Monthly Archives: September 2017

There are to our knowledge no reports of

There are, to our knowledge, no reports of effective, safe and simple inactivated vaccines against CyHV-1, 2 or 3. Regarding CyHV-1, there is no trial for a vaccine since CyHV-1 is not a major cause of mass mortality of carp Cyprinus carpio. There is no report of vaccination against CyHV-2 infection except for our previous report (Ito and Ototake, 2013) since sustainable propagation of this virus has previously been difficult due to the lack of the suitable cell lines for the viral culture. Moreover, for CyHV-3, although live attenuated virus has been used to vaccinate carp and protect the fish from viral challenge, a safe and effective vaccine is not currently widely accepted (Ilouze et al., 2011; OIE, 2013). CyHV-1, 2 and 3 belong to the same family Alloherpesviridae and sequence analyses of the genome of these viruses have shown that the CyHVs are closely related to each other (Davison et al., 2013; Waltzek et al., 2005). Although a simple inactivated vaccine against CyHV-3 infection in carp is not effective (Ilouze et al., 2011; OIE, 2013), the inactivated vaccine examined here shows efficacy for CyHV-2 infection in goldfish. Ito et al. (2013) reported that CyHV-2 DNA was detected from infected fish at 8 days post exposure. However, in another study CyHV-3 DNA was detected from infected fish at 1 day post exposure (Gilad et al., 2004; Ito et al., in press). These results indicate that the viral kinetics of CyHV-3 in carp differ significantly from those of CyHV-2 in goldfish. Moreover, we reported that most fish challenged with CyHV-2 at 13–15°C were resistant to infection, and fish infected at low temperatures were able to survive after the water temperature was raised to 24°C (Ito and Maeno, 2014). In rapamycin price to our findings with CyHV-2, carp infected with the closely related CyHV-3 at 12°C exhibited no mortality for 30 weeks, but began to die after the water temperature reached 21–23°C (St-Hilaire et al., 2005). In addition, the water temperature range over which koi herpesvirus disease caused by CyHV-3 infection is most prevalent is 18–23°C (Gilad et al., 2003), similar to the temperature range for HVHN. Taken together with the previous studies, the difference between efficacy of vaccines against CyHV-2 infection in goldfish and CyHV-3 infection in carp might reflect differences of the interactions between fish species and these pathogenic viruses.

Conclusions
The survival rate in the vaccinated groups was significantly higher than values in the respective negative control groups using Fisher\’s exact test. The RPS value for the Vaccinated-Booster groups was 63.6%, and the survival rates of Vaccinated-Booster groups were significantly higher (p=0.036) compared with the respective control groups using Student\’s t test. Also this study suggests that the effectiveness period of this vaccine against HVHN was 8 weeks at least. This report demonstrated that the simple formalin inactivated vaccine was effective against HVHN of goldfish and it was revealed that a booster shot of this vaccine was more efficient.

Acknowledgement
This study was funded by the Fisheries Research Agency in Japan.

Introduction
In aquaculture, Pseudomonas fluorescens is a common pathogen to both vertebrate and invertebrate animals. For fish, P. fluorescens is known to affect a wide range of farmed species. Currently, there is no effective means to control P. fluorescens infection in aquaculture, largely due to the lack of knowledge to the virulence mechanism of this pathogen.
Iron plays an important role in biology. However, iron is difficult to utilize by living organisms because it exists in various oxidized forms in the environments and, for pathogenic bacteria, is bound to iron-chelating proteins inside animal hosts. As a result, bacteria have developed various strategies for iron acquisition, such as production of siderophores, which are iron chelators that have extremely high affinity for Fe3+ (Braun and Hantke, 2011). However, since iron participates in the generation of hydroxyl free radicals that are deleterious to the cells, iron assimilation has to be tightly controlled (Andrews et al., 2003). In Gram-negative bacteria, this control is mediated primarily by ferric uptake regulator (Fur), a global transcriptional regulator involved in many cellular processes (Troxell and Hassan, 2013; Visca et al., 2002).

The samples were pooled by five in sampling order l

The samples were pooled by five (in sampling order: 50μl aliquots from five swabs (five pigs) per pool tube) to reduce the screening cost. RNA was extracted by using the QIAmp viral RNA minikit (QIAGEN), and influenza A – specific one-step real-time RT-PCR was carried out by using M52C/M253R influenza A primers and the QuantiFast SYBR® Green RT-PCR Kit (QIAGEN) according to the instructions of the manufacturer as previously described (Couacy-Hymann et al., 2012).
Influenza A virus genome was detected in 8 out of 65 positive pools: in samples collected in February, from June through September, and in November 2013. Subtyping real-time RT-PCR assays for HA detected only the A(H1N1)pdm09 virus genes in all the positive pools (WHO, 2009).

Discussion
Swine populations have been surveyed every month in Togo since 2009 within the framework of a large-scale animal influenza surveillance project, with no influenza A virus detected in nasal swabs and no influenza A agomelatine in swine sera (Couacy-Hymann et al., 2012). We have previously hypothesized that the combination of climate and animal density factors might be responsible for what appeared to be the absence of influenza virus in the backyard sector of Côte d’Ivoire, Benin, and Togo (Couacy-Hymann et al., 2012). Lowen et al. (2007) indeed, showed in a laboratory setting and in the guinea pig model that the lower the temperature and relative humidity, the higher the rate of aerosol transmission. In addition, Meseko et al. (2014) detected swine influenza virus in Nigeria throughout the 2 years of their study, with higher isolation rates from November through January, when the dry Harmattan wind is present. In the present study, we did not detect significantly more influenza virus in the Harmattan season (November through January in Togo as in Nigeria) than during the rest of the year. This finding could be explained by a much lower animal density in Togo than in Nigeria: 1.2million tons of pork meat was produced in Africa in 2012, with 10100 tons in Togo and 249000 tons in Nigeria (www.faostat.org). Data collected over a longer period of time would be necessary to properly test the seasonality hypothesis.
To better assess swine influenza virus prevalence in Togo, a surveillance of younger animals would be informative. In the Nigerian study, 78% of the swine influenza – positive samples were, indeed, from weaners and growing pigs (Meseko et al., 2014), which are thought to be more susceptible to infection; but we tested older pigs sampled at a slaughterhouse.
From October 2010 through January 2014, Togo reported sporadic influenza virus activity in humans to the World Health Organization (WHO). (H1N1)pdm09 was reported in the country in October and November 2010, from February through April 2011 and from June through August 2011, in February and July 2012 and from September through November 2012, and from April through July 2013 and October through December 2013 (WHO). Nzussouo et al. (2012) reported that (H1N1)pdm09 occurrence in most of West Africa was delayed compared to that of the rest of the world. The first countries in West Africa to report (H1N1)pdm09 circulation were Côte d’Ivoire in April 2009 and Ghana in August 2009 (WHO). Taken together, these data show (i) circulation of (H1N1)pdm09 virus in humans since at least 2010; (ii) low influenza virus activity (or reporting) without seasonality: a transmission event from human to swine is possible in Togo. The sporadic circulation of (H1N1)pdm09 in humans since 2010 might explain why no swine influenza virus was detected before 2013 in Togo although human-to-swine spillover was observed much earlier in many other countries. The absence of seasonality in human influenza reports in Togo is also in agreement with the irregular occurrence of the disease in swine.
The results of our phylogenetic analyses of A/swine/Togo/ONA32/2013(H1N1) genome suggest a human origin of the virus. Human-to-swine and swine-to-human transmission of influenza virus have been frequently reported (Brockwell-Staats et al., 2009), and proven human-to-swine transmission of (H1N1)pdm09 occurred several times in America at the early stages of the 2009 pandemic (Howden et al., 2009; Pereda et al., 2010; Weingartl et al., 2010). In Cameroon, Larison et al. recently studied the virus spillover from human to swine and found that free roaming of pigs is a significant risk factor. They hypothesized that virus transmission to swine may occur via consumption of human feces, which contains infectious particles (Larison et al., 2014). However, this mode of transmission seems relatively unlikely in the Togolese context, swine production in Togo consists of small farms and free range pigs, with little to no importation from abroad, a setup unfavorable for sustained swine influenza virus transmission (Saenz et al., 2006). Taken together, the literature and our phylogenetic analyses results support the hypothesis of human-to -swine transmission of (H1N1)pdm09 in Togo; however, epidemiological studies are warranted to show the transmission route definitively.

Previously we studied transmission of FMDV in groups of

Previously, we studied transmission of FMDV in groups of non-vaccinated and FMD O Manisa vaccinated sheep, infected with FMDV O/NET/2001 (Orsel et al., 2007). However, also strain specific differences transmission characteristics might exist. Because FMDV strain Asia-1 appears regularly in parts of the world where sheep play an important economic role, we now quantified transmission of FMDV strain Asia-1 in groups of non-vaccinated and vaccinated sheep. For this, we used the final sizes observed in the experiments. We evaluated whether the occurrence of contact transmission was distributed evenly over the infectious period of the sheep and if the viral load of a pen was correlated with the occurrence of transmission. In addition, the clinical data and laboratory results obtained in this experiment were used to evaluate which would be the best samples for detection of an FMDV infection in a sheep population.

Materials and methods

Results

Discussion
Transmission to contact sheep in the non-vaccinated sheep did occur with an R0 of 1.14, which is in accordance with our previous finding for another FMDV strain (Orsel et al., 2007). This indicates that an FMDV Asia-1 outbreak in sheep would not necessarily be self-limiting and that additional control measures might be needed to stop an epidemic. Vaccination is often used, either as emergency tool or in eradication programmes, to reduce virus transmission. The most important feature of a vaccine should be that after vaccination, the reproduction ratio R should be reduced below 1. Our results indicate that vaccination indeed reduces transmission, similarly as has been reported by others (Cox et al., 1999; Orsel et al., 2007; Parida et al., 2008; Madhanmohan et al., 2010). Vaccination however did not prevent infection of the vaccinated sheep, as 10 out of 12 inoculated vaccinated sheep shed virus in their OPF for a prolonged period of time. This is in DAPTinhibitor to studies where limited virus excretion after vaccination was reported (Cox et al., 1999; Parida et al., 2008; Madhanmohan et al., 2010). Maybe, the difference can be explained based on the sampling method that was used. The OPF swabs samples that we used, were taken relatively deep in the mouth in the oropharyngeal region. Burrows (1968) showed that in carrier sheep most of the virus can be found in the tonsillar area, which is close to the sampling area that we used. The non-complete protection of the vaccinated animals might be caused by the lack of match between field strain and vaccine strain match, indicated by the low r1-value of 0.1, as a values less than 0.3 suggest that the field isolate is so different from the vaccine strain that the vaccine is unlikely to protect (Rweyemamu, 1984). However, although vaccination did not stop virus excretion in the inoculated vaccinated sheep, it significantly reduced the virus excretion (AUCs) as compared to non-vaccinated sheep, which indicates that vaccination will lead to less environmental contamination with FMDV. More importantly, it prevented transmission of FMDV in the vaccinated groups. In field situations, where animals without vaccination titres (new born animals, young animals with a weak response to vaccination because of maternal immunity, animals with already waning vaccination titres) might be present in a vaccinated population, the reproduction ratio might be a higher than as estimated for a fully vaccinated population. However, to estimate the reproduction ratio for transmission in heterogeneous populations (in this case a mixed population of vaccinated and non-vaccinated animals), also other transmission parameters, e.g. for transmission between vaccinated and non-vaccinated animals and other mathematical methods are required (Diekmann et al., 2010). Because the reproduction ratio in the non-vaccinated population is only slightly >1, it can be expected however, that also vaccination of a part of the population especially in combination with control measures that will reduce contacts, already will be sufficient to reduce R to below 1.

For both AIV and NDV co infection affected

For both AIV and NDV, co-infection affected the titers of virus shed, and in the case of vNDV, the number of PR619 cost shedding. The effect on viral replication caused by one virus over another is known as viral interference, a phenomenon in which a cell infected by a virus does not permit multiplication of a second homologous or heterologous superinfecting virus (Dianzani, 1975). Viral interference can occur by different mechanisms including: competing by attachment interference therefore reducing or blocking receptor sites for the superinfecting virus; competing intracellularly for replication host machinery; and virus-induced interferon interference (Kimura et al., 1976). Replication of one virus might be affected by previous replication in the same site of another virus that has already activated antiviral immune responses including immunomodulators or recruitment of immune cells. Other studies examining co-infection of LPAIV and NDV with other respiratory viruses of poultry demonstrated that co-infections can either exacerbate clinical disease, or, like in our study, affect virus replication by lowering viral titers, serological conversion and virus transmission (Gelb et al., 2007; Haghighat-Jahromi et al., 2008; Hanson et al., 1956; Raggi and Lee, 1963; Turpin et al., 2002).

Acknowledgements
The authors appreciate the assistance provided by Aniko Zsak, Tim Olivier, Dawn Williams-Coplin, Rami Cha, Kira Moresco, Ronald Graham, and Roger Brock in conducting these studies. This work has been funded by the Agriculture Research Service CRIS Project 6612-32000-048 and with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health Department of Health and Human Services, under Contract No. HHSN266200700007C. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Introduction
Wild waterfowl are the natural reservoir of avian influenza viruses (AIVs) (Webster et al., 1992). While AIVs typically replicate efficiently in their natural hosts, there is evidence that AIVs can infect mammals, including dogs, cats, plateau pikas, rhesus macaques, and humans (Cheng et al., 2014b; Shinya et al., 2012; Songserm et al., 2006; Yu et al., 2014a; Zhang et al., 2013a, 2013b). Some AIVs are reported to be capable of transmission between mammals, suggesting that AIVs may possess pandemic potential (Belser et al., 2013; Gao et al., 2009; Zhang et al., 2013c).
H7N7 viruses circulate widely in areas including North America (Spackman et al., 2008) and Europe (Slavec et al., 2012), as well as southern China (Lam et al., 2013), Australia (Bulach et al., 2010), and New Zealand (Bulach et al., 2010). Zoonotic transmission of H7 influenza viruses to humans has been suggested by reports of significantly elevated titers of H7-specific antibodies among poultry workers in Italy (Di Trani et al., 2012), and documented cases of human H7N7 infection in the Netherlands in 2003 and H7N9 infection in China in 2013 (Gao et al., 2013; Koopmans et al., 2004). A previous report found that ferrets inoculated by the ocular aerosol route with an avian H7N7 virus were capable of transmitting the virus to naïve animals in direct-contact or respiratory-droplet models (Belser et al., 2014). Taken together, reports of human infection with H7N7 AIVs and a lack of pre-existing immunity against these viruses in humans suggest that H7N7 AIVs may pose a pandemic threat. Therefore, the molecular features involved in the mammalian adaptation of H7N7 AIVs should be further studied.
While mice are not a natural host for influenza and viruses that cause severe disease in mice usually show little virulence in other mammals, they are a conventional animal model in which to study the pathogenesis of AIVs (Belser et al., 2007, 2013; Belser and Tumpey, 2013; Cheng et al., 2014a; Gabriel et al., 2005; Hatta et al., 2001; Li et al., 2005, 2014; Song et al., 2009; Tumpey et al., PR619 cost 2002). To identify the possible changes that are associated with the adaptation of H7N7 AIV to mammals, we serially passaged a wild waterfowl-origin H7N7 virus in mice. After sequential passage of an H7N7 virus in mice, we obtained two viruses which displayed increasing virulence and enhanced replication kinetics in vitro and in vivo. Multiple amino acid substitutions involved in the adaptation of H7N7 avian influenza virus in mice. While the adaptive mutations identified in this study may contribute to mammalian adaptation of wild bird-origin H7N7 AIVs, the effects of these mutations on mammalian pathogenicity need to be studied in the future works.

A potential link even if still unproven could

A potential link, even if still unproven, could be provided by a fourth animal population: the Peccary (Family Tayassuidae). In particular the collared peccary (Pecari tajacu) and white-lipped peccary (Tayassu pecari) have been recently shown to be susceptible to PCV2 and to display high infection prevalence (de Castro et al., 2014). These species are commonly present in the Pantanal (Desbiez et al., 2011) and more generally share their distribution ranges with domestic pigs (including rural-extensively raised ones), wild boards and feral pigs. Consequently, peccaries harboring PCV2 may have infected feral pigs. However, the inadequate data regarding the domestic-rural pig and wild boar distributions and the PCV2 prevalence and order T0901317 distribution in peccaries, makes impossible to formulate a definitive hypothesis with a reasonable degree of confidence.
Overall, this study reveals an unexpected PCV2 variability within the previously neglected population of feral pigs in the Pantanal (Matto Grosso do Sul Brazil). Above all, this is the first report of currently existing PCV2c strains in Suidae species. These results open new avenues in the reconstruction of PCV2 history and in the understanding of the role of host and ecological niches and their interactions.

Acknowledgment

Introduction
The genus Corynebacterium contains many species recognized worldwide as pathogens of humans and/or animals. Currently, based on molecular classification and changes in taxonomy have been observed an increased interest in corynebacteria as opportunistic pathogens for humans (Trost et al., 2011) and animals (Greene, 2012). Corynebacteria have been increasingly related with zoonotic transmission from companion animals to humans (Funke et al., 2009; Hogg et al., 2009; Funke et al., 2010; Dias et al., 2011). Recently, Corynebacterium lactis was first isolated from milk of asymptomatic cows in Germany (Wiertz et al., 2013). This species shows phenotypic characteristics similar to other species of Corynebacterium. Consequently, reports of infections due to C. lactis before its description were probably attributed to Corynebacterium spp. In the present study the ability of C. lactis to cause cutaneous abscess in dogs was demonstrated. The results of this study will contribute to enhancing the knowledge about this recently described species.

Materials and methods

Results
Bacterial culture on agar base supplemented with 5% defibrinated sheep blood of clinical specimens revealed the presence of Corynebacterium-like colonies of different sizes, named 2447P and 2477G (Fig. 1C). On Columbia blood agar after 3 day incubation under aerobic conditions at 37°C colonies were white with uneven edge, dry and non-hemolytic. Gram-positive cells were non-spore-forming and non-motile short rods. API-Coryne profile number was 0 1 0 0 3 0 4 for both 2447P and 2477G isolates. The bacterial isolates produced catalase and alkaline phosphatase but were negative for esculin hidrolysis, pyrazinamidase, urease, nitrate reductase, gelatinase, pyrrolidonyl arylamidase, ß-glucuronidase, ß-galactosidase, α-glucosidase, N-acetyl-ß-glucosaminidase. Acid was produced from glucose, ribose but not from xylose, mannitol, maltose, lactose, sucrose and glycogen. Microorganisms also gave negative results for the tyrosine and DNAse tests and CAMP reaction. The classical biochemical tests and the API-Coryne system enabled phenotypic identification only as Corynebacterium sp. Both colony types were susceptible to all antimicrobial agents tested.
Comparative 16S rRNA gene sequence analyses showed that the clinical isolate 2447 was most related to Corynebacterium lactis (DSM 45799) type strain (similarity of 99.79%). Phylogenetic analysis with different methods based on 16S rRNA gene sequences showed that the clinical isolate 2447 occupied a distinct branch with C. lactis (DSM 45799) indicating that it belongs to the abovementioned species (Fig. 2). The 16S rRNA gene sequence of 2447 isolate was deposited in the GenBank with accession number KF661298.

Several studies of naturally and experimentally infected cattle

Several studies of naturally and experimentally infected cattle provide irrefutable evidence that the terminal rectum, especially the recto-anal junction (RAJ) is a predominant colonization site of E. coli O157:H7 and carriage of bacteria at this site is correlated with high levels of faecal shedding (Cobbold et al., 2007; Lim et al., 2007). Therefore, the rectal mucosa may be the ideal target for controlling EHEC in cattle. Administration of bovine lactoferrin to this location ceases E. coli O157:H7 shedding in cattle and seems to clear the colonization from the rectum and other more cranial compartments of the gastro-intestinal tract (Kieckens et al., 2015). Nevertheless, it is unknown which of the biological activities of lactoferrin are responsible for this clearance.
The objectives of this study were to: (i) compare the effect of lactoferrin treatment on faecal excretion and on mucosal colonization at the rectum following experimental infection with Shiga toxin-negative E. coli O157:H7 in cattle (ii) and establish if rectal treatment with lactoferrin may influence the local antibody response against EspA and EspB virulence proteins of E. coli O157:H7 and if this is correlated with protection against re-infection.

Materials and methods

Results

Discussion
In recent years, interest for intervention methods to control E. coli O157:H7 in ruminants has greatly increased (Callaway, 2010; Sargeant et al., 2007). Considering the different routes through which humans can become infected, interventions to reduce prevalence of EHEC should be applied at different levels: from the farm, through the slaughterhouse, to all stages of the food chain. The interventions at the farm level should be practical, economical and suitable from the animal welfare perspective. Since the terminal rectal mucosa has been identified as the predominant colonization site of E. coli O157:H7 in cattle, a possible intervention approach is to target this colonization site directly. Different in vivo studies to reduce the EHEC carriage in cattle have been tested including rectal immunization with purified H7 flagellin (McNeilly et al., 2008) or the rectal administration of O157-specific induced pluripotent stem cells (Rozema et al., 2009). However, these methods showed only limited success.
In a recent study, we demonstrated that daily, rectal application of 300mg bLF powder clears the infection in cattle within 3 weeks (Kieckens et al., 2015). With the intention to fasten clearance we used a 5-times higher dose of bLF (1.5g) in the present study, but this increase did not shorten the duration of excretion. The infection in the powder group still lasted 19 days. Nevertheless, the bLF treatment again completely cleared the bacterial colonization in all animals. Since a daily rectal administration during 19 days is not practical and oral administration was ineffective in cattle (Kieckens et al., 2015) in contrast to sheep (Yekta et al., 2011), a more efficient delivery system for LF has to be developed, which will release the protein in a highly dispersed and adhesive way over the rectal mucosa, improving its persistence in the rectum. Here, mucosal adhesive dosage forms, which can be rectally applied, could be an option.
An important finding of our study is that the application of bLF powder daily on the rectal mucosa cleared this site from bacterial colonization within 13 days. This suggests that the period of the rectal treatment could be shortened with at least a week. Nevertheless faeces were 6 days longer positive, what suggests that the bacteria temporarily persist in more cranial compartments of the gastrointestinal tract. Although the terminal rectum was shown to be the principal site of E. coli O157:H7 in cattle some studies indicate that rumen, small intestine, proximal colon or gallbladder are minor sites of colonization (Brown et al., 1997; Cray and Moon, 1995; Reinstein et al., 2007). Nart et al. (2008) reported that in a small number of animals bacteria can be found distributed throughout the large intestine. This finding suggest that there could be a different mechanism of colonization for a small number of animals, maybe due to the existence of multiple E. coli O157:H7 genetic types with different colonization strategy within one animal or different receptors within animals. Moreover, re-ingestion from a contaminated environment may contribute to persistence of bacteria in other sites of the gastro-intestinal tract (Naylor et al., 2003).

S aureus can not only invade

S. aureus can not only invade into mammary epithelial cells, but also alter cell junction protein expressions (Kwak et al., 2012), while vitamin D can protect epithelial barrier by regulating cell junction proteins (Chen et al., 2015b). Thus, we hypothesized that pre-treatment of 25(OH)D would increase the gene expression of occludin (a tight junction protein) and E-cadherin (a adherens junction protein). Nevertheless, inconsistent with the studies of Hsu et al. (2011) and Lopes et al. (2012), we did not find any transcriptional increase in the present study. The discrepancy may be partly attributed to different cell types used in the studies. Hsu et al. (2011) and Lopes et al. (2012) used cancer 4-ethylphenyl sulfate Supplier that are known for a reduced E-cadherin expression, whereas MAC-T cell used in our study is a non-tumorigenic cell line (Huynh et al., 1991). In addition, Hsu et al. (2011) and Lopes et al. (2012) treated their cells with 100nM 1,25(OH)2D (41.6ng/ml), which is unlikely to achieve by hydroxylase 100ng/ml 25(OH)D in our study. Considering that there is no potential VDR-responsive element in the E-cadherin promoter (Hsu et al., 2011), it is not surprising that this indirect regulation does not work at a lower concentration. On the other hand, many studies use differentiated cells to study the effects of vitamin D on cell–cell junctions (Chen et al., 2015a, 2015b), whereas MAC-T cells used in this study were undifferentiated. Although undifferentiated cells also have basal gene expression of junctional proteins, they may be insensitive to vitamin D variations compared with that of differentiated cells.
Tellez-Perez et al. (2012) reported that S. aureus downregulate CYP27B1 expression but upregulate VDR expression on primary bovine mammary epithelial cells, while our data are consistent for CYP27B1 but not for VDR. The reduction of VDR and occludin mRNA expression induced by S. aureus in our study is similar to that induced by lipopolysaccharide (Chen et al., 2015b). However, pre-treatment with 1,25(OH)2D did not attenuate the S. aureus-induced mRNA reduction in our study, which is in agreement with the finding that 1,25(OH)2D3 attenuate the lipopolysaccharide-induced reduction in Caco-2 cells (Chen et al., 2015b). These discrepancies could be attributed to the differences in experimental conditions such as cell type, bacterial strain, or treatment time. The universally transcriptional suppression may also due to the apoptosis induced by S. aureus after escape from the endosome (Bayles et al., 1998).
In conclusion, the present study indicate that vitamin D2 compounds have comparable effects on inhibiting cell viability and S. aureus invasion to their D3 analogues in vitro, and suggests that vitamin D2 and its metabolites may also be potential for controlling bovine mastitis. Further studies are needed to explore the mechanism of how vitamin D reduce S. aureus invasion, and how vitamin D can be exploited to control bovine mastitis in vivo.

Conflicts of interest

Funding

Acknowledgements

Introduction
Bovine Respiratory Disease (BRD) is a multifactorial disease characteristic of a viral-bacterial synergistic infection with predisposition from environmental stressors. The disease constitutes a major source of economic loss through mortality, clinical disease and associated treatments with long lasting reduced growth performance of infected young stock (Griffin, 1997). Bovine Parainfluenza Virus-3 (BPI3V) is one of the major viral pathogens of the BRD complex (Kahrs, 2001). BPI3V induced respiratory tract damage, resulting from the destruction of the ciliated respiratory epithelium (Bryson, 1985) and immunosuppression via depression of local cellular immunity by impairment of alveolar macrophage phagocytosis (Baker et al., 1997; Trigo et al., 1985), predisposes animals to more severe secondary bacterial and mycoplasma infections (Cusack et al., 2003; Kapil and Basaraba, 1997). With the absence of severe clinical symptoms (Vaucher et al., 2008), infected animals may not be detected prior to the onset of more severe infections (AFBI/DAFM, 2012). Furthermore, routinely employed BPI3V-antibody ELISA cannot differentiate between vaccinated and infected animals, and by the time infected animals convalesce the virus has been cleared from the system and respiratory tract damage has already occurred. Molecular diagnostic techniques are hindered by the presence of vaccine derived genetic material, often requiring on-going virus amplification in order to generate sufficient genetic material for accurate diagnosis. Consequently, there are no commercial tests available for differentiation between BPI3V vaccinated and non-vaccinated animals. Anti-mortem diagnostic tests for BPI3V such as immunohistochemistry and virus isolation provide limited information on the current health status of an animal and can only determine pathogen exposure but not the presence of diseased tissue (Fulton and Confer, 2012), further illustrating the need for the development of alternative diagnostics capable of detecting infected animals (and the presence of diseased tissue) at early stages of infection.

Concerning duration of treatment a mathematical simulation study concluded that

Concerning duration of treatment, a mathematical simulation study concluded that short duration of TET administration should be preferable in order to reduce selection for TET-resistance, since less time under treatment would reduce the competitive growth of the R-strains (Ahmad et al., 2015). The authors concluded that more prolonged treatment resulted in an increased occurrence of resistance and therefore it took longer to return to equilibrium (Ahmad et al., 2015). It has previously been demonstrated that it might also be possible to reduce the duration of treatment with a higher daily dose level to achieve the desired efficacy and lower resistance levels (Geli et al., 2012). Our results are in accordance with these predictions, but only at the specific time point 2dAT. Thus, in the field study the medium-dose for three days treatment regime (Do20.Dur3) led to the lowest TET-R bacteria at 2dAT compared to day followed by the high-dose regime (Do30.Dur2). By the end of the nursery period, all of the treatments can be assumed to be equal regarding TET-R development, and the number of TET-R coliforms was lower than when pigs entered the nursery period.

Conflict of interest

Acknowledgments
Authors would like to thank, P.R. Mortensen, A.B. Lind and M.Q. Pawlowski for technical assistance. The study was supported by the Danish Council (DFS) grant number 0603-00358B (project entitled “Minimizing Antibiotic Resistance Development-MINIRESIST”).

Introduction
Shiga-toxin Escherichia coli is a major etiologic agent of diseases in humans, whose clinical spectrum includes diarrhoea, haemorrhagic colitis, and haemolytic uremic syndrome (HUS), the leading cause of chronic renal failure in children in Argentina and several other countries (Karch et al., 2005; Repetto, 2005). The most frequently associated serotype is O157:H7 (Rivas et al., 2006), although six non-O157 O groups (O26, O45, O103, O111, O121, and O145) have been identified by the Centers for Disease Control and Prevention (CDC) as being responsible for over 70% of non-O157 STEC-associated illness in the USA. (Brooks et al., 2005).
Ruminants are the primary reservoir of EHEC O157:H7 (Yoon and Hovde, 2008)and have been recently recognized as important reservoirs for other STEC serotypes, some of them associated with human disease (Parul et al., 2016). In Argentina, cattle have been identified as a source of non-O157 STEC and some of these strains have been previously associated with hemolytic uremic syndrome (HUS), bloody and non-bloody diarrhea (Fernandez et al., 2013; Masana et al., 2011; Pizarro et al., 2014). Despite the carriage of Shiga purchase CM-272 genes, posing a significant threat to human health, the combination with other virulence factors needs to be further assessed in order to evaluate the pathogenic potential of STEC strains. In this sense, the mechanism of STEC adherence to intestinal epithelium is highly relevant for the understanding of the STEC-bovine relationship and for the design of vaccines and other antibacterial strategies.
One of the main features of E. coli O157:H7 is the Locus of Enterocyte Effacement (LEE), which is a 35.6kb pathogenicity island encoding a type three system (T3SS) apparatus, secreted proteins, an adhesin called Intimin and its translocated receptor Tir (Translocated Intimin Receptor) (Franzin and Sircili, 2015). These proteins are responsible for the formation of the attaching and effacing lesion and the effective colonization of cattle intestine (Dean-Nystrom et purchase CM-272 al., 2008). Despite the crucial role of Intimin in bacterial attachment, E. coli O157:H7 possesses many other proteins involved in adherence, some of them shared with non-O157 STEC. Among the fimbriae, the long polar fimbriae (Lpf) is relevant in this matter, which binds to extracellular matrix and is important for intestinal colonization in vivo. The hemorrhagic coli pilus (HCP) is a Type 4 pili (T4P) that has been described for several Gram-negative pathogenic bacteria and associated to several pathogenic processes. HCP is involved in adherence to bovine epithelium, biofilm formation and induction of proinflammatory cytokines and it has been tested as a vaccine component in goats (Zhang et al., 2014). Another virulence factor shared by O157 and non-O157 strains (Cadona et al., 2013) is the autotransporter EhaA. This protein has been implicated in bacterial attachment to extracellular matrix proteins, such as laminin and collagen, (Wells et al., 2008) and abiotic surfaces, and in promoting adhesion to primary epithelial cells of the bovine terminal rectum (Easton et al., 2011). Besides adhesins shared between STEC O157 and non-O157, several other virulence factors have been described only in LEE (−) strains. For example, Sab, the 146-kDa STEC autotransporter contributing to biofilm formation confers adherence to human epithelial cells and mediates also biofilm formation (Herold et al., 2009). However, this autotransporter adhesin has a low prevalence among isolates (Buvens and Pierard, 2012; Monaghan et al., 2011). The main characteristics of the above-mentioned adhesins were reviewed by Farfan and Torres (2011). In addition, Iha, an outer membrane protein adhesin related to the iron-regulated gene A (IrgA) of Vibrio cholerae, has been reported in non-STEC uropathogenic E. coli (Tarr et al., 2000) as well as in over 70% of the LEE-negative STEC strains associated with human clinical cases examined in studies in Germany (Hauser et al., 2013) and Argentina (Galli et al., 2010). Iha is present in many enterobacteria and it is associated with both adhesion and iron uptake (Colello et al., 2016). Furthermore, the STEC autoagglutinating adhesin (Saa), is expressed by LEE-negative STEC (Caprioli et al., 2005) and homologues of saa were found in several unrelated LEE-negative STEC serotypes associated with human disease (Paton et al., 2001).

THZ1 Limitations of this study that should

Limitations of this study that should be accounted for when extrapolating the findings to other situations include arena construction, THZ1 type, and type and shape of rubber pieces. Usage patterns, arena surface age, type and location could have also affected the results. There may be a manufacture effect, especially for the WSF arenas, as it has previously been shown that the wax content significantly affects surface properties (Orlande et al., 2012). Different sand types and sand particle sizes used by the different manufacturers for both surface types would also affect the stability of the surface (Barrey et al., 1991; Terzaghi et al., 1996) and its response to maintenance. Environmental factors, including temperature (Peterson et al., 2010) and moisture content (Ratzlaff et al., 1997), have been shown to alter the properties of racing surfaces. Adverse weather experienced during testing may have altered the surface properties of the surface of the outdoor arenas within this study.

Conclusions

Conflict of interest statement

Acknowledgements
The authors are grateful to the proprietors of the venues for use of their arenas and for funding from World Horse Welfare, the Swedish-Norwegian Foundation for Equine Research and UK Sport Lottery funding for the British Equestrian Federation World Class Programme.

Introduction
Correct saddle fit is important for optimal function of the equine back. A saddle that does not fit the horse may impair the structural development of the back and cause pain and muscle atrophy (Von Peinen et al., 2010). The shape of the horse\’s back influences the ease with which a saddle can be fitted correctly and thereby the distribution of the rider\’s weight. Horses working correctly with a well fitted saddle are more likely to exhibit a transient, exercise-induced increase in back width than horses not working correctly with an ill-fitting saddle (Greve and Dyson, 2014b).
Longitudinal studies have shown increases in muscle fibre cross-sectional area in horses in response to exercise (Rivero et al., 1992; Miyata et al., 1999). Repetitive immediate post-exercise changes in equine back dimensions may result in longer term changes (Greve and Dyson, 2014b). There are a number of other factors that may influence back dimension changes over time, including skeletal maturation, nutrition, alteration in bodyweight, season, conformation, duration and type/intensity of exercise, base line muscle development, core strength, lameness, back pain and the fit of tack. A better understanding of back dimension changes over time is important for determining how often saddle fit should be checked.

Materials and methods
A longitudinal study was performed using sports horses in regular work, presumed by the rider to be sound. The study was approved by the Ethical Review Committee of the Animal Health Trust (approval number AHT 12.14; date of approval 20 August 2012) and there was informed owner consent. The study population was a convenience sample of 506 horses (Greve and Dyson, 2014c), selected based on proximity to the authors\’ institute. Within the study population, stratified random sampling was used select 104 horses representing four work disciplines (dressage, eventing, showjumping and general purpose, the latter including horses used for unaffiliated competitions) and four age groups (3–5, 6–8, 9–12 and ≥ 13 years). Data were collected from October 2012 to December 2013. Each horse was assessed every 2 months over 1 year. Horses that were abroad competing could not be included in certain examinations. Horses lost to follow-up were attributed to horses moving yard, being sold or humanely destroyed because of lameness or other health problems.

Discussion
The seasonal changes are likely to have been influenced by diet (Cuddeford, 2013); the majority of the horses were turned out daily, with unlimited access to pasture, in addition to their usual source of nutrition. The carbohydrate contents of grasses and legumes fluctuate seasonally (Burns and Chamblee, 2000; Dubbs et al., 2003), with the highest content in late spring, lowest in the summer and winter, and intermediate in autumn (Vervuert et al., 2005). We observed peak changes in back dimensions in late spring and negative changes in January, probably reflecting the seasonal fluctuations in nutritional value of the pasture. In the cranial third of the saddle region (T8), the seasonal fluctuations were highly influenced by whether or not horses had undergone professionally assisted saddle-fitting. If the saddle did not fit correctly, these changes either did not occur or were negative.

GSK2606414 This three in one formulation CT which was marketed

This three-in-one formulation (CT 1341), which was marketed for both human (Althesin, GlaxoSmithKline) and veterinary (Saffan, GlaxoSmithKline) administration, caused severe side effects in numerous species. In cats the predominant adverse effects were hyperaemia and oedema of the pinnae and forepaws, urticaria and skin erythema (Dodman, 1980). CT 1341 caused an unacceptably high incidence of anaphylactoid reactions in dogs and humans, which subsequently saw Althesin withdrawn from human clinical practice in 1984 (Watt, 1975; Abraham and Davis, 2005). These adverse effects were mainly attributed to the Cremophor EL vehicle and, while Saffan continued to be available for veterinary use until 2002, it was contraindicated for use in dogs.
In 1999, a lyophilised powder of alfaxalone and cyclodextrin requiring reconstitution (Alfaxan-CD) was released; however, this product was only registered for use in cats. In 2001 a clear colourless, surfactant-free, aqueous formulation of 1% W/V alfaxalone dissolved with 2-hydroxpropyl-β-cyclodextrin (HPCD) was released for veterinary use in Australia (Alfaxan-CD RTU, Jurox) (Brewster et al., 1989; Estes et al., 1990); this new formulation has not demonstrated the side-effects observed with the previous (CT 1341) preparation (APVMA, 2010).
Cyclodextrins are ring-shaped chains of sugar molecules arranged so that their GSK2606414 domains face outwards and their lipophilic domains face inwards. They are soluble in water and provide, within their hydrophobic core, space for GSK2606414 interaction with hydrophobic molecules, such as steroids. The 1:1 molar HPCD:alfaxalone aggregate therefore behaves as one molecule to form an isotropic solution in water. This aggregate must dissociate in vivo, allowing the alfaxalone to obtain pseudo-equilibrium between its free (unbound) concentration and those molecules that are bound to plasma proteins and cell membranes (Brewster et al., 1989). The use of cyclodextrins in pharmaceutical formulations has been reviewed by Davis and Brewster (2004).
Although the newest formulation of alfaxalone (alfaxalone-HPCD) has been made available in many countries, including Australia, New Zealand, South Africa, Thailand, Canada and numerous European countries, the accumulating evidence for its use in cats has not been thoroughly reviewed. In September 2012, alfaxalone-HPCD was approved by the USA Food and Drug Administration (FDA) for induction and maintenance of anaesthesia in dogs and cats in the United States, although its market release was delayed by the Drug Enforcement Administration\’s (DEA) process for scheduling. Alfaxalone-HPCD provides an alternative in the face of anaesthesia drug shortages (i.e. propofol, thiopental).

Mechanism of anaesthetic effect
The primary mechanism of anaesthetic action of alfaxalone is attributed to positive allosteric modulation of the GABAA receptor, a ligand-gated chloride ion (Cl−) channel receptor for the neurotransmitter GABA, which universally inhibits neuronal excitability (Harrison and Simmonds, 1984; Albertson, 1992). Alfaxalone directly binds to GABAA receptors, potentiating the effects of endogenous GABA, causing movement of Cl− into the cell, hyperpolarisation of the neuron and inhibition of action potential propagation (Lambert et al., 2003). Investigations have also revealed a dual mechanism of action of alfaxalone. At low concentration, alfaxalone allosterically modulates the amplitude of GABA-induced ion currents, whereas, at higher concentrations, alfaxalone exerts an agonist effect, similar to barbiturates (Cottrell et al., 1987; Paul and Purdy, 1992; Lambert et al., 1995). The GABAA receptor is a pentameric transmembrane ion channel at which pharmacological properties of interacting drugs are determined by both the receptor subunit composition and by drug subunit selectivity. Within the central nervous system (CNS), neurones express numerous GABAA receptor subunit isoforms (e.g. α1–α6, β1–β3, γ1–γ3, δ, ε, θ, π, ρ1–ρ3) which determine the receptor\’s agonist affinity, chance of opening, conductance and other pharmacological properties (Lambert et al., 2003; Olsen and Sieghart, 2008). The variability in pharmacological properties of drugs that act at the GABAA receptor is due to variation in drug specificity for a particular subunit. The receptor subunit specificity for binding of alfaxalone has been evaluated in human recombinant GABAA receptors, and this work demonstrated that alfaxalone acts best as a positive allosteric modulator on the α1β1γ2L receptor isoform (Maitra and Reynolds, 1998).