Ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules in early and advanced phases of experimentally induced mucosal diseases in calves

OBJECTIVE: To investigate ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules (Peyer's patches) of calves with early and advanced phases of experimentally induced mucosal disease (MD). ANIMALS: Twenty 2.5- to 7-month-old Holstein-Friesian calves (11 females, 9 males). PROCEDURE: MD was induced in 13 of 18 calves that were persistently viremic with bovine viral diarrhea virus (BVDV). Eight of the 13 calves were euthanatized before the onset of clinical signs of MD, and 5 were euthanatized after becoming moribund with MD. Five persistently viremic calves and 2 calves without BVDV served as controls. Specimens of small-intestinal aggregated lymphoid nodules were prepared for transmission electron microscopy. RESULTS: The ultrastructure of follicles of small-intestinal aggregated lymphoid nodules from healthy calves was consistent with that in sheep. In the early phase of MD, changes were characterized by numerous apoptotic lymphocytes and macrophages with apoptotic bodies. In more advanced lesions, affected lymphoid follicles consisted of macrophages and variable numbers of follicular dendritic cells (FDC), whereas others did not contain FDC. In moribund calves, small follicles consisting predominantly of FDC and follicles with central cavities surrounded by macrophages, and few neutrophils were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The ultrastructural changes in lymphoid follicles of small-intestinal aggregated lymphoid nodules indicate apoptosis of lymphocytes as an initial event. The development of small follicles consisting predominantly of FDC or the complete loss of follicular architecture in advanced phases of MD is determined by the intensity of apoptosis of lymphocytes, the capacity of the macrophages for uptake, and the reorganization of a stromal network.     

Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus

One-day-old gnotobiotic piglets were inoculated intranasally with in vitro passaged porcine circovirus 1 (PCV-1), PCV-2, and porcine parvovirus (PPV) alone or in combination (PCV-1/PCV-2, PCV-1/PPV, and PCV-2/PPV). Piglets were evaluated for 1) the development of porcine postweaning multisystemic wasting syndrome (PMWS), 2) distribution of viral antigens by immunochemistry, and 3) viremia and the presence of viral DNA in nasal and ocular secretions and feces. All single agent-infected piglets and piglets infected with PCV-1/PCV-2 or PCV-1/PPV were clinically asymptomatic. They were transiently viremic and seroconverted to homologous virus(es). At termination of the study on postinfection day (PID) 35, microscopic lesions were restricted to focal inflammatory cell infiltrates in livers and myocardia. One piglet given PCV-1/PPV was PPV viremic for 2 weeks after infection and had lymphangiectasia of the spiral and descending colon associated with granulomatous inflammation. All four PCV-2/PPV-inoculated piglets developed PMWS, characterized by sudden onset of depression and anorexia, icterus, and submucosal edema. One piglet became moribund on PID 27, and the remaining three piglets were euthanatized between PID 27 and PID 30 because of severe disease. Lymph nodes were small and the livers were mottled. Disseminated angiocentric granulomatous inflammation was present in all tissues examined except the brain. Multiple lightly basophilic intracytoplasmic inclusion bodies were identified in macrophages and histiocytes. PCV-2 antigen was widely distributed within macrophages; PPV antigen was sparse. Hepatocellular necrosis and bile retention were prominent. PCV-2 DNA was identified in ocular, fecal, and nasal secretions. Terminal sera contained antibodies to PPV (4/4) and PCV-2 (3/ 4). Production of PMWS in gnotobiotic swine appears to require PCV-2 and additional infectious agents such as PPV for full disease expression in gnotobiotic piglets.     

Recrudescence of bovine herpesvirus-5 in experimentally infected calves

A latent infection of bovine herpesvirus-5 (BHV-5) was established in 4 calves. These calves, plus 2 controls, were given dexamethasone (DM) to reactivate the latent virus. The 4 principal calves developed antibodies to BHV-5 by postinoculation day (PID) 21. Antibody titers increased until PID 42 before decreasing to low levels of PID 75. After the first DM treatment (started on PID 76), an anamnestic antibody response was demonstrated in the 4 principal calves. Calves, 2, 3, and 4 were euthanatized and necropsied at PID 121, and their antibody titers were again decreasing. The virus BHV-5 was not isolated from the tissues by conventional techniques of viral isolation but was isolated from the trigeminal ganglion and spinal cord of calf 3 by explantation techniques. The BHV-5 was isolated, using conventional viral isolation techniques, from a nasal swab sample of calf 1 on PID 91 (15 days after the first DM treatment) and from the thoracic lymph node 6 days after the start of a 2nd DM treatment. Seemingly, BHV-5 may be latently harbored in the nerve tissues or calves and this virus may be reactivated from the upper respiratory tract following subsequent DM treatment.     

Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts

Eleven 3- to 50-day-old colostrum-deprived gnotobiotic calves and seven 25- to 63-day-old colostrum-deprived conventional calves were allotted into 3 groups. Each group was inoculated with a fecal isolate of bovine coronavirus via different routes: orally/intranasally OR/IN, No. 1 through 8, group 1 calves; OR, No. 9 through 13, group 2 calves; IN, No. 14 through 18, group 3 calves. Nasal swab specimens and fecal specimens were collected daily and were examined for coronavirus antigen by use of direct immunofluorescent staining (nasal epithelial cells) or by use of immune electron microscopy (fecal specimens). All but 4 calves (No. 11, 13, 17, and 18) were euthanatized on postinoculation days (PID) 3 to 7. Calves 11 and 17 became severely dehydrated and died at PID 5. Calves 13 and 18 were evaluated for nasal and fecal shedding of coronavirus through PID 14. Distribution of coronavirus antigen in the respiratory and intestinal tracts of the 14 euthanatized calves was evaluated by use of direct immunofluorescent staining. All calves developed profuse diarrhea by PID 2 to 4; however, calves did not develop clinical signs of respiratory tract disease before euthanasia or death. Inoculated calves shed coronavirus in their feces as detected by use of immune electron microscopy. Infected nasal epithelial cells were detected in all but 2 orally inoculated calves (No. 9 and 10). Route of inoculation influenced the sequence of initial detection of coronavirus antigen from fecal specimens or nasal swab specimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of viral antigen and development of lesions after experimental infection with highly virulent bovine viral diarrhea virus type 2 in calves

OBJECTIVE: To correlate tissue distribution with development of lesions after experimental infection with a virulent strain of noncytopathic bovine viral diarrhea virus (BVDV) type 2 in calves. ANIMALS: Ten 14-day-old and two 2-month-old colostrum-deprived calves. PROCEDURE: Calves were intranasally inoculated with BVDV type-2 strain 1373 from an outbreak of clinically severe bovine viral diarrhea (BVD).Two 14-day-old calves served as noninfected controls. Two calves each were euthanatized on postinoculation days 3, 6, and 12, and 1 each on days 8, 9, 13, and 14. Tissues were collected for immunohistologic and histologic examination. RESULTS: Inoculated calves developed nonspecific clinical signs characterized by high fever and decreased numbers of leukocytes and thrombocytes. Viral antigen was detected focally in lymphoid tissues on day 3. On days 6, 8, 9, 12, and 14, viral antigen became increasingly widespread throughout organs and tissues. Viral antigen in lymphoid tissues was associated with severe depletion of all compartments. Lesions in other tissues were not well correlated with distribution of viral antigen. Depletion of lymphoid tissues was observed in a calf on day 13, but viral antigen had been cleared from most tissues and was detected in vascular walls only. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with a virulent BVDV strain resulted in wide dissemination of viral antigen in host tissues. Severe lymphoid depletion developed in lymphoid tissues, whereas viral antigen was generally not associated with lesions in other tissues. Findings suggest that development of lesions in acute BVD is not solely a function of viral replication and is also attributable to host reaction to infection.     

Detection of Akabane viral antigens in spontaneous lymphohistiocytic encephalomyelitis in cattle.

A 5-month-old Japanese black bull calf and twenty-seven 1-27-day-old calves exhibiting neurological signs between August and October 1998 were examined. The bull calf exhibited rapid breathing, fever, hypersensitivity, and ataxia and was euthanized 4 days after the onset of symptoms. The 27 calves primarily exhibited ataxia, and 15 had arthrogryposis. Histological examination of the bull calf revealed perivascular infiltraction by mononuclear cells, diffuse to multifocal gliosis, and neuronal necrosis in the brain and spinal cord. Multiple malacic foci were found in the midbrain in 5 cases. In contrast, in the 15 calves necropsied in October, there were fewer inflammatory changes, but there was neuronal cell loss in the ventral horn and a decrease in myelinated axons in the lateral and ventral funiculi. Immunohistochemical examination using a rabbit antiserum against Akabane virus strain OBE-1 revealed a large amount of viral antigen in the degenerating neurons and glial cells of the bull calf, mainly in the spinal gray matter. Small amounts of viral antigen in swollen axons and a few glial cells were found in 5 of 27 calves. Thirteen of the 27 calves had high neutralization antibody titers against the Akabane virus, whereas there was no significant antibody titer in most of the calves necropsied during August. The present study revealed that viral antigen detection was very useful for the diagnosis of Akabane diseases in the 5-month-old bull calf that was suspected to be infected postnatally, while it had limited usefulness in the other young calves.     

Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus

Eight pigs were inoculated subcutaneously with a highly virulent hog cholera virus (HCV) strain ALD. The infected pigs developed severe illness and became moribund on postinoculation day (PID) 7 or PID 10. Histologic lesions were characterized by severe generalized vasculitis, necrosis of lymphocytes, and encephalitis. HCV antigen was detected in crypt tonsilar epithelial cells, macrophages, and reticular endothelial cells of lymphoid tissues. Antigen localization corresponded well with histologic lesions. Five pigs were inoculated with less virulent HCV Kanagawa/74 strain and were euthanatized on PID 30. All five infected pigs recovered from the illness but became stunted. They also had a slight follicular depletion of lymphocytes, histiocytic hyperplasia, and hematopoiesis in the spleen. Less virulent HCV antigen was observed in the tonsils, kidneys, pancreas, adrenal glands, and lungs. Although antigen localization was less associated with histologic lesions, immunoreactivity was stronger than that in the pigs infected with the ALD strain of HCV. An almost complete loss of B lymphocytes was recognized in pigs infected with the ALD strain and was correlated with follicular necrosis in lymphoid tissues. Loss of B lymphocytes was not prominent in the pigs infected with Kanagawa/74 strain. The number of CD4+ and CD8+ T lymphocytes was significantly higher than that in the noninfected control pigs.     

Nonsuppurative myocarditis caused by porcine circovirus type 2 in a weak-born piglet

Reproductive failure associated with porcine circovirus type 2 (PCV2) was observed in a farm, and a weak-born 8-day-old piglet was examined pathologically. Focal to locally extensive lesions, including infiltration of macrophages and lymphocytes, fibrosis and degeneration of the myocardium were observed in the heart. PCV2 antigens and nucleic acids were detected in degenerated cardiomyocytes and macrophages infiltrating the heart by immunohistochemical staining and in situ hybridization. Depletion of lymphocytes with occasional infiltration of multinucleated giant cells was seen in the lymphoid organs and PCV2 antigens were demonstrated in histiocytic cells. Crystalline arrays of viral particles were observed in infiltrated macrophages in the heart and, rarely, in cardiomyocytes by electron microscopy. Although myocarditis is a common finding in aborted or stillborn piglets in reproductive failure due to PCV2, it was also observed in the 8-day-old piglet with PCV2 association.     

Experimental model of type II bovine viral diarrhea virus-induced thrombocytopenia in neonatal calves.

Thrombocytopenia has been associated with type II bovine viral diarrhea virus (BVDV) infection in immunocompetent cattle, but the mechanism is unknown. The purpose of the present study was to develop and characterize a model of type II BVDV-induced thrombocytopenia. Colostrum-deprived Holstein calves were obtained immediately after birth, given a BVDV-negative and BVDV antibody-negative plasma transfusion, housed in an isolation facility, and randomly assigned to either control (n = 4) or infected (n = 5) groups. Infected calves were inoculated by intranasal instillation on day 3 of age with 10(7) TCID50 of the prototype type II isolate, BVDV 890, whereas control calves were sham inoculated. Blood counts and virus isolations from serum, white blood cells, and platelets were performed daily until day 12 after infection, at which time all experimental calves were euthanatized, and pathologic, virologic, and immunohistochemical examinations were performed. On physical examination, the control calves remained normal, but the infected calves developed pyrexia and diarrhea characteristic of type II BVDV infection. The platelet count decreased in all infected calves, and a statistically significant difference in the platelet count between control and infected calves was observed on days 7-12 after infection. In addition, the mean platelet volume and white blood cell counts also decreased. Examination of the bone marrow from the infected calves revealed immunohistochemical staining for BVDV antigen in megakaryocytes and evidence of concurrent megakaryocyte necrosis and hyperplasia.     

Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalin-fixed skin biopsy specimens.

The objective of this study was to evaluate the efficacy of immunohistochemical (IHC) staining for diagnosis of persistent bovine viral diarrhea virus (BVDV) infection using formalin-fixed, paraffin-embedded skin biopsy specimens. Skin from 41 of 42 calves shown to be persistently infected (PI) with BVDV by repeated virus isolation more than 3 weeks apart were immunohistochemically positive for BVDV antigen. Positive IHC staining was most pronounced in the keratinocytes and in hair follicle epithelium, hair matrix cells of the hair bulb, and the dermal papilla. All of the skin sections from 10 calves experimentally infected postnatally with BVDV (10(5) median tissue culture infective doses [TCID50]) and biopsied on days 0, 5, 7, and 9 postinfection were negative for viral antigen. Ten calves from a second group experimentally infected with a higher dose of BVDV (10(8) TCID50) were biopsied when viremic between 10 and 14 days postinfection and 4 calves exhibited positive IHC staining for BVDV; however, staining in these skin biopsies was confined to small foci in the nonfollicular epidermis and follicular ostia. This staining was distinct from that observed in skin obtained from PI cattle. Skin biopsy represents an effective method for identifying animals PI with BVDV.     

Experimental respiratory syncytial virus pneumonia in young calves: microbiologic and immunofluorescent findings

Young calves were inoculated with respiratory syncytial virus (RSV) intranasally or by a combined intranasal and intratracheal route and were killed between postinoculation (initial) days (PID) 1 and 14. Viral antigens were detected by immunofluorescence in nasopharyngeal cells from calves killed between PID 2 and 10. Evidence of infection of the trachea and lungs with RSV was obtained by immunofluorescence and virus isolation in calves inoculated by the combined route, but not in calves inoculated intranasally. Within the lungs, RSV antigens were observed in epithelial cells of bronchioli and alveoli. The only virus detected in inoculated calves was RSV. With the exception of 1 calf, bacteria or mycoplasmas were not isolated from the lower respiratory tracts of inoculated calves. Antibody to RSV was not detected in calves killed up to PID 5, but 4 of 5 colostrum-deprived calves killed between PID 10 and 13 had antibodies to RSV. Preexisting, maternally derived antibody to RSV did not protect the calves from infection. Seemingly, the clinical signs of pneumonia and pathologic lesions observed in inoculated calves were caused by RSV infection.     

Comparison of early splenic changes associated with replication of viruses in murine monocytic macrophages

An effect of replication of certain viruses in murine monocytic macrophages was manifested by depletion of cells through degenerative and necrotizing changes in thymus-dependent areas of lymphoid structures. In mice infected with murine hepatitis virus (MHV-3) or lactate dehydrogenase virus, these changes were transient in mice killed on postinoculation day (PID) 2. To study these morphologic changes due to viral replication, adult Swiss specific-pathogen-free homozygous nude mice (nu/nu) and their heterozygous haired littermates (nu/+) were inoculated with 10(5) LD50 of MHV-3, euthanatized, and necropsied on PID 1, 2, 4, 6, 8, and 10 along with noninoculated controls. The nu/+ and nu/nu mice killed on PID 2 had lymphocytic karyorrhexis and depletion of cells in the thymus-dependent area. In the heterozygote, these characteristic lesions were transient; whereas in the homozygote, lesions persisted and were present in survivors euthanatized and necropsied on PID 16. Although the intensity of lesions due to MHV-3 varied between nu/+ and nu/nu mice, virus titers determined on liver homogenates were similar for the homozygote and heterozygote during acute disease. Nude and nonnude mice given lactate dehydrogenase virus and killed on PID 2 had a transient depletion of lymphocytes; whereas mice given lymphocytic choriomeningitis virus and killed on PID 4 had a similar lesion. Lesions neither occurred when mice were treated with silica before inoculation, indicating that functional monocytic macrophages were required, nor occurred when another virus, herpes simplex virus type 1, was given.     

Distribution and cellular heterogeneity of bovine viral diarrhea viral antigen expression in the brain of persistently infected calves: a new perspective

Persistent infection following in utero exposure to bovine viral diarrhea virus (BVDV) early in gestation is a serious cause of morbidity and mortality in cattle industries worldwide. The brain is a primary target of persistent infection. In the current study, the types of cells infected and topography of viral antigen expression were examined in brain sections from 9 BVDV persistently infected crossbred calves, all less than 1 year of age, by immunohistochemical staining using the 15C5 primary monoclonal antibody. BVDV antigen was detected in the brains of all persistently infected calves. A variety of cell types was infected, including neurons, astrocytes, oligodendroglia, blood vessel-associated cells (pericytes, perivascular macrophages, smooth muscle cells), and cells in the leptomeninges (blood vessel-associated cells). Conclusive demonstration of viral antigen in vascular endothelial cells was elusive. The intensity and distribution of viral antigen staining in neurons were highly variable. Viral antigen staining was most consistent and intense in thalamic nuclei, most notably in dorsal and medial nuclear groups, followed by the hippocampus, entorhinal cortex, basal nuclei, and piriform cortex. Staining in other brain areas was often less intense and inconsistent. The variability in the intensity and topography of viral antigen in the brain may explain the heterogeneity in the clinical manifestations of BVDV-induced disease. Additionally, infection of the brain in persistently infected calves may underlie or at least contribute to endocrine disturbances and immunologic deficits that are protean manifestations of BVDV-induced disease.     

An attempt to protect calves against experimental bovine leukosis using allografts

Six calves sensitised by implanting skin from a calf were later inoculated with lymphocytes from the same calf after the calf had been infected with bovine leukosis virus (BLV). Two out of 6 calves challenged did not develop BLV antibodies and BLV was not isolated from these animals, whereas all of the 5 control calves became infected with BLV.     

Early platelet aggregation as a cause of thrombocytopenia in classical swine fever

Twenty pigs were inoculated with a virulent classical swine fever virus isolate to determine the mechanism responsible for thrombocytopenia using histopathologic, ultrastructural, and immunohistochemical (detection of viral antigens gp55 and FVIII-rag) techniques. In animals euthanatized at 2, 4, and 6 days postinoculation (dpi), clusters of granular material staining positive for FVIII-rag were observed in splenic cords, the marginal zone, hepatic sinusoids, and the perisinusoidal space. Moreover, numerous macrophages in these areas were swollen and displayed an intensely positive granular and cytoplasmic reaction. Cell alterations indicative of platelet activation and secretory and phagocytic activation of resident macrophages were also observed in these sites at 2 and 4 dpi. These results suggest that the thrombocytopenia observed in pigs is caused in the first instance by massive activation and subsequent phagocytosis of platelets secondary to the release of platelet-activating factors by activated macrophages.     

Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves.

A noncytopathic bovine viral diarrhea virus (BVDV), BVDV-890, isolated from a yearling heifer that died with extensive internal hemorrhages, was compared for virulence in calves with noncytopathic BVDV-TGAN, isolated from an apparently healthy persistently infected calf. After challenge exposure with BVDV-890, nonimmune calves (n = 7) developed fever > 40 C, diarrhea, leukopenia, lymphopenia, neutropenia, and thrombocytopenia. Most calves (n = 6) died or were euthanatized by 19 days after challenge exposure. Challenge exposure with BVDV-890 did not induce disease in 2 calves that had congenital persistent infection with BVDV or in 3 calves that had neutralizing antibody titer > 4 against BVDV-890. After challenge exposure with BVDV-TGAN, nonimmune calves (n = 7) developed fever > 40 C and, rarely, diarrhea or lymphopenia. All of those calves survived challenge exposure. The average maximal titer of BVDV-890 isolated from serum was 1,000 times that of BVDV-TGAN. In calves infected with BVDV-890, the average maximal percentages of lymphocytes and platelets associated with virus were greater than those found in calves infected with BVDV-TGAN. Additional findings of epidemiologic significance were prolonged shedding of virus and delayed production of viral-neutralizing antibody in 1 calf challenge-exposed with BVDV-890. Also, after production of neutralizing antibody, mutant virus that was refractory to neutralization was isolated from calves challenge-exposed with BVDV-TGAN.     

Fetal protection following exposure to calves persistently infected with bovine viral diarrhea virus type 2 sixteen months after primary vaccination of the dams

This study demonstrated that the bovine viral diarrhea virus (BVDV; types 1 and 2) fractions of a multivalent vaccine protected pregnant heifers and their fetuses at 149 to 217 days of gestation against exposure to calves persistently infected with BVDV type 2a. Eighty percent (eight of 10) of the control heifers were viremic at least 1 day following challenge, whereas all (20 of 20) BVDV-vaccinated heifers were virus isolation-negative on all postchallenge assessment days. Ninety percent (nine of 10) of the calves born to control heifers but only 5% (one of 20) of calves born to BVDV-vaccinated heifers seroconverted to BVDV type 2 before ingesting colostrum. One calf born to a control heifer was persistently infected. No calves from BVDV-vaccinated heifers were persistently infected.     

Idiopathic disseminated intracytoplasmic neuronal vacuolation in a neonatal Holstein calf born in the USA.

Histopathologic, immunohistochemical, and ultrastructural evaluations were made of a 6-day-old Holstein calf with severe vacuolation of the neuronal perikarya that was widely distributed throughout the central nervous system. No evidence of storage material within the vacuoles was revealed by histopathologic and ultrastructural examinations. Immunohistochemical and electron microscopic examinations were negative for protease-resistant prion protein and scrapie-associated fibrils, respectively. These results indicate that the clinical signs in this calf were not associated with transmissible spongiform encephalopathy. Neuronal vacuolation has not previously been documented in calves.     

Distribution of bovine virus diarrhoea viral antigens in the central nervous system of cattle with various congenital manifestations.

Distribution of bovine viral diarrhoea virus (BVDV) antigens in the central nervous system (CNS) of 26 cattle persistently BVDV infected, 11 cattle with mucosal disease (MD), and 32 calves with congenital brain malformations was studied using monoclonal antibodies against BVDV epitopes. In persistently infected cattle and in cattle with MD, a widespread infection of neurons was present. Predilection sites for BVDV antigens were the cerebral cortex and the hippocampus. In calves with congenital encephalopathies, viral antigen-containing neurons could only be detected in the CNS of four animals. From the topographical distribution of BVDV antigens in these four postnatal cases with end-stage lesions, no conclusions could be drawn concerning the pathogenesis of BVDV-induced encephalopathies.