High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations.

Reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (FRET-PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (A23403G in the spike protein; C14408T in the RNA-dependent RNA polymerase). Based on high-resolution melting curve analysis, the reverse transcription (RT) FRET-PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET-PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

Central Nervous System B-cell Lymphoma in a Bald Eagle ( Haliaeetus leucocephalus).

An adult bald eagle ( Haliaeetus leucocephalus) presented for nystagmus and an inability to fly. On physical examination, the eagle was open-mouth breathing and tachycardic at 200 beats per minute, had a wrinkled cere and sunken eyes, and was an estimated 10% dehydrated. Additionally, the eagle was extremely weak, with neurologic abnormalities including bilateral proprioceptive deficits, nystagmus, and no pupillary light reflex in the left eye. Despite aggressive treatment, the eagle continued to decline rapidly and subsequently died. On histologic examination, diffuse and widespread infiltration of neoplastic lymphocytes was present in the brain, optic nerves, and pecten. Immunohistochemical PAX-5 labeling confirmed B-cell lymphoma confined to the eye and nervous system. Test results for select avian retroviruses, Marek's disease, West Nile virus, avian influenza viruses, and Mycoplasma were negative. To our knowledge, this is the first report of B-cell lymphoma in a bald eagle. Although rare, this condition is a differential diagnosis in cases of neurologic or ocular diseases in birds.

Experimental infection of pregnant goats with bovine viral diarrhea virus (BVDV) 1 or 2.

Infections with bovine viral diarrhea virus (BVDV) of the genus pestivirus, family Flaviviridae, are not limited to cattle but occur in various artiodactyls. Persistently infected (PI) cattle are the main source of BVDV. Persistent infections also occur in heterologous hosts such as sheep and deer. BVDV infections of goats commonly result in reproductive disease, but viable PI goats are rare. Using 2 BVDV isolates, previously demonstrated to cause PI cattle and white-tailed deer, this study evaluated the outcome of experimental infection of pregnant goats. Pregnant goats (5 goats/group) were intranasally inoculated with BVDV 1b AU526 (group 1) or BVDV 2 PA131 (group 2) at approximately 25-35 days of gestation. The outcome of infection varied considerably between groups. In group 1, only 3 does became viremic, and 1 doe gave birth to a stillborn fetus and a viable PI kid, which appeared healthy and shed BVDV continuously. In group 2, all does became viremic, 4/5 does aborted, and 1 doe gave birth to a non-viable PI kid. Immunohistochemistry demonstrated BVDV antigen in tissues of evaluated fetuses, with similar distribution but reduced intensity as compared to cattle. The genetic sequence of inoculated viruses was compared to those from PI kids and their dam. Most nucleotide changes in group 1 were present during the dam's acute infection. In group 2, a similar number of mutations resulted from fetal infection as from maternal acute infection. Results demonstrated that BVDV may cause reproductive disease but may also be maintained in goats.

Hepatic Perisinusoidal Myofibroblast Proliferation and Systemic Inflammatory Response Precedes Sep/Tox Hepatitis in Broilers.

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.

Artículo regular­Proliferación de miofibroblastos perisinusoidales hepáticos y respuesta inflamatoria sistémica que precede a la hepatitis por septicemia y toxemia (sep/tox) en pollos de engorde. La septicemia-toxemia (sep/tox) se incluye en la Categoría 1 de seguridad alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA) y es la causa más común y económicamente significativa de decomisos de canales de pollos de engorde. Las lesiones hepáticas se consideran una posible consecuencia de la septicemia y de la contaminación bacteriana asociada con la canal. Por lo tanto, estas lesiones se consideran un indicador de septicemia/toxemia (hepatitis sep/tox). Este estudio se llevó a cabo para analizar las lesiones histológicas que preceden a las lesiones hepáticas muy visibles que conducen a los decomisos debido a septicemia/toxemia en la planta de procesamiento. Se utilizaron hígados de canales de pollos de engorde decomisados por los inspectores del USDA por septicemia/toxemia para establecer criterios microscópicos y generales de hepatitis en etapa terminal de la septicemia/toxemia. Después de la caracterización de la hepatitis por septicemia/toxemia, los pollos de engorde de una granja con un historial de decomisos por septicemia/toxemia se sometieron a examen post mortem e investigación bacteriológica en cuatro intervalos durante los últimos 20 días de producción. Se enviaron cinco pollos sanos y cinco clínicamente enfermos de cuatro casetas a los 18, 25, 32 y 38 días de producción (160 en total). Las lesiones microscópicas que representan la proliferación de miofibroblastos perisinusoidales hepáticos (HPMP), la granulopoyesis extramedular periportal (PEMG), la histocitosis folicular esplénica y la celularidad de la médula ósea (BMC) se clasificaron subjetivamente para cada ave, y la clasificación subjetiva se evaluó con técnicas cuantitativas digitales. La morfología de las células estrelladas hepáticas perisinusoidales y la transformación progresiva de estas células en miofibroblastos se confirmó mediante inmunohistoquímica para actina y desmina del músculo liso. Los cultivos aeróbicos de hígados y vesícula biliar de aves con septicemia/toxemia no produjeron crecimiento de bacterias asociadas con la septicemia. Se observó proliferación de miofibroblastos perisinusoidales hepáticos de leve a severa en todos los grupos de edad, lo que representa el 28% de las aves examinadas. Los aumentos en las células inflamatorias observados por granulopoyesis extramedular periportal y celularidad de la médula ósea se correlacionaron positivamente con proliferación progresiva de miofibroblastos perisinusoidales hepáticos y con hepatitis por septicemia/toxemia en etapa terminal en pollos de engorde.

Evaluation of hunter-harvested white-tailed deer for evidence of bovine viral diarrhea virus infection in Alabama.

Bovine viral diarrhea virus (BVDV) is one of the most relevant pathogens affecting today's cattle industries. Although great strides have been made in understanding this virus in cattle, little is known about the role of wildlife in the epidemiology of BVDV. While persistently infected cattle are the most important reservoir, free-ranging ungulates may become infected with BVDV as demonstrated by serosurveys and experimental infections. Therefore, free-ranging wildlife may maintain BVDV as the result of an independent cycle and may serve as a reservoir for the virus. Systematic studies on prevalence of BVDV-specific antibodies or frequency of persistent BVDV infection in North American wildlife are sparse, and no information is available from the southeastern United States. The objective of this study was to evaluate blood and skin samples from hunter-harvested white-tailed deer (Odocoileus virginianus) for evidence of BVDV infection. Virus-neutralizing antibodies were detected in 2 of 165 serum samples. Skin biopsy immunohistochemistry (IHC) was performed on samples from 406 deer using a BVDV-specific monoclonal antibody (MAb) (15c5), and BVDV antigen was detected in one sample. A similar IHC staining pattern was obtained using a second BVDV MAb (3.12F1). Viral antigen distribution in the skin sample of this deer resembled that found in persistently infected cattle and in a previously described persistently infected white-tailed deer; thus, the deer was presumed to be persistently infected. Evidence of BVDV infection in free-ranging white-tailed deer should encourage further systematic investigation of the prevalence of BVDV in wildlife.

Acute infection with bovine viral diarrhea virus of low or high virulence leads to depletion and redistribution of WC1(+) γδ T cells in lymphoid tissues of beef calves.

The objective of this study was to determine the abundance and distribution of γδ T lymphocytes in lymphoid tissue during acute infection with high (HV) or low virulence (LV) non-cytopathic bovine viral diarrhea virus (BVDV) in beef calves. This study was performed using tissue samples from a previous experiment in which thirty beef calves were randomly assigned to 1 of 3 groups: LV [n=10; animals inoculated intranasally (IN) with LV BVDV-1a (strain SD-1)], HV [n=10; animals inoculated IN with HV BVDV-2 (strain 1373)], and control (n=10; animals inoculated with cell culture medium). On day 5 post inoculation, animals were euthanized, and samples from spleen and mesenteric lymph nodes (MLN) were collected to assess the abundance of WC1(+) γδ T cells. A higher proportion of calves challenged with BVDV showed signs of apoptosis and cytophagy in MLN and spleen samples compared to the control group. A significantly lower number of γδ T cells was observed in spleen and MLN from calves in HV and LV groups than in the control calves (P<0.05). In conclusion, acute infection with HV or LV BVDV resulted in depletion of WC1(+) γδ T cells in mucosal and systemic lymphoid tissues at five days after challenge in beef calves. This reduction in γδ T cells in the studied lymphoid tissues could be also due to lymphocyte trafficking to other tissues.

Expression of type I interferon-induced antiviral state and pro-apoptosis markers during experimental infection with low or high virulence bovine viral diarrhea virus in beef calves.

The objective of this study was to compare the mRNA expression of host genes involved in type-I interferon-induced antiviral state (IFN-α, IFN-ß, Mx-1, PKR, OAS-1 and ISG-15), and apoptosis (caspase-3, -8, and -9), after experimental infection of beef calves with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV) strains. Thirty BVDV-naïve, clinically normal calves were randomly assigned to three groups. Calves were intranasally inoculated with low (LV; n=10, strain SD-1) or high (HV; n=10, strain 1373) virulence ncp BVDV or BVDV-free cell culture medium (Control, n=10). Quantitative RT-PCR was used to determine the target gene expression in tracheo-bronchial lymph nodes and spleen 5 days after infection. Interferon-α and -ß mRNA levels were up-regulated in tracheo-bronchial lymph nodes (P<0.05) in the HV group, but not in the LV group, compared with the control group. There was an up-regulation of type I interferon-induced genes in spleen and tracheo-bronchial lymph nodes of HV and LV groups, compared with the control group (P<0.01). mRNA levels of OAS-1 and ISG-15 were significantly higher in LV than HV calves (P<0.05). A significant up-regulation of caspase-8 and -9 was observed in tracheo-bronchial lymph nodes in the LV group (P=0.01), but not in the HV group. In conclusion, experimental infection with either high or low virulence BVDV strains induced a significant expression of the type I interferon-induced genes in beef calves. There was a differential expression of some interferon-induced genes (OAS-1 and ISG-15) and pro-apoptosis markers based on BVDV virulence and genotype.