Monthly Archives: July 2017

Considering the potential advantages of viruses with increased capsid stability

Considering the potential advantages of viruses with increased capsid stability for vaccine production, our results support that utilization of mutant such as VP1 N17D+VP2 H145Y could improve vaccine stability without exerting a major impact on its immunogenic properties. Moreover, the two amino rxr receptor replacements explored here can stabilize virions from at least three different FMDV serotypes (type C, O and Asia 1), which may be of interest for vaccine development in view of the highly genetic and antigenic diversity of FMDV (Liang et al., 2014; Park et al., 2016; Vazquez-Calvo et al., 2014; Wang et al., 2014). On the other hand, and regarding large-scale virus production for vaccine manufacturing, we have previously shown that the introduction of substitutions VP1 N17D+VP2 H145Y did not produce a major impact on viral fitness and did not alter viral growth properties on BHK-21 cells (Vazquez-Calvo et al., 2014), which are widely utilized to amplify viruses for FMDV vaccine production (Cao et al., 2016). In addition, viruses carrying these amino acid replacements are genetically stable (Vazquez-Calvo et al., 2014). Taken together, these properties can facilitate large-scale amplification of VP1 N17D+VP2 H145Y virus for vaccine stock production.
In summary, we have evaluated the immunogenicity in swine of a chemically inactivated vaccine based on a mutant FMDV with increased capsid stability. Our results showed that the presence of amino acid substitutions VP1 N17D and VP2 H145Y in the capsid of the virus did not compromise its immunogenic potential. These results support the feasibility of this kind of mutants with improved capsid stability as suitable candidates for the development of novel FMDV vaccines. However, the potential advantages of the usage of this kind of mutants in inactivated vaccines remain to be further tested by performing immunization and challenge experiments using the mutant viruses after storage at 4°C, at higher temperatures, or after acid pH treatment.

Acknowledgements

Introduction
Johne’s disease is a chronic, inflammatory intestinal disease affecting domestic and foreign ruminants that has a devastating impact on the dairy economy worldwide. rxr receptor The pathogen causing Johne’s disease is the acid fast and slow-growing Mycobacterium avium subspecies paratuberculosis (MAP) (Timms et al., 2011; Ott et al., 1999; Ahlstrom et al., 2016). Human Crohn’s disease has been linked to a potential causal role by MAP, but concrete evidence towards its zoonotic role in Crohn’s disease is still inconclusive (Hermon-Taylor et al., 2000; Nacy and Buckley, 2008). A possible infectious route of MAP into the human food cycle is by milk and dairy products (Donaghy et al., 2011; Grant et al., 2002). Therefore, research on MAP is needed due to its heavy economic impact on the bovine industry or due to a potential risk to human health. MAP epidemiology methods by molecular typing techniques have provided an improved representation of the genetic diversity, evolutionary relationships, interspecies transmissions of MAP and provided a better understanding of MAP spread among dairy herds (Stevenson et al., 2009; Leão et al., 2016; Nielsen and Toft, 2009). Molecular fingerprinting techniques, such as IS900 restriction fragment length polymorphism analysis (RFLP), multiplex PCR of the integration loci of IS900 (MPIL), multi-locus short sequence repeat (MLSSR), single nucleotide polymorphisms (SNPs) and whole genome sequencing (WGS) were all developed to assess the genetic variation of MAP strains (Amonsin et al., 2004; Motiwala et al., 2003; Bull et al., 2000; Leão et al., 2016; Ahlstrom et al., 2015). The mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) molecular typing technique was developed as a fast, reproducible and easy to use typing technique that targets eight established repetitive regions within the MAP genome. (Stevenson et al., 2009; Thibault et al., 2007; Radomski et al., 2010). The primary objective was to determine the genetic diversity of a large collection MAP isolates within the Republic of Ireland using the standardised molecular MIRU-VNTR genotyping method.

The age profile of OA of the TMJ in

The age profile of OA of the TMJ in the present sheep flock would strongly support wear and tear (primary OA) as an important contributing factor. OA of the TMJ in humans is usually caused by wear MS275 and tear leading to osteophytosis (Matsuura et al., 2001; Giannakopoulos et al., 2009). In humans, degenerative OA is an age-related disease with the severity of osseous changes in the condylar head and mandibular fossa increasing with age (Alexiou et al., 2009). There was no radiographic evidence of reduced bone density of the skull in the present study when compared to old sheep without OA affecting the TMJ. This finding is consistent with other studies that were unable to link broken MS275 (incisor tooth loss), even in the terminal stages, with any generalised loss of mandibular bone indicative of overall reduction in skeletal quality (Aitchison and Spence, 1984).
There is now a great deal of evidence that wild vertebrates show marked declines in their survival prospects and reproductive performance in later adulthood (Nussey et al., 2013). A recent study of wild moose found OA in leg joints to be highly prevalent in older animals and suggested this may affect their risk of predation (Peterson et al., 2010). Previous studies of the Soay sheep on St Kilda show clearly that survival and reproductive performance decline progressively in females >6 years of age (Colchero and Clark, 2012; Hayward et al., 2013). Jaw and dental pathologies could play an important role in the onset and rate of senescence in such wild ruminant populations, although few studies to date have explicitly made this link. However, the overall rarity of the TMJ pathology presented here means that it is unlikely to play a particularly important role in population-wide performance declines in later adulthood among Soay sheep on St Kilda. The low prevalence of TMJ OA meant there was very limited power available for us to test for associations between pathology and traits associated with health and fitness. There was a suggestive trend towards geriatric ewes with TMJ OA having reduced fecundity the year before their death, but this result was not statistically significant.

Conclusions

Conflict of interest statement

Acknowledgements
We thank the National Trust for Scotland and Scottish Natural Heritage for permission to work on St Kilda, and the Royal Artillery Range (Hebrides) and QinetiQ for logistical support. The Soay sheep project on St Kilda is currently funded by NERC, and we thank T. Clutton-Brock and M. Crawley for their contributions to the maintenance of the long term study, as well as the many field volunteers for their contributions to data and sample collection on St Kilda. We are grateful to Sophie Anderson for her help with the museum survey. DHN and KW are funded by a BBSRC David Philips Fellowship.

Disease transmission dynamics between wildlife and livestock are important and challenging issues. Farmyards may represent important bovine tuberculosis (bTB) transmission interfaces, since visits by badgers () can be frequent, involve a variety of building types and provide opportunities for direct and indirect contact between badgers and cattle (). Other opportunities for indirect contact are present at water troughs ().
Although badgers have been the primary focus of bTB studies, other free-ranging species that visit farmyards can be infected with and other pathogens (). Visits to farmyards by multiple species and interspecific interactions may increase the risk of disease transmission and breaches of biosecurity. The aim of this study was to quantify rates of visitation by cats (), red foxes (), rodents ( and spp.) and badgers () to farmyards in Northern Ireland using continuous surveillance.
The 1350 ha study area consisted of 11 cattle farmyards located in Northern Ireland (54°18′N, 5°38′W), in an area with a regionally high incidence of bTB in cattle (8.2% herd prevalence in 2013). Cattle over-wintered indoors from November to April and were outdoors on pasture for the remainder of the year. The number of cattle per farm was 20–320. At each farmyard, an infra-red motion activated camera (Bushnell Trailcamera) was placed at potential animal entry points to buildings from July 2012 to August 2013. Eighty-three cameras (1–17 per farmyard) were placed at least 3 m above ground and angled to provide a full view of each entry point.

There was no evidence for

There was no evidence for an infectious agent in the tiger cubs reported here. Infectious causes of vestibular disease in domesticated felids usually have a progressive course, or are accompanied by other signs of disease, neither of which was documented in these cases (Beatty et al., 2000; Gaskell et al., 2007; Gunn-Moore and Reed, 2011). No microbial agents were visualised on examination of the CSF and the lack of eosinophils in the CSF suggests that parasitic or protozoan infections are unlikely. Otoscopic, radiographic and MRI examinations of the external and middle ear did not support otitis media and interna as a contributing factor to this disease. Although no tigers were specifically tested for canine distemper virus (CDV), the lack of disease progression suggests that this is an unlikely cause and there was no evidence of suggestive lesions at necropsy. In exotic felids infected with CDV, signs of respiratory, gastrointestinal and CNS disease, including seizures and paresis, are common, and disease is usually fatal (Quigley et al., 2010).
Pedigree analysis was consistent with CVD being a familial disorder in Sumatran tigers. Furthermore, segregation analysis suggested an autosomal EPZ-6438 MOI, supported by the high frequency of the disorder within the pedigree, in combination with the predicted allele sequences based on that MOI and a Hardy–Weinberg segregation, as well as the presence of the syndrome in a hybrid tiger. Segregation analysis suggested that the trait has complete penetrance, although the disorder was not observed for two generations and then manifested in a subsequent generation, which is inconsistent with a fully penetrant dominant trait. Despite the segregation analysis indicating complete penetrance, there may have been too few animals included for a robust analysis. The research findings reported here reflect a common difficulty with segregation analysis, in that the data provided by the analysis may not be in agreement with the observations made or not compatible with any one type of inheritance. Errors in phenotype determination, genetic anticipation, phenocopy or the occurrence of new mutations could explain these discrepancies. If this genetic disorder is multi-factorial, there may also be little consistency between observations and segregation analysis results (Nicholas, 2010).
Congenital CVD in Sumatran tigers may be multi-factorial or polygenic. Interactions between genetic and environmental effects can be complex and may cause alterations in penetrance and the variation in expression of the phenotype seen in the tiger cubs. Modifier genes can also cause variation in phenotype and penetrance, with some genes having downstream effects, promoting the occurrence of the disease phenotype (Raj et al., 2010). Identification of the causative mutation could provide further clues to other genes that might modify expression of the mutated gene or could possibly identify allele variants of the mutation. The genes associated with CVD in other species might also be involved in CVD in tiger cubs, or there may be an as yet undefined gene or combination of genes responsible. Genetic mutations have been associated with malformation or malfunction of both peripheral and central vestibular components (Steel, 1995; Reardon et al., 2000; Zhao et al., 2008; Vernau et al., 2013).

Conclusions

Conflict of interest statement

Acknowledgements

Introduction
Prescription of non-steroidal anti-inflammatory drugs (NSAIDs) to dogs and cats is commonplace for perioperative analgesia (Farnworth et al., 2014; Hunt et al., 2015) and management of painful inflammatory conditions such as osteoarthritis (Sanderson et al., 2009; Sparkes et al., 2010). Concern exists amongst veterinary surgeons (Capner et al., 1999; Hugonnard et al., 2004) and pet owners about potential adverse effects (AEs) of NSAIDs in pet animals.
An AE is defined by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) as ‘any observation in animals, whether or not considered to be product-related, that is unfavourable and unintended and that occurs after use of a Veterinary Medicinal Product (VMP) (off-label and on-label uses)’. Most NSAID AEs are mild and self-limiting (Forsyth et al., 1998; Leong and Chan, 2006), although serious AEs, defined by the VICH as ‘an event which results in death, is life-threatening, results in persistent or significant disability/incapacity, or a congenital anomaly or birth defect’, coincident with NSAID administration are reported (Duerr, 2004; Enberg et al., 2006).

Taken together the lack of

Taken together, the lack of expression of neuronal markers and the scarcity of expression of DCx, combined with the diffuse expression of Olig2 and the presence of nestin and CD133 positive cells, suggest that tumour-initiating RG 7204 manufacturer demonstrate a glial progenitor-like phenotype in canine gliomas. This finding is partially in agreement with the description of lineage commitment of glioma cells in human patients. Glial progenitor-like phenotype has been described for all human gliomas but high-grade gliomas show additional expression of markers for neuronal lineage differentiation (Rebetz et al., 2008).
Owing to the heterogeneous representation of the various glioma types and the limited number of cases in our study, a statistical analysis was not appropriate. Despite the absence of statistically significant results, this proof of concept study shows the feasibility of detecting progenitor cell markers with IHC in spontaneous canine tumour samples and the presence of these cells in all grades of diffuse gliomas. The results are a further step in demonstrating the value of spontaneous models in comparative oncology. New therapeutic strategies specifically targeting CSCs are being developed (Cho et al., 2013) and the dog could serve as a translational model for such treatments. Additional studies with a larger cohort will be necessary to allow rigorous statistical analysis of pre-planned outcome measures. We plan to extend our investigation using a greater number of canine glioma samples, currently included in a multicentre study, to evaluate IHC as well as biological characteristics of primary culture cells (capacity for neurosphere formation, multi-lineage differentiation, and tumour initiation in immunocompromised xenografted rodents).

Conclusions

Conflict of interest statement

Acknowledgements
We gratefully acknowledge Ester Blasco and Lola Pérez for technical support, and Royal Canin Company, especially Isabelle Mougeot, for providing financial support for this study. Preliminary results were presented as an abstract at the 25th Symposium of the Spanish Society of Veterinary Pathology (SEAPV), Toledo, Spain, 19–21 June 2013, and as an oral communication at the Second Joint European Congress of the European Society of Veterinary Pathology, European Society of Toxicologic Pathology and the European College of Veterinary Pathologists, Berlin, Germany, 27–30 August 2014.

Introduction
Canine atlantooccipital overlapping (AOO) is defined by a diminished distance between the dorsal lamina of the atlas and the supraoccipital bone, with the dorsal lamina of the atlas located either immediately caudal to the foramen magnum or within it (Cerda-Gonzalez et al., 2009a; Dewey et al., 2009; Marino et al., 2012). This condition can cause cerebellar indentation, a reduction in cerebellomedullary cistern size, and may influence medullary position at the cervicomedullary junction (CCJ; Cerda-Gonzalez et al., 2009a; Marino et al., 2012). Prevalence of the condition is highest in small and toy-breed dogs (55%) and lower in Cavalier King Charles spaniels (CKCS; 20%; Marino et al., 2012).
Atlantooccipital overlapping resembles the human condition basilar impression/invagination (BI), in which the atlas and axis are displaced toward or through the foramen magnum, with variable involution of the foramen magnum margins (Chamberlain, 1939; Rao et al., 2002; Tassanawipas et al., 2005; Goel, 2009). Atlantooccipital overlapping and BI are both often seen alongside other CCJ anomalies, such as Chiari-like malformations, medullary elevation/herniation, and atlantoaxial instability (Goel et al., 1998; Rao et al., 2002; Cerda-Gonzalez et al., 2009a; Marino et al., 2012; Driver et al., 2013). In dogs, AOO has also been implicated as a cause of cerebellar indentation (Marino et al., 2012). However, the cause of AOO is unknown and it is unclear whether cranial displacement of the atlas or caudal bulging of the supraoccipital bone causes the overlap to occur.

br Introduction The prevalence of dementia increases

Introduction
The prevalence of dementia increases with age, and it purchase TIC10 was estimated that 24.3 million people had dementia worldwide in 2005 and the number will double every 20 years, reaching over 80 million by 2040. Dementia is a deteriorating disorder accompanied with various distressing neuropsychiatric symptoms. During the course of Alzheimer\’s disease (AD), up to 90% of patients develop the behavioral and psychological symptoms of dementia, of which psychosis and agitation present in more than half of all patients. Furthermore, dementia-related psychosis and agitation have been reported to be associated with decreased quality of life, more rapid cognitive decline, increased burden on caregivers, early institutionalization, and even higher mortality.
Although no pharmacotherapy has been approved by the US Food and Drug Administration (FDA) for patients with dementia-related agitation or psychosis, off-label use of antipsychotic agents is common. Before 2005, antipsychotic agents were recommended as the first-line pharmacotherapy for agitated dementia with delusions and as the high-second line for agitated dementia without delusions. Atypical antipsychotic agents are preferred over typical antipsychotic agents as they have fewer extrapyramidal side effects. Many clinical trials revealed a better efficacy for antipsychotic agents as compared with placebo, but there were also inconsistent results. A meta-analysis of 15 placebo-controlled, double-blind, parallel-group trials involving four atypical antipsychotic agents showed only modest efficacy on rating scales for risperidone and aripiprazole, but not for olanzapine. Another meta-analysis focusing on aggression and psychosis in AD showed significant improvements in aggression with risperidone and olanzapine treatment, but only significant improvements in psychosis with risperidone treatment. Unfortunately, antipsychotic agents were noted to be associated with increased risk for cerebrovascular accidents in elderly demented patients, so the FDA issued a “Dear Healthcare Professional” letter in April 2003. Further, meta-analysis found significantly increased mortality rate with 6- to 12-week atypical antipsychotic treatment in patients with dementia. Therefore, in April 2005, the FDA issued a black box warning that antipsychotics could increase mortality risk in demented patients. Considering both the efficacy and adverse effects, clinicians face a decision dilemma when treating patients with agitation or psychosis. Nonpharmacological interventions may be tried first, and when necessary, the lowest dosage and shortest duration of antipsychotic agent treatment are recommended. However, data about physicians\’ prescribing behaviors in real life practice are missing in Taiwan. Hence we conducted a study to investigate the dosage and duration of antipsychotic treatment in demented outpatients with agitation or psychosis. Specifically, we wanted to investigate: (1) the dosage and duration of antipsychotic treatment in demented outpatients with agitation or psychosis; and (2) whether the prescription behaviors changed after FDA warnings in 2003 and 2005.

Methods

Results
A total of 217 patients had achieved stable state, and 215 of them comprised the three antipsychotic groups (Table 1). The other two patients taking haloperidol and zotepine were excluded due to very small group size. There were significant differences in age, diagnosis, and distribution of clinics among the three groups.
Severity coded by methods defined in human chorionic gonadotropin (hCG) study had a fair correlation with BEHAVE-AD scores (Kendall\’s tau b correlation coefficient=0.566, p=0.001), supporting an acceptable accuracy of data coding. Table 2 shows the optimal dose, time to stable state, duration of stable state and total duration of target antipsychotic treatment. Although the definition of stable state only requires a duration of at least 4 weeks, the actual duration of “stable state” usually is much longer than 4 weeks. For example, the range of stable state for the 215 patients was 42 to 2703 days, with 99.5% of the patients having a stable state of ≥8 weeks, 90.2% ≥12 weeks and 82.8% ≥16 weeks. This means that the majority of this sample had been stabilized for 3 to 4 months, rather than 1 month only. The effectiveness of the antipsychotic agents was also reflected by a long median and mean duration of stable state (median 245 days, mean 374 days).

Yellow white laccases are rarely studied unlike blue

Yellow/white laccases are rarely studied unlike blue laccases. The major difference between yellow and blue laccases is the lack of an SCR 7 band at 610nm always found in blue laccases. As a matter of fact, yellow laccases are known to catalyze oxidation without the need for mediators and this makes yellow laccases a better biocatalyst than blue laccases [13]. In this study, Aureobasidium pullulans, a black-yeast-like fungus, of immense biotechnological application (such as the production of a battery of industrially important enzymes [14], polysaccharide (pullulans) and antimycotic agent, aureobasidin A [15]) was isolated from soil containing decayed plant litters at an unfarmed site (Latitude N 7°31.2006′ and Longitude E 4°31.5797′), in the Department of Botany, Obafemi Awolowo University Campus, Ile-Ife, Nigeria. Since yellow laccases are often less studied with more focus on the blue laccases, this study investigated the yellow laccase elaborated by A. pullulans NAC8, which was subsequently purified, biochemically characterized and the catalytic properties determined. Preliminary investigations on the utilization of this enzyme in decolorization of textile dyes and textile waste water effluents have been carried out in our laboratory [16]. The catalytic properties and laccase type of this enzyme from A. pullulans NAC8, has not been reported in any literature. The possible biotechnological applications of this yellow laccase such as in biocatalysis and possible utilization in the detoxification of textile dyes makes it necessary to explore its biochemical and catalytic characteristics.

Materials and methods

Results and discussion
The fungal strain, A. pullulans NAC8, shared 82% homology with A. pullulans HN2.3, 79% homology with Aureobasidium mansonii strain ATCC36276 and 100% homology with A. pullulans strains P-18, YY7. The phylogenetic tree was drawn using NJPLOT after alignment of the sequences with the Clustal X software (Fig. 1). The sequence was deposited in the gene bank of the NCBI (Accession No: KX023301). The choice of amplifying the 5.8S gene and the adjacent ITS regions 1 and 2 was to determine the phylogenetic position of the strains. ITS regions are highly variable and can only be aligned with confidence when comparing closely related taxa. Recently, laccase production by several strains of A. pullulans was carried out by Rich et al. [27].
Laccase from A. pullulans was purified using ion-exchange chromatography on DEAE-Sephadex and dialysis of the active pooled fractions against glycerol had a single peak (Fig. 1) of activity was obtained a final yield of 59.3% and a purification fold of 2.0. The summary of a typical purification is shown in Table 1. The enzyme bound to the ion exchanger and hence it is anionic. Laccase from Gaeumannomyces graminis had a 4.6% yield and 120-fold purification [21] using a combination of dialysis and ultrafiltration. Laccase from Bacillus sp. ADR was purified using DEAE-anion exchanger with 33% yield and 56 purification fold [22]. A distinct band was obtained corresponding to 68.4kDa (Fig. 2) hence the molecular weight determined on SDS–PAGE was 68.4kDa. Fungal laccases vary in their molecular weight but most fall within the range of 60–70kDa [23]. The molecular weight reported for A. pullulans laccase varies. Rich et al. [27] reported molecular weights above 70kDa after deglycosylation with Endo H and even a particular strain, NRRYL-2568 had a molecular weight of 100kDa after treatment with Endo H. This suggests that the degree of glycosylation affects the molecular weight from several strains of A. pullulans.
The concentrated enzyme was yellow in color, the absorption from 250 to 800nm gave just a single peak at 280nm (Fig. 4). There was no peak shown at 610nm. The ratio of absorption at 280nm (A280) to absorption at 610, (A610) was 26. This is characteristic of yellow laccases. The ratio of absorption at 280nm (A280) to absorption at 610, (A610) was determined to be 26.0. This was higher than (15–20) meant for blue copper laccases. A value of 36.0 was obtained for Ganoderma fornicatum laccase [24]. This suggests that A. pullulans NAC8 laccase is a yellow laccase rather than a blue one. Yellow laccases are able to catalyze the oxidation of non-phenolic aromatic compounds in the absence of exogenous mediators [25]. The atomic absorption spectrophotometry showed that the ratio of copper to manganese is 3:1. Laccases contain 4 copper atoms termed Cu T1 (where the reducing substrate binds) and trinuclear copper cluster T2/T3 (where oxygen binds and is reduced to water). The three copper atoms can be distinguished using UV/visible SCR 7 and electronic paramagnetic resonance (EPR) spectroscopy [1]. There have been reports of yellow laccases having less than four copper atoms with the copper atoms replaced with manganese or other metals. Telke et al. [22] reported the existence of 2 copper atoms in Bacillus sp. ADR laccase. Atomic absorption spectrophotometry indicated that laccase from A. pullulans has three copper atoms and one magnesium atom. Palmeiri et al. [26] reported the existence of just a copper atom instead of four in Pleurotus ostreatus. Most non blue laccases probably have the fourth copper atom replaced with another metal and it might be responsible for lack of absorption at 610nm. (See Fig. 4).

The efficiency of each mediator depended on the type

The efficiency of each mediator depended on the type, molecular structure of dye to be treated and enzyme inactivation [13,42] as in case of turnip peroxidase, [28] have shown that after addition of the mediator there was no significant effect on the decolorization of some dyes such as DR 23 and DR 239, while in DB 80 decolorization was continuously decreased with increase in the concentration of mediator. Also, among the six types of dyes, only reactive blue 38 was resistant to enzyme–mediator treatment. Acid blue 74, reactive blue 19 and aniline blue were partially decolorized by the enzyme alone, although decolorization was much more efficient and rapid in the presence of mediators, whereas reactive black 5 and azure B could be decolorized only in the presence of mediators [11]. It has been reported that the mediators could play a dual role, as mediator because they increased the rate of recalcitrant dye decolorization and acted as inhibitor for enzyme activity.

Conclusion
Here, it has been shown that POL would be effectively employed for the decolorization of industrially important synthetic dyes and, for the first time, natural dyes. Our results suggested that, the peroxidase from Ficus sycomorus latex has shown its potential in decolorization of all selected dyes. More than 90% of Punica granatum, Hibiscus, Diethyl-p-phenylenediamine hydrochloride, methylene blue and methyl green dyes were decolorized by employing more effective and cheaper redox mediators with POL. These findings suggested that the POL could be extended to the large-scale treatment of textile effluents as a potential option of dye decolorization.

Introduction
Proteases are one of the most important industrial S63845 produced by a wide range of microorganisms such as bacteria, yeasts, molds and are also found in plants and in various animal tissues [35]. Bacterial proteases are mostly extracellular, easily produced in larger amounts, thermostable, and active at a wider pH range. Protease from Bacillus has been purified and characterized, and significant activity, stability, broad substrate specificity, short period of fermentation, simple downstream purification and low cost have been demonstrated [15,24]. These properties make the bacterial proteases most suitable for a wider industrial application. Proteases represent one of the three largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes [3,12]. Proteases are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth, the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for their various applications [3,7]. Bacillus produces a wide variety of extra-cellular enzymes, including proteases. Several Bacillus species were involved in protease production i.e., Bacillus cereus, Bacillus sterothermophilus, Bacillus mojavensis, Bacillus megaterium and Bacillus subtilis[32,7,6,11]. The largest application of proteases is in laundry detergents, where they help in removing protein based stains from clothing [5,6]. For an enzyme to be used as a detergent additive it should be stable and active in the presence of typical detergent ingredients, such as surfactants, builders, bleaching agents, bleach activators, fillers, fabric softeners and other various formulation aids. In textile industry, proteases may also be used to remove the stiff and dull gum layer of sericine from the raw silk fiber to achieve improved luster and softness. Protease treatments can modify the surface of wool and silk fibers to provide new and unique finishes. Proteases have been used in the hide dehairing process, where dehairing is carried out at pH 8–10 [17]. Proteases are also useful and important components in biopharmaceutical products such as contact lens enzyme cleaners and enzymatic debriders [4]. The proteolytic enzymes also offer a gentle and selective debridement, supporting the natural healing process in the successful local management of skin ulcerations by the efficient removal of the necrotic material [31]. In this paper we aimed to purify and characterize neutral protease from local bacterial isolate and evaluate as dehairing enzyme.

Apart from biological importance of the

Apart from biological importance of the diurnal periodicity in the egg leukotriene receptor antagonists phenomenon, there is a practical diagnostic concern. As the parasite propagules are excreted to the external environment through feces, their presence and/or concentration in feces is of diagnostic importance. It is known that information on quality of fecal egg counts (FEC) is influenced by a couple of feces related factors, e.g., daily amount (Daş et al., 2011a), consistency (Le Jambre et al., 2007) and feces flow (Michael and Bundy, 1988). Furthermore, FEC are variable due to variation in worm fecundity, uneven distribution of eggs in feces, host resistance, and a possible low sensitivity of egg counting techniques (Michael and Bundy, 1988). In this study we investigated whether nematode egg excretion in naturally or experimentally infected chickens follow certain patterns within a day, which may allow determining the most appropriate sampling time for the highest parasite egg concentration.

Materials and methods

Results

Discussion
With natural and experimental infection trials, this study investigated patterns in nematode egg excretion in a chicken–host system. It was repeatedly quantified that both the amount of feces and the fecal egg concentration increased sharply from the early morning to early-noon (10.00a.m.) and remained reasonably stable at high levels during the daytime which thereafter decreased to their initial levels during the night both in naturally and in experimentally infected birds. These results demonstrate that egg excretion of the most common parasitic nematodes in chickens follow diurnal fluctuation patterns which result in higher egg excretion during the daytime than the nights.

Acknowledgments

Introduction
The problem of gastrointestinal trichostrongylid parasitism (GTP) in sheep production is well-known and has been intensely studied during adult worm infection. Regulating impacts of parasitism on production may be achieved by selection for parasite resistance or incorporation of breeds that possess parasite resistance (Vanimisetti et al., 2004). Genetic linkage studies indicate that regions of the genome associated with parasite resistance are those involved in the immune response (Coltman et al., 2001). Characterization of immune responses to GTP may therefore lead to identification of candidate genetic markers that could be useful to identify parasite-resistant individuals (Charon, 2004).
Acquired immunity to GTP in sheep has been well-defined as a classic Th-2 response with high levels of IgG, IgA and IgE, mastocytosis and eosinophilia at the site of infection (Lacroux et al., 2006). Unlike mouse models where susceptible mice have an inappropriate Th-1 response to GTP (Else et al., 1993), susceptible sheep are able to mount an appropriate type of immune response (Shakya et al., 2009). In sheep, susceptibility appears to result from a reduced level and delayed timing of immune responses (Rowe et al., 2009; Bowdridge et al., 2013) which facilitates establishment of the adult parasite. The difference between parasite resistance and susceptibility in sheep therefore lies in the magnitude of immune response to larvae during challenge infection.

In the Wroc aw Agglomeration the infection level of

In the Wrocław Agglomeration the infection level of I. ricinus ticks by Borrelia spp. is proven to be high (23.5%). Identification of B. afzelii, B. garinii and B. miyamotoi verifies results of a previous study in the Wrocław area (Kiewra et al., 2014). The infectious percentages of ticks from cats and dogs were not statistically different. There was also no significant difference in Borrelia infection between engorged ticks from domestic animals and questing ticks from vegetation. Hence, it appears that ticks feeding on pets do not lose their Borrelia infection. Borrelia infections in ticks from pets walking in different habitats show that there is no significant difference in ticks from LIAA, HIAA and mixed areas.
The present study also confirmed that B. burgdorferi s.l. can be transmitted by I. hexagonus (DNA sequencing proved the presence of B. afzelii), which is the second most common tick 5-alpha reductase inhibitors parasitizing dogs and cats in the Wrocław Agglomeration. These were collected from dogs that were walking in semi-natural areas, mostly along rivers. Thus, I. hexagonus may also contribute to the transmission of Borrelia in urban areas.

Conclusions

Acknowledgements
The authors would like to acknowledge all staff from veterinary clinics who have helped in collecting ticks and completing surveys for this research. Particular thanks are due to the following veterinary physicians: Szczepan Kawski from Dolittle’s Clinic, VMD Janusz and Wojciech Stańczyk from Dr. Hau Veterinary Emergency, Dariusz Rzepka from Ołtaszyńska Veterinary Clinic, Michał Senze from VETUS Veterinary Clinic, Tatiana Narajowska and Anna Staniewska from Veterinary Clinic, and Marcin Noczyński from Novet Veterinary Clinic. This task was partially financed by the European Union through the European Social Fund, as well as by the University of Wrocław (grant numbers 1145/M/IGM/13 and 2113/M/IGM/14).

Introduction
Ovine babesiosis is an endemic tick-borne disease of small ruminants in many European, African, Asian and Far Eastern countries. The disease causes significant economic losses in the livestock industry (Sayin et al., 1997; Yeruham et al., 1998; Fakhar et al., 2012; Ranjbar-Bahadori et al., 2012). Among the four main Babesia species including Babesia ovis, Babesia motasi, Babesia crassa and Babesia foliata; Babesia ovis is mostly responsible for ovine babesiosis, and the clinical infections caused by B. ovis are usually severe (Uilenberg, 2006). Mortality is unavoidable in untreated clinical cases. In cases of delayed diagnosis, some sick animals may die in spite of specific drug administration. The first clinical symptom is high fever which occurs in parallel with the appearance of the parasites in the blood. The parasitemia level increases quickly, and the other symptoms such as anemia, hemoglobinuria, dyspnea, tachycardia and impotence develop in the absence of specific treatment. A few days after the onset of high fever, pancytopenia also occurs in the cases of severe infection. The compensation of abnormalities in the hematological picture takes a long time in the animals treated for acute infection. Some recurrences can also occur in animals during the post-treatment period (Yeruham et al., 1998; Esmailnejad et al., 2012; Sevinc et al., 2013).

br Materials and methods br Results br Discussion We have

Materials and methods

Results

Discussion
We have purified a 64-kDa glycosylated antigen from the Venezuelan TeAp-N/D1 isolate of T. equiperdum. Tryptic peptides from p64 were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry, and the retrieved sequences yielded only one hit to a putative VSG from T. brucei TREU927 (Tb927.4.5460) after searching the NCBInr and the T. brucei brucei genome database (Camargo et al., 2015). This result indicated that p64 corresponds to the soluble form of the predominant VSG from T. equiperdum TeAp-N/D1. Moreover, p64 appeared to be very sensitive for diagnostic purposes (Uzcanga et al., 2004) and was recognized by anti-T. vivax bovine rho kinase (Uzcanga et al., 2002, 2004).
A high immunological cross-reactivity between trypanosome species such as T. evansi and T. vivax has been described (Desquesnes and Tresse, 1996). Accordingly, the OIE has reported that the use of whole cell lysates of T. evansi leads to strong cross reactions with T. vivax, T. congolense and even Trypanosoma (Schizotrypanum) cruzi (OIE, 2010). Although in vivo outbred murine models of trypanosomosis (CD-1, RjOrl:Swiss mice) have been developed using the IL 1392 strain of T. vivax that was originally derived from the Y486 isolate from Africa (Blom-Potar et al., 2010; Chamond et al., 2010; Leeflang et al., 1976), and in vitro non-infective T. vivax epimastigote axenic cultures have been reported using the same IL 1392 strain (D\’Archivio et al., 2011), the production of T. vivax antigens continues to be a limiting factor because most T. vivax stocks are restricted to large animals (cows, sheep, goats, pigs, etc.), and possess relatively low level parasitaemias. In contrast, rodents can be readily inoculated in the laboratory with T. equiperdum and T. evansi to acquire high quantities of parasites to prepare antigens for serological tests. Correspondingly, we have focused on the diagnosis of T. vivax-caused animal trypanosomosis by using cross-reacting antigens isolated from Trypanozoon trypanosomes (Camargo et al., 2004; Uzcanga et al., 2002, 2004; Velásquez et al., 2014). In the present study, further evidences were obtained confirming that p64 from T. equiperdum TeAp-N/D1 is a cross-reacting antigen suitable to be used as a tool to detect bovine trypanosomosis caused by T. vivax.
The time course of experimental infections of bovines with T. vivax were evaluated here by measuring whole anti-p64 antibodies and specific anti-p64 IgG and IgM antibodies in animal sera by indirect ELISA. Levels of parasitaemia in T. vivax-infected bovines showed characteristic regular oscillations, which represented the emergence, proliferation and immunological elimination of antigenically different populations of trypanosomes. High levels of whole anti-p64 antibodies and specific anti-p64 IgM and IgG isotypes were detected in sera from these bovines during the time course of infection, demonstrating that B-cells did experience immunoglobulin class switching to produce IgG isotypes in T. vivax-infected cows. These results further illustrate that p64 behaves as a cross-reacting antigen between Trypanozoon trypanosomes and T. vivax and revealed that p64 has common invariant epitopes that were immunorecognized by bovine sera throughout the whole infection period.