Characterization of Memphis 37 strain of human respiratory syncytial virus in neonatal lambs

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. Many animal models including rodents and several other species have been utilized to study RSV, but most studies investigate disease in adult animals and fail to address the unique physiology and immunology that makes infants more susceptible to severe disease. Our group and others have used the perinatal lamb to model infant RSV disease as lambs have a similar pulmonary development and are susceptible to RSV. Lambs develop mild clinical symptoms including fever, tachypnea, and malaise as well as mild to moderate gross and histologic lesions when experimentally infected with bovine or human RSV; preterm lambs develop more severe disease with bRSV. The lamb model has a strong foundation and forms a valid model for moderate disease as evidenced by clinical alteration of respiration as well as gross and histologic lesions, but there is need for a model of more severe disease. Human RSV A2 strain replicates in lambs and causes disease but other strains may have enhanced virulence. Memphis 37 is an RSV-A strain isolated from a pediatric case and used in studies in human adult subjects. The overarching goal of the studies undertaken was to develop a model of enhanced RSV that could be utilized for investigating the pathogenesis of enhanced disease as well as therapeutic regimens. The central hypothesis for these studies was that Memphis 37 causes moderate to severe disease in neonatal lambs as measured by alterations in respiration as well as gross and histologic lesions. Memphis 37 (M37) strain hRSV caused moderate disease in perinatal lambs as measured by clinical disease (expiratory effort or tachypnea), post-mortem gross lesions, and histologic lesions with abundant RSV-antigen immunoreactivity as well as significant viral mRNA levels. Nebulized Memphis 37 hRSV caused similar disease in lambs to hRSV strain A2. Memphis 37 hRSV grown in HEp-2 cells induced increased expiratory effort and greater lesion scores (gross and histologically) as well as increased antigen immunoreactivity as compared to Vero-grown M37. Nebulization and intranasal inoculation are reproducible methods of inoculation that caused a unique distribution pattern of lesions when compared to each other or fiberoptic intrabronchial inoculation. In conclusion, Memphis 37 hRSV will cause disease in perinatal lambs and shows promise for use in a model of severe RSV disease

Innate Immunology of Bovine Respiratory Disease

Pneumonia is a leading cause of loss to the cattle industry in the United States and Europe. Of cattle diseases, it has the greatest economic impact. Respiratory pathogens can cause serious outbreaks of acute pneumonia in neonatal, weaned and growing calves. Chronic infection leads to debilitation, decreased performance, and culling in older animals. The means to enhance effective and non-injurious immune responses are needed because of the high incidence of pneumonia in cattle, ubiquity of respiratory pathogens, the increasing frequency of antibiotic resistance, and the general expectation by consumers for producers to use antibiotics less frequently. The lung has a wide array of both innate and adaptive immune responses to airborne particulates, vapors, and microbial pathogens. Vaccines can effectively enhance resistance to some pathogens, but not all. More recently, additional attention has been given to innate immune responses and method/regimens that increase innate immune activity. Despite advances in managerial practices, vaccines, and clinical therapies, pneumonia remains a widespread problem and methods to enhance host resistance to pathogen colonization and pneumonia are needed. There are a variety of factors and conditions that pre-dispose cattle to pneumonia. Cattle have anatomic and cellular differences from humans and other species and are managed in groups/ herds all of which increases susceptibility to microbial pathogens. This review highlights the basic innate immune response of the respiratory tract and newer developments in the understanding of adaptive immune responses of the bovine respiratory tract placing special emphasis on features unique to cattle

Clinical presentations and antimicrobial susceptibilities of Corynebacterium cystitidis associated with renal disease in four beef cattle

Background Renal disease caused by Corynebacterium cystitidis in beef cattle may be misclassified as Corynebacterium renale, and limited information about C. cystitidis infections in beef cattle currently is available. Objective To describe clinical presentation, diagnosis, minimum inhibitory concentrations (MICs), and outcome of renal disease caused by C. cystitidis in beef cattle. Methods Retrospective case series. Animals Four client-owned beef cattle. Results All affected cattle had anorexia as a primary complaint. Of the 3 that had ante-mortem diagnostic tests performed, all had pyelonephritis based on azotemia in combination with urinalysis and ultrasonographic findings. Cultures yielded C. cystitidis which was identified by biochemical testing, 16S RNA sequencing, and mass spectrometry. All affected cattle deteriorated despite aggressive treatment, indicating that C. cystitidis infections in beef cattle may carry a poor prognosis. Bacterial isolates collected from the 4 cattle showed similarities in MICs for ampicillin, florfenicol, gentamicin, neomycin, sulfadimethoxine, trimethoprim sulfonamide, and tylosin. Conclusions and clinical importance Corynebacterium cystitidis should be considered in the differential diagnosis of cattle with renal disease. Definitive diagnosis of C. cystitidis as compared to C. renale may be challenging

Human Respiratory Syncytial Virus Memphis 37 Grown in HEp-2 Cells Causes more Severe Disease in Lambs than Virus Grown in Vero Cells

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells

Increased Concentration of Iodide in Airway Secretions is Associated with Reduced RSV Disease Severity

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN−), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN− yields the bactericidal hypothiocyanite (OSCN−) in airway surface liquid (ASL). Although SCN− is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I−) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN−, inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I− concentration, and this I− concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses

Human Respiratory Syncytial Virus Memphis 37 Causes Acute Respiratory Disease in Perinatal Lamb Lung

Respiratory syncytial virus (RSV) is the leading cause of hospitalization due to respiratory illness among infants and young children of industrialized countries. There is a lack of understanding of the severe disease mechanisms as well as limited treatment options, none of which are fully satisfactory. This is partly due to lack of a relevant animal model of perinatal RSV infection that mimics moderate to severe disease in infants. We and others have shown mild disease in perinatal lambs with either a bovine or a human A2 strain of RSV. The Memphis 37 clinical strain of human RSV has been used to produce mild to moderate upper respiratory disease in healthy adult volunteers. We hypothesized that the Memphis 37 strain of RSV would infect perinatal lambs and produce clinical disease similar to that in human infants. Perinatal (3- to 5-day-old) lambs were inoculated intranasally with 2 mL/nostril of 1×105 focus-forming units (FFU)/mL (n=2) or 2.1×108 FFU/mL (n=3) of RSV Memphis 37. Clinical signs, gross and histological lesions, and immune and inflammatory responses were assessed. Memphis 37 caused moderate to severe gross and histologic lesions along with increased mRNA expression of macrophage inflammatory protein. Clinically, four of the five infected lambs had a mild to severe increase in expiratory effort. Intranasally administered RSV strain Memphis 37 infects neonatal lambs with gross, histologic, and immune responses similar to those observed in human infants

Effects of Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants

Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets

AbstractPorcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2009–2010 and reported in United States swine for the first time in February 2014. However, diagnostic tools other than polymerase chain reaction for PDCoV detection were lacking and Koch׳s postulates had not been fulfilled to confirm the pathogenic potential of PDCoV. In the present study, PDCoV peptide-specific rabbit antisera were developed and used in immunofluorescence and immunohistochemistry assays to assist PDCoV diagnostics. The pathogenicity and pathogenesis of PDCoV was investigated following orogastric inoculation of 5-day-old piglets with a plaque-purified PDCoV cell culture isolate (3×104 TCID50 per pig). The PDCoV-inoculated piglets developed mild to moderate diarrhea, shed increasing amount of virus in rectal swabs from 2 to 7 days post inoculation, and developed macroscopic and microscopic lesions in small intestines with viral antigen confirmed by immunohistochemistry staining. This study experimentally confirmed PDCoV pathogenicity and characterized PDCoV pathogenesis in neonatal piglets

Full-Length Genome Sequences of Senecavirus A from Recent Idiopathic Vesicular Disease Outbreaks in U.S. Swine

Since July 2015, vesicular lesions affecting growing pigs and sows accompanied with neonatal mortality have been reported in multiple U.S. states. Senecavirus A has been consistently detected from these cases. The complete genome sequences of 3 recent U.S. Senecavirus A isolates were determined to further characterize this virus

Characterizing the rapid spread of porcine epidemic diarrhea virus (PEDV) through an animal food manufacturing facility

New regulatory and consumer demands highlight the importance of animal feed as a part of our national food safety system. Porcine epidemic diarrhea virus (PEDV) is the first viral pathogen confirmed to be widely transmissible in animal food. Because the potential for viral contamination in animal food is not well characterized, the objectives of this study were to 1) observe the magnitude of virus contamination in an animal food manufacturing facility, and 2) investigate a proposed method, feed sequencing, to decrease virus decontamination on animal food-contact surfaces. A U.S. virulent PEDV isolate was used to inoculate 50 kg swine feed, which was mixed, conveyed, and discharged into bags using pilot-scale feed manufacturing equipment. Surfaces were swabbed and analyzed for the presence of PEDV RNA by quantitative real-time polymerase chain reaction (qPCR). Environmental swabs indicated complete contamination of animal food-contact surfaces (0/40 vs. 48/48, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P \u3c 0.05) and near complete contamination of non-animal food-contact surfaces (0/24 vs. 16/18, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P \u3c 0.05). Flushing animal food-contact surfaces with low-risk feed is commonly used to reduce cross-contamination in animal feed manufacturing. Thus, four subsequent 50 kg batches of virus-free swine feed were manufactured using the same system to test its impact on decontaminating animal food-contact surfaces. Even after 4 subsequent sequences, animal food-contact surfaces retained viral RNA (28/33 positive samples/total samples), with conveying system being more contaminated than the mixer. A bioassay to test infectivity of dust from animal food-contact surfaces failed to produce infectivity. This study demonstrates the potential widespread viral contamination of surfaces in an animal food manufacturing facility and the difficulty of removing contamination using conventional feed sequencing, which underscores the importance for preventing viruses from entering and contaminating such facilities