The effects of polyhexamethylene biguanide (PHMB) and TLR agonists alone or as polyplex nanoparticles against Leishmania infantum promastigotes and amastigotes

Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 µM), miltefosine (9.455 µM), and allopurinol (0.124 µM). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF-α and IL-6 levels. Increased TNF-α was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF-α production

Erratum to: Leishmania infantum-specific production of IFN-γ and IL-10 in stimulated blood from dogs with clinical leishmaniosis

BACKGROUND: There is limited information available on cytokine profiles in dogs with different degrees of disease severity due to natural infection of Leishmania infantum. The aim of this study was to investigate L. infantum-specific IFN-γ and IL-10 production in blood from dogs with leishmaniosis at diagnosis and correlate these findings with disease severity, humoral immune response and blood parasitemia. METHODS: Sixty dogs were diagnosed based on physical examination, routine laboratory tests, L. infantum-specific antibody levels measured by quantitative ELISA and blood parasitemia by real-time PCR. Heparin whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ and IL-10 concentrations were measured in supernatants with sandwich ELISAs. RESULTS: The majority of dogs (n = 36) were classified as LeishVet stage II (moderate disease). The rest of the dogs were classified as stage I (n = 10), III (n = 10) and IV (n = 4). Dogs classified with stage I and IIa presented significantly higher (P = 0.02) LSA IFN-γ concentrations, lower (P <0.0001) antibody levels and a tendency for lower blood parasitemia (P = 0.1) than dogs classified with stages IIb, III or IV while no differences in ConA IFN-γ or IL-10 concentrations were observed among groups. Thirty-five dogs produced significantly higher LSA IFN-γ (mean ± SD: 2320 ± 3960 pg/ml) and ConA IFN-γ (mean ± SD: 7887 ± 7273 pg/ml) when compared with 25 dogs that did not produce detectable LSA IFN-γ but produced ConA IFN-γ (mean ± SD: 4917 ± 5233 pg/ml). IFN-γ producer dogs presented lower (mean ± SD: 5750 ± 14,082 ELISA units (EU), P = 0.001) antibody levels and blood parasitemia (mean ± SD:   5 ± 10 parasites/ml, P = 0.001) when compared with IFN-γ non-producers (mean ± SD: 19,638 ± 28,596 EU and 1100 ± 5112 parasites/ml), respectively. LSA IL-10 was not detectable in 34 dogs while 49 dogs secreted ConA IL-10 (mean ± SD of 90 ± 103 pg/ml). LSA IFN-γ concentration was negatively correlated with blood parasitemia and antibody levels and positively correlated with ConA IFN-γ and LSA IL-10 concentrations. CONCLUSIONS: The results of this study demonstrate that sick dogs lacking L. infantum specific IFN-γ production in stimulated whole blood produce a strong humoral response, have a high blood parasitemia and severe clinical disease. IL-10 does not appear to be a marker of disease severity

Leishmania infantum-specific IFN-γ production in stimulated blood from dogs with clinical leishmaniosis at diagnosis and during treatment

There is limited data regarding Leishmania infantum specific T cell mediated immunity in naturally infected sick dogs at the time of diagnosis and during anti-Leishmania treatment. Our aim was to investigate the kinetics of L. infantum specific IFN-γ production in dogs with leishmaniosis at the time of diagnosis and during treatment and to correlate it with specific L. infantum antibodies, blood parasitemia and clinicopathological findings. Thirty-four dogs were diagnosed with leishmaniosis based on physical examination, routine laboratory tests and L. infantum-specific antibody levels by quantitative ELISA. Heparinized whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ concentration was evaluated in supernatants of stimulated blood using a commercial sandwich ELISA. Leishmania real-time PCR was also performed for assessing blood parasitemia. Dogs were treated with meglumine antimoniate and allopurinol. Sixteen dogs were classified as IFN-γ non-producers after LSA stimulation (mean ± SD: 0 ± 0 pg/mL) and 18 dogs as IFN-γ producers (mean ± SD: 2885.3 ± 4436.1 pg/mL) at the time of diagnosis (P < 0.0001). IFN-γ non-producers were classified in a more severe clinical staging than IFN-γ producers that presented a mild to moderate clinical staging (P = 0.03). In the IFN-γ non-producer group, production of IFN-γ after LSA stimulation was significantly increased during treatment especially at day 365 (P = 0.018) together with clinical improvement when compared with day 0. In contrast, IFN-γ producers maintained their IFN-γ production after LSA stimulation and no statistically significant changes were found during treatment follow-up. At diagnosis, IFN-γ non-producers showed a significantly higher blood parasitemia versus IFN-γ -producers (P = 0.005). IFN-γ non-producers drastically reduced blood parasitemia to minimum values at day 365 when compared with day 0 (P = 0.017). No significant differences were found at day 365 in blood parasitemia of IFN-γ producers compared to pre-treatment. At diagnosis, L. infantum specific antibodies were higher in IFN-γ non-producers than IFN-γ producers (P = 0.014). A marked reduction of antibody levels was found at day 365 when compared with day 0 in IFN-γ non-producers (P = 0.005) and producers (P = 0.001). These results demonstrate that IFN-γ concentration increases with long-term anti-Leishmania treatment together with clinical improvement in dogs that do not produce IFN-γ at diagnosis. Together with clinical recovery, reduction in blood parasitemia and L. infantum specific antibodies, tracking IFN-γ concentration could constitute an important prognostic tool for immune monitoring in CanL

Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool.We thank Jaime Maldonado and HIPRA (Spain) for the A/Swine/Spain/ 54008/2004 (H3N2) strain, Edward J. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) IAV strain, T. M. Chambers and the University of Kentucky for the A/Equine/OH/1/03 (H3N8) IAV strain, and Hon Ip and the U.S. Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbor Seal/New Hampshire/179629/2011 (H3N8) IAV strains. We thank Sergio López, David Solanes, Francisco X. Abad, Jordi Alberola, Jaume Martorell, and Eduard J. Cunilleras for help in providing different samples and during the experimental infections, as well as the personnel in Cat3 laboratories and the animal house. We thank Adolfo García-Sastre for providing materials and for support as the principal investigator of the NIAID-funded Center for Research in Influenza Pathogenesis (HHSN266200700010C). The research leading to these results received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), the Research Infrastructures Action under grant FP7-228393 (a NADIR project), and projects AGL2010-22200-C02-01 and AGL2007-60274 of the Spanish Ministry of Science and Innovation

Prevalencia de lesiones de caries en pacientes tratados ortodoncicamente en Universidad de Talca: estudio piloto

37 p.Los tratamientos de ortodoncia fijos tienen muchos beneficios para los pacientes, ya que mejoran su estética, funcionalidad oclusal y calidad de vida. Sin embargo, los aditamentos que se utilizan pueden representar un factor de riesgo para desarrollar lesiones de caries, debido a que pueden facilitar el acúmulo de biofilm, lo cual sumado a una dieta cariogénica puede desencadenar el desarrollo de estas lesiones. En este estudio observacional descriptivo de corte transversal determinamos la prevalencia de lesiones de caries en 25 adolescentes de 12 a 15 años tratados en el Programa de Especialización en Ortodoncia y Ortopedia Dentofacial en el Centro de Clínicas Odontológicas de la Universidad de Talca. Se determinó la gravedad de las lesiones a través de los criterios ICDAS y la actividad de estas a través de los criterios Nyvad. Observamos que todos los participantes presentaron al menos una lesión de caries, principalmente lesiones de mancha blanca. El 67% de las lesiones de caries diagnosticadas estaban en contacto con un aditamento ortodóncico, donde el 77,7% de ellas estaban en contacto con brackets. Del total de pacientes evaluados, el 52% requirió tratamiento preventivo en conjunto con tratamiento rehabilitador. Nuestros resultados sugieren que la ortodoncia fija si es un factor que predispone a la aparición de nuevas lesiones de caries cuando existe un mal control del biofilm y la dieta del paciente. Palabras clave: ortodoncia, aparatos ortodóncicos fijos, caries denta

Humoral Responses and Ex Vivo IFN-γ Production after Canine Whole Blood Stimulation with Leishmania infantum Antigen or KMP11 Recombinant Protein

The effect of Leishmania infantum soluble antigen (LSA) and recombinant Kinetoplastid Membrane Protein 11 (rKMP11) on the induction of ex vivo specific IFN-γ (n = 69) and antibody responses (n = 108) was determined in dogs. All dogs were tested for serological response to both antigens and divided into Group 1: healthy (Asturias, Spain, n = 26), Group 2: sick (n = 46), Group 3: healthy Ibizan hounds (Mallorca, Spain, n = 22) and Group 4: healthy (Bari, Italy, n = 14). Antibody levels were higher for LSA when compared to rKMP11 (p = 0.001). Ibizan hounds were all seronegative to rKMP11 and 18% were low seropositive to LSA. Sick dogs presented higher antibody response to both antigens compared to the rest of the groups (p < 0.0001). All groups showed higher IFN-γ levels after LSA compared to rKMP11 responses (p < 0.05). The highest response to LSA was found in Ibizan hounds (p < 0.05). IFN-γ to LSA and rKMP11 stimulation was observed in 34% and in 2.8% of the sick dogs, respectively. Here, we demonstrated that anti-rKMP11 antibodies are mainly present in dogs with moderate to severe disease. Furthermore, cellular immune response measured by specific ex vivo IFN-γ production was more intense to LSA than stimulated to rKMP11

Association between feline immunodeficiency virus and Leishmania infantum infections in cats : a retrospective matched case-control study

Feline leishmaniosis caused by Leishmania infantum is often associated with feline immunodeficiency virus (FIV) infection; however, the role and clinical significance of this coinfection remain unknown. This study aimed to assess whether FIV is associated with L. infantum infection in cats from canine leishmaniosis endemic areas and to report the clinical signs and hematological alterations associated with coinfection. A retrospective matched case-control study (ratio 1:2) was conducted. Data of clinical examination and complete blood count (CBC) were selected from a cohort of 705 cats examined for epidemiological studies on feline leishmaniosis conducted between 2012 and 2019. Ninety-one FIV seropositive cases and 182 FIV seronegative control cats were selected. Matching was done according to age, sex, lifestyle and geographic provenience of case cats. Rapid ELISA devices were mainly used to detect anti-FIV antibodies. Anti- Leishmania IgG antibodies were detected by indirect-immunofluorescence test (IFAT). Leishmania DNA was searched in blood, oral and conjunctival swabs by quantitative real-time PCR. Feline immunodeficiency virus seropositive cats had no hematological abnormalities suggestive of an advanced stage of FIV infection and were statistically more frequently IFAT positive, and their risk of being L. infantum antibody positive was 2.8 greater than in the FIV seronegatives. The association of FIV seropositivity with L. infantum antibody positivity was confirmed in the univariable model of logistic regression. A multivariate model found FIV infection and L. infantum PCR positivity as predictors of a positive L. infantum IFAT result. Male outdoor cats from rural or suburban areas were at risk for FIV and L. infantum antibody positivity. Clinical signs more frequently associated with the coinfection were oral lesions, pale mucous membranes and low body condition score (BCS). This study documents that FIV seropositive cats with no hematological abnormalities suggestive of an advanced stage of FIV infection are more prone to be L. infantum seroreactive by IFAT in endemic areas. Therefore, FIV seropositive cats should be tested for L. infantum antibodies and treated for preventing sand fly bites. Pale mucous membranes, low BCS and oral lesions but no CBC abnormalities were significantly associated with the coinfection. The online version contains supplementary material available at 10.1186/s13071-022-05230-w

Exploring the Relationship Between Susceptibility to Canine Leishmaniosis and anti-Phlebotomus Perniciosus Saliva Antibodies in Ibizan Hounds and Dogs of Other Breeds in Mallorca, Spain

Background: Canine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response. Methods: This study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods. Results: Results indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT. Conclusions: To our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds

Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.This work was funded by the coordinated project RTA 2011-00111-C03 of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), by Instituto de Salud Carlos III (“Programa especial de investigación sobre la gripe pandémica” GR09/0023, GR09/0040, GR09/0039), and by the Servei de Diagnòstic de Patologia Veterinària (SDPV) of the Universitat Autònoma de Barcelona. This work was also partially funded by the CERCA program from Generalitat de Catalunya

Pulmonary transcriptomic responses indicate a dual role of inflammation in pneumonia development and viral clearance during 2009 pandemic influenza infection

Background: The interaction between influenza virus and the host response to infection clearly plays an important role in determining the outcome of infection. While much is known on the participation of inflammation on the pathogenesis of severe A (H1N1) pandemic 09-influenza virus, its role in the course of non-fatal pneumonia has not been fully addressed. Methods: A systems biology approach was used to define gene expression profiles, histology and viral dynamics in the lungs of healthy immune-competent mice with pneumonia caused by a human influenza A (H1N1) pdm09 virus, which successfully resolved the infection. Results: Viral infection activated a marked pro-inflammatory response at the lung level paralleling the emergence of histological changes. Cellular immune response and cytokine signaling were the two signaling pathway categories more representative of our analysis. This transcriptome response was associated to viral clearance, and its resolution was accompanied by resolution of histopathology. Discussion: These findings suggest a dual role of pulmonary inflammation in viral clearance and development of pneumonia during non-fatal infection caused by the 2009 pandemic influenza virus. Understanding the dynamics of the host's transcriptomic and virological changes over the course of the infection caused by A (H1N1) pdm09 virus may help identifying the immune response profiles associated with an effective response against influenza virus