Vaccination of turkeys with an avian pneumovirus isolate from the United States

Four-week-old poults obtained from avian pneumovirus (APV) antibody-free parents were vaccinated with different serial 10-fold dilutions of cell culture-propagated APV vaccine. The birds were vaccinated with 50 microl into each conjunctival space and nostril (total of 200 microl). Each poult of each group was vaccinated in groups that received doses of 4 x 10(4), 4 x 10(3), 4 x 10(2), 4 x 10(1), or 4 x 10(0) 50% tissue culture infective dose (TCID50) of APV vaccine, respectively. Respiratory signs were seen between 3 and 12 days postvaccination (PV) in the poults that were vaccinated with 4 x 10(4), 4 x 10(3), and 4 x 10(2) TCID50, respectively. In these groups, APV was detected from swabs collected at 5 days PV and seroconversion was detected at 2 wk PV. The groups that were originally vaccinated with 4 x 10(1) and 4 x 10(0) TCID50 developed mild clinical signs after vaccination, but neither virus nor antibody was detected PV. At 2 wk PV (6 wk of age), birds from each group, along with five unvaccinated controls, were challenged with APV. Upon challenge, the 4 x 10(4) and 4 x 10(3) TCID50 groups were protected against development of clinical signs and were resistant to reinfection. The group previously vaccinated with 4 x 10(2) TCID50 developed clinical signs after challenge that were considerably milder than those seen in the groups that had previously been vaccinated with lower doses or no virus. Even though 4 x 10(2) TCID50 vaccine dose administered by intranasal ocular route resulted in infection, incomplete protection resulted with this pivotal dose. Upon challenge, the 4 x 10(1) and 4 x 10(0) TCID50 groups exhibited milder disease signs than those seen in the challenged unvaccinated controls. In these groups, APV was detected in preparations of swabs collected at 5 days postchallenge (PC) and seroconversion was detected at 2 wk PC. These results indicate that the dose of APV vaccine that causes protection is higher than that required to produce infection.     

Foot-and-mouth disease: a review of intranasal infection of cattle, sheep and pigs

In an outbreak of foot-and-mouth disease (FMD) it is important to identify animals at risk from airborne virus. Investigations have been carried out over the years to determine the dose required to infect cattle, sheep and pigs by the intranasal route. This paper reviews the results of investigations for animals which have been infected by instillation or spraying a virus suspension into the nostrils or by exposure to affected animals through a mask or by indirect contact. The lowest doses were found by use of a mask. With virus from affected pigs given through a mask, doses of 18 infectious units (IU) in cattle and 8 IU in sheep were found to cause infection and give rise to lesions. Overall, cattle required the least amount of virus followed by sheep. Pigs required a dose of 22 IU to cause infection and a dose of 125 IU to give rise to lesions. In many experiments pigs failed to become infected. With all three species the dose varied with the individual animal and the virus strain. For modelling previous outbreaks and in real time, a dose of 8 IU or 10 and 50% infectious doses (ID50) could be used where cattle and sheep were involved. Experience in the field, combined with the results from experiments involving natural infection, indicate that pigs are not readily infected by the intranasal route. However, for modelling purposes a dose of about 25 IU should be used with care. Investigations are needed to determine doses for virus strains currently in circulation around the world. In addition, the nature of the aerosol droplets needs to be analysed to determine how the respective amounts of infective and non-infective virus particles, host components and, in later emissions, the presence of antibody affect the survival in air and ability to infect the respiratory tract. Further work is also required to correlate laboratory and field findings through incorporation of the doses into modelling the virus concentration downwind in order that those responsible for controlling FMD are provided with the best available assessment of airborne spread. Finally, the doses found for infection by the intranasal route could be applied to other methods of spread where virus is inhaled to assess risk.     

Intranasal infection of Getah virus in experimental horses.

Aerosol transmission in equine Getah virus (GV) infection was examined by intranasal inoculation with 10(3.0) to 10(7.0) TCID50 of the MI-110 strain in 7 experimental horses. The establishment of intranasal infection of GV was confirmed in all these horses by detecting serum neutralizing antibody against the MI-110 strain. Horses inoculated with more than 10(4.0) TCID50 of the virus manifested mild pyrexia, eruptions, serous nasal discharge, lymphopenia or monocytosis. Viremia ranging from 10(1.0) to 10(3.5) TCID50/0.2 ml occurred in horses inoculated with 10(5.0) TCID50, or more. Virus recovery from the nasal cavity was observed only in horses inoculated with 10(7.0) TCID50, and the viral titers recorded were 10(3.0) TICD50/ml or less. From these results, it is assumed that GV disseminated from the nasal cavity of naturally infected horses, except for intranasal infection with a lot of the virus, is probably very low in titer. So it seems to be rare that GV in natural cycles is spread from horse to horse by aerosol transmission.     

A Classical Live Attenuated Vaccine for Sheep Pox

A classical live attenuated sheep pox vaccine was prepared using the Ranipet strain of sheep pox virus (SPV) at the 50th passage in a secondary lamb testicular cell system. The TCID50 and RD50 were 10(9.63)/ml and 10(9.51)/ml. respectively. The SID50 of SPV challenge virus was 10(5)/ml. The vaccine was found to have no adverse effects in laboratory animals, and was safe and effective in SPV seronegative lambs. In the field, 660 sheep were vaccinated with an immunizing dose containing 1 x 10(2) TCID50. Randomly selected vaccinated sheep mounted good cell-mediated immunity and humoral responses as measured by glucose utilization test and serum neutralization test, respectively, for the study period of 6 months.     

Median infectious dose (ID₅₀) of porcine reproductive and respiratory syndrome virus isolate MN-184 via aerosol exposure

The median infectious dose (ID(50)) of porcine reproductive and respiratory syndrome (PRRS) virus isolate MN-184 was determined for aerosol exposure. In 7 replicates, 3-week-old pigs (n=58) respired 10l of airborne PRRS virus from a dynamic aerosol toroid (DAT) maintained at -4°C. Thereafter, pigs were housed in isolation and monitored for evidence of infection. Infection occurred at virus concentrations too low to quantify by microinfectivity assays. Therefore, exposure dose was determined using two indirect methods ("calculated" and "theoretical"). "Calculated" virus dose was derived from the concentration of rhodamine B monitored over the exposure sequence. "Theoretical" virus dose was based on the continuous stirred-tank reactor model. The ID(50) estimate was modeled on the proportion of pigs that became infected using the probit and logit link functions for both "calculated" and "theoretical" exposure doses. Based on "calculated" doses, the probit and logit ID(50) estimates were 1 × 10(-0.13)TCID(50) and 1 × 10(-0.14)TCID(50), respectively. Based on "theoretical" doses, the probit and logit ID(50) were 1 × 10(0.26)TCID(50) and 1 × 10(0.24)TCID(50), respectively. For each point estimate, the 95% confidence interval included the other three point estimates. The results indicated that MN-184 was far more infectious than PRRS virus isolate VR-2332, the only other PRRS virus isolate for which ID(50) has been estimated for airborne exposure. Since aerosol ID(50) estimates are available for only these two isolates, it is uncertain whether one or both of these isolates represent the normal range of PRRS virus infectivity by this route.     

Semen changes in boars after experimental infection with pseudorabies virus

Two groups of adult boars were inoculated with a field strain of pseudorabies virus (PRV) by intranasal droplet; one group was given 5 x 10(5) median tissue culture infective doses (TCID50), and the other, 5 x 10(6) TCID50. (A third group was maintained as controls.) Ejaculates were examined twice a week for volume, sperm numbers, sperm morphology, and presence of PRV. Severe clinical disease with fever followed administration of the larger virus dose. Death (one boar), testicular degeneration, and transient elevation in spermatozoa with proximal cytoplasmic droplets were seen in different members of this group. The smaller dose resulted in seroconversion, but did not produce signs of disease. In this group, volume, sperm numbers, and sperm morphology did not decline when compared with base-line values or data of control animals. The virus was not isolated from semen. Effects of PRV infection on semen quality in boars seem to be related to the associated clinical signs of systemic disease.     

Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys

Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.     

Study of the potential involvement of pseudorabies virus in swine respiratory disease.

In order to investigate the potential involvement of pseudorabies virus (PRV) in swine respiratory disease, nine week old pigs were intranasally inoculated with the PRV strain 4892. Two doses of infection were used: 10(4.5) median tissue culture infectious doses (TCID50)/pig and 10(3.5) TCID50/pig, with ten pigs per group. In the group of pigs inoculated with 10(4.5) TCID50, seven out of ten pigs died within six days after inoculation. The mortality rate in the group of pigs inoculated with the lower dose was only two out of ten and, there were several pigs in this group that showed signs of respiratory distress besides some mild nervous signs. Pseudorabies virus was isolated from various tissues collected postmortem, including alveolar macrophages. Virus localization in tissues was also detected by in situ hybridization. The histopathological examination of the respiratory tract tissues revealed a pathological process that was progressing from mild pneumonia to severe suppurative bronchopneumonia. The isolation of virus from alveolar macrophages provides support to the hypothesis that replication of PRV during the course of infection produces an impairment of the defense mechanisms in the respiratory tract.     

Vaccination of sheep against Aujeszky's disease with a gI-deleted mutant]

The use of gl deleted live vaccines against Aujeszky's disease (AD) facilitates to differentiate vaccinated from field-virus infected animals. In this study different modes of vaccination were tried to find out how sheep can be protected from a lethal infection with ADV. It could clearly be demonstrated that Aujeszky disease virus (ADV) is spread by horizontal transmission from infected pigs to sheep. The nasal discharges of infected pigs contained a maximum of 10(8.75)TCID50/g mucus at days 3 and 4 p.i. and those of the contact-pigs 10(8.5)TCID50/g mucus at days 6 and 7 after contact. Non-vaccinated contact sheep were infected horizontally by the pigs. The highest titres ranged from 10(6.25) to 10(7.5)TCID50/g mucus. These animals were sacrificed at day 5 p.i. exhibiting acute symptoms of AD. The nasal discharge of vaccinated sheep contained much lower amounts of ADV (maximum: 10(4.25)TCID50/g mucus). All surviving animals had developed antibodies. Following challenge with the ADV-strain NIA3, no febrile response or virus-shedding was observed in sheep vaccinated 2x s.c. or 2x i.m. with a gl deleted live vaccine, whereas sheep, vaccinated only 1x i.m. (4 out of 4 animals) or 1x i.m. (3 out of 4 animals) or 1x i.n. and 1x i.m. (1 out of 4 animals) had to be sacrificed after showing acute symptoms of AD. In conclusion it can be stated that a double parental vaccination with a gl deleted live vaccine protects sheep against a field-virus AD infection.     

Canine parvovirus: environmental effects on infectivity

Effects of various environments on the infectivity of canine parvovirus-2 (CPV-2) were studied. When CPV-2 was subjected to several controlled indoor environments, the virus remained infective at approximate initial inoculation amount (median tissue culture infective dose [TCID50] = 10(5.5)/ml) for 12 months at temperatures less than -20 C, decreased to TCID50 of 10(2.3)/ml by 12 months at 4 C, and had a TCID50 of less than 10(1)/ml at room temperature (20 C) or higher in less than 2 months. The CPV-2 subjected to outdoor environments was not infective beyond 5 months, except that kept in areas protected from sunlight and drying conditions. The virus surviving in the outdoor environments was not infective for study dogs, whereas the virus maintained at less than 20 C was.     

Experimental Maedi Infection in Sheep: 2. Antibody Response

All 4 sheep inoculated via the respiratory tract with 7×10⁶ TCID50 af maedi M88 strain developed complement fixing (CF) antibodies within 3 months after inoculation, and a gradual rise in CF titers was found during the first year. The antibody titers have been maintained, though with some fluctation, through the following year, and the titers vary from 64 to 256. Virus neutralizing activity against maedi M88 strain was detected in the sera of all intrapulmonarily inoculated sheep within 8 months after inoculation. Titers have been maintained or have slightly increased. The level of titers, ranging from 8 to 256, was clearly different between individual sheep.One of the 4 sheep inoculated intracerebrally with 5×10⁵ TCID50 of maedi M88 strain developed CF antibodies 1 month after inoculation, but no neutralizing antibodies until death 11 months after inoculation. The rest of the intracerebrally inoculated sheep displayed no evidence of CF or neutralizing antibodies within 18 months after inoculation in spite of numerous virus isolations from peripheral blood leukocytes. The absence of antibodies might perhaps be attributed to phenomena such as differences in tropism, provirus state, immunological tolerance and size of inoculum.One sheep hyperimmunized with repeated s.c. and i.v. injections of maedi M88 strain developed high CF antibody titers but lower neutralizing antibody titers.The 2 uninoculated control sheep developed no CF or neutralizing antibodies within 18 months after inoculation.     

In vitro and in vivo detection of infectious pancreatic necrosis virus in fish by enzyme-linked immunosorbent assay

A double antibody enzyme-linked immunosorbent assay (ELISA) was used to detect infectious pancreatic necrosis virus (IPNV). The ELISA detected VR299 strain of IPNV at a dose of 10 to 20 ng of purified IPNV protein or 10(4) TCID50 in tissue culture fluid. Specificity of ELISA was demonstrated by an ELISA inhibition test. The ELISA did not detect infectious hematopoietic necrosis virus. Normal cell culture fluid and virus-non-inoculated rainbow trout (Salmo gairdneri Richardson) homogenate did not react in the test system. The IPNV was detected in rainbow trout fry inoculated with IPNV. Although infective virus titer in fish decreased rapidly 1 week after inoculation, IPNV antigen was detected by ELISA for 15 days. The IPNV antigen was detected in the fish tissue after inactivation of infective virus. The ELISA is a rapid and reliable method for the diagnosis of IPNV infection.     

IgG from acutely infected cats blocks mucosal feline immunodeficiency virus infection

We have previously shown an absence of detectable systemic or local infection in cats exposed to an infectious (100 TCID(50)) feline immunodeficiency virus (FIV) plasma inoculum via either the rectal or vaginal mucosa. In contrast, this same plasma inoculum was infectious via parenteral inoculation. Moreover an equivalent dose of cell-free tissue culture-origin virus inoculum infected 100% of cats by either the rectal or vaginal exposure route. To evaluate this phenomena, we used a tissue culture system to identify a heat-stable factor in the plasma of cats acutely (3 weeks) infected with FIV that blocked infection of naive peripheral blood mononuclear cells (PBMC) by either cell-free or cell-associated FIV in vitro. A single application of as little as a 1:200 dilution of either heparinized or Alsevier's anticoagulated plasma effectively inhibited production of FIV p26 in culture over a 21-day co-culture period. Depletion of antibody using a protein A column abrogated the inhibitory effect of FIV plasma against in vitro FIV infection. Co-inoculation of heat-inactivated plasma with 400 TCID(50) FIV-B-2542 cell-free supernatant virus onto the vaginal mucosa of two cats resulted in complete inhibition of infection in one cat and increased time to infection in the second. Thus, antibody found in the plasma of cats acutely infected with FIV blocks cell-associated and cell-free infection, inhibits virus production in previously infected cells, and reduces mucosal transmission efficiency in vivo. Extrapolation may help explain the relatively inefficient mucosal transmission of human immunodeficiency virus-1 (HIV) and other lentiviruses.     

Replication and transmission of porcine circovirus type 2 in mice

Little information is known about infection, replication and transmission of porcine circovirus type 2 (PCV2) in species other than swine. Two sets of animal experiments were carried out to investigate the susceptibility of mice to PCV2 and to study their possible role in maintaining and transmitting the virus. In the first experiment 14 mice were inoculated with PCV2 by the intraperitoneal route with 5 x 10(2) TCID50 of the PCV2-ROM strain (Cadar et al., 2007). In a second experiment 24 mice were divided into two groups (A and B); mice in Group A (n = 18) were inoculated orally with 1 x 10(5) TCID50 PCV2-ROM and mice in Group B (n = 6) were left uninoculated until day 12 post inoculation (p.i.), when they were mixed with Group A. The animals were sacrificed at intervals for postmortem investigation and virus genome detection by polymerase chain reaction (PCR). The PCR results indicated that PCV2 could replicate in mice infected intraperitoneally or by the oral route, and that the virus can be transmitted directly from mouse to mouse.     

Quantitative aspects of the transmission of African swine fever

The contagiousness of pigs during different stages of infection with African swine fever virus was assessed by measuring the amount of virus excreted and the amounts of virus in the blood and other tissues, as well as determining the infectious dose of the virus by various routes. The virus was present in substantial amounts in secretions and excretions of acutely infected pigs for only 7 to 10 days after the onset of fever and was present in the greatest amount in the feces. Virus persisted in the blood of some recovered and clinically normal pigs for 8 weeks and in the lymphoid tissues for at least 12 weeks. The intranasal/oral ID50, and the IV/IM ID50 of a moderately virulent isolate of African swine fever virus were determined to be 18,500 and 0.13, 50% headsorbing units, respectively. A highly virulent isolate required approximately 10-fold more virus to cause infection by the intranasal/oral route.     

Transmission and pathogenicity of encephalomyocarditis virus (EMCV) among rats

Due to the probable role played by rodents as a reservoir for the transmission of the EMC virus to pigs, the experiment reported here was performed in order to assess the transmission rate of EMCV within a rat population. Twenty-five eight-week-old Wistar rats housed in individual plastic cages were experimentally infected either with a Greek myocardial EMCV strain (5 rats with a 0.2 x 10(6) TCID50 dose per rat and 10 rats with a 0.5 x 10(4.5) TCID50 dose per rat, oronasally) or a Belgian myocardial EMCV strain (10 rats with a 0.5 x 10(4.5) TCID50 dose per rat, oronasally). Two to five days later, each inoculated rat was moved to a new clean cage and coupled with a contact rat to compare the pathogenicity of the two strains and to estimate the basic reproduction ratio R0, indicating the level of EMCV transmission. During the experiments, faecal virus excretion was measured as well as the serological response against EMCV. After euthanasia, virus isolation was attempted from different rat tissues. Neither strains produced mortality, nor clinical signs and only low titres of neutralising antibodies were found. All contact rats, however, were infected and the virus was isolated from their faeces and from various tissues. Both 10-pair experiments revealed a point estimate for the R0 of infinity (95%-CI for both the Greek and Belgian EMCV strains = 4.48 - infinity), as did the 5-pair experiment with a higher dose of the Greek strain (95%-CI = 1.83 - infinity). Combining the results from the two 10-pair experiments resulted in an estimate for R0 of infinity (95%-CI: 9.87 - infinity). These results indicate that the EMC virus can spread very easily within a rat population by horizontal rat-to-rat transmission (R0 > 1).     

Infection studies in kittens, using feline infectious peritonitis virus propagated in cell culture

The propagation of feline infectious peritonitis virus (NW1-FIPV strain) in cell culture is described. Tissue culture-propagated virus was used to inoculate specific-pathogen-free kittens intraperitoneally, intratracheally, or orally. Intraperitoneal inoculation caused seroconversion and effusive peritonitis in 100% of the kittens. Intratracheal inoculation produced disease in 60% of the kittens, and oral inoculation in only 20%. Seroconversions without production of disease occurred in 10% of the kittens inoculated by either the intratracheal or the oral route. The remainder of the kittens inoculated by the intratracheal (30%) and oral (70%) routes did not develop serum antibodies or disease.     

Dose response studies of acute feline immunodeficiency virus PPR strain infection in cats

The effects of virus dose on host response were evaluated for the PPR strain of feline immunodeficiency virus (FIV-PPR). Specific pathogen-free cats were inoculated intravenously with 50, 250 or 1250 TCID(50) of FIV-PPR. Two weeks after inoculation, virus was detected in 10(6) peripheral blood mononuclear cells (PBMCs) of all infected animals, and the CD4(+):CD8(+) T lymphocyte ratios fell from greater than 2 to approximately 1 in all infected animals within the first 8 weeks after infection. Provirus detected in all groups using PCR and 10(3) PBMC was biphasic. Nine of 15 animals were positive between weeks 2 and 4 p.i. and 14 of 15 were positive by week 8 p.i. Transient lymphadenopathy was detected in most cats receiving 1250 TCID(50) and the 250 TCID(50) of virus, whereas no lymphadenopathy was detected in the 50 TCID(50) group or the five uninfected cats. Animals that had received the largest dose seroconverted earliest (on average at week 4.0) and those receiving the least seroconverted last (on average at week 5.6). Neither neutropenia nor lymphopenia were detected. FIV-specific CTL responses of memory effector cells could be detected in animals receiving all three doses but was highly variable among individual animals. Neurological manifestations determined after 15 weeks p.i. were observed in most infected cats, including two of the three that had received 50 TCID(50) of virus. However, the observed neurologic abnormalities were markedly less severe in the animals receiving the least amount of virus. Therefore, lymphadenopathy and neurologic signs of illness were less severe and seroconversion was slower in the animals that received the lowest dose compared with those receiving the 250 and 1250 TCID(50) doses of the FIV-PPR strain.     

Comparison of type I and type II bovine viral diarrhea virus infection in swine.

Some isolates of type II bovine viral diarrhea virus (BVDV) are capable of causing severe clinical disease in cattle. Bovine viral diarrhea virus infection has been reported in pigs, but the ability of these more virulent isolates of type II BVDV to induce severe clinical disease in pigs is unknown. It was our objective to compare clinical, virologic, and pathologic findings between type I and type II BVDV infection in pigs. Noninfected control and BVDV-infected 2-month-old pigs were used. A noncytopathic type I and a noncytopathic type II BVDV isolate were chosen for evaluation in feeder age swine based upon preliminary in vitro and in vivo experiments. A dose titration study was performed using 4 groups of 4 pigs for each viral isolate. The groups were inoculated intranasally with either sham (control), 10(3), 10(5), or 10(7) TCID50 of virus. The pigs were examined daily and clinical findings were recorded. Antemortem and postmortem samples were collected for virus isolation. Neither the type I nor type II BVDV isolates resulted in clinical signs of disease in pigs. Bovine viral diarrhea virus was isolated from antemortem and postmortem samples from groups of pigs receiving the 10(5) and the 10(7) TCID50 dose of the type I BVDV isolate. In contrast, BVDV was only isolated from postmortem samples in the group of pigs receiving the 10(7) TCID50 dose of the type II BVDV isolate. Type I BVDV was able to establish infection in pigs at lower doses by intranasal instillation than type II BVDV. Infection of pigs with a type II isolate of BVDV known to cause severe disease in calves did not result in clinically apparent disease in pigs.     

Studies on the live-virus vaccine against infectious laryngotracheitis of chickens. II. Evaluation of the tissue-culture-modified strain C7 in laboratory and field trials

Laboratory and field tests were conducted to evaluate the immunogenicity of a live-virus vaccine against infectious laryngotracheitis (ILT) of chickens that was prepared using tissue-culture-modified strain C7. Eighty-three percent or more of chickens vaccinated by the ocular (OC) or intranasal (IN) route with 10(4.75) TCID50 of the attenuated strain C7 at 50 days of age were protected against challenge with a virulent strain of ILT virus without showing any clinical signs for 4 weeks post-vaccination (PV). When vaccine was administered by aerosol, however, only 65% of vaccinated chickens were protected against challenge. Fifty-seven percent of chickens vaccinated at 70 days of age maintained immunity for 6 months PV. Immune response in younger chickens was inferior to that in older ones. In the field trials, clinical observation revealed no adverse reactions caused by the vaccination, and 60% or more of broilers and 80% or more of layers vaccinated by the OC route were protected against challenge at 4 weeks PV. These results confirmed the safety and efficacy of the vaccine.