An inactivated whole cell vaccine and LppQ subunit vaccine appear to exacerbate
the effects of CBPP in adult cattle

R. A. J. Nicholas^[17], G. Tjipura-Zaire^[18], R. S. Mbulu^[19], M. Scacchia^[20], F. Mettler^[21], J. Frey^[22], I. Abusugra^[23] and O. J. B. Huebschle^[24]

Introduction

The limitations of the live T1 vaccine have been well documented (Rweymamu et al., 1995). They include the provision of only short term and incomplete immunity and contain residual virulence. A continual appeal from affected countries is the need for better vaccines but these seem a long way off. Inactivated vaccines have been reported for a number of mycoplasma diseases including enzootic pneumonia in pigs, contagious agalactia (Tola et al., 1999) and contagious caprine pleuropneumonia (Rurangirwa et al., 1995). Indeed dead vaccines for CBPP were tried in Africa and shown to be relatively efficacious (Gray et al., 1986; Garba and Terry, 1986). Recent work has shown that a saponin inactivated vaccine against M. bovis, the cause of calf pneumonia, provided significant protection against a strong challenge both by aerosol and contact (Nicholas and Ayling, 2003). Lung lesions and mycoplasma dissemination were significantly reduced in vaccinated calves compared to infected controls. We decided to use the same approach to develop an inactivated vaccine for CBPP. In addition some cattle were vaccinated with the purified recombinant LppQ lipoprotein to see whether this could provide protection (Abdo et al., 2000). Cattle infected by intubation were used to challenge all vaccinated cattle by contact.

Materials and methods

Saponin vaccine

The Afadé strain of Mycoplasma mycoides subspecies mycoides SC (Mmm SC) was cultured in bulk, distributed into ampoules of 1 ml and stored at -70°C to provide a standardised source of inocula. The master seed culture was thawed and dispensed into 10 ml quantities of Eaton’s broth media. After 48-72 h 10 ml broths were subcultured into 100 ml, and after the same time these broths were inoculated into vessels of greater or equal to 1 l and incubated for a further 2 days. The precise protocol (incubation times, final culture volumes) was established in preliminary experiments. Washed cells were inactivated with 2% saponin (Sigma, UK) at 37°C for 2 h. To check successful inactivation, 0.5ml of saponised cells were inoculated into 5 × 20 ml of Eaton’s broth; saponised cells were also directly plated onto Eaton’s medium containing agar. After five days, 0.5 ml of incubated broths were subcultured into fresh broths and plates of above media. This procedure was followed every five days for a total of 30 days. Plates were examined daily for 7 days then discarded. No mycoplasma growth was seen at any time.

LppQ

The production of the recombinant N’-terminal half of the lipoprotein Q was described by Abdo et al., (2000).

Vaccination

All animal experiments were carried out in the Mashere district of Northern Namibia where CBPP is endemic. Four 7-8 year old cattle were subcutaneously vaccinated with 1 ml of the saponised vaccine on two separate occasions 6 weeks apart. The same numbers of cattle were inoculated with 1 ml of recombinant LppQ in ISCOM construct at the same time intervals. Approximately 4 months later all animals were placed in contact with intubated cattle (see below).

Challenge

Ten 7-8 year old cattle were intubated tracheo-bronchially, and the procedure which was monitored with the aid of a bronchoscope, was performed by inserting a horse stomach tube into the trachea till the bifurcation and kept in position until completion of the intervention. Each animal was infected with 10 ml of Mmm SC culture from the 2^nd passage, titre 109/ml suspended in 25 ml of 2% agarose. Forty ml of culture medium was used to flush down the suspension to the target site.

To simulate natural disease the saponin and LppQ vaccinated cattle were placed into the same paddock where the intubated cattle were held. In addition 8 unvaccinated control cattle of the same age were also introduced.

For the entire observation period, all cattle were fed with lucerne and had a common drinking trough. They were monitored clinically, serologically and bacteriologically. All testing was done at the Central Veterinary Laboratory, Windhoek. The LppQ ELISA was performed according to Bruderer et al., (2002).

Results

Intubated cattle

On day 10 post-intubation a serological response was detected by CFT in all cattle. Peak titres ranged from 1:1000 to 1:10 000 and the response was maintained at high levels for about 75 days. Although a gradual decrease of CF titres was then recorded all animals still showed a clear serological reactivity (± 1:80) at the time of slaughter. Temperatures exceeding 39.0°C were recorded between day 7 and 15 post-intubation and persisted over a period varying between 2 and 10 days. Animals showed respiratory distress characterised by coughing from day 15 post-intubation onwards. No mortality was observed during the observation period. At slaughter 149 days post-intubation, 5/10 animals showed small but specific CBPP pulmonary changes and Mmm SC was isolated from lung tissues (see Table 1).

In contact control cattle

On day 35 post-exposure (p.e.) one cow (9442) died, no lesions due to Mmm SC were observed during post-mortem examination and no mycoplasma was isolated from either lung or lymph node specimens collected at post-mortem. Death was due to causes other than CBPP. In the 3 cows that died later, in the course of the experiment, and in the remaining 4 animals that were sacrificed at the end of the experiment, low and sporadic CF antibody response was detected from day 91 onwards but rose massively in the survivors three weeks before the end of the experiment. Temperatures ranging between 39.1°C and 40.3°C were first recorded on day 124. The only exception was cow 9610 that was febrile since day 103 p.e. (not shown) showed serological reactivity on sample taken on day 105 and died on day 119. Mmm SC was isolated and antigen detected by PCR and immunocytochemistry (ICC) testing in lung and/or lymph node tissue samples taken from all 7 cattle.

Pathological changes observed either after death or at slaughter were: increased consistency of the lung, enlargement and marmorisation affecting the apical and/or diaphragmatic lobes and in some instances the entire lung, adhesion between visceral and parietal pleura. Sequestra when present were small. In one case both lungs were affected. Peribronchial and mediastinal lymph nodes were hyperplastic, of increased consistency and in some instances haemorrhagic (see Table 1).

Table 1. Pathological findings of vaccinated and contact cattle following exposure to CBPP

Abreviations:

Saponin vaccinated cattle

Antibody titres as detected by CFT developed within two weeks of the first vaccination to significant levels of 1/40-1/320 (Table 2). A second vaccination 6 weeks later boosted 3 of the 4 cows to 1/320. Titres then declined in all cattle to 1/10-1/20 just before exposure to challenge. Titres remained at these levels for the next 4 months, and then there was a sudden massive increase just prior to post mortem which coincided with the death of one of the cows (see Table 1).

Table 2. Serological response of cattle vaccinated with saponin vaccine

* 1^st vaccination
** 2^nd vaccination
*** animals exposed to infection

LppQ ISCOM vaccinated cattle

LppQ antigen does not elicit CFT antibodies so the homologous ELISA test was performed. Antobody to LppQ was detected within two weeks of the first vaccination and remained high until challenge. Following exposure, titres remained high until 1 month before postmortem when titres rose rapidly. Sporadic low CFT titres were seen over the four months prior to exposure to challenge and remained at this state until one month before post mortem when another massive increase was seen which also saw the death of two cows (Table 3). The two remaining cows had titres of over 1/2560 at post mortem (see Table 1).

Table 3. Serological response of cattle vaccinated with LppQ vaccine

* 1^st vaccination
** 2^nd vaccination
*** animals exposed to infection

Discussion

While live vaccines have always been used for CBPP control, there have been a few reports of experiments using inactivated vaccines. Adult cattle that received two large doses of a heat inactivated Blenheim vaccine strain containing complete Freund’s adjuvant received better protection than when given a single dose of the same vaccine or T₁ broth vaccine (Gray et al., 1986). A formalised preparation of 10¹⁰ CFU/ml of the Gladysdale strain emulsified with liquid paraffin gave similar protection to the T₁ broth vaccine whether the vaccine was used immediately after inactivation or stored for 1 and 2 years or challenged 3 or 6 months after vaccination (Garba and Terry 1986). In spite of these encouraging results no further work on inactivated vaccines has been published.

The present work took two different approaches to vaccination against CBPP: first, the use of a whole cell mycoplasma vaccine inactivated with saponin, an approach which had previously been found to be protective for calf pneumonia, contagious agalactia and CCPP; and second, the use of a recombinant subunit vaccine prepared from the highly immunogenic lipoprotein LppQ. In spite of two vaccinations at 6 weekly intervals there was no evidence in the small number of animals used of any protection afforded by either preparation; indeed, although it was difficult to quantify, there appeared to be an exacerbation of pathology in the vaccinated animals compared to unvaccinated contact animals. Lesions and fibrin were most extensive and pleural fluid more abundant in vaccinated animals. In the LppQ group 2 of 4 cattle died before the end of the experiment while 1 of 4 died in the saponin group. This compares to 3 of 7 that died in the control group.

An interesting aspect of this experiment was the length of time it took the unvaccinated groups to seroconvert: following challenge apart from the odd sporadic positive CFT titres; it was three months after exposure before cattle seroconverted and then titres rose rapidly. Strong vaccine antibody titres were seen in the saponin group two weeks after the second vaccination and these declined slowly but remained detectable for a further 6 months before rising rapidly prior to post mortem. It would have been interesting, had resourced permitted, to have seen how the T1/44 vaccine performed under these conditions.

References

Abdo, E.-M., Nicolet, J. and Frey, J. (2000). Antigenic and genetic characterization of lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides SC. Clinical Diagnosis and Laboratory Immunology 7, 588-595.

Bruderer, U., Regalla, J., Abdo, E. M., Huebschle, O. J. B. and Frey, J. (2002). Serodiagnosis and monitoring of CBPP with an indirect ELISA based on LppQ of Mycoplasma mycoides subsp. mycoides. Veterinary Microbiology 84, 195-205.

Garba, S. A. and Terry, R. J. (1986). Immunogenicity of iol based CBPP vaccines in cattle. Vaccine 4, 266-270.

Gray, M. A., Simam, P. and Smith, G. R. (1986). Observations on experimental inactivated vaccines for CBPP. Journal of Hygeine (Camb) 97, 305-315.

Nicholas, R. A. J. and Ayling, R. D. (2003). Mycoplasma bovis, disease, diagnosis and control. Research in Veterinary Science 74, 105-112.

Rurangirwa, F. R., McGuire, T. C., Kibor, A. and Chema, S. (1987). An inactivated vaccine for contagious caprine pleuropneumonia. Veterinary Record 121, 397-402.

Tola, S., Manunta, D., Rocca, S., Rocchiagiani, A. M., Idini, G., Angioi, P. P. and Leori, G. (1999). Experimental vaccination against Mycoplasma agalactiae using different inactivated vaccines. Vaccine X, 2764-2768.