Oral Swabs Research Articles

Active surveillance of antimicrobial resistance in companion animals: A pilot study in a Spanish Veterinary Teaching Hospital

The role of small animal veterinary hospitals in the onset and dissemination of antimicrobial-resistant organisms (AMROs) is still not clear, and the implementation of an internal surveillance systems is a cost-effective tool to better understand their impact. The aim of this study was to describe a pilot program of active surveillance in a Spanish Veterinary Teaching Hospital, developed to estimate the detection frequency of AMROs in the commensal flora of patients and in the environment. Surveillance was focused on Methicillin-resistant Staphylococci (MRS), third generation cephalosporins resistant gram-negative bacteria (3GCR-GNB), and carbapenems-resistant gram-negative bacteria (CR-GNB). Oral and perirectal swabs were collected in the same dogs and cats hospitalized > 48 h, at their admission and before their discharge. Out of 50 patients sampled, 24% (12/50) were carriers at admission of at least one of the three investigated AMROs. Twenty-eight percent of patients (14/50) acquired at least one AMRO during the hospital stay. MRS detection frequency at admission was 12% (6/50), while acquisition was 6% (3/50). 3GCR-GNB detection frequency was 14% at admission (7/50) and acquisition 22% (11/50), while CR-GNB detection frequency was 2% at admission (1/50) and acquisition 2% (1/50). Environmental surveillance (98 samples) showed a total detection frequency of 22.4% for MRS (22/98), 2% for 3GCR-GNB and CR-GNB (2/98). Clinical staff’ shoe soles showed high detection frequency for MRS (50%). 3GCR Escherichia coli was the most isolated species in patients (n = 17). The results show how active surveillance can be used as a tool to assess the impact of AMROs in veterinary hospitals to subsequently build up tailored control plans based on specific issues.

Molecular epidemiology and temporal dynamic of SARS-CoV-2 imported from Hong Kong to mainland China

Tracking the genome diversity and evolution of virus like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the hotspot of current researches, which were essential for the developing of prevention strategies and therapeutic vaccines. In the current study, a total of 206073 oral or nasopharyngeal swab specimens were collected in 2022 from entry populations originated from Hong Kong, among which 141 whole genome sequences were obtained through next-generation sequencing and analyzed. Two major Omicron sub-lineages were identified: BA.2.2 (70.21%, 99/141) and BA.5 (24.11% 34/141). An average of 53.22 mutations was emerged in the genome, with S protein and ORF1a harboring the highest number of mutations among structure proteins and non-structure proteins (p<0.001), respectively, making them the most variable regions. The temporal changes of the mutations among each protein were unique, indicating the distinct evolution pattern of these proteins. A novel mutation in the Nsp5 (3 C-like proteinase) was reported for the first time. As it may influence the viral replication and transcription, further monitoring is needed. The data reported here highlighted the importance of monitoring the SARS-CoV-2 genome and would be useful for the generation of evidence-based prevention programs and effective vaccines to COVID-19.

Association of Helicobacter Pylori in Chronic Tonsillitis.

The objective of the study was to detect the presence of Helicobacter pylori in adenotonsillar tissue and to assess the association between the presence of H pylori with Chronic Tonsillitis or Adenotonsillitis. This was a cross sectional study conducted among 60 patients diagnosed with chronic tonsillitis and adeno tonsillitis undergoing tonsillectomy or adeno tonsillectomy meeting the paradise criteria in a tertiary care hospital, Pondicherry. Rapid urease test was done in the intraoperative period immediately after the specimens were taken.The rapid urease test kit was observed for color change from yellow to pink within 4h. The tissue was sent for histopathological examination for staining with H&E and Giemsa stain to detect the presence of helicobacter pylori. The mean age of the study participants was 15.75 ± 8.46 and majority of the study participants were females. (61.7%). 66.7% and 33.3% of the study participants had chronic tonsillitis and adeno tonsillitis respectively. Oral swab showed normal flora, yeast and methicillin resistant Staphylococcus aureus in 96.6%, 1.7% and 1.7% respectively. 1.7% (1),13.3% (8) and 20% (12) of the study participants showed positive in card test immediate, at 15min and 1h respectively. Giemsa stain showed that 11.7% was positive for H.pylori. Out of the 7 patients with positive Giemsa stain, 4 had chronic tonsillitis and 3 had adenotonsillitis. Colonisation of adenoids and tonsils byH.pyloriis a novel forefront with contradictory results dependent on the precision of the detective techniques used and population studied. Further research may be warranted to establish the varied colonisation depending on the geographical locations.

Use of the Investigator ESSplex SE QS Kit (QIAGEN) at Half PCR Reaction Volumes for the Analysis of Forensic Samples

The Investigator ESSplex SE QS Kit (Qiagen) is a next-generation polymerase chain reaction (PCR) kit that, in 60 min, amplifies 17 Short Tandem Repeat (STR) markers, including the five European Standard Set (ESS) loci (D10S1248, D12S391, D1S1656, D22S1045, D2S441), the SE33 marker, and the locus Amelogenin for sex determination. Two quality sensors (QS1 and QS2) are also co-amplified to check PCR performance. Since forensic laboratories carry out hundreds of DNA typings annually, we verified the kit’s performance using half reaction volumes with the aim of improving the number of samples that may be amplified with a single kit and consequently reducing laboratory costs. In the present study, intended as a technical note rather than internal validation, some control samples (oral swabs) with known DNA profiles and 40 real casework samples were analyzed. We observed that reducing the total reaction volume, while keeping all component ratios unaltered, yields DNA profiles comparable to those obtained using standard reaction volumes and with allele peaks higher than those with regular volumes. Using half volumes for PCR amplification enables the analysis of a larger number of samples compared to the standard protocol, thereby reducing laboratory costs without compromising the quality of the analysis.

SARS-CoV-2 presence in recreational seawater and evaluation of intestine permeability: experimental evidence of low impact on public health.

Coastal seawater pollution poses a public health risk due to the potential ingestion of contaminated water during recreational activities. Wastewater-based epidemiology has revealed the abundant presence of SARS-CoV-2 in seawater emitted from wastewater outlets. The objective of this research was to investigate the impact of seawater on SARS-CoV-2 infectivity to assess the safety of recreational activities in seawater. Wild SARS-CoV-2 was collected from oral swabs of COVID-19 affected patients and incubated for up to 90 min using the following solutions: (a) standard physiological solution (control), (b) reconstructed seawater (3.5% NaCl), and (c) authentic seawater (3.8%). Samples were then exposed to two different host systems: (a) Vero E6 cells expressing the ACE2 SARS-CoV-2 receptor and (b) 3D multi-tissue organoids reconstructing the human intestine. The presence of intracellular virus inside the host systems was determined using plaque assay, quantitative real-time PCR (qPCR), and transmission electron microscopy. Ultrastructural examination of Vero E6 cells revealed the presence of virus particles at the cell surface and in replicative compartments inside cells treated with seawater and/or reconstituted water only for samples incubated up to 2 min. After a 90-min incubation, the presence of the virus and its infectivity in Vero E6 cells was reduced by 90%. Ultrastructural analysis performed in 3D epi-intestinal tissue did not reveal intact viral particles or infection signs, despite the presence of viral nucleic acid detected by qPCR. Indeed, viral genes (Orf1ab and N) were found in the intestinal luminal epithelium but not in the enteric capillaries. These findings suggest that the intestinal tissue is not a preferential entry site for SARS-CoV-2 in the human body. Additionally, the presence of hypertonic saline solution did not increase the susceptibility of the intestinal epithelium to virus penetration; rather, it neutralized its infectivity. Our results indicate that engaging in recreational activities in a seawater environment does not pose a significant risk for COVID-19 infection, despite the possible presence of viral nucleic acid deriving from degraded and fragmented viruses.

Examination of Oral Candida Infection in Primary Sjögren's Syndrome Patients

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due to the hyposalivation experienced by pSS patients, oral dysbacteriosis often occurs. A common complication of pSS is the oral Candida infection. In this article, the authors describe systematic methods that can effectively diagnose oral Candida infection and identify the Candida strains using saliva, oral mucosal swabs, or mouthwash from pSS patients. The Sabouraud's Dextrose Agar (SDA), hyphal formation assay, potassium hydroxide (KOH) smear test, and calcofluor white (CFW) staining assay are used for the diagnosis of oral Candida infection. A Candida diagnostic agar is used for the identification of Candida strains. Finally, antifungal susceptibility testing is used to determine appropriate antifungal drug treatment. This standardized method can enhance the diagnosis, treatment, and future research of pSS-related oral Candida infections. Early diagnosis, using this method, can also prevent any complications arising due to delay in receiving appropriate treatment.

Newer insights of H1N1: Swine Flu Virus

Swine flu, caused by the H1N1 influenza virus, is a subtype of influenza A that affects both the upper and lower respiratory tracts. It is primarily found in pigs and can be transmitted to humans through genetic variations in the virus. The 1918 Spanish flu pandemic resulted in the deaths of 50 to 100 million individuals. In 2009, the pandemic affected 178 countries, resulting in an estimated 43 to 89 million cases and 1799 deaths. The pathophysiology of H1N1 involves inflammation of the respiratory tract, with an incubation period of 1 to 4 days and a contagious period lasting 5 to 7 days. The signs and symptoms of swine flu include cough, sore throat, fever, myalgia, congestion, headache, rhinorrhoea, dizziness, sneezing, loss of appetite, fatigue, abdominal pain, shortness of breath, and in rare cases, vomiting and diarrhoea. The most common cause of death is respiratory failure, and neurological symptoms can occur due to high fever. To diagnose swine flu, various tests such as haematological, biochemical, and microbiological tests are conducted, including the collection of nasal or oral swabs for reverse transcriptase polymerase chain reaction (RT-PCR). Prevention and control measures include managing swine flu in pigs through herd management, hygiene practices, and vaccination. Treatment options vary based on the severity of the case. Mild to moderate cases can be managed with rest, antipyretics, NSAIDs, antihistamines, and oral rehydration therapy. Severe cases may require intravenous hydration, antibiotics for bacterial infections, antiviral therapy, and respiratory support.

Human buccal epithelial cells as a model system for molecular analysis of DNA, RNA and protein

We report use of human buccal epithelial cells as an easy model system for isolation and molecular analysis of genomic DNA, RNA, and protein to study any gene of interest by Polymerase Chain Reaction (PCR), RNA expression by Reverse Transcription-PCR (RT-PCR), protein-profiling, and expression by western blot as well as DNA-methylation by Msp I/Hpa II-restriction digestion. We used simple methods to isolate genomic DNA, RNA and protein from human buccal cells collected by oral swab and cultured them in-vitro. The microscopic observation of haematoxylin and eosin (EA-50) stained cells, genomic PCR of house-keeping genes (GAPDH and β-actin), RT-PCR of GAPDH and β-actin mRNAs and whole cell protein-profiling by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) were carried out. Expression of β-actin protein in supernatant and pellet fractions of the cells was determined by western blot analysis. MTT-assay was carried out to assess the cell viability and cell growth. Green Fluorescence Protein (GFP)-DNA was expressed in these cells by transient transfection. DNA-methylation in the genome was analyzed by Msp I/ Hpa II restriction digestion and agarose gel electrophoresis. Thus these methods can be used for molecular analysis of DNA, RNA and protein from the human buccal epithelial cells for studying and monitoring health, disease, population genetics/genomics and epidemiology under different conditions.

Isolation and diagnosis of Streptococcus mutans and Streptococcus sorbinus from patients with tooth decay and gingivitis

The Streptococcus bacteria Streptococcus sobrinus and Streptococcus mutans are one of the most common types in the mouth, where tooth decay, necrosis and gingivitis are among the health problems that the Iraqi society suffers from, as the treatment of bacteria has become a challenge due to their ability to resist many antibiotics. The study aimed to isolate and diagnose Streptococcus mutans and Streptococcus sobrinus and study their resistance to a number of antibiotics, and evaluate the susceptibility of Streptococcus mutans and Streptococcus sobrinus isolates in biofilms. Using biochemical tests and phenotypic diagnostics. The study included the collection of (275) oral swabs from patients with tooth decay from the area of caries on the surface of the tooth and from cysts for infected patients and visitors at the Medical City Hospital in Baghdad Governorate with ages ranging between (6-70) years and of both sexes for the period from 1/9/2022 to 1/11/2022. the number of isolates was 6 (2.18%) belonging to Streptococcus mutans and only one isolate (0.36%) of Streptococcus sobrinus, the sensitivity of all the isolates of the bacteria under study was tested towards 10 antibiotics, and the results showed that the isolates were resistant to most of the antibiotics used in this study, and all the isolates were chosen to test their ability to form biofilms using the microtitration plate, and the results showed that most of the isolates were strong in the formation of biofilms.

Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics

Molecular diagnostics is an increasingly important clinical tool, especially in routine sampling. We evaluated two non-invasive methods (oral swabs and mouthwashes) for sampling nucleic acids from the oral/pharyngeal area. We created a workflow from sample collection (n = 59) to RT-qPCR based analysis. The samples were further characterized in terms of their cellular composition as well as the purity, degradation and microbial content of the derived DNA/RNA. We determined the optimal housekeeping genes applicable for these types of samples. The cellular composition indicated that mouthwashes contained more immune cells and bacteria. Even though the protocol was not specifically optimized to extract bacterial RNA it was possible to derive microbial RNA, from both sampling methods. Optimizing the protocol allowed us to generate stable quantities of DNA/RNA. DNA/RNA purity parameters were not significantly different between the two sampling methods. Even though integrity analysis demonstrated a high level of degradation of RNA, corresponding parameters confirmed their sequencing potential. RT-qPCR analysis determined TATA-Box Binding Protein as the most favorable housekeeping gene. In summary, we have developed a robust method suitable for multiple downstream diagnostic techniques. This protocol can be used as a foundation for further research endeavors focusing on developing molecular diagnostics for the oropharyngeal cavity.

Comparative evaluation of assay performance for SARS-CoV-2 detection in animal oral samples, lung homogenates, and phosphate-buffered saline using the TaqPath COVID-19 Combo kit.

A One Health approach has been key to monitoring the COVID-19 pandemic, as human and veterinary medical professionals jointly met the demands for an extraordinary testing effort for SARS-CoV-2. Veterinary diagnostic laboratories continue to monitor SARS-CoV-2 infection in animals, furthering the understanding of zoonotic transmission dynamics between humans and animals. A RT-PCR assay is a primary animal screening tool established within validation and verification guidelines provided by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), World Organisation for Animal Health (WOAH), and the U.S. Food and Drug Administration (FDA). However, differences in sample matrices, RNA extraction methods, instrument platforms, gene targets, and cutoff values may affect test outcomes. Therefore, targeted validation for a new sample matrix used in any PCR assay is critical. We evaluated a COVID-19 assay for the detection of SARS-CoV-2 in feline and canine lung homogenates and oral swab samples. We used the commercial Applied Biosystems MagMAX Viral/Pathogen II (MVP II) nucleic acid isolation kit and TaqPath COVID-19 Combo kit, which are validated for a variety of human samples, including nasopharyngeal and oropharyngeal swab samples. Our masked test showed a high detection rate and no false-positive or false-negative results, supporting sample extension to include feline oral swab samples. Our study is a prime example of One Health, illustrating how a COVID-19 assay designed for human testing can be adapted and used to detect SARS-CoV-2 in oral swab samples from cats and likely dogs, but not lung homogenates.

Clinical epidemiology, determinants, and outcomes of viral encephalitis in Ghana; a cross-sectional study.

Viral encephalitis is a rare, yet severe neurological disorder. It poses a significant public health threat due to its high morbidity and mortality. Despite the disproportionate burden of the disease in impoverished African countries, the true extent of the problem remains elusive due to the scarcity of accurate diagnostic methods. The absence of timely and effective diagnostic tools, particularly Real-time Polymerase Chain Reaction, has led to misguided treatment, and an underestimation of the disease burden in Ghana. We conducted a prospective cross-sectional study to determine the viral aetiologies of encephalitis among patients presenting to a major referral hospital in Ghana from May 2019 and August 2022. The study aimed at providing a comprehensive information on the clinical epidemiology, and outcomes of viral encephalitis in Ghana. Clinical samples were collected from patients presenting with signs and symptoms of encephalitis and tested for viral agents using real-time polymerase chain reaction. We assessed the clinical epidemiology, risk factors and outcome of individuals using descriptive and logistic regression analysis. Seventy-seven (77) patients were enrolled unto the study. The participants frequently presented with fever (85.7%), seizures (80.5%), lethargy (64.9%) and headache (50.6%). Viruses were detected in 40.3% of the study participants in either cerebrospinal fluid, rectal or oral swab samples. The most frequently detected viruses were cytomegalovirus (48.4%), enteroviruses (38.7%) and HSV (29.0%). Twenty-one (27.3%) of the patients died while on hospital admission. Gender (OR = 5.70 (1.536-1.172), p = 0.01), and negative polymerase chain reaction test results were identified as significant factors associated with death. Antiviral treatment increased the chance of survival of viral encephalitis patients by 21.8%. Our results validate the crucial role of molecular tools as essential for the rapid diagnosis of viral encephalitis, enabling effective treatment and improved patient outcomes. This study contributes valuable epidemiological and clinical insight into viral encephalitis in Ghana.

Utility of Oral Swab in the Diagnosis of Pulmonary Tuberculosis

Utility of Oral Swab in the Diagnosis of Pulmonary Tuberculosis

Novel herpesvirus in the critically endangered Galapagos pink land iguana

Virus surveillance in wildlife is important to understanding ecosystem health, taxonomy, and evolution. Nevertheless, viruses in reptiles, and specifically in squamates, continue to be understudied. Herein, we conducted a health assessment on the critically endangered Galapagos pink land iguana (Conolophus marthae) and the vulnerable Galapagos land iguana (Conolophus subcristatus). We collected oral and/or cloacal swabs from 47 clinically healthy iguanas and tested for adenovirus (cloacal swabs, n = 47) and herpesvirus (oral swabs, n = 45) using broad-spectrum PCRs. Two out of 38 (5.3 %) Galapagos pink land iguanas tested positive for herpesvirus, while no herpesvirus was detected in all Galapagos land iguanas (n = 7). Both herpesviral sequences were identical between them and divergent (61.9 % amino acid identity) when compared to the closest herpesvirus sequences available in GenBank/EMBL/DDBJ. The genetic distance between this and other herpesviruses is consistent with its classification as a novel virus species. All iguanas were negative for adenovirus. This is the first description of a herpesvirus in iguanas of the Galapagos islands, and the first report of a potential pathogen for the iconic Galapagos pink land iguana. Further research is needed to understand the implications of this virus in the conservation and management of one of the most endangered iguana species in the world.

Change of epidemiological characteristics of four respiratory viral infections in children before and during COVID-19 pandemic

ObjectivesViruses are the main infectious agents of acute respiratory infections (ARIs) in children. We aim to describe the changes in epidemic characteristics of viral ARIs in outpatient children before and during the COVID-19 pandemic. Patients and methodsFrom 2017 to 2022, the results of viral detection in oral pharyngeal swabs in 479,236 children with ARIs in the outpatient department of Children’s Hospital, Zhejiang University School of Medicine, were retrospectively analyzed. Viral antigens, including adenovirus (ADV), influenza A (FLUA), influenza B (FLUB) and respiratory syncytial virus (RSV) were detected by the colloidal gold method. ResultsThe median age was 3.4 (1.6–5.6) years. Among all the children, 159,895 cases (33.4 %) were positive for at least one virus. The total positive rate for ADV, FLUA and FLUB during the pandemic period was lower than during the pre-pandemic period in every season (pre-pandemic period vs. pandemic period11.7 % vs. 4.7 %, 13.9 % vs. 9.2 %, 7.0 % vs. 5.2 %, respectively, with overall p value < 0.001). However, the positive rate fir RSV was not significantly different between the pre-pandemic period and the pandemic period (5.6 % vs. 5.8 %, p = 0.117). Atypical timing of RSV (summer-autumn 2021) and FLUA (summer 2022) was noted. ConclusionsPublic health interventions for different pathogens are maximally effective. While positive rates for ADV, FLUA and FLUB decreased during the COVID-19 pandemic period, positive rates for RSV remained similar. In RSV and FLUA, off-season outbreaks were observed. Measures need to be taken to protect children from possible infection surges due to immunity debt having accrued over the last three years.

Can medication-related osteonecrosis of the jaw be attributed to specific microorganisms through oral microbiota analyses? A preliminary study

BackgroundMedication-related osteonecrosis of the jaw (MRONJ) can cause significant pain and loss of aesthetics and function if not treated properly. However, diagnosis still relies on detailed intraoral examinations and imaging. Prognosis varies even among patients with similar stages or conditions of MRONJ, emphasizing the need for a deeper understanding of its complex mechanisms. Thus, this study aimed to identify the oral microbiota of patients with MRONJ.MethodsThis single-center prospective cohort study included patients with confirmed MRONJ who visited the Department of Oral and Maxillofacial Surgery at Yonsei University Dental Hospital between 2021 and 2022. Oral swab samples were collected from the affected and unaffected sides of each patient. The composition and enumeration of the microbial communities were analyzed, and the diversity was compared to verify ecological changes in the groups using a next-generation sequencing-based 16S metagenomic analysis. A statistical analysis was performed using Wilcoxon signed-rank test with SPSS version 22, and values of P less than 0.05 were considered statistically significant.ResultsThe final study sample included 12 patients. The mean age was 82.67 ± 5.73 (range, 72–90) years. Changes in microbial composition were observed at different taxonomic levels (phylum, genus, and species). The identified microorganisms were commonly associated with periodontitis, gingival disease, and endodontic infection, suggesting a multifactorial etiology of MRONJ.ConclusionsAlthough this study is based on a small number of cases, it shows that MRONJ is not caused by a specific microorganism but can rather be caused by a variety of factors. By addressing these findings in large-scale studies, the significance of oral microbiome in pathogenesis can be further elucidated and can facilitate the development of effective therapeutic interventions for patients with MRONJ.

Comparative efficacy of Moringa oleifera and coconut oil-based mouthwashes versus chlorhexidine in reducing human oral microbial populations in healthy adults: a single blind clinical trial

IntroductionDentists have considered chlorhexidine as the gold standard antimicrobial solution against oral microbes for decades. The side effects of chlorhexidine and our historical reliance on natural remedies create the need for herbal alternative. This study evaluated the effectiveness of chlorhexidine, coconut oil, and moringa on the prevalence of human oral bacteria using a single-blind, randomised, clinical trial method. MethodsA total of 90 oral swabs were collected from consenting human participants on day 0 of the clinical trial. The participants were given either chlorhexidine (group A), coconut oil (group B), or Moringa oleifera (group C) mouthwashes and instructed to rinse for 14 days, twice daily. The participants were sampled again on day 14. Total plate count (TPC) and differential plate count (DPC) were done on both samples of each participant collected on day 0 and day 14. Bacteria were identified by standard biochemical tests. ResultsStreptococcus mitis, Veillonella parvula, Klebsiella aerogenes, Streptococcus mutans, Bacteroides fragilis, Aggregatibacter actinomycetecomitans, Streptococcus pneumoniae, and Staphylococcus aureus were isolated from the plaque. All 3 types of mouthwash exhibited statistically significant differences in TPC and DPC of day 0 and day 14 when compared using a paired t-test (P value = 0.03, 0.01, and <0.01 respectively). No statistically significant difference was noted in the antimicrobial activity of the three mouthwashes when compared using one-way ANOVA, post-hoc Tukey’s test. ConclusionOur results show that M. oleifera and coconut oil have a bactericidal effect on oral microbial populations associated with caries and periodontal disease, comparable to chlorhexidine.

Performance Evaluation of the STANDARD i-Q COVID-19 Ag Test with Nasal and Oral Swab Specimens from Symptomatic Patients.

We evaluated the diagnostic performance of the STANDARD i-Q COVID-19 Ag Test, which was developed to detect viral antigens, using nasal and oral swabs. Sixty positive and 100 negative samples were analyzed. We determined the distribution of the Ct values according to the day of sample collection after symptom onset, the diagnostic performance of the total samples and subgroups separated by Ct value or time of sample collection, and the Ct value at which maximal accuracy was expected. No differences were observed in Ct values, except for the samples obtained on the day of symptom onset. The diagnostic sensitivity and specificity of the oral swabs were 75.0 and 100.0%, respectively, whereas those of the nasal swabs were 85.0 and 98.0%, respectively. The sensitivity was higher in samples with a high viral load collected earlier than those collected later, although the difference was not significant. False-negative results were confirmed in all samples with a Ct value ≥ 30.0. These results indicate that tests using oral and nasal swabs are helpful for diagnosing acute symptomatic cases with suspected high viral loads. Our tests exhibited relatively low sensitivity but high specificity rates, indicating the need to assess negative antigen test results.

Inefficient Transmission of African Swine Fever Virus to Sentinel Pigs from an Environment Contaminated by ASFV-Infected Pigs under Experimental Conditions

Knowledge about African swine fever virus (ASFV) transmission and its survival in the environment is mandatory to develop rational control strategies and combat this serious disease in pigs. In this study, the risk that environmental contamination poses for infection of naïve pigs was investigated. Naïve pigs were introduced as sentinels into contaminated pens kept at ambient temperature (about 18–22˚C) either on the same day or up to 3 days after ASFV-infected pigs were removed. Three experiments were carried out in which four to six pigs per pen were inoculated with virulent ASFV isolates OURT88/1 (genotype I), Georgia 2007/1, or POL/2015/Podlaskie (genotype II), respectively. The majority of the inoculated pigs developed acute disease but with no evident haemorrhagic lesions or haemorrhagic diarrhoea and were culled at the predefined humane endpoint. The levels of ASFV DNA detected in the blood of the infected animals reached 107−9 genome copies/ml before euthanasia. Environmental swabs were taken from different surfaces in the animal rooms, as well as from faeces and urine, close to the time of introduction of the naïve animals. Relatively low quantities of virus DNA were detected in the environmental samples, in the range of 103−7 genome copies per swab or per gram and ml of faeces and urine. No infectious virus was recovered from these environmental samples. Neither clinical signs nor virus genomes were detected in the blood of any of the sentinel pigs over a period of 2 to 3 weeks after exposure, indicating that transmission from the ASFV-contaminated environment did not occur. Interestingly, viral DNA was detected in nasal and oral swabs from some of the sentinel animals at early days of exposure (ranging between 103.7−5.8 genome copies per swab), though none of them developed ASF. The results indicate a relatively low risk of ASFV transmission from a contaminated environment under the conditions provided in these experimental studies and in the absence of bloodshed from infected animals.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.