Partial characterization of an adenovirus-like virus isolated from broiler chickens with transmissible viral proventriculitis

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.     

Physical and genomic characteristics identify chicken proventricular necrosis virus (R11/3 virus) as a novel birnavirus

Chicken proventricular necrosis virus (CPNV), isolate R11/3, previously was isolated from transmissible viral proventriculitis-affected chickens and was determined to be the likely etiology of this disease. CPNV was identified as a birnavirus on the basis of virion size and morphology (icosahedral, approximately 75 nm in diameter, nonenveloped); buoyant density in cesium chloride (1.32 g/ml); a genome comprising bisegmented, double-stranded RNA (approximately 3.8 and 3.4 kilobase pairs); and nucleotide sequence analyses. Nucleotide sequencing of CPNV RNA, segment B, identified a single large open reading frame that encodes a 903-amino acid protein. The 903-amino acid protein was identified as the putative VP1, the viral RNA-dependent RNA polymerase (RdRp), on the basis of sequence homologies with other birnavirus VP1 proteins. The CPNV VP1 possessed the unique permuted RdRp sequence motif arrangement characteristic of birnaviruses; however, phylogenetic analyses based on VP1 demonstrated that CPNV is deeply divergent from other birnaviruses.     

Experimental infection of laying chickens with egg-drop syndrome 1976 virus

Brown layer hens (BC and HC strains) and white layer hens (WL strain) orally infected with the H-162 strain of the egg-drop syndrome 1976 virus developed few clinical signs except for abnormal egg production. Depressed and/or aberrant-egg production was observed for 3 days or longer in 17 of 18 BC hens, 13 of 15 HC hens, and 10 of 17 WL hens. On the average, abnormal egg production began 8.8, 10.3, and 12.2 days after infection of the BC, HC, and WL hens, respectively. Egg production was depressed in the WL hens, but little depression was observed in the BC and HC hens. Aberrant-egg production was much less frequent in the WL hens than in the BC and HC hens. Aberrant eggs were shell-less, soft-shelled, thin-shelled, and/or discolored. No eggs of abnormal internal quality or shape were observed. The virus spread from infected BC and WL hens to contact hens.     

Experimental infection of sheep and goats with transmissible mink encephalopathy virus.

In a study to learn more about the pathogenicity of transmissible mink encephalopathy virus for the natural hosts of scrapie, 20 Cheviot sheep and 19 dairy goats were inoculated intracerebrally with the Idaho strain of the virus. Five sheep and nine goats became affected with a progressive neurological disease. The incubation period in the sheep varied from 45 to 80 months (mean, 65 months) and in the goats from 31 to 40 months (mean, 35 months). Except for degeneration of the cerebral cortex (neocortex), the disease was indistinguishable clinically and neurohistologically from scrapie. During two more passages of the virus in goats, the incubation period was shortened to 12 to 15 months, the morbidity rate rose to 100% (6/6 dairy goats and 3/3 African pygmy goats), and the cortical lesion became constant and more pronounced. By the intracerebral inoculation of pastel mink, transmissible mink encephalopathy virus was detected in the brains of several affected sheep and goats but not in extraneural sites (lymphoid tissues and intestine), except for a trace amount in the proximal colon of one goat. Even after two passages in goats, the virus remained nonpathogenic for the laboratory mouse. Despite the essential likeness of the experimental disease and scrapie, the common identity of their causal viruses remains to be determined. Even so, the results of this study are still compatible with the view that transmissible mink encephalopathy virus almost certainly is scrapie virus whose biological properties became altered by chance passage in mink, a carnivore and an aberrant host.     

Experimental infection of rabbits with bovine viral diarrhoea virus by a natural route of exposure

Bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle that can naturally infect a wide range of even-toed ungulates. Non-bovine hosts may represent reservoirs for the virus that have the potential to hamper BVDV eradication programs usually focused on cattle. Rabbits are very abundant in countries such as the United Kingdom or Australia and are often living on or near livestock pastures. Earlier reports indicated that rabbits can propagate BVDV upon intravenous exposure and that natural infection of rabbits with BVDV may occur but experimental proof of infection of rabbits by a natural route is lacking. Therefore, New Zealand White rabbits were exposed to a Scottish BVDV field strain intravenously, oro-nasally and by contaminating their hay with virus. None of the animals showed any clinical signs. However, the lymphoid organs from animals sacrificed at day five after exposure showed histological changes typical of transient infection with pestivirus. Most organ samples and some buffy coat samples were virus positive at day five but saliva samples remained negative. Development of antibodies was observed in all intravenously challenged animals, in all of the nebulised group and in four of six animals exposed to contaminated hay. To our knowledge this is the first report of BVDV propagation in a species other than ruminants or pigs after exposure to the virus by a natural route. However, to assess the role of rabbits as a potential reservoir for BVDV it remains to be determined whether persistent infection caused by intra-uterine infection is possible and whether BVDV is circulating in wild rabbit populations.     

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.     

Experimental infection of laying chickens with adenovirus 127 and with a related virus isolated from ducks

Infection of commercially reared white leghorn and white rock hens with adenovirus 127 was associated with decreases in total egg production, external egg quality, egg weight (P less than 0.01), and eggshell thickness (P less than 0.01). The egg-production and egg-quality disturbances were transient, and production returned to normal approximately 4 weeks postinfection. Infection of white leghorns with a hemagglutinating adenovirus isolated from Missouri ducks did not adversely affect egg production, external egg quality, or eggshell thickness, but it was associated with decreased egg weight (P less than 0.01). Prior infection with the duck adenovirus prevented the adverse egg-production effects of adenovirus 127 infection. Mean hemagglutination-inhibition antibody titers of chickens infected with adenovirus 127 or with duck adenovirus ranged from 1:588 to 1:10809, and mean titers of uninfected chickens did not exceed 1:2.     

Vaccines against infection with bovine viral diarrhea virus/mucosal disease (BVDV/MD): a short overview

Various vaccine preparations against an infection with Bovine Viral Diarrhea Virus (BVDV) have been used since more than 30 years. To prevent reproduction failure and the generation of persistently infected animals, protection of heifers and cows against transplacental infection is the most important aim of BVDV vaccination. In principal, BVD vaccines with replication competent, attenuated BVDV (modified live vaccines) and vaccines with inactivated BVDV preparations (killed vaccines) are used. In Germany, modified live vaccines as well as killed vaccines are registered, however, only BVDV type I strains are included in both types of vaccines. This paper presents an short overview about the different BVD vaccines and their efficacy and safety. In addition, new vaccine types are mentioned and final conclusions are drawn.     

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.     

Experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus: Koch's postulates fulfilled

Aerosol exposure of eight pregnant sows to cell-culture- propagated Lelystad virus resulted in clinical signs characteristic of so-called mystery swine disease. After an incubation of 4-7 days, all sows were inappetant and listless for 6-9 days. Two sows developed a transient red-blue discolouration of the ears ('abortus blauw' or blue ear disease) accompanied by abdominal respiration, and two had a fever for one day only. One sow aborted at 109 days of gestation. The other seven sows, farrowing between 113 and 117 days of gestation, gave birth to numerous mummified, dead, and weak piglets. Of these seven, the mean number of piglets born dead to each sow was 4.6 and the mean number born alive was 7.7; 3.1 piglets per sow (40%) died within the first week. Lelystad virus was isolated from 31 piglets, which were born dead or died shortly after birth. Antibody was detected in precolostral blood samples or ascitic fluids of 23 piglets, a finding which demonstrated transplacental passage of the virus in six out of eight litters. We conclude that Lelystad virus is the causal agent of mystery swine disease. Since its aetiology is no longer a mystery, we propose the more appropriate name 'porcine epidemic abortion and respiratory syndrome (PEARS)'.     

Experimental disease in young chickens induced by a Mycobacterium paratuberculosis isolate from a patient with Crohn's disease.

The susceptibility of young chickens to infection with an isolate of Mycobacterium paratuberculosis which had been recovered from diseased tissues of a patient with Crohn's disease was determined. Two-week-old Leghorn-Cochin chicks were inoculated with strain Linda. Six birds received 10(7) organisms orally, six intraperitoneally, and five intracardially. Six uninoculated birds served as contact controls. Birds from each group were necropsied at two-week intervals. Focal granulomatous lesions occurred in two of six chickens which were inoculated orally, in six of six inoculated intraperitoneally, in three of five inoculated intracardially, but in none of six controls. Of the 11 birds with lesions, acid-fast bacilli were demonstrated in five. Immunoperoxidase reactivity paralleled the presence of acid-fast bacilli.     

Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone

Five 13- to 18-month old Belgian Blue bulls were used in this experiment. Four bulls (Nos. 2, 3, 4 and 5) were inoculated intratesticularly with 10(5) plaque-forming units of bovine herpesvirus-4 (BHV-4) in each testicle (Day 0). The challenge BHV-4 strain was previously isolated from testicle cells of a bull exhibiting orchitis and azoospermia. The fifth bull (No. 1) was used as a control and received the same volume of uninfected cell culture supernatant. For 5 days, beginning on Day 51 post-infection, two bulls (Nos. 4 and 5) and the control bull (No. 1) received 0.1 mg kg-1 of dexamethasone. Unilateral castrations were then performed at regular intervals for viral examination. Treatment with dexamethasone reactivated latent BHV-4, but no clinical signs were observed in treated bulls until the end of the experiment (Day 93). Only Bull 3 showed conjunctivitis and temporary azoospermia. The virus was recovered from various samples showing that: (i) BHV-4 can be present in a latent state in the testicles and mononuclear blood cells; (ii) dexamethasone reactivates the virus; (iii) the virus is excreted by nasal and ocular routes. Each infected bull seroconverted and a booster antibody response appeared after dexamethasone treatment as shown by immunofluorescence. Neutralizing antibodies were detected in each bull by complement-dependent neutralization test with titres higher than those obtained by a classical neutralization test. No booster response of neutralizing antibodies was observed after dexamethasone treatment. The antigenically relevant envelope BHV-4 proteins were identified by Western blotting using sera samples from the animals. DNA restriction endonuclease profiles of viruses reisolated after primary infection and reactivation showed only small differences.     

Experimental infection of C3H/HeJ mice with the NY18 isolate of Anaplasma phagocytophilum

Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum. Small animal models of A phagocytophilum in laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum. Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophilum infections.