Mouse pneumonia model by Acinetobacter baumannii multidrug resistant strains: Comparison between intranasal inoculation, intratracheal instillation and oropharyngeal aspiration techniques

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation.

Tk-deleted pseudorabies virus retains high pathogenicity in rats

Introduction The pseudorabies virus (PRV) gene encoding thymidine kinase (tk) is an important virulence-associated factor. Attenuation of PRV in susceptible animals is a frequent result of tk deletion. The aim of the study was to assess the pathogenicity of tk-deleted PRV in rats. Material and Methods Sprague Dawley rats were infected with the tk-deleted PRV strain SuHV-1 ΔTK:247via intranasal or intramuscular inoculation. PRV loads in ten tissues from dead and euthanised rats were determined using real-time PCR. Results Infection with SuHV-1 ΔTK:247 could cause death in rats. The 50% lethal dose (LD50) of SuHV-1 ΔTK:247 via intranasal inoculation was 103.16 TCID50 in rats. Intramuscular inoculation required a higher dose of SuHV-1 ΔTK:247 (105.0 TCID50). A high SuHV-1 ΔTK:247 titre was observed in the trigeminal ganglia or spinal cord of dead rats. Conclusion The results of this study show that rats are highly susceptible to PRV infection, and tk deletion did not completely diminish the pathogenicity of PRV in rats.

A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

The objective of this study was to compare the virulence of three different porcine circovirus type 2 (PCV2) genotypes (PCV2a, PCV2b, and PCV2d) in pigs infected with either one of these three PCV2 genotypes versus pigs dually inoculated with Mycoplasma hyopneumoniae and PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by another intranasal inoculation at 6 weeks of age with one of three PCV2 genotypes. Dual infection with two pathogens produced moderate and severe dyspnea, lethargy, and reduced weight gain in pigs regardless of the PCV2 genotype evaluated compared with pigs only inoculated with PCV2. The overall levels of PCV2d viremia and severity of lymphoid lesions, and PCV2-antigen within lymphoid lesions were significantly higher in pigs dually inoculated with M. hyopneumoniae/PCV2d when compared with all other dually inoculated groups. The level of PCV2 viremia and the production of PCV2-associated lymphoid lesions did not differ significantly among PCV2a, PCV2b, and PCV2d single-inoculated pig groups. The results of this study demonstrated that M. hyopneumoniae potentiated the replication of PCV2d more than it did with the other PCV2 genotypes as measured by lymphoid lesion severity.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) – the SARS-CoV-2 receptor – shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus. IMPORTANCE Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) – the SARS-CoV-2 receptor – shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Intranasal Administration of ACIS KEPTIDE™ Prevents SARS-CoV2-Induced Acute Toxicity in K18-hACE2 Humanized Mouse Model of COVID-19: A Mechanistic Insight for the Prophylactic Role of KEPTIDE™ in COVID-19

Previously, we have demonstrated that ACIS KEPTIDE™, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE™ attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*106) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 μg/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities. In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo, significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice.HighlightsACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 & 8). Intranasal inoculation of SARS-CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6) and kidney tubular cells (Fig.8). ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8).ACIS KEPTIDE™ completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8).ACIS KEPTIDE™ improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult.Intranasal administration of 0.05% Beta-propiolactone (βPL)-inactivated SARS-CoV2 (1 *106) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation (Supplemental videos) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality.The peptide backbone of ACIS KEPTIDE™ provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8). Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8) of SARS-CoV2-treated K18-hACE2 mice.