The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus.

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.

Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus.

Sixteen 20 day old pigs, devoid of neutralizing antibody to pseudorabies virus (PRV), were divided into two groups of eight, an the animals of each group were housed in a separate unit. In each group 6 pigs were inoculated intranasally with the thymidine kinase (TK-) mutant (Group 1) or the field strain of PRV (Group 2), each pig receiving an inoculum of 4 ml. The remaining 2 pigs in each group served as uninoculated controls. The only clinical sign observed in the pigs of Group 1 was a transient febrile reaction, in the case of six pigs inoculated with the TK- mutant of PRV, whereas no signs of disease were seen in the uninoculated controls. The virus was isolated from the 6 infected pigs of the group only on post infection day (PID) 2, whereas it was never isolated from the controls. By contrast, the pigs of Group 2, had a severe clinical response and one, among those that were inoculated with the field strain of the PRV, died on PID 9. Virus was consistently isolated from all pigs of Group 2, inoculated and control. On PID 30 all pigs, i.e. the 8 of Group 1 and 7 of the Group 2 which survived to the infection, were subjected to dexamethasone (DMS) treatment. After DMS treatment virus was never isolated from the nasal swabbings obtained from the pigs of Group 1, whereas it was consistently isolated from pigs of Group 2. After 30 d from the start of DMS treatment the pigs were killed and several tissues were collected from each pig for virus detection, by isolation in tissue culture and by PCR analysis. At necropsy no lesions were found in pigs of Group 1, whereas acute pneumonia and gliosis in the trigeminal ganglia were observed in pigs of Group 2. Virus was never isolated from any of the tissues taken from pigs of both, Group 1 and Group 2, nevertheless sequences of PRV were detected by PCR analysis in the trigeminal ganglia of the pigs of both Groups.

A study on the characteristics of La Sota strain of Newcastle disease virus adapted to grow in a cell line from bovine embryo kidney (BS/BEK).

The lentogenic La Sota strain of Newcastle disease virus (NDV) was adapted to grow in the BS/BEK cell line of bovine embryo kidney origin with a infectious titre similar to that in chicken embryos. No modification in the biological properties was detected after serial passages in the cell line, as indicated by CPE and by size and shape of the plaques. By contrast, the intracerebral pathogenicity index test, determined in 1-day-old chicks, was lower than for La Sota grown in chicken embryos. However, the immunogenicity of the cell culture adapted virus did not show any variation as demonstrated by serological response and by protection following challenge with virulent NDV. Accordingly, it appears that La Sota grown in cell cultures has retained its biological and immunological characteristics and if these results are confirmed by field trials and long term protection tests, the use of the BS/BEK cell line could be an alternative to chicken embryos for the cultivation of NDV.

A study of two mutant strains of pseudorabies virus (PRV) unable to express thymidine kinase (TK) function.

Two mutant strains of pseudorabies virus (PRV) were selected from the virulent 86/27V virus treated with chemical drugs. The viruses, named 6A1 and 6C2, respectively, appeared to be unable to express thymidine kinase function, as demonstrated by the autoradiography test. They showed a reduced virulence for some susceptible animal species (chickens, mice, rabbits, calves, lambs and piglets) and virus was isolated sporadically. The mutant viruses appeared to be able to protect animals against infection with the virulent strain of PRV. At gross, as well as at histological examination, no lesions in apparatus, system and tissues were detected in pigs inoculated with 6A1 and 6C2 viruses. By contrast, rabbits treated with 6C2 mutant strain presented lymphomononucleated cuffs, microgliosis, and neuronophagia in some areas of the brain. This focal spreading, together with the absence of neuronal necrosis and intranuclear inclusions, suggest an infection induced by a modified strain of PRV.

The response of pregnant gilts previously given an inactivated preparation of porcine parvovirus (PPV) to challenge infection with a fully virulent PPV.

Eight 40-day pregnant gilts, previously treated with an adjuvanted-inactivated viral preparation (AIVP) obtained with a field strain of porcine parvovirus (PPV) together with 4 pregnant untreated controls, were subjected to challenge infection with a virulent strain of PPV at the 40th day of gestation. After challenge, all controls became febrile for 2 to 8 days, whereas only one gilt among those which had been treated with the AIVP experienced fever which lasted 4 days. Virus was consistently recovered from fecal swabs obtained from the controls and only sporadically from feces of AIVP-treated gilts. When the gilts were killed 53 days after challenge infection, no macroscopic lesions were found in any of the gilts in either groups, but fetal death was observed in the two groups of animals. However, the rate of dead fetuses was much higher among the control (70.5%) than among those from the AIVP-treated gilts (10.1%). Virus was recovered from 23 of the 24 dead fetuses in the control group and only from 3 among the 8 dead fetuses which were reported for the AIVP-treated gilts.

Protection tests in pigs vaccinated with the lapinized Chinese strain of hog cholera virus (HCV) previously adapted in a minipig kidney (MPK) cell line, to challenge infection with virulent HCV.

Pigs which had been vaccinated with the Lapinized Chinese strain of Hog Cholera Virus previously adapted in a minipig cell line cultures (MPK-LC-HCV), resultet to be protected when they were subjected to challenge infection with virulent Hog Cholera Virus (HCV) 6 or 11 months later. The challenge virus was never isolated from any of the vaccinated pigs. The MPK-LC-HCV vaccine induced a significant rise of the antibody titer to the HCV in pigs kept under field conditions.

A characterization study of a cell line (BS/BEK) from bovine embryo kidney.

A cell line (BS/BEK) which was obtained from bovine embryo kidney tissue, when studied at its 140th passage level it showed the following properties: 1. An epithelial-like morphology, possessing a heteroploid karyotype with a modal chromosome number ranging between 70 and 75 chromosomes. 2. It failed to produce tumors in mice and in hamster. 3. It was shown to be ready susceptible to the replication of several viral agents originated from a variety of animal species. 4. It was not contaminated by mycoplasma or other bacterial spp.

The lapinized Chinese strain of hog cholera virus (HCV) previously adapted in minipig kidney (MPK) cell line cultures, would also protect pigs to experimental infection with virulent HCV [corrected].

Two-month old piglets previously inoculated with different dilutions of the Lapinized Chinese (LC) strain of Hog Cholera Virus (HCV), adapted in a minipig kidney (MPK) cell line, resisted challenge infection with virulent HCV. All the animals remained healthy and the challenge virus was never recovered from any of them. In contrast, the pigs which served as controls for the challenging virus underwent the clinically lethal form of the disease and HCV was consistently recovered from their tissues.

Cultivation of foot-and-mouth disease virus in BHK21 cells grown in microcarrier culture system.

When BHK21 cells were grown according to the microcarrier's system, they reached the highest concentration in 72 hrs. The infectivity of Foot-and-Mouth Disease (FMD) virus in BHK21 microcarrier culture increased 10 times compared with the conventional monolayer culture in rolling bottles.

General characteristics and viral susceptibility of a newborn pig kidney (NPK) continuous culture.

We have developed a fibroblastic-like continuous culture of newborn pig kidney (NPK). The current cell line was serially passaged 160 times and appeared to be well suited for production and assay of a number of viruses affecting pigs, such as pig parvovirus, pseudorabies and transmissible gastroenteritis. The cell line appeared aneuploid, with a modal chromosome number of 36 and induced tumors, classified as fibrosarcoma, in athymic mice.

A serological survey of swine parvovirus infection in Italy.

A serological survey to detect the presence of porcine parvovirus (PPV) infection in Italy and its geographic distribution was conducted. 1,332 samples of serum collected in 1983/1984/1985 were taken from pig breeding herds and, to a lesser extent, from fattening piggeries of representative regions of Italy. They were tested using the hemagglutination inhibition test (HIT). The results of the serological study indicate that parvovirus infection is widespread in Italian herds having 70.3% of sera antibodies to this virus; the most frequent titer detected was 1:8192 or higher. Sera from adult sows showed a higher rate (73.1%) of positive titers than those from 3-6 month-old pigs (40.7%). Positive samples were more relevant in the regions of northern Italy when compared with those from the southern Italy, Sicily and Sardinia.

Characterization of a parvovirus isolated from a pig fetus.

A small hemagglutinating virus belonging to the Parvoviridae Family was isolated from a 70 day-old pig fetus in a breeding herd in which infertility, metritis and abortions were reported. The virus, named 85/193L, was isolated either in primary pig kidney (PK) cells or in a continuous cell line of minipig kidney origin (MPK), both cell cultures actively growing. It produced a typical cytopathic effect (CPE) starting from the 3rd passage and intranuclear inclusions surrounded by a halo were observed in stained preparations. The isolate was completely resistant to ether, chloroform and to pH 3; it was not inactivated after treatment at 56 degrees C for 4 h and at 70 degrees C for 2 h, while it was completely inhibited by the 80 degrees C/30 min temperature. It contained deoxyribonucleic acid (DNA). The highest infectious titer was reached at 96 h post infection. The infectivity and the hemagglutinating activity of the isolated strain were both inhibited by the reference immune serum against NADL-2 pig parvovirus. This further confirmed that the 85/193L isolate belongs to the parvovirus genus.

A serologic survey of encephalomyocarditis virus infection in pigs in Italy.

Neutralizing antibodies to encephalomyocarditis virus (EMCV) were found in 69.13% of the serum samples obtained from pigs from representative regions of Italy. The antibodies are distributed fairly uniformly throughout the swine populations with all age groups being equally involved.

A study on the susceptibility of minipig kidney (MPK) and rabbit kidney (RK13) cell line cultures to the lapinized Chinese strain of hog cholera virus.

The susceptibility of two established cell lines of pig (MPK = minipig kidney) and rabbit (RK13 = rabbit kidney) origin to the lapinized Chinese (LC) strain of hog cholera virus (HCV) was studied. Spleen cells from rabbits infected with the virus under study were inoculated to cell cultures of either MPK and RK13 cells and subsequent passages were made by culturing the trypsinized infected cells with the normal cells. Only the MPK cell line appeared to be susceptible to virus replication. Since no cytopathic effects (CPE) were observed, the presence of the viral antigen in the inoculated cultures was detected by immunofluorescence tests. The virulence of the virus for rabbits was enhanced after its cultivation in MPK cell cultures. When the MPK cell culture system adapted virus was tested in neutralization trials in the presence of an HCV reference immune serum it was found that the virus did not modify its antigenic structure in any extent. Finally, the culture adapted virus appeared to be more immunogenic for rabbits than the original rabbits adapted virus. Based on these results, it seems reasonable to suggest the use of MPK cell line for the propagation of the LC strain of HCV as an alternative to the use of rabbits for the preparation of HCV vaccine.

The ability by different preparations of porcine parvovirus to enhance humoral immunity in swine and guinea pigs.

The capacity of different preparations of inactivated porcine parvovirus to stimulate antibody response was studied. The adjuvants chosen were aluminium hydroxide gel [Al(OH)3, 30-50%], water-in-mineral-oil emulsion (w/Mo, 50%) alone or combined with Al(OH)3, and two synthetic products known as dimethyldioctadecylammoniumbromide (DDA, 0.16%) and an acrylic acid polymer resin (Carbopol 934P, 0.02%) respectively. For each preparation, swine and guinea pigs devoid of antibody to porcine parvovirus were inoculated. Among all the preparations tested, two of them i.e. emulsified Al(OH)3 adsorbed antigen and Carbopol 934P emulsified virus, gave the most significant antibody response in animals, without any difference being noted in the behaviour of the two animal species.

Neonatal calf diarrhea induced by rotavirus.

This presentation summarizes the results of a comprehensive study on rotaviruses isolated in Italy from calves and rabbits affected by neonatal diarrhea. The results clearly indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. The evidence of differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by three rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits, respectively. When rotavirus strains of bovine, simian, porcine and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. On the other hand, a reciprocal antigenic correlation was found between bovine and rabbit isolates. Finally, it was proven that feeding newborn calves with colostrum of their dams, previously vaccinated with an inactivated rotavirus vaccine, could prevent the neonatal diarrhea from occurring.

Cultivation of a pig parvovirus in various cell cultures.

The susceptibility of several established cell lines of pig (LLC-PK1 = pig kidney; MPK = minipig kidney; PK15 = pig kidney; ESK = embryonic swine kidney), bovine (EBTr = embryonic bovine trachea), monkey (MA-104 = fetal rhesus monkey kidney) and human (HEL-299 = embryonic human lung) origin to porcine parvovirus was studied. The primary pig kidney cell cultures (pPK) were included in the study as the reference cell system. From the results it appeared that the virus only replicated in cell lines originated from swine. In particular the MPK and ESK cell lines showed a susceptibility similar to that observed for pPK cell cultures. Intranuclear inclusions and plaques were also induced in these cell systems. It appeared therefore that MPK and ESK cell lines both possess all the requirements for use in pig parvovirus studies.

Isolation of cytophatic strains of rotavirus from pigs.

Twenty-two rotavirus isolates were recovered from piglets suffering from diarrhea. The isolates readily propagated in MA-104 cell cultures where they induced typical cytopathic effects (CPE) and possessed the physicochemicals properties of the members of rotavirus genus, Family Reoviridae. The electron microscopy study, conducted in MA-104 infected cells, revealed virus particles which possessed the peculiar morphology of rotavirus. The isolates were neutralized by a reference porcine rotavirus antiserum: all were devoid of hemagglutinating activity for red cells of swine, cattle, sheep, rabbit, chicken and guinea pig.

A study on the sensitivity of bovine rotavirus to some chemical agents.

The behaviour of bovine rotavirus, strain 81/36 F, to some chemical agents was studied. The chemicals tested were all more or less effective, depending on their concentration and time of exposure under room temperature. It is suggested therefore, that they could be used as disinfectants in the case of rotaviral contamination.

A serologic survey of rotavirus infection in pigs.

Neutralizing antibodies to rotavirus were found in 97.18% of the serum samples obtained from pigs from representative regions of Italy. The antibodies are distributed fairly uniformly throughout the swine population with all age groups being equally involved.