Statistical analyses revealed no significant differences

Statistical analyses revealed no significant differences of seropositivity to T. gondii for categories within gender, breed, age group, hair length, housing, ingestion of raw or undercooked meat and/or viscera, contact with other animals and presence of clinical signs compatible with toxoplasmosis and/or leishmaniosis (Table 1). By univariable logistic regression, the odds of a cat being seropositive to T. gondii increased by an average factor of 1.58 (95% CI: 1.17–2.15; p=0.003) for each 1-year increase in age.

Discussion
Information from Angola on T. gondii and Leishmania spp. infections in animals and humans is scarce. The present sero-survey represents the first epidemiological study of T. gondii and Leishmania spp., two important zoonotic protozoan parasites, carried out in cats from Luanda and also from Angola. Other international studies corroborate the results obtained for T. gondii infection. Also by using the MAT, low seroprevalence figures of 9.3% (14/150) and 10.1% (35/348) were also reported in domestic cats from Durango, Mexico (Dubey et al., 2009) and from Bangkok, Thailand (Sukhumavasi et al., 2012), respectively. This low seroprevalence was attributed to low T. gondii positivity in the local small animal populations. We cannot exclude that this can also be the case of the present study. However, further studies are necessary in other host species from Luanda.
Using an enzyme-linked immunosorbent assay (ELISA), Lobetti and Lappin (2012) described a seroprevalence of 17.6% in 102 cats from South Africa. We had previously found a 35.8% seroprevalence in domestic cats from Portugal using the same serological test (Lopes et al., 2008). More recently, Cyclosporin A also by using the MAT, considerably higher figures of 85.4% (Tiao et al., 2013) and 91.7% (Dubey et al., 2013) were reported in cats from Ethiopia, and of 95.5% in Cairo, Egypt (Al-Kappany et al., 2011). Differences in the feline T. gondii seroprevalence levels among countries may be due to ecological and geographical factors, as well as to feeding and lifestyle of the studied animals, since most cats from Ethiopia and Egypt roamed freely (Dubey et al., 2013). According to Dubey (2010), factors affecting the prevalence of T. gondii in cats are not fully understood and need further investigation.
By using MAT and a cut-off titre of 20, Cyclosporin A to T. gondii were found in 15.5% out of 103 dogs from Luanda (Lopes et al., 2014), representing a statistically significant difference to the present results in cats (3.9%; p=0.005). On the other hand, seropositivity of Leishmania spp. in cats (0.0%) is in line with that detected in the same 103 dogs (1.9%; Vilhena et al., 2014), since no significant difference could be found (p=0.157).
Only three reports on human T. gondii infection are available from Angola (Martins and Abranches, 1976; López et al., 1992; Lobo et al., 2016), with no data available from animal species other than dogs and now cats (this study). Seroprevalence varied from 71.5% in young people aged 13–16 years old (López et al., 1992) to 27.3% in pregnant women from Luanda (Lobo et al., 2016), complementarily suggesting that 72.7% of women in childbearing age are susceptible to primo-infection by the parasite. The climate of different geographical locations affects the transmission dynamics of T. gondii in the environment, as oocysts cannot become sporulated, survive and remain infectious without favorable conditions (Dubey, 2010).
No statistically significant different seroprevalence values could be demonstrated for T. gondii among the categories within gender, breed, age group, hair length, housing, access to raw and undercooked meat and/or viscera, contact with other animals, and the presence of clinical signs suggesting clinical toxoplasmosis or leishmaniosis (Table 1). Although there were only a small number of seropositive animals, these observations suggest that those categories would not represent risk factors for T. gondii and that all cats are equally exposed to infection. Similar findings were obtained by Bresciani et al. (2007), Coelho et al. (2011) and Cardia et al. (2013), in cats from Brazil. Nevertheless, seroprevalence of infection in domestic cats has been found to vary with their lifestyle (wild, stray or domestic), age, serological test used and geographical location (Gauss et al., 2003). Cats of all ages, gender and breeds are susceptible to infection (Dubey et al., 1977; Dorny et al., 2002). In the present study, the odds of a cat being seropositive increased by an average factor of 1.58 for each 1-year increase in age. It can be assumed that increasingly older cats have had more chances to eat tissues of infected animals or to have contact to the surrounding environment potentially contaminated with T. gondii oocysts (Lopes et al., 2014).

Eighteen isolates of the novel Campylobacter were

Eighteen isolates of the novel Campylobacter were tested using the duplex real time PCR and gave negative results for C. jejuni and Campylobacter coli.
Sequencing of 16S rRNA revealed that 19 novel Campylobacter isolates cultured from four separate farms shared identical sequence over a common 1290bp fragment. Phylogenetic analysis (Fig. 3), and comparison of sequences with equivalent regions of the type strains of all currently described Campylobacter species, revealed that the isolates appeared to represent a hitherto unknown sublineage within the group.
Electron micrographs revealed the five isolates of the novel Campylobacter examined showed typical Campylobacter morphology: curved rods approximately 0.5μm in diameter and 2–3μm in length. As is typical for other Campylobacter species, the novel Campylobacter had single, unipolar or bipolar (Fig. 4), unsheathed flagella, although two isolates appeared aflagellate.
The experimental SPF Peptone were negative for Campylobacter sp. at the start of the experiment. Half of the four-weeks-old chickens were challenged intraperitoneally with 5×108 cfu the NCTC novel Campylobacter isolate and colonisation and pathology were determined at 2, 4, 7 and 14 days post-infection (dpi). None of the experimental birds died or showed signs of illness during the course of the experiment and no gross lesions were visible on post mortem examination. At 2 dpi, only 1 of 4 challenged birds was detectably colonised by the novel Campylobacter (Fig. 5). No Campylobacters were recovered from any tissue at 4 dpi, but by 7 dpi and 14 dpi, novel Campylobacter were recovered from the livers and spleens of 5/8 of the challenged birds. Caecal colonisation with the novel Campylobacter sp. was also detected in two birds at 7 dpi and one bird at 14 dpi. No novel Campylobacter sp. were recovered from the control birds.
The experimentally infected birds showed mild to severe multifocal necrotising hepatitis with the presence of fibrinogranulocytic foci and infiltration of macrophages and heterophils (Fig. 6). Microscopic examination and semi-quantitative scoring of liver lesions showed there was more evidence of hepatitis in challenged birds (G2) than in the control group of birds (G1) (Fig. 7 upper panel), with two infected birds showing a severe multifocal necrotising hepatitis with fibrinogranulocytic infiltration. The difference in liver lesion scores between challenged and control birds was found to be statistically significant at 2 dpi and 14 dpi (p=0.0286 for each time point), and approaching significance at 7 dpi (p=0.0571). In addition, there was considerable evidence of lymphoid depletion in the spleens of challenged birds, with none seen in the controls (Fig. 7 lower panel). Representative bacteria from livers, spleens and caeca were analysed by PFGE, and all were found to identical to the challenge strain (results not shown).

Discussion
An infectious and bacterial aetiology has been suspected for SLD/SLS in view of the epidemiology, reports of reductions in mortality following antimicrobial treatment (Grimes and Reece, 2011) and the gross and microscopic pathology. The experimental reproduction of an indistinguishable microscopic pathology using the novel Campylobacter species and its successful reisolation from liver and other tissues of the experimentally infected domestic fowl suggest that it is the causal organism for SLD/SLS.
Past failures to isolate this novel Campylobacter can be explained by its fastidious and slow-growing nature which mean it is easily missed on initial isolation if faster growing bacterial contaminants are present. It was fortuitous that in 2009 we isolated the novel Campylobacter in pure growth from two birds submitted live using the culture method on liver described above and this prompted us to adopt the method for subsequent investigations. The birds sampled were either very freshly dead or were culled immediately prior to post mortem examination the novel Campylobacter isolates were recovered from less than half of the farms investigated and in half or less of the tissues cultured. In many cases the failure to culture the novel Campylobacter organism was attributed to overgrowth by bacterial contaminants as there were a much higher proportion of contaminated cultures where isolation failed. It is important to note that on initial isolation the novel Campylobacter did not grow in Exeter broth, on Skirrows agar or on modified charcoal cefoperazone desoxycholate agar (mCCDA Oxoid) which are commonly used to isolate Campylobacter species. This may explain why this organism was not isolated in recent reported attempts which used mCCDA (Jennings et al., 2011; Muller et al., 2011).

Because S microti was extremely rarely encountered so

Because S. microti was extremely rarely encountered so far, three independent diagnostic approaches were employed in the present study in order to unequivocally confirm the identity of the bovine isolates. These approaches included: phenotypic identification using commercial identification kits as well as some conventional tests, genotypic methods (nucleotide sequence analysis of the 16S rRNA gene and four housekeeping genes that have been proved to be reliable for recognition of staphylococci) and whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry. The methods used varied with regard to efficacy and reliability.
Identification of our S. microti isolates using phenotypic methods turned out to be impossible. The digit codes obtained on both API STAPH and ID 32 STAPH were not included in the reference databases. In addition, some biochemical traits of the bovine isolates differed from those of the reference strains CCM 4903T and CCM 4904. Our strains failed to hydrolyze esculin and to produce lactacystin from mannose. Negative results for β-galactosidase and arginine arylamidase were contradictory to those presented in the original description of the species (Nováková et al., 2010) but consistent with observations made by Riesen and Perreten (2010). The phenotypic identification of S. microti could also be affected by variable results of the Voges–Proskauer test. Our isolates gave a positive reaction on the API STAPH strip and negative one on ID 32 STAPH and in the tube method. Given the reported weak reaction of S. microti for acetoin (Nováková et al., 2010), this phenomenon may have been associated with a different sensitivity of the identification systems used in the present study.
In contrast to phenotypic tests, genotype-based methods proved to be much better in the identification of the microorganism. Initially, our isolates were assigned to S. muscae based on sequence of the tuf gene and to S. microti while comparing nucleotide sequences of the 16S rRNA gene. To address this discrepancy, nucleotide sequence analyzes of three additional genes (sodA, rpoB and dnaJ) were performed, all of them indicating S. microti as the best match in the BLAST program. The failure of the tuf gene-based identification resulted from the fact that the gene sequence of S. microti has never been deposited in the GenBank database. In our isolates, the sequence similarity of the tuf gene to that of S. muscae was relatively high, however, and amounted to 98.8%. Because in less conserved genes (such as the tuf gene) homology values above 97% are often considered reliable (Heikens et al., 2005; Zadoks and Watts, 2009), this might lead to a misidentification of our strains if the tuf gene-based identification was the only method used. The use of sequence-based methods for the identification of CNS may also have some other drawbacks, such as limited discriminating capacity (as for the 16S rRNA gene) or difficulties to find conserved regions for designing primers (reported for the sodA gene) (Heikens et al., 2005). Therefore, microbiologists often need to analyze the combined sequence data for a definitive species identification.
For these reasons, as well as owing to its convenience, ease and speed of sample analysis, MALDI-TOF MS has become a promising tool for bacterial identification and classification (Welker and Moore, 2011). Although this method has been successfully used for the identification of staphylococci, its performance may be poor if mass spectra of particular species are not included into the MALDI Biotyper database (Dubois et al., 2010). In addition, microorganisms may display some degree of intraspecific variation, including biochemical properties and spectral patterns, so that various strains of a given species should be represented in the MALDI software (Welker and Moore, 2011). This may be difficult in the case of newly described or rarely encountered species, such as S. microti, of which only a few isolates are known.

br Acknowledgements This work was supported by Key Technologies Research

Acknowledgements
This work was supported by Key Technologies Research and Development Program of China Grant (2013BAD12B01), National Science Fund for Distinguished Young Scholars (31025029), and Chinese Universities Scientific Fund (2015DY004)

Introduction
Over the last decade, LA-MRSA have emerged in the agricultural setting and are considered a public health concern worldwide (Price et al., 2012). Two lineages of particular note have been identified: CC398 being nonreceptor tyrosine kinase in mainland Europe and CC9 in Asia (EFSA, 2009; Dhup et al., 2015). These clones have been reported in diverse livestock hosts such as pigs, veal calves and poultry where they are mainly associated with asymptomatic colonisation, although disease has been reported sporadically (Verkade and Kluytmans, 2014). Epidemiological studies have shown that LA-MRSA CC398 does not readily transmit between humans, but reports of infections occurring in people having direct contact with livestock (including farm workers, abattoir workers and veterinarians) are increasing (Verkade and Kluytmans, 2014).
Although LA-MRSA was not found in pig holdings in the UK in a European survey conducted in 2008 (EFSA, 2009), LA-MRSA CC398 has subsequently been reported sporadically in horses (Loeffler et al., 2009), bulk tank milk (Paterson et al., 2012), pigs (Hartley et al., 2014; Hall et al., 2015), turkeys (GOV.UK, 2013) and retail pork (Hadjirin et al., 2015); in addition, CC9 has been recovered from retail chicken meat (Dhup et al., 2015). Herein we report the first isolation of a novel LA-MRSA CC30 strain encoding a recognised marker of virulence (lukM) from fattening pigs in Northern Ireland identified as a result of passive surveillance.

Materials and methods

Results
Tissues from all three pigs tested negative for PCV2 by immunofluorescence and all other viruses screened for. However, bacterial isolates from a total of nine different tissues from the three pigs were confirmed as S. aureus; eight of which identified as mecA positive MRSA (Table 1).
All nine S. aureus isolates were resistant to penicillin and tetracycline (MICs ≥0.5 and >8μg/ml, respectively). Moreover, the eight mecA positive isolates showed low level resistance to cefoxitin (MIC 8μg/ml) and the remaining isolate was susceptible (MIC 2μg/mL) by Etest.
Genetic analysis of these isolates showed they identified as ST30, spa type t1749 and encoded the tetracycline resistance marker tet(K); all isolates except that recovered from the lung of pig B were mecA- and czrC-positive and harboured SCCmecVt (Table 1). In addition, all nine isolates encoded the lukM and lukF-P83 genes, a marker of virulence restricted to animal lineages (Schlotter et al., 2012; Simpson et al., 2013); tst and lukSF-PV were not detected but the enterotoxin gene cluster was present. Further, they lacked the genes associated with the immune evasion cluster (IEC) associated with ΦSa3, a recognised marker of human adaptation (Price et al., 2012). SNP-based analysis clustered the pig isolates into the same clade, distant by more than 627 SNPs from CC30CA-MRSA (lukS/lukF-PV positive) and MSSA from unrelated human cases included for comparative purposes (Fig. 1). All eight MRSA from the pig samples were highly related (0–7 SNPs, mean 3.5); the MSSA differed from these by 15-–21 SNPs.

Discussion
All nine isolates of S. aureus recovered from three pigs on the same farm in NI belonged to the same clone; the absence of the IEC genes and presence of lukM suggest they originate from an animal-associated lineage of S. aureus. The lukM gene encodes a gamma haemolysin which is cytotoxic for neutrophils. It has been associated with multiple S. aureus lineages including CCs 49, 133, 151 and 479 from animal species such as ruminants, pigs and squirrels and has been implicated in the pathogenesis of mastitis in cattle and exudative dermatitis in squirrels (Schlotter et al., 2012; Simpson et al., 2013). Although S. aureus was recovered from a variety of tissues in variable numbers from all three pigs suggesting systemic spread, it is not known whether a lukM-mediated mechanism may have contributed to the reduced growth rate in the affected pigs.

br Conclusion br Acknowledgements br

Conclusion

Acknowledgements

Introduction
The U.S. swine industry is the third largest producer of pork in the world, and respiratory disease in pigs is the most important health concern for swine producers today (U. S. Department of Agriculture, 2008). According to the recent survey conducted by the National Animal Health Monitoring System (NAHMS), respiratory disease is on the rise and is the greatest cause of mortality in nursery and grower/finisher swine, making the development of efficacious vaccines and therapeutic interventions that can protect against these infections a top research priority (U. S. Department of Agriculture, 2008). Bordetella bronchiseptica colonization is pervasive in swine herds and is an important contributor to respiratory disease in pigs. It is a primary cause of bronchopneumonia in young pigs, while it additionally contributes to secondary pneumonia in older pigs. It is also the primary etiologic agent of nonprogressive atrophic rhinitis, a mild to moderately severe reversible condition that promotes colonization by toxigenic strains of Pasteurella multocida promethazine hcl and can lead to severe progressive atrophic rhinitis (Brockmeier et al., 2012). In swine exhibiting pneumonia, B. bronchiseptica is often isolated in combination with other pathogens (Brockmeier et al., 2012). Previous studies have demonstrated that co-infection with B. bronchiseptica increases colonization and exacerbates the severity of disease caused by both viral and bacterial pathogens, including swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), Haemophilus parasuis, P. multocida and Streptococcus suis(Brockmeier et al., 2012).
Various typing methods have been applied to B. bronchiseptica in an effort to elucidate the population structure of the bacterium. One of the approaches found to be highly discriminatory is multilocus sequence typing (MLST) based on comparison of DNA sequences from seven housekeeping promethazine hcl (Diavatopoulos et al., 2005). The virulent B. bronchiseptica strain KM22 was originally isolated in Hungary in 1993 from a swine herd with atrophic rhinitis. Similar to the majority of B. bronchiseptica strains isolated from pigs exhibiting disease, KM22 is sequence type (ST) 7 in Clonal Complex 1 of an MLST-based Bordetella phylogeny (Diavatopoulos et al., 2005), and it harbors a PvuII ribotype (Register and Magyar, 1999). Additionally, KM22 contains a pertactin repeat region variant (Register, 2001) that is shared with the majority of strains isolated from swine. KM22 has been successfully used by our laboratory to develop a reproducible swine respiratory disease model reflective of clinical B. bronchiseptica infections within swine herds and host-to-host transmission (Brockmeier and Register, 2007; Nicholson et al., 2009, 2014a, 2012).
The complete genome sequence of the laboratory reference B. bronchiseptica strain RB50 was first made available in 2003 by Parkhill et al. (Parkhill et al., 2003). Recently Park et al. published the genome sequence of five B. bronchiseptica strains isolated from both human and non-human hosts (strains 253, 1289, MO149, Bbr77, and D445) (Park et al., 2012) and our laboratory reported the genome sequence of KM22 (Nicholson et al., 2014b). The goal of this study was to compare the genome sequence of KM22 to the complete genome sequence of the laboratory reference strain RB50 to identify genetic features unique to KM22. To gain a broader perspective of the genetic relationship of KM22 among other B. bronchiseptica strains, genetic sequences from KM22 were subsequently compared to five other B. bronchiseptica strains.

Materials and methods

Results and discussion
B. bronchiseptica isolate KM22 was originally isolated from a pig, and this isolate, along with KM22-derived mutants, have been used in many infection studies in its natural swine host to characterize the associated disease phenotypes. The phenotypes of some KM22-derived mutants used in these studies did not always concur with reciprocal studies using the well characterized rabbit RB50 isolate, along with RB50-derived mutants, in rodent infection models (Nicholson et al., 2009; Nicholson et al., 2012). To determine genomic differences that could account for such virulence-associated phenotypic differences, we compared the genomes from KM22 and RB50, as well genomes from five additional B. bronchiseptica strains (1289 isolated from a monkey, 253 from a dog, and MO149, Bbr77, and D445 from humans).

Taken experimental and field data together and in agreement

Taken experimental and field data together, and in agreement with the results reported here, it can be concluded that the APP response during PPRSV infection is variable depending on the specific strain. However, Hp is the most generally increased APP by this viral infection, suggesting that it may play a role in the pathogenesis of the disease and the modulation of the immune response, as suggested by other authors (Gomez-Laguna et al., 2010). In this sense, Hp is able to bind monocytes (El Ghmati et al., 1996) and B-cells (Langlois et al., 1997), and has immunomodulatory effects (Arredouani et al., 2003; Huntoon et al., 2008). Furthermore, it interacts with CD163, one of the receptors for PRRSV, increasing BLZ945 of the anti-inflammatory cytokine IL-10 (Van Gorp et al., 2008).

Acknowledgements
We would like to thank Dr. Paolo Martelli, Dr. José M. Castro and Dr. Michael Roof for providing EU-17 and EU-18, EU-10 and JA-142 PRRSV isolates respectively, and Dr. Tomasz Stadejek for sending the clinical samples from which EU-12 isolate used in this study was obtained. Besides, we would like to thank Belén Prieto Suárez for her technical assistance. This study was supported by grants AGL2004-06850-C02-01 and CSD-2006-00007 from the Spanish Government (to C.P.) and AGL2006-02364/GAN (to A.B.).

Introduction
Nocardia spp. are Gram-positive, nonmotile, aerobic, facultatively intracellular actinomycetes which are common in organic material, soil, water and plants and induce pyogranulomatous inflammatory diseases in healthy and immunocompromised humans and animals (Beaman and Beaman, 1994). In animals, the majority of reported cases is caused by species of the Nocardia (N.) asteroides complex. Pathogenetically, pulmonary infections occur after inhalation and cutaneous abscesses or mycetoma formations are sequels of wound infections, respectively (Kirpensteijn and Fingland, 1992). Other common pathogenic species include N. brasiliensis, N. pseudobrasiliensis, N. nova and N. otitidiscaviarum (Ribeiro et al., 2008). Since their first description in 2001 (Gürtler et al., 2001), N. veterana isolates have been associated with various infections in humans (Dua and Clayton, 2014) and bovine mastitis in dairy cattle from Brazil (Condas et al., 2013), whereas links to nocardiosis in companion BLZ945 animals are still missing so far.
In the present study, N. veterana was isolated from a three-month-old dog, which suffered from systemic nocardiosis along with a canine distemper virus (CDV) infection. The results of this study underline the risk of systemic infections by N. veterana in dogs, which frequently live in close contact to humans and may represent a risk factor for immunocompromised humans as well (Edwards, 2007).

Materials and methods

Results

Discussion
Nocardiosis is considered as an emerging disease among humans (Pottumarthy et al., 2003) and domestic animals throughout the world (Ribeiro et al., 2008). Until now, approximately 90 species have been described [NCBI taxonomy for Nocardia, (Poisnel et al., 2015)]. Since molecular methods, relying on 16S rDNA sequencing, were used for identification, some previously unknown Nocardia species, such as N. veterana, have been discovered (Gürtler et al., 2001). In humans, the majority of patients affected by nocardiosis suffers from a primary immunocompromising condition with deficient cell-mediated immunity and develops nocardiosis as a secondary disease (Pottumarthy et al., 2003; Valdezate et al., 2015). In immunocompetent hosts, nocardiosis has been described only rarely (Pottumarthy et al., 2003). Comparable to the situation in humans, nocardiosis in dogs occurs mainly in immunosuppressed individuals, with most of them suffering from CDV infection (Ribeiro et al., 2008). CDV is a morbilliviral disease and causes bronchointerstitial pneumonia, demyelination of the central nervous system, ocular disease, pustular and/or hyperkeratotic cutaneous lesions (“distemper exanthema”/“hard pad disease”), abortions and profound immunosuppression, which boosts susceptibility for opportunistic infections (Beineke et al., 2009).

br Material and methods br Results Regarding the

Material and methods

Results
Regarding the study of the five concatenated sequences, phylogenetic analysis using the neighbour-joining method is displayed in Fig. 1. This phylogenetic tree shows the presence of three main groups, except for LC53, LC33, LC45 and LC91 which are visibly correlated with the geographical origin of the studied strains. However, there is not apparent correlation between the phylogenetic origin and year of isolate of the strains. Additionally, in this study, multiple Mmc strains coming from the same farms were analysed, revealing that even when these isolates had been obtained during the same year, phylogenetic differences sometimes appeared between them.
Concerning the individual study of each housekeeping gene, the phylogenetic trees based on their individual gene sequences are presented in the Supplementary Figs. 1 (fusA), 2 (glpQ), 3 (gyrB), 4 (lepA) and 5 (rpoB). The Simpson’s index for the individual purchase oxyntomodulin were in range from 0.819 (fusA) to 0.945 (glpQ) while the variability for the concatenated genes study was 0.953 (Table 2). None of the individual gene trees was able to correlate the phylogenetic origin of the isolates with their geographical area as the concatenated tree did. Nevertheless, the phylogenetic tree obtained for lepA was able to show the common origin of all the strains coming from the Canary Islands. In this sense, the phylogenetic trees obtained by the individual study of fusA, qlpQ, gyrB and rpoB were not capable to reproduce any of the main phylogenetic groups reported by the study of the concatenated genes.
As shown by the pairwise distance analysis (Fig. 2), the maximal divergence between the assessed isolates was 1.7%, with the reference strain (PG3) being the most divergent strain within the present study. In addition, the mean intragroup distance for our 39 field isolates and PG3 was 0.9%. Analysing these data within the three main investigated geographical areas, the isolates from the Canary Islands were the most divergent, presenting a mean intragroup distance of 0.8%. The Mmc strains from Murcia and Andalusia showed a mean intragroup distance of 0.77% and 0.76%, respectively. Moreover, strains coming from Andalusia and Murcia were less divergent (0.9%) between each other, than when both groups were compared with field isolates coming from the Canary Islands. Still, when the strains were studied according to their year of isolation, no correlation was found between this factor and the resulting pairwise distances, even when these isolates were as well sorted by geographical origin.

Discussion
Epidemiological investigations, which are essential for disease surveillance and control, need suitable typing tools to carry out phylogenetic studies with enough resolution. In this sense, sequenced-based typing methods are technically simple, robust and portable (van Belkum et al., 2007). Thus, previous studies have employed MLST schemes to monitor and assess evolutionary differences between different strains of other ruminant mycoplasmas, such as M. agalactiae or M. bovis (Manso-Silvan et al., 2012; McAuliffe et al., 2011; Rosales et al., 2015). To our knowledge, this is the first MLST phylogenetic analysis performed on Mmc since a scheme was developed for the M. mycoides cluster (Manso-Silvan et al., 2007).
Simpson\’s indices of diversity for the five assessed loci ranged from 0.819 to 0.945, whereas the index obtained for the scheme was 0.953. This result indicates that the MLST scheme applied in the present study (Manso-Silvan et al., 2007) provides adequate discriminatory power as, although there are no previous references of this index concerning Mmc, our results are comparable to the genetic diversity values yielded after studying other mycoplasma species such as Mycoplasma arginini and Ma (Olaogun et al., 2015; McAuliffe et al., 2011)
Contrary to the outcomes of those studies (Manso-Silvan et al., 2012; McAuliffe et al., 2011; Rosales et al., 2015), our results show a correlation between the geographical origin of the different isolates assessed and their phylogenetic distance. Consequently, our phylogenetic tree (Fig. 1) shows a common origin for almost all the assessed strains, determined by the geographical area where they were isolated. Notwithstanding, this geographical distribution might be an effect of the major commercial relationships (livestock movements) between farms within the same areas. In fact, all the studied farms from Murcia reared the same caprine breed (Murciano-Granadina) and, except for LC72, all the strains coming from Andalusia were isolated from farms which belong to the same association of Malagueña goat breed. Moreover, taking into consideration that LC45 came from Catalonia but from a Murciano-Granadina herd, and the fact that LC72 was isolated from the only studied farm in Andalusia rearing Murciano-Granadina goats, it is understandable why both strains (LC72 and LC45) are genetically closely related to LC53 and LC33, which are from Murcia. Thus, our data suggest that MLST studies should be performed in connection with the analysis of possible commercial relationships between the studied herds.

br Materials and methods br Results br

Materials and methods

Results

Discussion
Currently, there are series of live attenuated vaccines (LaSota, Hitchner B1 and AV324/96) for controlling Newcastle disease. Naturally occurred and attenuated live vaccine LaSota has been used for more than half century in China or other areas of the world. The safety and efficacy of LaSota strain have been widely accepted (Ahmed et al., 2003; Huang et al., 2001; Peeters et al., 1999). LaSota strain is the parent strain of clone30, the virus does not undergo detectable genetic recombination which would make this vaccine much more stable and safe (Duan et al., 2015; Nagai, 1999; Schirrmacher and Fournier, 2009). In the past, due to the lack of effective and specific reagents, the vaccine adjuvants for livestock industries have been hampered (Guo et al., 2013). Recently, with the development of animal models and the release of the chicken genome many candidate reagents are being provided, especially the capability of cytokines to enhance immune response across livestock including poultry (Asif et al., 2004; Johnson et al., 2000).
The adjuvant, as an important measure to enhance the efficacy of vaccination (Petrovsky and Aguilar, 2004; Salgaller and Lodge, 1998), is able to improve innate immune responses to a given antigen (Gupta et al., 1993; Sivakumar et al., 2011). Thus, adjuvant is an important part in vaccines development. Currently, to meet the needs of the development of new vaccines, adjuvants have been transformed from single and traditional forms to new and diversified forms. Molecular adjuvants, which induce humoral and cell-mediated immune responses, have become a research hotpot in vaccine development (Mbow et al., 2010; Sivakumar et al., 2011). The recombinant Rabies viruses expressing cytokines GM-CSF or fliC are effective vaccines for both intramuscular and oral immunizations, these vaccines could recruit/active more DC cells and CHIR-99021 Supplier to enhance the adaptive immune responses (Zhou et al., 2013). In the development of novel DNV vaccine, cytokine chIL18 enhances the protection of NDV vaccine by inducing interferon-gamma production and promoting Th1 immunity. The DNA vaccine with chIL18 could enhance the cell-mediated and humoral immunity (Hung et al., 2010). The effect of chIL4 and chIFN-γ were also studied. The experiment showed that humoral immunity is enhanced by IL4 and CMI by IFN-γ. At the same time, IL4 is preferable than IFN-γ as a molecular adjuvant for NDV DNA vaccine (Sawant et al., 2011). These molecular adjuvants provide a more feasible and effective vaccine strategy in developing a new generation of NDV vaccines. However, there is no report that adjuvant vaccines induce quick immune response in birds with high level of maternal antibodies. Based on above studies, in the present study, we generated the four recombinant NDVs expressing chIL2, chIL15, chGM-CSF or fliC and investigated their immunological characteristics in both SPF chicken and birds with different level of maternal antibodies.
In present study, the CD4+ and CD8+ T cells of the four adjuvant groups were significantly higher than that of the parent virus group during 8day’s experiment, suggesting that the insertion of the foreign adjuvant gene enhances the maturation of T cells, particularly CD4+ T cells, which contribute to stimulate B cell maturation and antibody responses. In response to T-dependent antigens, such as NDV, HSV-1 or other vaccines (Bromberg et al., 1982), helping B cells to produce antibody is the major function of CD4+ T cells (Zhu and Paul, 2010; Zhu et al., 2010). After antigenic stimulation, CD4+ T cells can activate B cells to enter germinal center (GC) and direct B-cell differentiation to plasma cells and memory B cells (Batista and Harwood, 2009; Ha et al., 2014). The cell-mediated stimulation can be detected as early as 2–3days post NDV infection (Kapczynski et al., 2013; Reynolds and Maraqa, 2000), quick and strong CMI responses were also observed in our study. The present study showed that the insertion of the adjuvant genes, especially chGM-CSF or fliC, could potently induce the maturation of the T cell to activate B cell responses, which is consistent with the studies been reported before (Chen et al., 2012; Mizel and Bates, 2010; Sanders et al., 2006; Song et al., 2015). It is the adjuvant genes in the recombinant NDV that contributes to produce antibody quicker than routine vaccine. These enhancement effects were also confirmed by the up-regulation of chIL1, chIL4 and chIL6. As the result, the birds immunized with these adjuvant vaccines could take shorter time than birds immunized with parent vaccines to acquire the antibody at protection level 4log2 (Yosipovich et al., 2015) (Fig. 4). These results suggest that the recombinant NDV adjuvant vaccines can induce quicker and stronger cell and humoral responses, which are helpful for chicken flocks with individuals containing low HI antibodies facing natural NDV challenge (Yosipovich et al., 2015).

br Discussion Most studies conducted in companion

Discussion
Most studies conducted in companion animals focused on E. coli isolated from dogs and cats (Liu et al., 2012; Pomba et al., 2009), and few studies have investigated the prevalence of PMQR in other canine or feline Enterobacteriaceae (Gibson et al., 2010; Shaheen et al., 2013). Moreover, prevalence-type studies are rare; most work is based on selected culture collections (e.g., cephalosporin-resistant strains, ST131 clones, multidrug-resistant strains) to study the mechanisms of resistance.
In the ComPath project, more enterobacterial species were studied. The overall prevalence of PMQR in Enterobacteriaceae amounted to 2.0% (12 out of 604 isolates), assuming that the wild type Enterobacteriaceae (535 isolates not included in this PMQR study) do not harbour PMQR genes. Among the 69 non-wild type isolates studied, the occurrence of PMQR amounted to 17.4% (12 out of 69 isolates). Of the 57 non PMQR-strains, in four strains no QRDR mutations were detected. Contribution of efflux and reduced outer membrane permeability has been reported as an important mechanism of fluoroquinolone resistance in Enterobacteriaceae isolates from not only humans but also animals (Liu et al., 2012; Davin-Regli and Pagès, 2015). It is likely such contribution may explain increased enrofloxacin MICs in those four strains. In the current study, qnr gene was the most prevalent PMQR determinant detected (1.5%; 9 out of 604 isolates), followed by aac(6′)-Ib-cr (0.7%; 4 out of 604 isolates). In rotenone to some other studies (Gibson et al., 2010; Shaheen et al., 2013), qepA genes were neither detected in the current study nor in a Dutch study on companion animals isolates (Dierikx et al., 2012). In addition, in a study conducted between 2008 and 2009 in USA on 54 ESBL-producing E. coli from cats and dogs, aac(6′)-Ib-cr was more prevalent (56%) than qnr (43%) or qepA (9%) (Shaheen et al., 2013). In a collection of multidrug-resistant E. coli strains isolated in Australia between 1999 and 2004 from dogs, 83% of qnr were isolated and 73% of qepA (Gibson et al., 2010). Among cephalosporin-resistant Enterobacteriaceae isolated from dogs in China between 2006 and 2007, 41.7%, 30.6% and 8.3% were positive for aac(6)-Ib-cr, qepA and qnr genes respectively (Ma et al., 2009). Among enrofloxacin-resistant uropathogenic E. coli strains isolated from cats and dogs in China between 2008 and 2010, 28% and 10% were positive for qnrS and aac(6′)-Ib-cr, respectively (Liu et al., 2012).
According to other results reported from human clinical strains, K. pneumoniae isolates studied here have been shown to carry OqxAB as a native household efflux pump without association to the transposon Tn6010 (Bialek-Davenet et al., 2015; Guillard et al., 2015b). The transposon-borne version is associated with boosted expression because of the lack of rarA that normally down regulates oqxR and the presence of several plasmid copies of the plasmid-borne transposon version. Nonetheless, in K. pneumoniae that naturally encode OqxAB-encoding, it has been shown that the OqxAB overexpression increases the antibiotic resistance phenotype, but also the virulence (Bialek-Davenet et al., 2015). Conversely to other studies focusing on PMQR in bacterial strains recovered from animals, we did not find any E. coli isolates carrying OqxAB. Indeed, a high oqxAB prevalence of around 30% has been recently reported in E. coli isolated from companion and domesticated animals in China (Yang et al., 2014; He et al., 2015). OqxAB decreases susceptibility to quinoxalines, such as mequindox and olaquindox (Hansen et al., 2007). The high prevalence of OqxAB in China might be explained by the extensive use of mequindox to treat E. coli infections in food-producing animals for the last 30 years.
The low prevalence of PMQR is in alignment with other European findings (Dierikx et al., 2012). PMQR determinants, however, might be more prevalent in countries outside Europe (e.g., Shaheen et al., 2013). It should also be noted that caution is required for the comparability of different studies; most studies refer to a selection of isolates for resistance mechanism studies, and the selected strain collections do not reflect the prevalence of resistance, in contrast to our study. Indeed, Larsen et al. (2015) have compared the susceptibility of clinical isolates from skin tissues of dogs not treated with antibiotics with isolates from clinical specimens of dogs with unknown treatment history. They concluded that antimicrobial susceptibility data based on routine diagnostic specimens are generally biased in favour of resistance and are not representative for patients without a history of antimicrobial therapy. In addition, the evaluation of the results of various studies is following different guidelines or strains that are epidemiologically related.

br Materials and methods br Results br Discussion

Materials and methods

Results

Discussion
In the present study, the MAR susceptibility profiles of S. Typhimurium clinical isolates from pigs were determined using a new ciprofloxacin breakpoint, which was recently revised to reduce technical difficulties and to increase the reliability of clinical laboratories (Humphries et al., 2012). MAR showed the highest potency against all isolates, including ACSSuT (36.4%) (Table 1). The MAR resistance rate in isolates was similar to that of previous reports in Korea and lower than those in other countries (Lim et al., 2009; Kuang et al., 2015; de Jong et al., 2012). Regarding the relatively small exposure to MAR than is used for other drugs in veterinary use in Korea (QIA, 2014), the quantity is based on the strategy for treatment with MAR to prevent the emergence of resistant S. Typhimurium strains.
Dosing above the MPC has been suggested to minimize the selection of mutants during antibiotic treatment (Drlica and Zhao, 2007). Recently, a one-dose regimen for MAR based on the MPC was reported to result in no emergence of bacterial resistance in clinical susceptible pathogens (Vallé et al., 2012). Furthermore, our laboratory suggested that adjusting the AUC24h/MPC ratio of MAR using an in vitro dynamic model for a 3-d treatment could prevent the selection of resistant S. Typhimurium (Lee et al., 2016). Therefore, to reduce chance for the development of MAR resistance by a single or repeated exposure to FQ, an optimized dosage based on the MPC concept will be required. Overall, MPC-based approaches were used in this study, suggesting a higher efficacy of MAR in preventing the selection of single-step mutants of S. Typhimurium induced by molecular resistance mechanisms, target mutation, and overexpression of multi-drug efflux pumps.

Conclusion
Our findings suggest that a single-exposure to a sub-MPC of MAR was sufficient to reduce susceptibility in Salmonella clinical isolates without leading to high-level FQ resistance. Our results confirmed that gyrA mutations play a critical role in the development of FQ resistance in both resistant isolates and single-step mutants. In addition, the acrAB-tolC-mediated efflux mechanism was found to contribute to decreased susceptibility to MAR in the presence or absence of gyrA mutations. This mechanism was associated with the Exendin-4 manufacturer of global regulators (marA/soxS/ramA) and partially associated with a local regulator (acrR). Therefore, to reduce chance for the development of MAR resistance, an optimized dosage based on the MPC concept and continuous monitoring of S. Typhimurium resistance on farms will be required.

Conflicts of interest

Ethical approval

Introduction
In general, avian influenza A viruses (AIVs) have host-species barriers (Yoon et al., 2014). While the wild birds are the natural host of AIVs (Choi et al., 2017; Jiang et al., 2017; Webster et al., 1992), AIVs can also infect mammals including tigers, leopards, dogs, cats, plateau pikas, rhesus macaques, and humans (Cheng et al., 2014b; Gao et al., 2013; Keawcharoen et al., 2004; Shinya et al., 2012; Songserm et al., 2006; Subbarao et al., 1998; Yu et al., 2014; Zhang et al., 2013a, 2013b). Considering AIVs are reported to be capable of infection with mammals and transmission between mammals (Belser et al., 2013; Gao et al., 2009; Kimble et al., 2011; Sang et al., 2015b; Wu et al., 2010; Zhang et al., 2013c), AIVs may possess pandemic potential.
H3N2 AIVs circulate widely in areas including North America (Corzo et al., 2012; Guo et al., 2015; Pasick et al., 2010), Europe (Campitelli et al., 2002; Jonassen and Handeland, 2007), Asia (Choi et al., 2012; Gerloff et al., 2016; Li et al., 2016; Yang et al., 2015), and Oceania (Peroulis and O\’Riley, 2004). Transmission of H3N2 AIVs to mammals has been reported on previous studies (Lee et al., 2009; Lei et al., 2012; Song et al., 2008; Su et al., 2012). In 1968, 1957 H2N2 pandemic influenza virus was replaced by 1968 H3N2 pandemic influenza virus that possessed an H3 HA gene and a PB1 gene of H3 AIVs origin (Neumann et al., 2009). Additionally, avian-origin H3N2 swine influenza strains were isolated from pigs in Italy in 1985 that appeared to result from reassortment between the genes encoding the HA and NA proteins of the human-like H3N2 swine influenza virus and the internal proteins of the avian-origin H1N1 swine viruses (Castrucci et al., 1993). From then on, avian-origin H3N2 swine influenza viruses continue to circulate in swine in the world (Campitelli et al., 1997). A previous report found that dogs inoculated with an avian-origin H3N2 virus were capable of transmitting the virus to naïve animals in direct-contact models (Song et al., 2009a). Considering reports of mammals’ infection with H3N2 AIVs and a lack of pre-existing immunity against H3N2 AIVs in mammals and humans, H3N2 AIVs may pose a pandemic threat. Therefore, the pathogenesis involved in the mammalian adaptation of H3N2 AIVs should be further studied.