and small mammals in eastern Slovakia.

In a survey of game animals and small mammals, the sera of 185 animals were examined for the presence of immunoglobulin G antibodies to the spirochete Borrelia burgdorferi sensu lato, which causes Lyme borreliosis. These animals comprised 59 fallow deer (Dama dama), 56 mouflons (Ovis musimon) and 70 small mammals of six different species. The sera of the fallow deer and the mouflons were examined by indirect haemagglutination assay. The sera of the small mammals were examined by modified enzyme-linked immunosorbent assay, which is available in a commercial kit. The sera of the 59 fallow deer demonstrated positivity of 40.77% (titres 1:40-1:80). The 56 mouflons demonstrated seropositivity of 17.8% (1:40-1:80). The sera of the small mammals were highly positive in the yellow-necked field mouse (Apodemus flavicollis) at 42.1% (titres 1:200-1:1,600), followed by the bank vole (Clethrionomys glareolus) at 14.3% (1:400-1:800), the common vole (Microtus arvalis) at 12.5% (1:200) and the black-striped field mouse (A. agrarius) at 10.0% (1:200-1:400-800). The authors also report the rate of infestation of these small mammals by the tick Ixodes ricinus, as these mammal species are potential reservoirs for this vector. The study focuses on the relationship between the possibility of infestation by I. ricinus and the reservoir competence of the different species under study, as well as the possible spread of disease. The detected rate of seroprevalence indicates that all the investigated animals have had contact with infected ticks.

Clinical evaluation of three different gonadotrophin-releasing hormone analogues in an IVF programme: a prospective study.

The efficacy and safety of short acting buserelin and nafarelin intranasal spray were compared to long acting leuprorelin depot intramuscular or subcutaneous injection in this prospective study of 157 women undergoing controlled ovarian hyperstimulation (COH) for in-vitro fertilisation (IVF). Patients were allocated to three groups to receive buserelin 150 microg nasal spray three times daily (Group B), nafarelin nasal spray 400 microg twice daily (Group N), or leuprorelin depot 3.75 mg once by intramuscular or subcutaneous injection (Group L) for pituitary desensitisation prior to commencing COH with human menopausal gonadotrophins (hMG) according to the Centre's protocol. The mean (+/-S.D.) age (years) (32.6+/-3.8: Group B, 32.1+/-3.3: Group N versus 32.1+/-3.3: Group L); mean (+/-S.D.) total dosage of hMG (ampoules) (37.5+/-16.1: Group B, 39.8+/-14.2: Group N versus 41.9+/-12.6: Group L) and mean daily dosage of hMG (ampoules) (3.1: Group B, 2.8: Group N versus 3.0: Group L) seen were not statistically significantly different. The duration between starting the different gonadotrophin-releasing hormone (GnRHa) and the beginning of the next menstrual period was also not seen to be statistically significantly different between the three groups (Group B: 10+/-5.5, Group N: 9.1+/-4.1 versus Group L: 8.2+/-3, days). The number of abandoned cycles was higher in Group L (17% versus 11.8%: Group B and 11.3%: Group N) but this difference did not reach statistical significance. The clinical pregnancy rates per oocyte retrieval and per embryo transfer procedure were respectively, 31.1, 35% in Group B, 12.8, 14% in Group N versus 20.5, 23.7 in Group L and were not seen to be statistically significantly different even when ongoing pregnancy rates were compared. Apart from a statistically significantly greater incidence of allergic nasal reactions in the nafarelin group (P=0.001), all other side-effects were not shown to be statistically significantly different between the three groups. We conclude that a single dose of leuprorelin depot can be considered to be as an equally effective alternative to multiple doses of buserelin or nafarelin for pituitary desensitisation in women undergoing COH for IVF.

Presensitization of microorganisms by acid treatments to low dose gamma irradiation with special reference to Bacillus cereus.

Effect of acid presensitization to low dose gamma irradiation on Bacillus cereus in sheep/goat meat stored at refrigeration temperature was assessed. Food grade organic acids (viz. propionic, lactic and acetic acids) were used as presensitizers followed by treatment with 1, 2 and 3 kGy irradiation doses (Co(60)). Two percent acetic acid plus 3 kGy irradiation elicited most effective to lower the total viable count and B. cereus count. Combination of treatments availed to reduce the dose required for elimination of radioresistant B. cereus and also masked the irradiation odour developed due to radiolysis. The study also revealed substantial increase in shelf life of mutton after combination treatment than single treatment without any adverse effect on acceptability of meat.

Skin incision recurrence of adenocarcinoma of the cervix five years after radical surgery for stage 1A disease.

A 31-year-old para 1 had a radical hysterectomy for stage 1A adenocarcinoma of the cervix. She developed skin incision metastasis five years later in spite of normal regular vault smears.

Tuberculosis of the lacrimal gland.

A case of tuberculous dacryoadenitis with abscess formation resolving completely with anti-tuberculous chemotherapy is presented. The growth of acid-fast bacilli in culture from the pus is noteworthy.

Thiourea as a xenobiotic, showing its adverse effects on mortality, behaviour and metamorphosis and on histopathological and cytological changes in the developing ovaries of Dysdercus similis.

The effect of thiourea (applied topically, in different concentrations, to the eggs and all the nymphal instars, and to adults also by feeding) on the red cotton-bug Dysdercus similis was studied. The eggs were severely affected (i.e. mortality was high), while the nymphal instars showed an adverse effect on ecdysis and adults which emerged from the treated last nymphal instar were characterized by high mortality, abnormal behaviour and reduced fecundity and viability. They were also smaller, weaker and morphologically abnormal compared with the controls and were differently coloured. They were likewise unable to reproduce, owing to atrophy of the gonads. The ovaries contained a large number of immature, pathological oocytes with degenerating follicular epithelium, which was thin with a regular outline in the early stages, but later became multilayered, with pyknotic nuclei, and displayed active destruction of glycoproteins and lipoproteins. Fibrogenesis and thickening of the tunica propria were clearly discernible. Although treated females were kept together with untreated males, the two showed no signs of being attracted to one another and displayed no mating behaviour, although some females showed a very low degree of ovulation. Only a few survived the Ist nymphal instar and none survived up to adult emergence, despite being kept under normal laboratory conditions.

BHC as a xenobiotic, showing histopathological changes in the developing ovaries and variability of the behaviour of Poekilocerus pictus.

The leaf-hopper P. pictus is a well-known pest of Calotropis plants. Adult females and males were treated with a sublethal BHC concentration to control fecundity and viability. Marked histological changes occurred in the follicular epithelium of both pathological and immature oocytes. Vitellogenesis was completely arrested and the PYP bodies present prior to treatment disintegrated and were resorbed by the pycnotic follicular epithelium. Fibrogenesis and thickening of the tunica propria gradually decreased. The sublethal BHC concentration injected into the haemolymph severely damaged the fat bodies and thus adversely affected the developing ovaries. When adults were treated and kept in separate pairs, mating was prolonged; it was followed by ovulation, but the number of eggs was always smaller than in the control females and they did not develop any further, although provided in the laboratory with all normal conditions.