Brazilian Clinical Strains of Actinobacillus pleuropneumoniae and Pasteurella multocida: Capsular Diversity, Antimicrobial Susceptibility (In Vitro) and Proof of Concept for Prevention of Natural Colonization by Multi-Doses Protocol of Tildipirosin.

One hundred Actinobacillus pleuropneumoniae (App) and sixty Pasteurella multocida subsp. multocida serogroup A (PmA) isolates were recovered from porcine pneumonic lungs collected from eight central or southern states of Brazil between 2014 and 2018 (App) or between 2017 and 2021 (PmA). A. pleuropneumoniae clinical isolates were typed by multiplex PCR and the most prevalent serovars were 8, 7 and 5 (43, 25% and 18%, respectively). In addition, three virulence genes were assessed in P. multocida isolates, all being positive to capA (PmA) and kmt1 genes, all negative to capD and toxA, and most of them (85%) negative to pfhA gene. The susceptibility of both pathogens to tildipirosin was investigated using a broth microdilution assay. The percentage of isolates susceptible to tildipirosin was 95% for App and 73.3% for PmA. The MIC50 values were 0.25 and 1 µg/mL and the MIC90 values were 4 and >64 µg/mL for App and PmA, respectively. Finally, a multiple-dose protocol of tildipirosin was tested in suckling piglets on a farm endemic for both pathogens. Tildipirosin was able to prevent the natural colonization of the tonsils by App and PmA and significantly (p < 0.0001) reduced the burden of Glaesserella parasuis in this tissue. In summary, our results demonstrate that: (i) tildipirosin can be included in the list of antibiotics to control outbreaks of lung disease caused by App regardless of the capsular type, and (ii) in the case of clinical strains of App and PmA that are sensitive to tildipirosin based on susceptibility testing, the use of this antibiotic in eradication programs for A. pleuropneumoniae and P. multocida can be strongly recommended.

Phenotypic Characterization of Encephalitis in the BRAINS of Badgers Naturally Infected with Canine Distemper Virus.

Canine distemper virus (CDV) affects a huge diversity of domestic and wild carnivores, with increasing numbers of mortality events worldwide. The local cell-mediated immune response elicited against a natural infection is an important factor in determining the outcome of CDV infection. Therefore, the purposes of this study were to describe the local immune response within the central nervous systems (CNSs) of seven badgers naturally infected with CDV in Asturias (Atlantic Spain) and to determine the phenotype and distribution of microglial cells, T and B lymphocytes, and astrocytes in the foci of gliosis located in the thalamus and cerebellum using immunohistochemistry. The immunohistochemical assessment demonstrated the presence of Iba1-positive microglia and GFAP-positive astrocytes in the foci of gliosis, whereas T (CD3-negative) or B (CD20-negative) lymphocytes in those same lesions were absent. Our results also revealed that the badgers with natural CDV encephalitis presented lesions mostly located in the white matter of the thalamus and cerebellum, suggesting a CDV-specific tropism for the white matter of badger brains in those locations. The knowledge gained in the field of the immunopathogenesis of distemper disease affecting the CNSs of badgers could help to clarify CDV disease patterns in this species.

Mortality Causes in Captive Cantabrian capercaillie (Tetrao urogallus cantabricus) in Spain.

The Cantabrian capercaillie (Tetrao urogallus cantabricus) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli, Clostridium perfringens, or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.

Evaluation of the COVID-19 Lockdown-Adapted Online Methodology for the Cytology and Histology Course as Part of the Degree in Veterinary Medicine.

The COVID-19 pandemic and lockdown brought numerous teaching challenges requiring innovative approaches to teaching and learning, including novel modes of content delivery, virtual classrooms, and online assessment schemes. The aim of this study is to describe and assess the efficacy of the methods implemented at the University of León (Spain) to adapt to lockdowns in the context of the Cytology and Histology (CH) course for veterinary medicine undergraduate students. To evaluate the success of lockdown-adapted methodologies, we used inferential statistical analysis to compare the academic outcomes of two cohorts: 2018-2019 (traditional face-to-face-presential-learning and evaluation) and 2019-2020 (some face-to-face and some online lockdown-adapted learning and online lockdown-adapted evaluation). This analysis considered scores in both theoretical and practical exams and students' final subject score. We also evaluated the number of logs onto the Moodle platform throughout the 2019-2020 period, as well as performing a student satisfaction survey in both courses. The use of explanatory pre-recorded lectures, continuous online self-assessment tests, and virtual microscopy (VM) may have produced significant improvements in the acquisition of histology competencies among students in the lockdown cohort. However, we need to implement further strategies to improve the assessment of students' true level of knowledge acquisition. According to the student feedback, VM is a well-accepted resource that is perceived as a flexible and enjoyable tool to use. However, while students found that the resource enhances their ability to learn about microscopic structures, they felt that it should not completely replace optical microscopy.

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.

Anatomical Site, Typing, Virulence Gene Profiling, Antimicrobial Susceptibility and Resistance Genes of Streptococcus suis Isolates Recovered from Pigs in Spain.

A set of 207 Streptococcus suis isolates were collected from ten autonomous communities from Spain in 2019 to 2020 from pigs with meningitis, pneumonic lungs, arthritic joints or other swollen viscera, to a lesser extent. Thirteen capsular types were detected being the most prevalent serotype 2 (21.7%), followed by serotypes 1 (21.3%), 9 (19.3%) and 3 (6.3%). Serotypes 2 and 9 were recovered mainly from the central nervous system (CNS), while serotype 1 was isolated mostly from swollen joints and serotype 3 from the lungs. Twenty-five isolates (12.1%) could not be typed. The most prevalent pathotype was epf + mrp + sly + luxS (49 isolates, 23.8%), and it was related mainly to serotypes 1 and 2. Serotypes 1-3 and 9 were significantly associated with anatomical sites of isolation and virulence factors, serotype 9 (CNS) and serotypes 3 and 9 (lungs) being associated with virulence profiles without the epf gene. S. suis isolates showed globally high antimicrobial resistances, but ampicillin followed by spectinomycin and tiamulin resulted in the highest activities, while the greatest resistances were detected for sulphadimethoxine, tetracyclines, neomycin, clindamycin and macrolides. A total of 87.4% isolates were positive to the tetO gene, 62.4% to the ermB gene and 25.2% to the fexA gene, while 14.6% were positive to all three genes simultaneously. A significative association between isolate resistances to tetracyclines and macrolides and the resistance genes tested was established, except for phenicol resistance and the fexA gene. A set of 14 multiresistance patterns were obtained according to the number of antimicrobials to which the isolates were resistant, the resistances to 12 or more agents being the most prevalent ones. A remarkable amount of multiresistance profiles could be seen among the S. suis serotype 9 isolates.

Immunohistochemical detection of p53 and pp53 Ser392 in canine hemangiomas and hemangiosarcomas located in the skin.

BACKGROUND: p53 protein is essential for the regulation of cell proliferation. Aberrant accumulation of it usually occurs in cutaneous malignancies. Mutant p53 is detected by immunohistochemistry because it is more stable than the wild-type p53. However, post-translational modifications of p53 in response to ultraviolet radiation are important mechanisms of wild-type p53 stabilization, leading to positive staining in the absence of mutation. The aims were: 1) to analyze the immunohistochemical expression of p53 and phospho-p53 Serine392 in canine skin endothelial tumours; and 2) to determine if any relationship exists between p53 and phospho-p53 Serine392 overexpression and cell proliferation. RESULTS: p53 and phospho-p53 Serine392 immunolabeling was examined in 40 canine cutaneous endothelial tumours (13 hemangiomas and 27 hemangiosarcomas). Their expression was associated with tumour size, hemangiosarcoma stage (dermal versus hypodermal), histological diagnosis and proliferative activity (mitotic count and Ki-67 index). Statistical analysis revealed a significant increase of p53 immunoreactivity in hemangiosarcomas (median, 74.61%; interquartile range [IQR], 66.97-82.98%) versus hemangiomas (median, 0%; IQR, 0-20.91%) (p < .001) and in well-differentiated hemangiosarcomas (median, 82.40%; IQR, 66.49-83.17%) versus hemangiomas (p = .002). Phospho-p53 Serine392 immunoreactivity was significantly higher in hemangiosarcomas (median, 53.80%; IQR, 0-69.50%) than in hemangiomas (median, 0%; IQR, 0.0%) (p < .001). Positive correlation of the overexpression of p53 and phospho-p53 Serine392 with mitotic count and Ki-67 index was found in the cutaneous vascular tumours (p < .001). The Ki-67 index of the hemangiomas (median, 0.50%; IQR, 0-2.80%) was significantly lower than that of the hemangiosarcomas (median, 34.85%; IQR, 23.88-42.33%) (p < .001), and that specifically of well-differentiated hemangiosarcomas (median, 24.60%; IQR, 15.45-39.35%) (p = .001). Immunolabeling of 18 visceral hemangiosarcomas showed that the p53 (median, 41.59%; IQR, 26.89-64.87%) and phospho-p53 Serine392 (median, 0%; IQR, 0-22.53%) indexes were significantly lower than those of skin (p = .001; p = .006, respectively). CONCLUSIONS: The p53 and phospho-p53 Serine392overexpression together with high proliferative activity in hemangiosarcomas versus hemangiomas indicated that p53 might play a role in the acquisition of malignant phenotypes in cutaneous endothelial neoplasms in dogs. The Ki-67 index may be useful in distinguishing canine well-differentiated hemangiosarcomas from hemangiomas.

Immunohistochemical Assessment of Immune Response in the Dermis of Sarcoptes scabiei-Infested Wild Carnivores (Wolf and Fox) and Ruminants (Chamois and Red Deer).

Sarcoptic mange is caused by the mite Sarcoptes scabiei and has been described in several species of domestic and wild mammals. Macroscopic lesions are predominantly hyperkeratotic (type I hypersensitivity) in fox, chamois and deer, but alopecic (type IV hypersensitivity) in wolf and some fox populations. To begin to understand the immune processes underlying these species differences in lesions, we examined skin biopsies from wolves (Canis lupus), foxes (Vulpes vulpes), chamois (Rupicapra rupicapra) and red deer (Cervus elaphus) naturally infested with S. scabiei. Twenty skin samples from five animals per species were used. Sections were immuno-stained with primary antibodies against Iba1 to detect macrophages, lambda chain to detect plasma cells, CD3 to detect T lymphocytes and CD20 to detect B lymphocytes. Skin lesions contained significantly more inflammatory cells in the fox than in the wolf and chamois. Macrophages were the most abundant inflammatory cells in the lesions of all the species studied, suggesting a predominantly innate, non-specific immune response. Lesions from the wolf contained higher proportions of macrophages than the other species, which may reflect a more effective response, leading to alopecic lesions. In red deer, macrophages were significantly more abundant than plasma cells, T lymphocytes and B lymphocytes, which were similarly abundant. The fox proportion of plasma cells was significantly higher than those of T and B lymphocytes. In chamois, T lymphocytes were more abundant than B lymphocytes and plasma cells, although the differences were significant only in the case of macrophages. These results suggest that all the species examined mount a predominantly innate immune response against S. scabiei infestation, while fox and chamois may also mount substantial humoral and cellular immune responses, respectively, with apparently scarce effectiveness that lead to hyperkeratotic lesions.

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.

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.

Acute phase protein concentrations in colostrum-deprived pigs immunized with subunit and commercial vaccines against Glässer's disease.

Haemophilus parasuis is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P<0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Glässer's disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectiveness.

Development and characterization of protective Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin and comparison with other subunit and commercial vaccines.

Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108 CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuis invasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Glässer's disease outbreaks caused by serovar 5.