Development and distribution of rinderpest virus antigen in experimentally infected goats

Three goats, experimentally infected with rinderpest virus were examined for the development and distribution of precipitating antigens in various tissues and secretions using the agar gel immunodiffusion test. Virus antigens were detected in ocular secretions and lymph node biopsies from the second to the fourth and fifth days of pyrexia, respectively, but were not detected in nasal secretions. Precipitating antigens were demonstrated in various lymphoid organs, the lung and abomasum of a goat killed on the fourth day of pyrexia. These findings are discussed in relation to the epidemiology of rinderpest in goats in Africa.     

The use of the direct immunoperoxidase test to detect the multiplication of rinderpest virus in bovine kidney cell culture

The direct immunoperoxidase test has been used to detect rinderpest virus antigens in infected bovine kidney cell cultures to study the multiplication of the virus. Infected bovine kidney coverslip cultures were sequentially tested with peroxidase-labelled, anti-rinderpest globulins at 3, 6, 24, 48, 72, 96, 120 and 144 h post-infection. The progressive virus-specific cytopathic changes compared well with the increase in the number of cells showing the presence of viral antigens when tested by the direct immunoperoxidase test. The specificity of the reaction was confirmed by using negative and antiserum-blocked BK cell cultures on coverslips.     

Detection of African malignant catarrhal fever virus antigens in cell cultures by immunofluorescence

A fluorescent antibody (FA) test for antigens of African bovine wildebeest-derived malignant catarrhal fever virus was developed. Serum from one of the few survivors of the experimental disease in steers was used to prepare the conjugate. Both a virulent and an attenuated strain of malignant catarrhal fever virus were used to infect bovine thyroid cell cultures. Cells infected with both strains were readily detected by FA staining as early as 24 h post infection, whereas cytopathic effect could be observed by bright-field microscopy only after days 5 or 6 post infection. Controls consisting of normal bovine thyroid cells or infected cells treated with conjugated normal globulins did not show autofluorescence. The reaction was blocked by treatment of infected cells with homologous positive antisera but not by treatment with normal bovine serum or antisera to foot-and-mouth disease, rinderpest, bovine virus diarrhea, Ibaraki, infectious bovine rhinotracheitis, or bovine herpes mammilitis viruses. Treated with African malgnant catarrhal fever virus conjugate did not react.     

Antigenic variation between three strains of rinderpest virus detected by kinetic neutralisation and competition ELISA using early rabbit antisera

Strains of rinderpest virus (RV) with high and low virulence were compared with cell culture-attenuated RV for antigenic differences. Sera collected from rabbits 1 week after inoculation with live virus distinguished between homologous and heterologous strains by kinetic neutralisation and competition ELISA.     

Serological studies with peste des petits ruminants and rinderpest viruses in Nigeria

One hundred and ninety-five goat and 67 sheep sera collected from various parts of southern Nigeria were screened for neutralising antibodies to both the peste des petits ruminants (PPR) and rinderpest viruses. Neutralising antibodies against both viruses were found in the sheep and goat sera examined. Parallel titration of samples which neutralised both viruses indicated a primary infection with the PPR virus (PPRV). However, some samples which failed to neutralise PPRV neutralised the rinderpest virus (RV) indicating RV activity in sheep and goats in Nigeria. These findings are discussed in relation to the diagnosis of PPRV infection and the recent reappearance of bovine rinderpest in Nigeria.     

Improved isolation of rinderpest virus in transformed bovine T lymphoblast cell lines.

Bovine T lymphoblast cell lines transformed by the protozoan Theileria parva were compared with bovine kidney (BK) and Vero cells for their ability to isolate various strains of rinderpest virus from tissues and infected secretions. All of the strains of rinderpest virus that were tested, including attenuated cell-culture, caprinised and lapinised vaccines, and both mild and virulent pathogenic strains, readily induced syncytial cytopathic effect (cpe) in T lymphoblasts. The cpe could often be detected within one day of inoculation of lymphoblasts, whereas it took three to 14 days to appear in Vero and BK cells. Using lymphoblasts it was possible to reisolate rinderpest virus from nine of 42 swabs collected from three cattle experimentally infected with an isolate from a recent outbreak of mild disease whereas the same swabs yielded only one reisolate on BK cells. It was also possible using the lymphoblasts to detect infectious virus in the ocular, nasal and oral secretions of goats and rabbits infected with caprinised and lapinised virus, respectively. Peste des petits ruminants virus appeared to grow as rapidly as rinderpest virus in the lymphoblasts whereas canine distemper virus readily induced cpe on first passage but less readily on subsequent passage. Measles virus induced relatively little cpe when inoculated into lymphoblasts and did not appear to passage in these cells. The lymphoblasts are easy to maintain in culture and since they rapidly recovered 11 isolates from 37 diagnostic samples could prove useful in laboratories carrying out rinderpest diagnosis.     

Enzootic Pneumonia of Pigs: Identification of a Causative Mycoplasma in Infected Pigs and in Cultures by Immunofluorescent Staining

Immunofluorescent staining has been used to identify Mycoplasma hyopneumoniae in smears of broth cultures, in infected pig testicle cell cultures, and in frozen cut sections of pneumonic lungs from field and experimentally produced cases of enzootic pneumonia. In the pneumonic pig lung, fluorescent staining was limited to the surface of the bronchial and bronchiolar epithelium and to the contained exudate. In a series of trials using experimentally infected pigs fluorescence was not detected until 25 days post-infection and was regularly seen in pigs killed thereafter. Porcine immune globulin precipitated from the serum of experimentally infected pigs and conjugated with fluorescein isothiocyanate was reactive and specific for the detection of M. hyopneumoniae. Immune globulin conjugates prepared from the serum of hyperimmunized rabbits were reactive but in some cases produced a faint non-specific staining of frozen tissue sections. No such non-specific reactions were noted on stained culture smears or cell cultures.

Fluorescence was not seen in known positive preparations stained with non-immune pig globulin conjugates or in preparations from uninoculated cell cultures or pigs, stained with non-immune or immune globulin conjugates.

Mycoplasma hyorhinis was detected by immunofluorescent staining with homologous conjugates, in smears of broth cultures and in tissue sections from pigs with polyserositis.

Immunofluorescent staining was found to be species specific and useful for the early species identification of mycoplasma isolated from pigs.

     

Herpes viral hepatitis in a toucan

Herpesvirus infection was diagnosed in a toucan. The herpesvirus was isolated from the liver and identified by electron microscopy in the liver and in cell culture. A negative immunofluorescent reaction was obtained when virus-infected cell cultures were reacted with a conjugate to the herpesvirus of Pacheco's disease. The main pathologic finding in the toucan consisted of a severe necrotizing hepatitis with intranuclear inclusions in the liver and spleen. A presumptive diagnosis of chlamydiosis was also made, based on a positive direct fluorescent monoclonal antibody reaction to chlamydial antigens in impression smears of liver and spleen. Chlamydial isolation attempts were unsuccessful. The toucan had been in contact with two macaws that had died 5 days before the toucan died and were diagnosed by histology as having herpesvirus hepatitis.     

The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis

Hyperimmune sera were produced by serial inoculation of rabbits with Vero cell-adapted, sucrose gradient-purified Nigerian peste des petits ruminants virus (PPRV) isolate. Two antisera produced, neutralized the homologous PPRV but not the heterologous rinderpest Kabette "O" virus. The antisera gave strong precipitin lines with purified PPRV antigens and were used to detect PPRV and rinderpest virus antigens from ante-mortem secretions and post-mortem tissue homogenates from PPR and rinderpest virus infected goats and cattle by the agar gel precipitation tests (AGPT). The hyperimmune sera gave good titration curves with both purified Nigerian goat and the United Arab Emirate wildlife PPRV isolates in the indirect enzyme linked immunosorbent assay (ELISA). Results of indirect ELISA showed that although there were some cross reactions with the rinderpest, canine-distemper and measles viruses, at 1:100 dilution, the antisera would give a positive signal with only the homologous PPR virus.     

The Use of Antigen-capture Enzyme-linked Immunosorbent Assay (ELISA) for the Diagnosis of Rinderpest and Peste des Petits Ruminants in Ethiopia

Rinderpest had been reported in most parts of Ethiopia when the Pan African Rinderpest Campaign (PARC) was launched. As a result of intensive disease investigation and strategic vaccination, most parts of the country are now considered provisionally free, and widespread vaccination has been replaced by clinical and serological surveillance. Details of any episodes of disease are recorded and followed up after laboratory confirmation of suspected cass using antigen-capture ELISA. This paper is based on observations on the performance of the antigen detection ELISA compared to the agar gel immunodiffusion (AGID) test, which also differentiates rinderpest from peste des petits ruminants (PPR). The stability of the specific viral antigen was monitored for 4 days, and rinderpest and PPR antigens were still detected, depending on the type of specimen. Antigen capture ELISA is more rapid, sensitive and virus specific than the AGID. Even if the cold chain of the specimen is compromised for a day or two during sample collection and submission, the specimen may still be suitable for testing by ELISA.     

Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela

Background: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. Objective: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. Methods: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. Results: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. Conclusions: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.     

An immunoperoxidase monoclonal antibody stain for rapid diagnosis of infectious bursal disease

Cell smears of chicken-embryo-fibroblast (CEF) cultures and bursa of Fabricius from chickens experimentally infected with six different strains of infectious bursal disease virus (IBDV) were examined for the presence of IBDV by the avidin-biotin-peroxidase complex method of immunoperoxidase (IP) staining using a monoclonal antibody specific for IBDV designated BK70. IBDV of different strains and serotypes were readily detected by the IP method in cell smears prepared from infected CEF cultures and from bursas. Bursal cells were positive for IP stain in most of the infected bursas (87.5%), despite their mild IBD lesions. Positive IP staining of bursal smears was well correlated with the recovery of IBDV from the bursas and with IBD lesions in the bursas. IP stain with a monoclonal antibody (BK70) appeared potentially useful for rapid and definitive diagnosis of IBD.     

Proliferation of chicken lymphoblastoid cells after in vitro infection with Marek's disease virus.

Activated, thymus-derived (T) lymphoblasts were exposed to Marek's disease virus and cultivated in attempts to induce in vitro transformation. After 9 to 15 days, colonies or small clusters of proliferating lymphoblasts were observed in cultures from three of a total of 122 attempts. These developed into proliferating cell cultures that resembled conventional Marek's disease (MD) lymphoblastoid cell lines in terms of growth characteristics and morphology. All proliferative cultures were unusual in that 1) the expression of viral internal antigens consistently or periodically was very high (up to 30% of all cells) and 2) the cells deteriorated and/or proliferation ceased in all cases after culture periods of 45-176 days. The proliferative cultures were all characterized as CD2+ and CD3+, Ia-bearing T cells; one was CD4+/CD8- and TCR2+, the other two were CD4-/CD8- and TCR1+. The latter two are the only cultures of MD-infected cells known to be TCR1+.     

Colostral Transfer of Rinderpest Neutralizing Antibody to Offspring of Våccinated Dams

The transfer of maternal antibodies to Friesian and buffalo calves born of dams vaccinated against rinderpest was through colostrum only. Colostral antibody titers at the time of parturition were higher than the serum titer. Two hours after suckling, a high level of rinderpest neutralizing antibodies was detected in the sera of newborn animals. The half-life of maternal antibodies in buffalo and Friesian calves was found to be approximately 33 and 29 days respectively. By the age of 7-8 months, 60 per cent of buffalo calves and 80 per cent of Friesian calves had no detectable levels of rinderpest neutralizing antibody.     

Sheep erythrocyte and bluetongue virus antibody responses of spleen cell cultures from mice.

The optimum conditions for the culture of cells from dissociated spleens were determined. Routinely, 10(7) cells were seeded per ml of RPMI 1640 medium supplemented with 20% pre-tested foetal calf serum. For the assay of the immune response, cultures were supplemented with 30 muMolar mercaptoethanol. The immune responses to sheep erythrocyte and bluetongue virus antigens were determined by the haemolytic plaque-forming cell assays described by Oellermann (1974) and Oellermann, Carter & Marx (1976a). The optimum sheep erythrocyte antigen concentration was 2 X 10(6) erythrocytes per 10(7) spleen cells and maximum IgM plaque-forming cells were detected after 4 days in culture. Successful stimulation of the immune response to bluetongue virus was achieved in spleen cell cultures from mice previously primed with bluetongue virus. The optimum antigen concentration was 30-40 ng bluetongue virus per 10(7) spleen cells and the maximum plaque-forming cell response was observed after 4 days in culture.     

Time dependent decreases of maternal canine virus antibodies in newborn pups

Levels of passively transferred maternal antibodies to three canine viruses, rabies virus (RV), canine distemper virus (CDV) and infectious canine hepatitis (ICH) virus, in serum specimens from 14 fetal pups and in serial serum specimens collected up to 45 days after whelping from 14 neonate pups were compared with levels of antibodies to these viruses in milk and sera collected concurrently from their respective dams. Radioimmunoassays using RV-, CDV- and ICH virus-specific antigens showed that sera from all fetal pups had detectable levels of antibodies to all three canine viruses and ICH neutralising antibodies were detected in sera from 10 of the 14 fetal pups. As the time after whelping increased, titres of RV-, CDV- and ICH virus antibodies measured by radioimmunoassay and ICH virus neutralising antibody tests in serially collected specimens of milk from dams rapidly decreased, while titres of the antibodies in serum specimens from newborn pups in their litters steadily decreased. Individual fetal and newborn pups with a high titre of antibody to one virus also had high titres to the other two viruses, although a wide range of titres was observed among pups in each of the litters studied. Markedly higher titres of antibody to all three viruses were observed in serially collected specimens of sera from dams than in sera from fetal and newborn pups in their litters. Results show that maternal RV, CDV and ICH virus antibodies are transferred from dams to pups in utero and by nursing. Levels of these maternal virus-specific antibodies in newborn pup sera decreased at similar rates as time after whelping increased.     

Re-infection of wildlife populations with rinderpest virus on the periphery of the Somali ecosystem in East Africa

We report surveillance for rinderpest virus in wildlife populations in three major ecosystems of East Africa: Great Rift Valley, Somali and Tsavo from 1994 to 2003. Three hundred and eighty wild animals were sampled for detection of rinderpest virus, antigen or genome and 1133 sampled for antibody in sera from Kenya, Uganda, Ethiopia and Tanzania from 20 species. This was done modifying for wildlife the internationally recommended standards for rinderpest investigation and diagnosis in livestock. The animals were selected according to susceptibility and preference given to gregarious species, and populations were selected according to abundance, availability and association with livestock. Rinderpest virus, antigen and/or genome were detected in Kenya; within Tsavo, Nairobi and Meru National Parks. Serological results from 864 animals (of which 65% were buffalo) from the region were selected as unequivocal; showing the temporal and spatial aspects of past epidemics. Recent infection has been only in or peripheral to the Somali ecosystem (in Kenya). Our evidence supports the hypothesis that wildlife is not important in the long-term maintenance of rinderpest and that wildlife are infected sporadically most likely from a cattle source, although this needs to be proven in the Somali ecosystem. Wildlife will continue to be a key to monitoring the remaining virus circulation in Africa.     

An immunofluorescence diagnostic test for feline viral rhinotracheitis.

Hyperimmune serum against feline viral rhinotracheitis was produced in a goat and conjugated with a fluorescent dye. Cell cultures infected with rhinotracheitis virus had positive immunofluorescence. Cell cultures infected with other feline viruses and herpesviruses of other species did not fluoresce. In cats experimentally infected with rhinotracheitis virus, the virus was isolated from nasal and conjunctival swabs 1 to 9 days after inoculation. Nasal smears stained with the conjugated antiserum fluoresced 1 to 9 days after inoculation when clinical disease was most apparent. Conjunctival smears had positive immunofluorescence 1 to 6 days, but not 9 days, after inoculation. On postinoculation day 23, rhinotracheitis virus was not isolated from nasal or conjunctival swabs and nasal and conjunctival smears did not fluoresce. Rhinotracheitis virus or feline calicivirus was isolated from naturally infected cats with upper respiratory tract disease. Nasal and conjunctival smears from rhinotracheitis virus-infected cats had positive immunofluorescence in all cast showing clinical illness. Smears from 1 clinically normal cat from which rhinotracheitis virus was isolated did not fluoresce. Nasal and conjunctival smears from calicivirus-infected cats did not fluoresce.     

A Comparison of the Viruses of Infectious Bovine Rhinotracheitis (IBR), Infectious Pustular Vulvovaginitis (IPV), and Rinderpest: Part I. Studies of Antigenic Relationships

The viruses of infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV), and rinderpest were compared by specific methods. The results further confirmed that IBR and IPV are caused by agents with common antigens. No antigenic relationship was found between these viruses and rinderpest virus, which confirms earlier work.