In Vitro Antimicrobial Susceptibilities of Francisella tularensis subsp. holarctica Isolates from Tularemia Outbreaks That Occurred from the End of the 20th Century to the 2020s in Spain.

A collection of 177 Francisella tularensis subsp. holarctica clinical isolates (29 from humans and 148 from animals, mainly hares and voles) was gathered from diverse tularemia outbreaks in the Castilla y León region (northwestern Spain) that occurred from the end of the 20th century to the 2020s. Along with four F. tularensis subsp. holarctica reference strains, all of these clinical isolates were tested using a broth microdilution method to determine their susceptibility to 22 antimicrobial agents, including ß-lactams, aminoglycosides and one member each of the tetracycline, glycylcycline, quinolone and sulphonamide classes. Many multi-resistance profiles were found among the tested isolates, but especially among those of human origin (all but two isolates showed resistance to at least 13 of 18 antimicrobial agents). Even so, all human isolates were susceptible to gentamicin and tobramycin, while more than 96% of animal isolates were susceptible to these two aminoglycosides. Ciprofloxacin showed activity against more than 92% of animal and human isolates. However, almost 21% of human isolates were resistant to tetracycline, and more than 65% were resistant to tigecycline. Finally, a quite similar activity to other F. tularensis subsp. holarctica isolates collected 20 years earlier in Spain was observed.

Tularemia Outbreaks in Spain from 2007 to 2020 in Humans and Domestic and Wild Animals.

In this study, tularemia outbreaks associated with humans and several domestic and wild animals (Iberian hares, wild rabbits, voles, mice, grey shrews, sheep, dogs, foxes, wolves, ticks, and river crayfish) are reported in Spain from 2007 to 2020. Special attention was paid to the outbreaks in humans in 2007-2009 and 2014-2015, when the most important waves occurred. Moreover, positive rates of tularemia in lagomorphs were detected in 2007-2010, followed by negative results in 2011-2013, before again returning to positive rates in 2014 and in 2017 and in 2019-2020. Lagomorphs role in spreading Francisella tularensis in the epidemiological chain could not be discarded. F. tularensis is described for the first time infecting the shrew Crocidura russula worldwide, and it is also reported for the first time infecting wild rabbits (Oryctolagus cuniculus) in Spain. Serological positives higher than 0.4% were seen for sheep only from 2007-2009 and again in 2019, while serological rates greater than 1% were revealed in dogs in 2007-2008 and in wild canids in 2016. F. tularensis were detected in ticks in 2009, 2014-2015, 2017, and 2019. Lastly, negative results were achieved for river crayfish and also in environmental water samples from 2007 to 2020.

TLR2, Siglec-3 and CD163 expressions on porcine peripheral blood monocytes are increased during sepsis caused by Haemophilus parasuis.

TLRs, Siglecs and CD163 are cell surface receptors that play an important role in immune response and sepsis. The objective of this study was to assess changes in the expression levels of several of these receptors (TLR2, TLR4, CD163, Siglec-1, Siglec-3, Siglec-5 and Siglec-10) on the surface of peripheral blood mononuclear cells from pigs with sepsis caused by Haemophilus parasuis. Flow cytometry was employed to analyze samples from an experimental infection and from cell cultures. A significant increase in CD163, TLR2 and Siglec-3 expression during infection was seen. However, in vitro exposure of peripheral blood monocytes to bacteria or sera from infected pigs did not increase the expression of these receptors. These changes may be due to recruitment of monocytes into the blood compartment in response to H. parasuis-induced sepsis.

Immunogenic characterization of vaccines based on Haemophilus parasuis Nagasaki strain, OmpP2, OmpP5 and OmpD15, in colostrum-deprived pigs experimentally challenged with the same strain.

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.

New insights about functional and cross-reactive properties of antibodies generated against recombinant TbpBs of Haemophilus parasuis.

Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund's adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.

A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection.

This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässer's disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response.

In vitro efficacy of several disinfectants against Salmonella enterica serovar Enteritidis and Escherichia coli strains from poultry / Eficácia de vários desinfetantes in vitro contra cepas de Salmonella entérica serovar Enteritidis e Escherichia coli aviária

ABSTRACT The efficacy of 28 individual or blended disinfectants against avian Salmonella enterica serovar Enteritidis and Escherichia coli strains was determined. An in vitro test in the presence and absence of serum as source of organic material was conducted. Povidone-iodine (releasing 1% available iodine), 1% potassium permanganate, 70% ethanol, 2% chlorhexidine digluconate and three commercial formulations based on quaternary ammonium compounds + formaldehyde or cresol derivates were the most effective against all strains tested and reduced bacterial counts by more than 106 times (6-log10) regardless of the presence of organic matter. These commercial compounds as well as ethanol and chlorhexidine among the individual substances tested might be helpful in the adoption of environmental control measures against these two enterobacteria in poultry industry.

RESUMO: A eficácia de 28 desinfetantes individuais ou combinados sobre cepas de Salmonella enterica serovar Enteritidis e Escherichia coli foi determinada. Um teste in vitro em presença e ausência de soro como fonte de matéria orgânica foi realizado. Iodopovidona (contendo 1% de iodo ativo), permanganato de potássio a 1%, etanol a 70%, digliconato de clorexidina e três formulações comerciais, baseadas em compostos de amônia quaternária + formaldeído ou em derivados de cresóis, foram mais eficazes contra as cepas bacterianas testadas, reduzindo em mais 106 vezes (6-log) a contagem bacteriana, independente da presença de matéria orgânica. Esses compostos comerciais, bem como o etanol e a clorexidina entre as substâncias químicas individuais avaliadas, podem ser úteis para a implementação de medidas de controle ambiental contra estas duas enterobacterias de importância para a indústria aviária.