Bovine mastitis, an evolving disease: Application of infrared thermography for the diagnosis of subclinical forms and presence of methicillin resistant Stahpylococcus aureus in dairy farms

Subclinical mastitis in dairy cows is a big economic loss for farmers. The monitoring of subclinical mastitis is usually performed through Somatic Cell Count (SCC) in farm but there is the need of new diagnostic systems able to quickly identify cows affected by subclinical infections of the udder. In the last years the fight against mastitis has seen the emergence of mastitis cases caused by multiresistant bacteria which poorly respond to antibiotic treatments. One of the most important pathogens because of this aspects of antimicrobial resistance and for its potential as zoonotic pathogen is the Methicillin Resistant Staphylococcus aureus (MRSA). The aim of this thesis is to investigate two important aspects of bovine mastitis, in a first study we evaluated the potential of infrared thermography in the diagnosis of subclinical mastitis and in a second study it was investigated the presence and the prevalence of MRSA in the territory of the Veneto Region. In the first study we evaluated the udder health status of 98 Holstein Friesian dairy cows with high SCC in 4 farms. From each cow a sample of milk was collected from all the functional quarters and submitted to bacteriological culture, SCC and Mycoplasma spp. culture. Thermographic images were taken from each functional udder quarter and nipple. Pearson’s correlations and Analysis of Variance were performed in order to evaluate the different diagnostic techniques. The most frequent pathogen isolated was Staphylococcus aureus followed by Coagulase Negative Staphylococci (CNS), Streptococcus uberis, Streptococcus agalactiae and others. The Somatic Cell Score (SCS) was able to discriminate (p<0.05) cows positive for a pathogen from cows negative at the bacteriological culture except for cows with infection caused by CNS. Infrared thermography was correlated to SCS (p<0.05) but was not able to discriminate between positive and negative cows. Thermographic imaging seems to be promising in evaluating the inflammation status of cows affected by subclinical mastitis but seems to have a poor diagnostic value. In the second study we investigated the presence and prevalence of MRSA in the Veneto Region. In order to do that 70 farms holding at least 100 animals were selected. In each farm were collected composite milk samples and nasal swabs from lactating cows. The samples collected were submitted for specific bacteriological culture for MRSA. The isolated strains were characterized to discover the presence of genes codifying for the resistance to several antimicrobial classes and to evaluate the epidemiological characteristics. 15 MRSA strains were isolated from 6 farms. 13 from milk samples and 2 from nasal swabs. The prevalence found was similar to what obtained in previous studies conducted in other European countries. The prevalence inside the farms was low and no farm was simultaneously positive for MRSA in milk and in nasal swabs. The low prevalence of colonization of the upper respiratory tract in somewhat surprising, considered the high prevalence of colonization in veal calves farms, in which are raised male calves coming from dairy farms. The genetic characterization has determined the belonging of the isolated strains to well known clones (CC1, CC97, CC398) adapted to livestock (Livestock Associated MRSA) and different from the more dangerous strains associated to healthcare environment (Hospital Associated MRSA). The clones found has anyway already proven their zoonotic potential and represent not only a new challenge in the fight against bovine mastitis but also a risk for the exposed personnel

Survey of Methicillin-resistant coagulase positive Staphylococcus spp. carriage in healthy dogs and dogs with skin disease

Canine skin diseases (SD) are very common in the veterinary practice and are often complicated by recurrent bacterial infection. Affected dogs usually need multiple cycles of antibiotic treatments (AT) that can lead to development of multidrug resistant strains [1]. Coagulase-positive staphylococci (CPS) are the most often isolated pathogens from canine SD. The aim of this study is to assess the prevalence of multidrug and methicillin resistant coagulase-positive staphylococci (MRScp) isolated from healthy dogs and dogs with SD, and to evaluate the correlation with clinical scores and previous AT. Forty-nine dogs were enrolled: 25 healthy and 24 with SD. Clinical history and previous AT were recorded. After a complete physical examination, clinical scores (CADESI-3 and pruritus) were calculated. Skin swabs from mouth, ear, genitalia, axilla and skin lesions, when present, were cultured in a nutrient and in a selective medium for MRScp. Suspected Staphylococcus colonies were identified by Maldi-Tof MS and specific PCR; methicillin resistance was confirmed by a PCR targeting mecA gene. Susceptibility tests and genetic typing, including spa-typing, SCCmec-typing and MLST were performed on isolates. Normal distribution of data was tested with Shapiro-Wilk test. Data were analyzed using ANOVA and z-test if normally distributed, otherwise with Mann-Whitney Test. Correlations between bacterial resistance and clinical scores or previous AT were assessed by Spearman test. P-value <0.05 was considered significant. Ninety-five strains of CPS were isolated from 229 samples. A total of 13/95 strains were MRScp and were identified as Staphylococcus pseudintermedius. Among them, 10 were multidrug resistant and two were isolated from healthy dogs. The Sequence Type 71, spa-type t02 e SCCmec type II-III, which represents the main clonal strain in Europe [2], was the most frequently identified genetic type (11/13) also in this study. Staphylococci were more commonly isolated from axilla, genitalia and ear conduct of dogs with SD compared with healthy dogs (p<0.001). Four out of the 6 MRScp positive dogs had received AT in the previous 6 months. No significant correlations between clinical scores or previous AT and methicillin resistance was found. Although the low number of dogs included in the study could have affected the results of the investigated correlations, this study confirms the role of Staphylococcus pseudintermedius in canine pyoderma and shows that pet dogs may play a significant role as MRScp carriers. Furthermore, close attention should be also paid also to the control of healthy dogs

Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV's major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered

Protective Efficacy of H9N2 Avian Influenza Vaccines Inactivated by Ionizing Radiation Methods Administered by the Parenteral or Mucosal Routes

H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection

Virological and immunological features of SARS-CoV-2-infected children who develop neutralizing antibodies

As the global COVID-19 pandemic progresses, it is paramount to gain knowledge on adaptive immunity to SARS-CoV-2 in children to define immune correlates of protection upon immunization or infection. We analyzed anti-SARS-CoV-2 antibodies and their neutralizing activity (PRNT) in 66 COVID-19-infected children at 7 (\ub12) days after symptom onset. Individuals with specific humoral responses presented faster virus clearance and lower viral load associated with a reduced in&nbsp;vitro infectivity. We demonstrated that the frequencies of SARS-CoV-2-specific CD4+CD40L+ T&nbsp;cells and Spike-specific B cells were associated with the anti-SARS-CoV-2 antibodies and the magnitude of neutralizing activity. The plasma proteome confirmed the association between cellular and humoral SARS-CoV-2 immunity, and PRNT+ patients show higher viral signal transduction molecules (SLAMF1, CD244, CLEC4G). This work sheds lights on cellular and humoral anti-SARS-CoV-2 responses in children, which may drive future vaccination trial endpoints and quarantine measures policies

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission