Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine

Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.     

Antibody response to an anti-rabies vaccine in a dog population under field conditions in Bolivia

Rabies remains an important public health issue in Bolivia, South America. Public concern and fears are most focussed on dogs as the source of rabies. The objective of the present study was to assess immunity of an inactivated suckling mouse brain vaccine against canine rabies used for the official vaccination campaigns under field conditions in an endemic area of rabies in Bolivia. A total of 236 vaccinated and 44 unvaccinated dogs in Santa Cruz de la Sierra, selected using stratified random sampling, were investigated in order to obtain owned dog characteristics and antibody titres against rabies in April 2007. The proportion of vaccinated dogs with an antibody titre exceeded the protection threshold value of 0.5 EU/ml was 58% [95% confidence intervals (CI): 52-65], indicating that vaccination is likely to elicit an antibody response (odds ratio 6.3, 95% CI: 1.2-11.5). The range of geometric mean of antibody titre for vaccinated dogs (0.89 EU/ml; 95% CI: 0.75-1.04) was considered to meet the minimal acceptable level indicating an adequate immune response to the vaccine. However, the titre level was not satisfactory in comparison with the results from other field investigations with inactivated tissue culture vaccines. It is recommended for public health authorities to (1) consider modernizing their vaccine manufacturing method because the level of immunity induced by the current vaccine is comparably low, (2) conduct frequent vaccination campaigns to maintain high levels of vaccination coverage, and (3) actively manage the domestic dog population in the study area, which is largely responsible for rabies maintenance.     

Prevalence,geographic distribution and phenotypic differences of Piscirickettsia salmonis EM‐90‐like isolates

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM‐90 apart from the prototypic LF‐89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM‐90‐like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM‐90‐like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM‐90‐like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.     

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89^T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89^T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.     

The mutagenesis of a type IV secretion system locus of Piscirickettsia salmonis leads to the attenuation of the pathogen in Atlantic salmon,Salmo salar

Piscirickettsiosis is a threatening infectious disease for the salmon industry, due to it being responsible for significant economic losses. The control of outbreaks also poses considerable environmental challenges. Despite Piscirickettsia salmonis having been discovered as the aetiological agent of the disease more than 25 years ago, its pathogenicity remains poorly understood. Among virulence factors identified so far, type four secretion systems (T4SS) seem to play a key role during the infection caused by the bacterium. We report here the genetic manipulation of P. salmonis by means of the transference of plasmid DNA in mating assays. An insertion cassette was engineered for targeting the icmB gene, which encodes a putative T4SS‐ATPase and is carried by one of the chromosomal T4SS clusters found within the genome of P. salmonis PM15972A1, a virulent representative of the EM‐90‐like strain. The molecular characterization of the resulting mutant strain demonstrated that the insertion interrupted the target gene. Further in vitro testing of the icmB mutant showed a dramatic drop in infectivity as tested in CHSE‐214 cells, which is in agreement with its attenuated behaviour observed in vivo. Altogether, our results demonstrate that, similar to other facultative intracellular pathogens, P. salmonis’ virulence relies on an intact T4SS.     

Can camels disperse seeds of the invasive tree Prosopis juliflora?

Endozoochory has been recognised as the most important dispersal mechanism in invasive Prosopis species, because their sugary, tasty pods attract animals and because some of their seeds remain intact after passing through some animals' digestive systems. In this study, we evaluated the role of the camel (Camelus dromedarius) as a potential disperser of the seeds of invasive tree Prosopis juliflora. Four camels of similar weight (ca. 400 kg) and age (ca. 2 years) were each fed with 70 fruits (1000 seeds approximately) of P. juliflora, which were retrieved from the camels' dung at 24‐h intervals for 4 days. The seeds retrieved were tested for germination and viability, along with seeds not eaten by the camels, with and without pericarp. Less than 3% of the seeds eaten were retrieved from the camels' dung. Most of the seeds (74%) were retrieved between 24 and 72 h after ingestion. The passage through the camel gut significantly accelerated and increased seed germination of P. juliflora in comparison with uneaten seeds covered with pericarp (48–75% and 15% respectively). While gut passage liberated P. juliflora seeds from the pericarp, increasing and accelerating their germination, the viability of seeds which had not germinated after germination trials were decreased (ca. ~20%) relative to uneaten seeds that had also not germinated. Our results show that, despite the low recorded seed recovery, camels can potentially disperse seeds of P. juliflora, which is in line with field observation showing P. juliflora expansion along the camels' routes in Gebel Elba National Park, south‐east Egypt.     

Leaf-trait responses to irrigation of the endemic fog-oasis tree Myrcianthes ferreyrae: can a fog specialist benefit from regular watering?

Myrcianthes ferreyrae is an endemic, endangered species, with a small number of individuals located only in hyperarid, fog-oases known as lomas along the Peruvian desert in southern Peru, where fog is the main source of water. Following centuries of severe deforestation, reforestation with this native species was conducted in the Atiquipa lomas, Arequipa-Perú. On five slopes, five 2-year-old seedlings were irrigated monthly with water trapped by raschel-mesh fog collectors, supplementing natural rainfall with 0, 20, 40, 60 and 80 mm month(-1) from February to August 2008. We measured plant growth, increment in basal diameter, height and five leaf traits: leaf mass area (LMA), leaf carbon isotope composition (δ(13)C), nitrogen per leaf area, total leaf carbon and stomatal density; which are indicative of the physiological changes resulting from increased water supply. Plant growth rates, estimated from the variation of either shoot basal diameter or maximum height, were highly correlated with total biomass. Only LMA and δ(13)C were higher in irrigated than in control plants, but we found no further differences among irrigation treatments. This threshold response suggests an on-off strategy fitted to exploit pulses of fog water, which are always limited in magnitude in comparison with natural rain. The absence of a differential response to increased water supply is in agreement with the low phenotypic plasticity expected in plants from very stressful environments. Our results have practical implications for reforestation projects, since irrigating with 20 mm per month is sufficient to achieve the full growth capacity of this species.     

Effects of experimental infection by Caligus rogercresseyi (Copepoda: Caligidae) on the common jollytail, Galaxias maculatus (Osmeriforme: Galaxiidae) (Jenyns, 1842) in Chile

Galaxias maculatus, the common jollytail, is a native smelt fish with a lacustrine or diadromous life cycle. In Chile, the rearing cycle of this fish includes a freshwater and a marine phase. Several diseases and parasites reported for the freshwater phase could be avoided by rearing the fish in salty or brackish water. Nonetheless, this alternative could result in Caligus rogercresseyi infections. The objective of this study was to evaluate experimentally the capacity of C. rogercresseyi to infect the common jollytail and the effects this parasite may cause to the fish. Prevalence and intensity of experimental C. rogercresseyi infections on G. maculatus were estimated and the effects of this parasitosis on the fish were evaluated on fish survival, weight loss, swimming behavior, and skin damage. Two experiments were carried out with adult fish taken from the Maullín Estuary. Fish were acclimatized to a salinity of 32 ppt and divided into two groups of 20 fish each. The experimental group was infected with copepodid stages of C. rogercresseyi and the other was used as a control. The fish were kept in individual tanks until observing ovigerous female copepods. C. rogercresseyi recognizes G. maculatus as a host under experimental conditions and optimal salinity for the parasite. Results show that under experimental condition copepodid stage can successfully infect the fish, developing to the adult stage, mating, and producing eggs (females). C. rogercresseyi caused alterations in fish behavior, superficial body damage, weight loss, and mortality. Rearing the common jollytail in salty water should take into consideration measures to control these infections as they can damage fish and affect surrounding wild fish populations. There are no records of this parasite in natural common jollytail populations; however, our understanding of the ecological role played by the common jollytail in C. rogercresseyi transmissions among sympatric host species could be greatly improved through further research related to host preferences and epidemiological data for different sympatric host species.     

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut‐off values that were used to characterize the pathogen population. This work represents the first large‐scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.