Interferon-gamma in the serum and effusions of cats with feline coronavirus infection

The aim of this study was to quantify and compare interferon-γ (IFN-γ) concentrations in the serum of clinically normal cats infected with feline coronavirus (FCoV) with its concentration in the sera and effusions of cats with feline infectious peritonitis (FIP), a disease associated with infection with a mutated form of FCoV.Clinically normal FCoV-infected cats living in catteries with a high prevalence of FIP had the highest serum IFN-γ concentrations. The serum concentration of IFN-γ was not significantly different in cats with FIP compared with clinically normal FCoV-infected animals living in catteries with a low prevalence of the disease. Moreover, the concentration of IFN-γ was significantly higher in the effusions than in the serum of cats with FIP, probably due to IFN-γ production within lesions. These findings support the hypothesis that there is a strong, ‘systemic’ cell mediated immune response in clinically normal, FCoV-infected cats and that a similar process, albeit at a tissue level, is involved in the pathogenesis of FIP.     

Antibody-mediated enhancement of disease in feline infectious peritonitis: Comparisons with dengue hemorrhagic fever

Non-immune kittens passively immunized with feline serum containing high-titered antibodies reactive with feline infectious peritonitis virus (FIPV) developed a more rapid disease after FIPV challenge than did kittens pretreated with FIPV antibody-negative serum. Antibody-sensitized, FIPV challenged—kittens developed earlier clinical signs (including pyrexia, icterus, and thrombocytopenia) and died more rapidly than did non-sensitized, FIPV-challenged kittens. Mean survival time in sensitized kittens was significantly (P < 0.05) reduced compared to non-sensitized kittens (mean ± SEM, 10.0 ± 0.6 days vs. 28.8 ± 8.3 days, respectively). Lesions induced included fibrinous peritonitis, disseminated pyogranulomatous inflammation and necrotizing phlebitis and periphlebitis. FIPV antigen, immunoglobulin G, complement (C3) and fibrinogen were demonstrated in lesions by immunofluorescence microscopy.The pathogenesis of dengue hemorrhagic fever (DHF) in persons bears striking resemblance to that of FIP in experimental kittens. In both FIP and DHF, non-neutralizing antibody may promote acute disease by enhancement of virus infection in mononuclear phagocytes or by formation of immune complexes, activation of complement and secondary vascular disturbances.     

Detection of feline coronavirus antibody, feline immunodeficiency virus antibody, and feline leukemia virus antigen in ascites from cats with effusive feline infectious peritonitis

To investigate the usefulness of ascites as a material for viral tests in cats with effusive feline infectious peritonitis (FIP), we attempted to detect anti-feline coronavirus antibody, anti-feline immunodeficiency virus antibody, and feline leukemia virus antigen in ascites from 88 cats clinically suspected with effusive FIP. In each of these three viral tests, all cats positive for serum antibody/antigen were also positive for ascitic antibody/antigen, while cats negative for serum antibody/antigen were also negative for ascitic antibody/antigen. This finding indicates that ascites is useful for these viral tests.     

A comparison between immunofluorescence staining on smears from Membrana nictitans (M3 test), immunohistopathology and routine pathology in cats with suspected feline infectious peritonitis (FIP).

An indirect immunofluorescence method using smears from membrana nictitans (M3 test) to diagnose feline corona virus (FCV) infection was compared with immunohistopathology (using indirect immunofluorescence assay (IFFA) performed on organs (IFO], and routine pathology (RP) in cats with suspected feline infectious peritonitis (FIP). A close correlation between the 2 immunofluorescence methods (IFO and M3) was observed. Although the M3 test requires samples from only 1 organ per animal, both the sensitivity and specificity were high (80%), when compared to IFO (using samples from an average of 5 organs per animal). In 21% of the cats with suspected FIP typical pathological lesions were found. As the M3 test is relatively easy to perform, it could reduce work-load of pathology laboratories and provide valuable data for clinical and epidemiological use.     

After nasopharyngeal infection,foot-and-mouth disease virus serotype A RNA is shed in bovine milk without associated mastitis

Foot-and-mouth disease (FMD) is a highly contagious aphthoviral infection of cloven-hoofed animals, inducing vesiculopustular stomatitis, pododermatitis, and thelitis. Vesicular fluid represents a major pathway of virus excretion, but bovine milk is another important source of virus shedding. We describe here the time course of FMD virus (FMDV) excretion in the milk and characterize associated lesions in the mammary gland. Three dairy cows were infected by nasopharyngeal instillation of FMDV and monitored over 12 d. Autopsy was performed at the end of the study, and specimens were collected for histopathology, IHC, and RT-qPCR. All 3 cows developed fever, drooling, vesiculopustular stomatitis, interdigital dermatitis, and thelitis. FMDV RNA was detectable in whole milk until the end of the trial, but only transiently in saliva, nasal secretions, and blood serum. Although histology confirmed vesiculopustular lesions in the oral and epidermal specimens, the mammary glands did not have unequivocal evidence of FMDV-induced inflammation. FMDV antigen was detectable in skin and oral mucosa, but not in the mammary gland, and FMDV RNA was detectable in 9 of 29 samples of squamous epithelia but only in 1 of 12 samples of mammary gland.     

猫传染性腹膜炎病毒核衣壳蛋白的表达及间接ELISA法的建立

本研究对猫传染性腹膜炎病毒（FIPV）N蛋白的编码基因进行克隆和原核表达,并在纯化重组N蛋白的基础上,建立了FIPV抗体间接ELISA检测方法。研究结果显示,该重组纯化的N蛋白具有良好的抗原反应性,可用于FIPV阳性血清的筛查,为我国出入境检疫部门监控FIPV疫情提供技术支撑。     

Toggenburg Orbivirus, a new bluetongue virus: Initial detection, first observations in field and experimental infection of goats and sheep

A novel bluetongue virus termed “Toggenburg Orbivirus” (TOV) was detected in two Swiss goat flocks. This orbivirus was characterized by sequencing of 7 of its 10 viral genome segments. The sequencing data revealed that this virus is likely to represent a new serotype of bluetongue virus [Hofmann, M.A., Renzullo, S., Mader, M., Chaignat, V., Worwa, G., Thuer, B., 2008b. Genetic characterization of Toggenburg Orbivirus (TOV) as a tentative 25th serotype of bluetongue virus, detected in goats from Switzerland. Emerg. Infect. Dis. 14, 1855–1861].In the field, no clinical signs were observed in TOV-infected adult goats; however, several stillborn and weak born kids were reported. Although born during a period of extremely low vector activity, one of these kids was found to be antibody and viral genome positive and died 3.5 weeks postpartum.Experimental infection of goats and sheep, using TOV-positive field blood samples, was performed to assess the pathogenicity of this virus.Goats did not show any clinical or pathological signs, whereas in sheep mild bluetongue-like clinical signs were observed. Necropsy of sheep demonstrated bluetongue-typical hemorrhages in the wall of the pulmonary artery. Viral RNA was detected in organs, e.g. spleen, palatine tonsils, lung and several lymph nodes of three experimentally infected animals.Unlike other bluetongue virus serotypes, it was not possible to propagate the virus, either from naturally or experimentally infected animals in any of the tested mammalian or insect cell lines or in embryonated chicken eggs.In small ruminants, TOV leads to mild bluetongue-like symptoms. Further investigations about prevalence of this virus are needed to increase the knowledge on its epidemiology.     

成都地区猫传性腹膜炎病毒ORF3、7b以及S基因七肽重复区1序列的分析

猫传性腹膜炎(FIP)是由猫冠状病毒引起的猫科常见致死性传染病。为分析成都地区猫传性腹膜炎病毒(FIPV)部分基因的序列特征,本研究从11只FIP患猫的腹水中提取总RNA,采用普通PCR或套式PCR对FIPV的非结构蛋白基因ORF3 (3a、3b和3c)、7b以及S基因七肽重复区1 (HR1)序列进行PCR扩增并测序。结果显示:检测到FIRV的上述几个基因均呈遗传多样性,序列存在很大的个体间差异。3a和3b基因主要表现为氨基酸突变,3c基因在约89%的样品中存在截短。7b基因有7个位点的氨基酸被完全替换。S基因HR1序列融合肽的1045位甲硫氨酸被亮氨酸替换,其下游的1057位丝氨酸被丙氨酸替换。遗传进化树显示,本实验扩增的FIRV 7b及S基因HR1序列与GenBank中国外FIPV相应基因序列亲缘性较高。推测3c基因的截短和/或某些氨基酸的替换的共同作用与病毒的致病性有关。本研究结果为FIP的临床确诊提供了分子诊断依据,增加了临床诊断结果的可靠性。     

The kinetics of feline leukaemia virus shedding in experimentally infected cats are associated with infection outcome

Feline leukaemia virus (FeLV) infection in felids results mainly from oronasal exposure to infectious saliva and nasal secretions, but the potential for viral transmission through faeces and urine has not been completely characterized. In order to assess and compare potential FeLV transmission routes, we determined the viral kinetics in plasma, saliva, faeces and urine during early experimental FeLV infection (up to week 15 post-exposure) in specific pathogen-free cats. In addition to monitoring p27 antigen levels measured by ELISA, we evaluated the presence of infectious particles by cell culture assays and quantified viral RNA loads by a quantitative real-time TaqMan polymerase chain reaction. RNA load was associated with infection outcome (high load-progressive infection; low load-regressive infection) not only in plasma, but also in saliva, faeces and urine. Infectious virus was isolated from the saliva, faeces and urine of infected cats with progressive infection as early as 3-6 weeks post-infection, but usually not in cats with regressive infection. In cats with progressive infection, therefore, not only saliva but also faeces and to some extent urine might represent potential FeLV transmission routes. These results should be taken into account when modelling FeLV-host interactions and assessing FeLV transmission risk. Moreover, during early FeLV infection, detection of viral RNA in saliva may be used as an indicator of recent virus exposure, even in cats without detectable antigenaemia/viraemia. To determine the clinically relevant outcome of FeLV infection in exposed cats, however, p27 antigen levels in the peripheral blood should be measured.     

Biochemical changes in blood and uterine fluid of fowl following experimental EDS'76 virus infection

The pH, pCO2, and pO2 values and the concentrations of sodium, potassium, calcium, magnesium, chloride, phosphorus, bicarbonate, base excess, protein, glucose, as well as the activity of alkaline phosphatase and malate dehydrogenase were determined in the venous blood and uterine fluid of control and EDS'76 virus-infected fowl. Moreover, the pH of the mucosa of different parts of the oviduct was measured. Hens were examined in the period from 10 to 24 days following infection; blood and uterine fluid samples were collected approximately 14 hours after oviposition, provided a plumped egg was present in the uterus. Examination of blood and pH measurement of oviduct mucosa did not yield significant differences between infected and noninfected hens. In comparison with noninfected control birds, the mean sodium concentration of the uterine fluid of infected hens producing soft shelled or shell-less eggs had evidently increased, while the mean concentration of potassium, calcium, magnesium and glucose had decreased. Similar differences were also observed between infected hens producing normally shelled eggs and infected hens producing abnormally shelled eggs. No significant differences between infected and not infected hens were observed concerning the other values determined in the uterine fluid. It is concluded that functional disturbances which account for shell aberrations following EDS'76 virus infection are located in the surface epithelial cells of the uterine mucosa. These disturbances are very probably initiated by a depressed function of the sodium pump. All changes observed in the uterine fluid of infected hens could be explained by this depressed function.     

Quantitative real-time PCR assays for the concurrent diagnosis of infectious laryngotracheitis virus,Newcastle disease virus and avian metapneumovirus in poultry

Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log₁₀ dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%–98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.     

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes ( n  = 4), orbital tissue biopsy ( n  = 1) and ocular evisceration contents ( n  = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin–biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.