Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 g of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10^4±0.15 TCID₅₀/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre >1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.     

Involvement of spleen components of mature fowl in the primary and secondary humoral antibody response following experimental EDS'76 virus infection

Summary

Cellular changes in spleens of mature fowl in relation to both the primary and secondary humoral antibody response following experimentalEDS'76 virus infection were studied. The influence of splenectomy on humoral antibody response was also examined.

Experimental fowl had been naturally infected with fowl adenovirus (FAV) but did not possess precipitins to these viruses at the time of EDS’ 76 virus infection. Since EDS'76 infection provokes a recall of the group antibody to FAV, this infection simultaneously induces a primary response against EDS’ 76 virus and a secondary response due to the recall of the group antibody to FAV.

HI and precipitating antibodies toEDS'76 virus (primary response) werefirst detected at 6 and 8 days p.i. respectively. Curves of HI, precipating and neutralising antibody titres were biphasic; the first peak (IgM peak) occurred at 10–11 days p.i., the second (IgG peak) at 16–28 days p.i.

Precipitating antibodies to FAV (secondary response) were demonstrated from 4 days p.i. The curve of these antibody titres was also biphasic, with peaks at the same times as in the primary response.

Based on HI and AGP testing of primary and secondary immune response in both splenectomised and non‐splenectomised fowl it is concluded that in the primary response the spleen of the adult fowl is involved significantly in only IgM secretion, while in the secondary response it is likely that bothIgM and IgG are secreted in considerable amounts.

Clusters of lymphoblasts and plasmablasts were observed at 3 days p.i. in the red pulp. It is very likely that antigen‐antibody complexes are formed from that time and circulate bound to the surface of lymphocytes. These antigen‐loaded lymphocytes are ‘picked up’ from the blood stream by

– red pulp macrophages, leading to enhanced formation of lymphoblasts in the red pulp. Great numbers of these cells (which are very probably IgM secreting cells) were present on days 6 and 7 p.i., but were no longer detectable after day 10 p.i.

– macrophages of the macrophagalellipsoidal corona (MEC), leading to significant enlargement of the periellipsoidal lymphoid tissue(PELT) by an increase of the number of lymphocytes observedfrom days 4–12 p.i. The MEC was significantly enlarged from 7–12 days p.i., very likely due to an increased number of macrophages.

Following deposition of antigen in the white pulp, formation of follicles begins. The number of small, intact follicles includingfollicle precursors increasedfrom 6 days p.i. From 15 days p.i. to the end of the experiment both the number and size of follicles increased significantly.

Uptake and processing of antigen by macrophages is probably accompanied by death of some of these cells. This might explain the degenerative changes observed in large mesenchymal cells, probably macrophages, at 3 and 5 days p.i. in the red pulp and at 5 and 6 days especially in the MEC. Splenitis which was present at 3 and 5 days p.i. and oedema observed in and around ellipsoidal cells at 5 days p.i. may be due to mediators released from these degenerative macrophages.

A significantly increased number of follicles with lymphoblasts was seen from 2–15 days p.i. while lymphoblasts and plasmablasts were present in the PELT from 5–15 days p.i., but predominantly at 6 and 7 days p.i. It is likely that disruption of follicles and blast transformation of white pulp lymphoid cells are secondary response events. White pulp lymphoblastsand plasmablastsare probably IgG secreting cells.

Splenomegaly was observed at 3, 5 and 6 days after infection and was mainly due to swelling of red pulp macrophages and infiltration of granulocytes in the red pulp. Ellipsoidal and periellipsoidal changes could contribute to the splenomegaly at 5 and 6 days p.i.     

Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies

Summary

Direct diagnosis of swine influenza infection by an indirect immunofluorescence technique using anti‐nucleoproteine monoclonal antibody was compared with virus isolation. Five 8‐week‐old pigs were inoculated with 2 × 107 EID₅₀ of strain A H₁N₁ Sww//4115/85. Clinical signs developed in only three pigs. Antigen was detected in nasal epithelial cells obtained from all animals the first day after inoculation; the antigen was detected in one pig 6 days after the infection. Fluorescence was present in the nucleus, nucleolus and cytoplasm of infected cells. The indirect immunofluorescence test was specific and as sensitive as virus isolation in embryonated eggs, allowing a rapid diagnosis that could be achieved within hours.     

Evaluation of transmission of swine influenza type A subtype H1N2 virus in seropositive pigs

OBJECTIVE: To examine clinical signs, virus infection and shedding, and transmission of swine influenza virus (SIV) subtype H1N2 among seropositive pigs. ANIMALS: Eighteen 3-week-old pigs with maternal antibodies against SIV subtypes H1N1, H3N2, and H1N2. PROCEDURE: Ten pigs (principal) were inoculated intranasally with subtype H1N2 and 2 groups of contact pigs (n = 4) each were mixed with principal pigs on day 7 (group 1) or 28 (group 2). Two principal pigs each were necropsied on days 4, 14, 21, 28, and 42 days after inoculation. Four pigs in each contact group were necropsied 35 and 14 days after contact. Virus excretion was evaluated after inoculation or contact. Lung lesions and the presence of SIV in various tissues were examined. RESULTS: Mild coughing and increased rectal temperature were observed in principal pigs but not in contact pigs. Nasal virus shedding was detected in all principal pigs from day 2 for 3 to 5 days, in group 1 pigs from day 2 for 4 to 9 days after contact, and in group 2 pigs from day 4 for 2 to 6 days after contact. Trachea, lung, and lymph node specimens from infected pigs contained virus. Antibody titers against all 3 subtypes in all pigs gradually decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Protection from viral infection and shedding was not observed in pigs with maternal antibodies, but clinical disease did not develop. Vaccination programs and good management practices should be considered for control of SIV subtype H1N2 infection on swine farms.     

Inhibition of African swine fever infection in the presence of immune sera in vivo and in vitro

In assay cultures, sera from African swine fever convalescent pigs inhibited infection by homologous African swine fever virus. The infection-inhibition capacity did not correspond with the virus-neutralizing capacity. The serum did not prevent infection by heterologous virulent viruses. Sera from pigs challenge inoculated with the homologous virulent virus and later with a heterologous virulent virus inhibited the infection by different heterologous virulent viruses. These sera did not interfere with the infection by pseudorabies virus. The specificity of the reaction indicated that the infection inhibition was caused by antibody.     

Production of High Potency Anti-Teschen Disease Serum From Rabbits and Guinea Pigs

An antiserum of high antibody content against Teschen disease Konratice virus was obtained by inoculating rabbits and guinea pigs with an antigen composed of aluminum gel and virus propagated in swine kidney cell cultures. The rabbit and guinea pig serums neutralized 1,500 TCID₅₀ of virus at dilutions of 1:5,120 and 1:2,048, respectively. The antibody level in the rabbit serum was tenfold greater than that in convalescent swine serum. Rabbit serum neutralized 2.8 x 10⁶ plaque-forming units of the Konratice virus. At a dilution of 1:5,120, this serum neutralized 1,500 TCID₅₀ of the Reporyje virus. The methods used to prepare and assay the serum are described.     

The Response of Ponies to Myxovirus influenzae A-equi 2 I. Serum and Antibody Titres Following Exposure

The antibody response in serum and nasal secretions of groups of ponies vaccinated or infected with Myxovirus influenzae A-equi 2 was examined. Following infection by aerosol with live virus, a weak antibody response was recorded in both serum and secretions. Antibody levels were undetectable in secretions at 31 days after infection.

After primary intramuscular vaccination with killed virus, using sodium alginate as an adjuvant, antibody was detected only in the serum. However, following revaccination, a pronounced antibody response was demonstrated in both serum and secretions. Antibody was still detectable in all four ponies when tested 135 days later.

Only a serum antibody response was detected in ponies after primary intramuscular vaccination with a commercial vaccine. Upon revaccination nasal antibody occurred in all ponies but this only persisted for about 30 days.

Neither serum nor nasal antibody response occurred following intranasal vaccination and revaccination with a killed virus vaccine.

     

A congenital persistent swine fever infection. I. Clinical and virological observations

A congenital swine fever infection is described. The infection is characterized by persistent viraemia, continuous virus excretion and late onset of disease, with death occurring 2–11 months after birth.A “carrier sow” from a field outbreak gave birth to one dead, one slightly abnormal and nine normal piglets, six of which were allowed to live. Disease symptoms were not observed before the age of 9–28 weeks (average 20 weeks) and the average survival time was over 6 months. Virus titres in blood plasma fluctuated between 10^5?0 and 10^6?9 plaque forming units/ml and virus was excreted throughout the lives of the pigs. Leucocyte counts showed normal to subnormal values, falling below 10 000/mm³ at about the onset of illness. Lesions characteristics of swine fever were not observed, but severe atrophy of the thymus was seen in all animals. Cryostat sections revealed the presence of viral antigen in lymphoidal, reticulo-endothelial and epithelial tissues. The difference between this persistent congenital infection and the chronic swine fever described by other authors, and the importance of “healthy” virus carriers for the epizootiology of the disease are discussed.     

Serological examination and egg production of progeny of fowl experimentally infected with Egg Drop Syndrome 1976 virus

Summary

Following EDS'76 virus (BC14 virus) infection of breeder chickens by the conjunctival route, vertical transmission occurred in the first week after infection. In the progeny which had been infected with EDS'76 virus by the vertical route, increasing haemagglutination inhibiting (HI) litres to BC14 virus and increasing numbers of birds with HI litres were observed from 3 weeks to 15 weeks of age. Sixty‐one per cent of the hens and 77 per cent of the cocks had ²log HI BC14 virus litres exceeding 4 at an age of 15 weeks.

Some birds which had been serologically negative throughout the rearing period, seroconverted between 25 and 28 weeks of age. This phenomenon occurred in hens as well as in cocks. Simulation of stress twice during the laying period by injection of corticosteroid hormone did not increase the number of birds serologically positive to EDS'76 virus.

EDS'76 was observed in the group of hens that was vertically infected, since egg production was significantly depressed between 28 and 34 weeks of age. Probably this was mainly the result of a production drop in the hens showing seroconversion at 27 or 28 weeks of age.

In the group of fowl vertically infected with EDS'76 virus, serologically positive birds appeared to be protected for the greater part to BC14 virus challenge at 50 weeks of age, while negative birds seemed to be fully susceptible. Chicks hatched from eggs collected in the third and fourth week after infection of the dams had maternal antibodies. Fertility and hatchability of apparently normally shelled eggs seemed not to be affected after BC14 virus infection of the dams. Intensive contact with contaminated faeces is probably an indispensable condition for lateral transmission of the virus.     

Upper respiratory infection of lactating sows with transmissible gastroenteritis virus following contact exposure to infected piglets.

Ten breeding sows were left in direct contact with their newborn piglets that had been experimentally infected with transmissible gastroenteritis (TGE) virus. All sows became infected with the virus. The sows developed fever and showed mild clinical signs of the disease for a few days. The sows excreted virus in the nasal secretion, feces, and milk during the acute febrile phase of illness. Virus was isolated from the nasal secretion of one sow as early as 20 hours after contact exposure to the infected piglets. At necropsy, the virus was more frequently isolated from the tissues of the upper respiratory tract than from small intestines; this finding indicated that the TGE coronavirus replicated in the upper respiratory tract and induced an acute respiratory infection in susceptible adult swine. Neutralizing antibody was present in the sera 8 sows after 12 to 36 days during the convalescent period. From these results, we conclude that susceptible sows in direct contact with ill piglets can become infected and by excreting virus can serve as a source of TGE virus for other susceptible pigs on the premises.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.     

II. Detection of the Virus in Swine Tissues by Means of the Modified Direct Complement-Fixation Test

The modified direct complement-fixation test, supplemented with unheated normal calf serum, was used to demonstrate antibodies in sera of swine immunized to African swine fever virus. These antibodies did not react in the ordinary direct non-supplemented complement-fixation test.

African swine fever complement-fixing antigen in infected swine tissue is not denatured by extraction with fat solvents. Consequently, good antigens devoid of non-specific reactivity were obtained by extraction with a mixture of acetone and ether.

The virus was detected in infected swine tissue harvested one day after beginning of pyrexia. The modified direct complement-fixation test demonstrated cross-reactions between the six strains of virus studied.

     

Precipitation Reactions in Agar Between Swine Serum and Homologous Pancreas Extracts

Hyperimmune anti-hog cholera and nonimmune swine sera yielded approximately 50% more precipitation reactions in agar-gel diffusion tests with pancreas extracts from SPF noninfected swine than with extracts obtained from swine experimentally infected with virulent hog cholera virus. The pancreas-reacting property of swine serum was determined to be relatively heat stable, withstanding 68 C for 30 minutes.

Of various swine serum fractions tested, the only one that reacted with pancreas extracts contained gamma, beta and alpha-globulins. In the absence of alpha-globulin, precipitation reactions were not observed.

Sera of newborn SPF piglets, containing 50% alpha-2 globulin, formed more intense precipitation lines with swine pancreas extracts than were formed by the sera of their dams with the same extracts.

The pancreas-reacting activity of swine sera was completely removed by absorption with pancreatic tissue. This property was not removed by absorption with guinea pig kidney, or beef, swine or human erythrocytes.

Maceration of pancreatic tissue released reactive substances in a polydispersed form. This was demonstrated by the ability of almost all supernates and sediments from differential centrifugation of such preparations to form precipitation lines with swine sera. Reactive substances from swine pancreas were found to be relatively heat labile, being inactivated in one hour at 56C.

No evidence was obtained in this study to indicate that the observed precipitation reactions were related to hog cholera virus and its corresponding antibody. The reactions are believed to have resulted from the interaction of protein-related substances present in normal swine pancreas with a relatively heat stable component, possibly alpha-globulin, in swine serum.

     

An Experimental Viral Pneumonia in Swine

Pneumonia resulted when a porcine entero-virus, ECPO-6, was inoculated intranasally into four and six week old, colostrum deprived, specific pathogen free piglets.

Clinical signs consisted of mild depression, rapid breathing with rectal temperatures reaching 104-105 F five to six days after infection.

Pneumonic lesions, both gross and microscopic were present 21 days post infection. Although the virus was readily isolated from the cecal contents of infected pigs three weeks after infection the virus was not recovered from infected lungs after the first 7-10 days.

     

Virus isolation and immune responses in susceptible swine exposed with pseudorabies virus (Shope strain).

Eighteen seronegative swine weighing from 9 to 11 kg were exposed intranasally with the Shope strain of pseudorabies virus (PRV) and were observed for 21 days in an experiment to detect virus shedding and immune responses. All swine had PRV in their nasal passages at 7 days after exposure; they also had precipitating antibodies to PRV as determined by the microimmunodiffusion test (MIDT) and very low levels of virus-neutralizing (VN) antibodies. The PRV was isolated from only 2 swine at postexposure day 14; all swine were MIDT positive, and VN titers ranged from 4 to 128. Virus was not isolated from the swine at 21 days after exposure, but all were MIDT positive; VN titers ranged between 8 and greater than or equal to 256.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

Humoral and cellular responses in swine exposed to transmissible gastroenteritis virus.

Swine exposed to attenuated transmissible gastroenteritis virus had higher virus-neutralizing antibody titers than did swine exposed to virulent virus. The cellular response, measured by the direct leukocyte migration-inhibition (LMI) procedure, was greater in swine exposed to virulent virus than in swine exposed to the attenuated virus. Leukocytes from exposed swine were inhibited more in the LMI procedure in the presence of the homologeous sensitizing antigen than in the presence of the heterologous viral antigen. The humoral response measured by virus neutralizing reached a peak 21 days after exposure, and the cellular response measured by LMI reached a peak 28 days after exposure.     

两种不同猪瘟疫苗对育肥猪的免疫效果比较

为比较猪瘟疫苗E2基因工程亚单位疫苗和猪瘟活疫苗对育肥猪免疫效果和生产成绩的影响,以便为规模猪场在选择猪瘟疫苗时提供参考。本试验从汉中某代养场选取了一批健康的断奶仔猪分成三组,A组免疫猪瘟E2基因工程亚单位疫苗（简称E2亚单位疫苗）,B组免疫猪瘟活疫苗（脾淋源）,C组为对照组不做猪瘟疫苗免疫。免疫60 d后各组随机抽取10、15和12份血样用猪瘟病毒抗体检测试剂盒检测猪瘟抗体水平。结果表明接种的两种猪瘟疫苗对育肥猪的免疫效果差异极显著（P<0.01）,其中E2亚单位疫苗相比于猪瘟活疫苗能够明显提高育肥猪抗体阻断率和降低离散度,并且有助于育肥猪的健康生长。     

Neurological disorders,virus persistence and hypomyelination in calves due to intra‐uterine infections with bovine virus diarrhoea virus

The clinical and pathological findings after a natural intra‐uterine infection with BVD‐virus in a Friesian dairy herd are described. The virological and serological aspects will be discussed in a separate paper (30).

In a period of 4 years, 11 calves were hum with the following nervous symptoms: more or less serious incoördination, tremor, oscillating nystagmus, and a negative blinking reflex. The pupillary and sucking reflexe's were normal. No ocular defects, such as lenticular opacity or retinal atrophy were observed.

The first calf was born in 1979. Within 6 months the symptoms disappeared. After a normal conception and pregnancy this animal gave bⁱrth to 2 clinically normal calves in 1981 and 1982. The second calf died at the age of 2 months, due to an ulcerating enteritis.

In 1980, again 8 calves with the same nervous symptoms were born within a period of 3 months. Two calves died at the age of 3 days and 5 weeks respectively; 2 calves were sold when 10 days and 3 weeks old; one calf did not improve and was necropsied at the age of 17 days. The remaining 3 calves showed only a slight hypermetria when examined after 6 months. At that time nystagmus was only visible with ophthalmoscopy. Two calves were slaughtered when 10 months old. The last one, a bull, proved to be sterile and was necropsied at the age of 1 ½> year.

A calf, born in 1981, recovered within a week and was necropsied at the age of 15 days. The last calf, born in 1982, did not improve at all and was necropsied at the age of 14 days.

During these 4 years none of the other animals in the herd showed any symptoms due to an acute or chronic BVD‐virus infection.

At post mortem examination of 6 animals no macroscopically visible malformations were found. Hypomyelination and abnormal glial cells were evident in 5 cases, especially in the two youngest calves which did not show any improvement. One of them had an obvious thymic hypoplasia. The calf which recovered within a week showed only very slight changes. In one of the calves slaughtered at 10 months, inflammatory lesions were found in the brain. The diagnosis was confirmed by virological investigations.

Clinically as well as pathologically there was a close resemblance to Border disease in lambs and congenital tremor in piglets after prenatal exposure to Hog cholera virus.     

Infection of the Bovine Udder with Bovine Herpesvirus

Infectious bovine rhinotracheitis — infectious pustular vulvovaginitis (bovine herpesvirus) grown in tissue culture was used as inoculum in trials to infect the lactating bovine udder. Six experiments were undertaken in which one or more quarters were infused with 1 ml. of tissue culture fluids containing 10⁶ to 10⁷ tissue culture infectious doses (TCID) of virus. In four of the experiments the inoculated quarters showed marked evidence of infection in the form of acute inflammation, swelling, reduced milk secretion and profound changes in the physical appearance of the milk. In each case virus was recovered in high titres in the milk from about the second until the tenth to fifteenth days following exposure. Uninfected quarters remained normal in appearance and virus could not be recovered from the milk.

In three of the experiments it was shown that serum and milk antibodies appeared shortly after the disappearance of virus from the milk. One experiment involving two animals showed that about 1000 TCID of virus were required to produce infection. In one experiment a cow having a pre-inoculation serum titre for bovine herpesvirus proved resistant to infection.

The experiments indicate that the bovine udder is readily susceptible to bovine herpesvirus in non-immune animals, and that the virus produces an acute, limited infection leading to a temporary disfunction of the gland. It appears that natural invasion of the udder through the teat canal is not readily accomplished by the virus.