Isolation and characterization of epizootic hemorrhagic disease virus from white-tailed deer (Odocoileus virginianus) in eastern Washington.

A virus was isolated from the spleen of a white-tailed deer (Odocoileus virginianus) that had died during an epizootic in Washington state in 1967. Inoculation of a 10% spleen suspension from the deer caused hemorrhagic disease in normal white-tailed deer. Studies were conducted on the biological, physicochemical, and serologic properties of the Washington isolate. An in vitro assay system, utilizing a cultured primary of white-tailed deer fetal cells from an entire fetus, was employed for isolation and propagation of the virus. Cytopathic effect was characterized by focal development of rounded and clumped cells. Propagation was unsuccessful in suckling mice, BHK-21, and Vero cell cultures. The virus was resistant to treatment with ether, sodium deoxycholate, trypsin, oxytetracycline hydrochloride, and was sensitive to chloroform. Virus yield was not affected when infected cultures were treated with 5-iodo-2'-deoxyuridine, but dactinomycin (actinomycin D) treatment of infected cultures reduced virus yield. The virus was inactivated when heated at 70 C for 5 minutes or when exposed to pH 5 for 18 hours at 4 C. The virus was completely excluded from the filtrate by a 0.10- micronm (APD) membrane filter. Staining of infected cells with acridine orange indicated the presence of double-standard nucleic acid in the cytoplasm. Serum-neutralization tests with antiserums against the homologous virus and the New Jersey and Alberta strains of epizootic hemorrhagic disease virus resulted in neutralization of the Washington isolate. The Washington virus was not neutralized by bluetongue virus antiserum. Cells infected with the Washington isolate exhibited intracytoplasmic fluorescence by the indirect fluorescent antibody method with New Jersey and Alberta epizootic hemorrhagic disease antiserums but not with bluetongue antiserum.     

Characteristics of the herpesvirus of malignant catarrhal fever isolated from captive wildebeest calves

A herpesvirus was isolated from buffy coat cells from a newborn wildebeest (Connochaetes gnou) and from tissues of a 12-day-old wildebeest during the 1982 calving season of a captive, inbred herd maintained in a zoologic collection. Both wildebeests were clinically healthy, and there was no herd record that malignant catarrhal fever (MCF) existed. Each viral isolate produced cytopathologic changes in bovine kidney cell cultures (intranuclear inclusions and massive syncytia). The viral-infected cell cultures contained antigens of MCF virus detected by immunofluorescence. The morphology of each viral isolate as determined by electron microscopy was that of a herpesvirus. Suspensions of 4 to 5 ml of disrupted cell culture material which contained virus from each wildebeest were inoculated (IV) into white-tailed deer (Odocoileus virginianus). Each deer became clinically ill within 28 days. Both deer had mucoid catarrh and a febrile response (40.5 to 41 C). Each also seroconverted to MCF virus. The histopathologic change in the tissues from the 2 inoculated deer was vasculitis. At 16 to 17 days after the deer were inoculated, a syncytial-forming virus was isolated from each deer from buffy coat cells fused with polyethylene glycol (1000) to bovine fetal kidney cells. The virus was identified as MCF virus by immunofluorescence and production of antibody to MCF virus. The presence of virus in the inbred wildebeest herd established this species as a reservoir or latent carrier of African MCF virus at the zoologic park.     

Different isolates from Leishmania braziliensis complex induce distinct histopathological features in a murine model of infection

The aim of this study was to evaluate the histopathological features in tissues of mice infected by human isolates (I, II, and III) or the reference M2903 strain of Leishmania braziliensis complex. BALB/c and C57Bl/6 mice were infected in the hind footpad with 10⁶ stationary-phase promastigotes of L. braziliensis complex. The evolution of lesions was observed for 10 weeks and the animals were then euthanized and liver, spleen and popliteal lymph nodes were collected. Tissues were stained with hematoxylin and eosin and analyzed by immunohistochemistry assay. Increased thickness of infected footpads was observed in all animals, lesions were nodular and non-ulcerated. Mice infected with isolate I presented inflammatory infiltrates consisting predominantly of mononuclear cells in all tissues examined, and also a great number of megakaryocytes, compared with other isolates. Infection with isolate II led to an infected footpad enlargement not seen in other isolates. In addition, mononuclear infiltrates in the liver and hemosiderin in spleen were noted. Conversely, mice infected with either isolate III or M2903 strain only showed an increased number of megakaryocytes in spleen. All tissues examined had detectable amastigote forms of Leishmania by immunohistochemistry in all groups. Taking together, our results showed an unforeseen behavior of different isolates of L. braziliensis complex that led to diverse pathological findings.     

Violet mink develop an acute disease after experimental infection with Aleutian disease virus (ADV) isolate ADV SL3

Six-Aleutian (aa)-genotype violet mink were infected intraperitoneally with the Aleutian Disease Virus (ADV) bone marrow derived isolate ADV SL3. All animals developed virus-specific antibodies and hypergammaglobulinaemia. Mortality during the fourteen week duration of the infection was 50%. The virus induced (histo)pathological lesions typical for Aleutian Disease. By immunohistochemical examination using a virus capsid-specific monoclonal antibody viral antigen was detected in lymph nodes, spleen, kidneys and once in hepatic Kupffer cells. By Southern blot and in situ hybridization studies with strand-specific RNA probes able to distinguish viral replicative forms from merely sequestered genomic DNA, ADV replication was detected in mesenteric lymph nodes and spleen. In one mink DNA replicative forms were also found in bone marrow cells or mononuclear cells of the peripheral blood, respectively. Only single-stranded viral DNA was detected in liver, kidney, gut and lung of infected animals. From Southern blot hybridization results a different, possibly organ-specific permissiveness of ADV in vivo is suggested.     

Pathogenesis of 'sheep-associated' malignant catarrhal fever in rabbits

Pathogenesis studies of experimentally produced sheep-associated malignant catarrhal fever (MCF) in laboratory rabbits are described. Animals were examined at intervals after inoculation. The principal change was a proliferation of lymphoid cells which began as soon as three days and became quite pronounced by 13 days after inoculation. The appendix, mesenteric lymph node and spleen were most obviously affected. The reason for this was a progressive increase in T-lymphocytes, which appeared to be a hyperplasia rather than neoplasia in T-dependent areas of these organs. Lymphoid cells also accumulated in interstitial spaces of non-lymphoid organs. The use of cyclosporin-A suppressed the lymphoid proliferation but rabbits still developed clinical MCF after a similar incubation period. It is suggested that the agent of MCF might produce its effect by infecting and causing a dysfunction of lymphoregulatory cells, resulting in benign polyclonal T-lymphocyte proliferation. Terminal necrosis could be due to natural killer cell activity.     

Malignant catarrhal fever: experimental transmission of the 'sheep-associated' form of the disease from cattle and deer to cattle, deer, rabbits and hamsters

Attempts to transmit malignant catarrhal fever (MCF) from 16 bovine cases of the 'sheep-associated' form of the disease are described. On two occasions disease was transmitted to bovine calves but transmission to red deer (Cervus elaphus) was not achieved. In addition, MCF was transmitted from one experimentally affected calf to a rabbit and on another occasion directly to rabbits with material from a field case which failed to transmit to a bovine calf or red deer. Subsequently each of these isolates was readily passaged through rabbits and one was also passaged to Syrian hamsters. Tissue from MCF-affected red deer consistently produced disease on inoculation into rabbits and deer but failed to cause disease in bovine calves. Contact infection between red deer occurred once and roe deer (Capreolus capreolus) were also shown to be susceptible to infection by inoculation. Passage of MCF in rabbits with an isolate from red deer failed to produce evidence of further adaptation even after 125 serial passages. Despite the failure to transmit disease from cattle to deer or from deer to cattle it is considered probable that there is only one sheep-associated agent which causes MCF in both species. The reasons for the anomalies in transmission of this form of the disease are discussed.     

Histopathology of avian adenovirus group II splenomegaly of chickens

Chickens were infected with an avian adenovirus group II isolate previously obtained from chickens exhibiting splenomegaly in commercial broiler flocks. The isolate was inoculated orally in 6-week-old experimental chickens, which were euthanatized and necropsied 6 days postinoculation. The primary gross lesions found were splenomegaly and splenic mottling. Numerous tissues from 12 chickens were taken for histologic evaluation. Histologic lesions included splenic reticuloendothelial cell hyperplasia with intranuclear inclusions. Lymphocytic degeneration was seen in the lungs of most chickens examined. Lung hemorrhage and edema with endothelial disruption and congestion of pulmonary arterioles were found less frequently. The splenic lesions in the chickens were similar to those seen in turkeys naturally infected with hemorrhagic enteritis, and the lung lesions resembled those seen in pheasants naturally infected with marble spleen disease.     

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.     

In vitro antibody response to the tetrapeptide repeats of Plasmodium falciparum circumsporozoite protein by splenocytes from mice infected with P. yoelii yoelii

The antibody response to the recombinant protein, R32tet32, which contained the repetitive sequence (NANP)n of Plasmodium falciparum CSP was determined in C57BL/6 mice during the course of nonlethal infection with Plasmodium yoelii 17X. Marked suppression of the IgG antibody response to R32tet32 occurred when mice were immunized at peak parasitemia (on day 16). In vitro antibody responses of spleen cells from acutely infected mice to R32tet32 were similarly suppressed. Stimulation of normal spleen cells cultured for 5 days with 100 ng/ml of R32tet32 gave an optimal IgG antibody response, but spleen cells from infected mice obtained at peak parasitemia failed to respond to a broad range of antigen concentrations. Cocultivation studies employing enriched lymphocyte populations from infected and uninfected C57BL/6 mice indicated that both T and B cells from infected mice were defective in their response to R32tet32. The response to the repetitive region was restored by the addition of recombinant mouse interleukin-2 (IL-2) at a dose of 50 U/ml to cultures of spleen cells from infected mice.     

Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.     

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5?×?10⁵ TCID₅₀ of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5′-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.     

Isolation and characterisation of lymphoblastoid cells from cattle and deer affected with 'sheep-associated' malignant catarrhal fever

Cells with the histological and ultrastructural characteristics of large granular lymphocytes (LGL) have been obtained in culture from both cattle and red deer (Cervus elaphus) reacting with 'sheep-associated' malignant catarrhal fever (MCF). Such cells have been derived from thymus, lymph node and spleen suspensions as well as from cerebrospinal fluid cells and cultured cornea. On most occasions their presence was observed only transitorily but by providing the cells with feeder monolayers and, or, interleukin-2, several lines were maintained indefinitely, and some became independent of these factors after prolonged culture. A similar cell line was also derived from a Père David's deer affected with MCF at Whipsnade zoological park. Functionally, cultured LGL were cytotoxic to both primary cell cultures and cell lines and their cytotoxicity was not restricted to histocompatible target cells. These findings suggest that the cultured cells have natural killer cell-like activity and that they are important targets for the agent of MCF in cattle and deer. One cell line derived from a red deer transmitted the disease but none of the cells generated from cattle did.     

Antigens and antibodies of malignant catarrhal fever herpesvirus detected by immunodiffusion and counter-immunoelectrophoresis

Using hyperimmune rabbit and cattle sera, immunodiffusion (ID) and counter-immunoelectrophoresis (CIEP) tests detected three or four and two or three malignant catarrhal fever (MCF) virus antigens, respectively, in infected cells. The ID test detected precipitating antibodies to MCF virus in $\text{3}\text{9}$ experimentally infected rabbits, $\text{0}\text{14}$ experimentally infected cattle, $\text{1}\text{13}$ naturally infected cattle, $\text{62}\text{176}$ wildebeest and $\text{3}\text{20}$ hartebeest. The CIEP test detected specific antibodies in $\text{3}\text{9}$ rabbit sera, but non-specific reactions prevented its use with bovine sera.The CIEP test was 2 to 4 times more sensitive than ID for detecting antibodies to MCF virus, but both tests were less sensitive than indirect immunofluorescence.The ID test demonstrated an antigenic relationship between wildebeest and hartebeest strains of MCF virus. Neither ID nor CIEP detected MCFV antigens in tissues infected with MCF virus.     

Heartwater. The artificial transmission of Cowdria ruminantium in domestic ruminants and mice

The artificial transmission of Cowdria ruminantium with infected blood, organ homogenates, peritoneal macrophages, tick stabilate and tissue culture cells is discussed. Organ homogenates prepared from the myocardium, spleen, kidneys and liver of diseased animals are commonly used to infect mice. The efficacy of organ homogenates as a source of C. ruminantium depends on factors such as the route of inoculation and the heartwater isolate used. Heartwater is artificially transmitted with infected tick stabilate, haemocytes, rectal ampules and hypodermal homogenates. The infectivity of saliva collected from Amblyomma hebraeum female ticks was very low compared to the ground-up suspensions prepared from the same group of ticks.     

Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symptomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicaspase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P<0.05). Apoptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P<0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F=20.45; P=0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis.     

Effects of Bordetella avium on lymphoid tissue monoamine concentrations in turkey poults

Six hours post-hatch, large white turkey poults were inoculated intranasally with 5 x 10(7) cells of the W isolate of Bordetella avium. Three hours after inoculation and subsequently on days 7, 10, 14, 21, and 28 postinoculation, poults from infected and control groups were killed by cervical dislocation. Thymus, spleen, and bursa of Fabricius were removed, weighed, and frozen until assayed for norepinephrine (NE), dopamine (DA), and serotonin (5HT). B. avium infection caused a reduced concentration of NE, DA, and 5HT in the spleen, thymus, and bursa of Fabricius. The reduced concentrations of these monoamines in lymphoid tissues of diseased poults may be a normal response during the course of a disease or during the mounting of an immune response.     

Experimental infection of cats with Chlamydophila felis

Cats experimentally infected with a British isolate of Chlamydophila felis (C. felis), B166 strain, by droplet into the eye and nose developed conjunctivitis, mild rhinitis and fever. The chlamydophila were first isolated from conjunctiva, nictitating membrane and then from lung, tonsil, liver, spleen, kidney, nasal and vaginal swabs and blood. These results indicate that C. felis B166 strain first infected and replicated in the conjunctiva and nictitating membrane in cats with symptoms which were mostly limited to conjunctivitis, and then pervaded the whole body by bacteremia.     

Effect of infection of turkeys with haemorrhagic enteritis adenovirus isolate on the selected parameters of cellular immunity and the course of colibacillosis

The aim of the study was to determine the effect of a Polish low-virulence isolate of haemorrhagic enteritis adenovirus (HEV) on the immune system in turkeys and on the course of colibacillosis in birds infected under laboratory conditions. Turkeys were infected per os with HEV at the dose of 10(4.3)EID50/mL and with E. coli (APEC) (serotypes 078:K80:H9) at the dose of 4x10(9)CFU/mL by injection to the thoracic air sac. The birds infected with the HEV were infected with the APEC either simultaneously or after 5 days. Five days after HEV infection, the percentages of subpopulations of the CD3+CD4+ and CD3+CD8alpha+ T cells and the IgM+ B cells were determined in blood and spleens of the HEV-infected turkeys and in the control (uninfected) birds. The course of colibacillosis was more severe in turkeys infected with the APEC 5 days after infection with the HEV than in those infected with the HEV and APEC simultaneously and than in those infected only with APEC. Five turkeys out of the 18 infected with the APEC 5 days after infection with HEV, died. Their body weights were statistically significantly lower with higher FCR values 41 days after the infection in comparison to turkeys in the other groups. A considerable decrease in the percentage of the T and B cells subpopulations in the blood were found in turkeys infected with the HEV and while the percentage of CD3+CD4+ T cells subpopulation in the spleen increased significantly, the contribution of the CD3+CD8alpha+ T cells and IgM+ B cells subpopulations were decreased. These changes in the immune system of turkeys, occurring 5 days after infection with the HEV, made them more susceptible to infection with the APEC.     

犬细小病毒HZ0761株的分离与鉴定

采集疑似细小病毒(CPV)感染犬的粪便,采用同步培养法接种胎猫肾细胞(F81)进行病毒分离鉴定。通过PCR检测、HA试验、IFA鉴定、电镜观察和空斑纯化,获得1株犬细小病毒,并命名为HZ0761。感染的F81细胞48h后出现明显的细胞病变;在病料和感染的F81细胞中均扩增出CPV VP2基因的特异性片段(221 bp);病毒液可凝集猪红细胞,血凝价为1∶28,其血凝性能被特异性抗体抑制;IFA可见特异性亮绿色荧光;电镜观察感染的F81细胞核内可见20 nm左右的病毒颗粒;病毒液的TCID50为10-4.8/mL,VP2基因序列分析显示该毒株为CPV-2 a型。     

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation,propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.