Bacterial communities from vagina of dairy healthy heifers and cows with impaired reproductive performance.

Vaginal microenvironment plays a significant role in bovine fertility since its resident microorganisms interact with the host mucosa and constitutes the first barrier against ascending pathogens in the reproductive tract. In this study, the vaginal microbiome of healthy heifers (H) and cows with impaired reproductive performance, metritis complex (MT) or repeat breeders (RB), was assessed using a 16S rRNA gene sequencing approach. Analysis revealed that even though a vaginal microbiological guild (Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes, Fusobacteria and Actinobacteria) was shared among healthy heifers and cows with uterine disease; further analysis at genus level showed significant differences depending on the reproductive health status. The relative abundances of recognized uterine pathogens such as Bacteroidetes, Fusobacterium and Helcococcus were higher in MT when compared with H and RB; therefore, their presence in vagina can be considered as a risk factor for fertility. The present study describes for the first time, the composition of native bacterial communities in the vagina of cows undergoing the repeat breeding syndrome (RBS), and reports an association between this disease and the presence of Porphyromonas and unassigned genera of the Pasteurellaceae family. In addition, this work highlights the bacteria associated with a healthy vagina: genera from the families Lachnospiraceae, Rikenellaceae and the genera Acinetobacter, Bacillus, Oscillospira, CF231 and 5-7NS. Results highlighted herein, signify the potential of the evaluation of the bovine vaginal microbiome to future design therapeutic interventions to improve pregnancy rates however, further research is needed to elucidate the balance of bacterial species resulting in an optimal reproductive health.

Vaginal bacterial diversity from healthy gilts and pregnant sows subjected to natural mating or artificial insemination.

The profitability of commercial pig farms largely depends on the reproductive performance of gilts and sows. The aim of this study was to identify differences in the composition and diversity of vaginal microbiota between gilts (G) and pregnant (P) sows, both artificially inseminated (AI) and natural mating (NM). Samples were collected by scraping the vaginal mucosa of G (n = 10) and P (NM, n = 10 and AI, n = 7) sows. Samples were analysed by culture-dependent techniques and 16S-rRNA gene High-Throughput-Sequencing. The profiles of the cultured microbiota showed two distinctive clusters, one of them grouped four samples of P sows from the AI group. The vaginal microbiota from P had lower richness than G sows (Mann-Whitney/Kruskal-Wallis test, p < 0.01), but all vaginal samples had a similar diversity. The PERMANOVA analyses revealed significant differences (p < 0.01) between the microbial communities' structures from G and P sows. The bacteria phyla with the highest relative abundances were Proteobacteria (33.1%), followed by Firmicutes (32%), Cyanobacteria (13.3%) and Actinobacteria (13.2%). The relative abundance for phyla, families and genera was estimated and Proteobacteria was significantly higher (p = 0.038) in P than in G sows; Firmicutes was significantly lower in AI than G and NM sows. A "core microbiota" included Lactobacillus, Bacillus, Enterococcus, Acinetobacter and Pseudomonas. The results presented highlight the differences in the bacterial composition between G and P sows, as well as the changes in the microbial populations associated with the breeding method.

Bacterial communities associated to the urethra of healthy gilts and pregnant sows undergoing different reproductive protocols.

Nowadays, it is known that the urogenital microbiota plays a key role in the urinary health of mammalians. Despite the urinary infections affect the health and the welfare of breeding sows, the urethral microbiota of healthy sows remains unknown. Therefore, this work evaluates the urethral bacterial communities of healthy gilts and sows to determine the presence of Enterobacteriaceae populations, and the structure of this microbiota in gilts (G) and pregnant (P) sows. Samples were collected by scraping the urethral mucosa of G (n = 9) and P sows, which included natural mating (NM, n = 9) and artificial inseminated (AI, n = 7) sows. Samples were analyzed by culture-dependent techniques and 16S-rRNA gene high-throughput-sequencing. All females were positive for Enterobacteriaceae culture, without significant differences (Kruskal-Wallis) between G and P groups (median values: 2.78 and 3.09 log CFU/mL, respectively; P = 0.497). Also, the rate of Enterobacteriaceae/total mesophilic microorganisms was individually calculated, without significant differences between G and P sows (median values: 0.61 and 0.66, respectively; P = 0.497). When analyzing the bacterial communities, it was found similar richness in G, NM, and AI; however, diversity was lower in P sows than G (Mann Whitney/Kruskal-Wallis test, P < 0.01). The dominating phyla that constituted a "core microbiome" included Firmicutes, Proteobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes, which were common for all the studied females. The relative abundance for phyla, families, and genera was estimated, and Firmicutes was significantly higher in NM than AI sows (P = 0.02, Mann-Whitney/Kruskal Wallis test for univariate statistical comparisons); Pseudomonadaceae and Enterobacteriaceae were higher in AI than in NM (Mann-Whitney/Kruskal-Wallis, P < 0.05). Lactobacillus and Pseudomonas were among the dominant genera; however, only Pseudomonas sp. was significantly higher in AI than NM (Mann-Whitney/Kruskal-Wallis, P = 0.006). The results represent the first evidence about the existence of a urethral microbiota that includes Enterobacteriaceae, as well as the patterns of this microbiota in G and P sows. The knowledge of this urethral microbiota might allow for future research to develop innovative protocols to restore and/or preserve the healthy ecology of the urinary microbiome to prevent diseases ensuring the welfare of breeding sows.

Characterization of native Escherichia coli populations from bovine vagina of healthy heifers and cows with postpartum uterine disease.

Even though Escherichia coli are common bacteria of the bovine vaginal microbiota, they represent an important pathogen that causes diseases in the reproductive tract and subfertility. However, the actual endometrial virulence profile of E. coli is poorly understood. The present study aims to characterize the phylogenetic structure and virulence potential of native vaginal populations of E. coli from healthy heifers (H), and cows with postpartum uterine diseases (PUD), such as metritis/endometritis (MT) or repeat breeder cows (RB). To this end, the virulence repertoire of 97 E. coli isolates was genotypically and phenotypically assessed. Most of them were assigned to phylogenetic group A (74%), followed by B1 (17%) and D (9%); RB strains were significantly (p < 0.05) more represented by B1. Seven of the 15 evaluated virulence genes (VFG) were detected and the most prevalent were fimH (87%), agn43 (41%) and csgA (35%); while traT (27%), fyuA (11%), hlyA (5%) and kpsMT II (5%) were observed in a lower proportion. Particularly, fyuA was significantly higher (p < 0.05) in MT cows whereas csgA showed the same behavior in PUD animals (p < 0.05). When comparing H and PUD strains, these last ones were associated to positive expression of biofilm, fimbriae curli/cellulose and motility; yet RB strains did not show motility. Vaginal B1 E. coli populations, that possess VFG (fyuA and csgA) as well as the expression of motility, curli fimbriae/cellulose and biofilm, may represent risk factors for endometrial disorders; specifically, those that also, have kpsMT II may have a pathogenic potential for causing the RB syndrome. Future research focusing on the detection of these strains in the vaginal microbiota of cows with postpartum uterine diseases should be done since the control of their presence in vagina could reduce the risk that they access the uterus during the postpartum period.

Antimicrobial resistant Escherichia coli in the reproductive tract microbiota of cows and sows.

Escherichia coli is a natural colonizer of the urogenital mucosa of healthy females; however it is one of the pathogens associated to reproductive failures in cows and sows. A better knowledge about the characteristics of native E. coli will allow us to differentiate them from pathogenic strains. Ninety autochthonous isolates from the reproductive tract of sows and cows were characterized to determine the phylogenetic profile, antibiotic resistance and virulence factors; also, comparisons between different breeding systems were performed. Vaginal colonization of E. coli was statistically higher in cows (57.5%) than sows (23.8%), and most isolates belonged to the phylogenetic group A: 79.69 and 80.77%, respectively; moreover phylo-groups B1 (12.5 and 11.54%) and D (7.81 and 7.69%) were significantly lower; however, none was classified as B2. Positive associations between virulence factors and group D were found. Isolates with antimicrobial susceptibility were associated with group A and the MDR (Multiple Drug Resistance) was related to the porcine source. These results contribute to the knowledge of extra-intestinal E. coli populations; which could affect the reproductive performance of females.

[Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate]. / Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica.

Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate. In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 IU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 IU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary interest.

Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica / Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate

En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.

In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.