F.R. Rurangirwa1,2 and T.C. McGuire11 Department of Veterinary Microbiology and Pathology
Washington State University, Pullman, USA
2 Small Ruminant CRSP, P. O. Box 58137, Nairobi, Kenya
Contagious caprine pleuropneumonia is a disease with high morbidity (80-100%) and mortality (6080%) rates. Rapid diagnosis of this devastating disease is essential for its immediate containment. A pen-side diagnostic test - latex agglutination - based on the carbohydrate elaborated by Mycoplasma capricolum subsp. capripneumoniae (Mccp) has been described. One way to contain and control the disease is through vaccination. A liquid vaccine is presently in use. However, freeze-dried saponin killed Mccp has been shown to confer protective immunity of over 12 months. The freeze-dried antigens are stable one year after storage at 4°C or 22°C. This indicates that a freeze-dried vaccine is feasible and indeed preliminary field trails have indicated that a freeze dried vaccine confers protective immunity.
Contagious caprine pleuropneumonia (CCPP) is a cause of major economic losses to goat production in at least 30 countries in Africa and Asia containing a total goat population of more than 300 million. Classical, acute CCPP is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp) (MacOwan and Minette 1976) originally known as Mycoplasma F38. This mycoplasma was first isolated from the lungs of goats with pleuropneumonia in Kenya and demonstrated to cause CCPP (MacOwan and Minette 1976). Subsequently, Mccp has been isolated in other countries including Chad, Ethiopia, India, Oman, Sudan, Tunisia, Turkey and Uganda, and pleuropneumonia disease indistinguishable from naturally occurring CCPP has been experimentally reproduced with Mccp by several research groups. The disease is characterised by fibrinous pleuropneumonia with increased straw-coloured pleural fluid. Morbidity and mortality rates may reach 100% and 80%, respectively. Close contact among goats is necessary for spread of the disease (MacOwan and Minette 1976).
The diagnosis of outbreaks of CCPP is complicated by other infectious agents causing similar syndromes. Pleuropneumonic disease resembling Mccp-associated CCPP can also be produced by Mycoplasma mycoides subsp. capri (Mmc) and caprine variants of M. mycoides subsp. mycoides (Mmm). Mmc was originally considered to be the cause of CCPP, but its full importance as a pathogen of goats has now become doubtful, both because of the discovery of the Mccp and because many isolates previously classified as Mmc have subsequently been found to be caprine variants of Mmm. Mmc has been isolated from several countries in Africa and Asia, and from Australia. The disease reproduced experimentally with Mmc is largely restricted to the thoracic cavity, with or without a septicaemic phase and death. In contrast, caprine variants of Mmm generally causes a syndrome which may include not only pleuropneumonia but also mastitis, polyarthritis, keratoconjunctivitis, acute septicaemic death, sometimes with symptoms of the central nervous system, and abortion. Mmm is a major cause of disease in goats in USA, France, Israel and India. Experimentally, the disease caused by Mccp differs from that produced by Mmc and Mmm in: being readily contagious and fatal to susceptible goats; not affecting sheep or cattle; not producing local oedematous reactions when injected subcutaneously; and being characterised histo-pathologically by an interstitial, intralobular oedema of the lung, compared with the thickening of the interlobular septa which is seen with Mmc and Mmm (Kaliner and MacOwan 1976). Pasteurella haemolytica (both biotypes A and T) and P. multocida have also been associated with pleuropneumonia in goats, although experimental evidence of their pathogenicity in this host is meagre.
Diagnosis of CCPP can be divided into field and laboratory diagnosis. Because this devastating disease occurs in epidemics, it is essential that the diagnosis be achieved very quickly so that control measures such as treatment of the sick goats and vaccination of those at risk is carried out immediately. Therefore, proper field diagnosis is paramount.
Field diagnosis is achieved through:
· Observation of the presenting clinical signs which include fever (40-42°C), coughing, dyspnea, high morbidity (about 100%) and high mortality of about 70%. Post-mortem examination will reveal fibrinous pleuropneumonia accompanied with increased pleural fluid, and enlarged and oedematous mediastinal lymph nodes.
· Latex agglutination test (LAT) (Rurangirwa et al 1987b). This test is based on a polysaccharide isolated from Mccp (Rurangirwa et al 1987a) which is used to sensitise latex beads. The sensitised latex beads are then used to detect serum antibodies from goats infected with CCPP (Rurangirwa et al 1987b). The specificity of LAT was assessed using WM25 monoclonal antibody which is specific for Mccp (Rurangirwa et al 1987c; Belton et al 1994) and reacts with the polysaccharide (Rurangirwa et al 1992). The specificity of LAT was further confirmed by evaluating specific growth inhibiting rabbit antisera to various mycoplasma isolates (Rurangirwa et al 1987c).
The sensitised latex beads are stable at 4°C, room temperature and 37°C for over one year. Thus the long shelf-life of the beads at different temperatures makes it possible to prepare large amounts which can be stored until used. The latex agglutination test is an excellent procedure for the diagnosis of CCPP and can be run in two minutes on samples of whole blood or serum, requires no sophisticated equipment or storage facilities and is adaptable to any laboratory or field conditions - an example of a pen-side diagnostic test. The test is carried out by mixing a drop of the sensitised beads with a drop of blood or serum from the suspected animal on a glass slide for one minute and the results read visually and recorded as positive or negative. LAT combined with presenting clinical signs and necropsy indicating fibrinous pleuropneumonia is confirmatory of Mccp associated CCPP.
Several serological tests including complement fixation (MacOwan and Minette 1976) passive haemagglutination (Muthomi and Rurangirwa 1972) and enzyme-linked immunosorbent assay (Wamwayi et al 1989) detect serum antibodies to Mccp. However, routine use of these tests is constrained by lack of equipment and trained personnel. Similar constraints are encountered in the use of DNA probes (Taylor et al 1992; Thiaucourt et al 1992) and PCR (Bascunana et al 1994).
Culture, isolation and characterisation of Mccp. This procedure which takes a minimum of 10 days is too slow to be of any practical use in the containment of CCPP outbreak.
Since the disease occurs in epidemics, antibiotic treatment, as the only control measure would be very uneconomical. Therefore, efforts have been directed towards controlling the disease by vaccination. This section documents developmental stages of the CCPP vaccine presently in use. The first immunisation trial using Mccp was carried out by MacOwan and Minnette (1978). They inoculated 20 goats intratracheally with a high-passage culture of Mccp. On contact challenge of the inoculated goats one month later, 11 of the 20 vaccinated goats were protected, whereas all 20 control goats contracted CCPP. The results provided an indication that goats could be protected against CCPP by using Mccp.
While investigating the effect of streptomycin in goats with natural and experimental CCPP caused by Mccp (Rurangirwa et al 1981a), it was found that goats treated with streptomycin on the 3rd day of temperature elevation (>4°C) recovered from the disease and became completely immune to reinfection with Mccp (Rurangirwa et al 1981a). Serum samples from such recovered goats caused in vitro growth inhibition of Mccp (Rurangirwa et al 1981 a). This observation led to a series of experiments in which goats were immunised with sonicated F38 antigens in different adjuvants which included Freund's complete adjuvant, Freund's incomplete adjuvant and aluminium hydroxide (Rurangirwa et al 1981b; Rurangirwa et al 1984).
It was demonstrated that protective immunity in goats could be induced by using sonicated Mccp antigens in Freund's complete and/or incomplete adjuvants. The immunity was present for at least six months after vaccination (Rurangirwa et al 1984). Aluminium hydroxide, like antigen alone, provided only 20% protection (Rurangirwa et al 1984). Thus, an unequivocal protection of goats against CCPP with sonicated Mccp antigens in an oil adjuvant was demonstrated. Although oil adjuvants are not suitable for use in food animals, use of Freund's complete and/or incomplete adjuvants demonstrated that Mccp organisms disrupted by sonication and injected with an appropriate adjuvant would induce protective immunity in goats against CCPP.
A pilot experiment revealed that saponin could be used as an adjuvant for Mccp vaccines. This finding was very important because saponin does not cause obvious tissue damage. Thus, a series of experiments were carried out using saponin as an adjuvant to assess:
· immunogenic stability of Mccp after Iyophilisation
· immunogenicity after storage at 22°C and 4°C
· the duration of immunity induced by a single dose of the Iyophilised Mccp.
Lyophilisation trials (Rurangirwa et al 1987d)
Since Iyophilised vaccines are easy to store and transport, a preliminary study was undertaken to find out if Iyophilised Mccp would induce protective immunity in goats against CCPP. Eight goats were immunised with 8 mg/ml of freeze dried Mccp in saponin to inactivate the organism and serve as adjuvant. The goats received a similar immunisation 21 days later. Two months after initial immunisation, the vaccinated and control goats were challenged by contact exposure. Six of the control goats and only one of eight vaccinated goats died of CCPP. These results led to the conclusion that Iyophilised Mccp antigens could protect goats against CCPP caused by Mccp, when an optimal immunising dose is used with an appropriate adjuvant.
Determination of minimum immunising dose of Iyophilised Mccp (Rurangirwa et al 1987c; Rurangirwa et al 1987d)
Seven groups of five goats each were immunised with different doses containing 0.015, 0.075, 0.15, 0.20, 0.375, 0.750, and 1.50 mg protein of Iyophilised Mccp using saponin as an adjuvant. An eighth group of five goats was immunised with the highest dose (1.50 ma) without adjuvant. A ninth group of control goats was immunised with saponin in phosphate-buffered saline (PBS), pH 7.4. All the goats were immunised again four weeks later. The immunised goats, along with control goats, were contact challenged four weeks after the second immunisation.
The mean time for the control goats to develop pyrexia of 40°C was 14+1.5 days after exposure to infected goats, and all the control goats died of CCPP. The goats that were immunised with 1.5 mg Mccp antigen without saponin had an incubation period similar to that of the control group. Two goats immunised with the lowest amount of Iyophilised Mccp (0.015 mg in saponin) died of CCPP. One goat given 0.075 mg in saponin also died of CCPP. There was no pyrexia nor any signs of disease in the goats that were immunised with >0.15 mg in saponin, except in one of the group immunised with 0.375 ma, which had a temperature reaction 40 days after contact exposure and died of CCPP six days later. The results indicated that the minimum immunising dose that fully protected all goats in a group was 0.15 ma.
Immunogenic stability of Iyophilised Mccp stored at 22°C and 4°C for 3, 9 and 14 months (Rurangirwa et al 1987c)
In order to check the stability of the antigen upon storage, antigen (Iyophilised Mccp) was stored at 4 and 22°C for up to 14 months. Goats were immunised with two doses of the minimum immunising dose (0.15 ma) of the antigen after storage for 3 and 14 months at 22°C and after 3, 9 and 14 months at 4°C. The immunised goats, together with corresponding controls, were challenged by contact exposure three weeks after the booster dose. Lyophilised Mccp kept for 3, 9 and 14 months induced protective immunity in all the immunised goats. The average mean time for the non-immunised control groups of goats, at 3, 9, and 14 months, to develop pyrexia were 15,17 and 22 days after contact exposure to the infected goats, respectively. There was no indication of CCPP in the immunised goats.
Duration of immunity induced by a single dose (Rurangirwa et al 1987c)
Three groups of 30 goats each were immunised with single doses of 0.15, 0.30 and 1.2 mg of the Iyophilised Mccp containing saponin as an adjuvant. A fourth group was injected with saponin in PBS. Ten goats from each group were subsequently exposed to virulent challenge by contact 3, 6 and 12 months later. All the goats immunised with a single dose of 0.15 mg were protected. Those immunised with a single dose of 0.3 mg were protected, except in one case when at 12 months a goat reacted and died of CCPP. Two goats in the group immunised with 1.2 mg also died of CCPP - one from the group challenged at 6 months, and the other from the group challenged at 12 months.
Three major conclusions were drawn from the results of these immunisation trials:
1. Lyophilisation and storage of Mccp at 22°C or 4°C did not affect its immunogenicity.
2. Large quantities of Iyophilised Mccp could be produced and stored at 22°C or 4°C for <14 months without affecting immunogenicity.
3. Duration of immunity induced by a single minimum immunising dose of 0.15 mg Iyophilised F38 was >12 months. Thus, the vaccine developed for CCPP can be Iyophilised, has a minimum dose of 0.15 mg and a shelf-life of >14 months when stored at 22°C or 4°C, and induces immunity lasting over 12 months.
Preliminary field test of the Iyophilised vaccine (Rurangirwa et al 1991)
One hundred and fifteen, one-year-old goats from a single farm were screened for antibodies to Mccp using a latex agglutination test (Rurangirwa et al 1987b) and 100 of them which lacked antibody to Mccp were purchased. Fifty of the goats were immunised subcutaneously with a single dose (0.15 ma) of the Iyophilised, saponin killed CCPP vaccine (Rurangirwa et al 1987e) while the remaining 50 were left as unimmunised controls. All the goats were maintained on the same farm for two months and then transported to Kabete Veterinary Laboratory for contact challenge. The challenge was carried out by housing the 100 goats with five goats infected with Mccp by endobronchial intubation (MacOwan and Minette 1976). During contact challenge, the two groups were grazed during the day and housed at night together with the five experimentally infected goats. All goats were examined daily and their rectal temperatures recorded. An autopsy was performed on any goat that died; the gross lesions recorded and specimens of affected organs taken for isolation of mycoplasma and bacteria. The trial was carried out for four months after challenge and the surviving goats were euthanased and autopsies carried out. Mycoplasma were isolated using Newing's tryptose broth (Gourlay 1964) and their identity as Mccp was confirmed by growth inhibition (Rurangirwa et al 1987c).
Twelve vaccinates and 14 controls died of diarrhoea due to salmonella infection during the acclimatisation period of three weeks at the Kabete Veterinary Laboratory leaving 38 vaccinates and 36 controls for contact challenge. None of the dead goats had lung lesions and no Mccp were isolated from lung or lymph node tissues. Thirty control goats developed pyrexia at a mean of 39 (±14.3 SD) days after exposure. Twenty-seven of the 30 affected control goats died of CCPP at a mean 8.1 (±2.33 SD) days after pyrexia began and three recovered. Four months after contact challenge, the three recovered control goats were euthanased; they had adhesions of the cardiac and apical lobes to the thoracic wall but Mccp organisms were not isolated from their tissues. The remaining six control goats had no signs of disease and when they were euthanased and examined at the end of four months, there were no lesions and Mccp organisms were not isolated from their tissues.
Failure of six of 36 control goats to contract CCPP is in contrast to containment experiments in which almost 100% infection occurred in the contact challenges (Rurangirwa et al 1987e). A morbidity and mortality rate of 60 to 80% has been reported for the field outbreaks of CCPP (MacOwan and Minette 1977). The experimental conditions for the present study were set to mimic field conditions as opposed to containment throughout the experimental period. Therefore, the failure of six controls to show any signs of disease may be attributed to a less severe challenge than was previously used (Rurangirwa et al 1987e). The recovery of the three control goats from CCPP after showing clinical signs indicates that a small number of goats may recover from CCPP, especially if they are not subjected to movement stress in search of food and water.
All the surviving vaccinates withstood the challenge. There was a temperature elevation in two of the goats (41 and 62 days after challenge) which returned to normal after two days. There was no pyrexia and no signs of disease in the remaining 36 vaccinated goats during the four-month experimental period. At the end of the experiment active lesions were not seen in the 36 euthanased vaccinated goats without clinical signs and Mccp was not isolated from their tissues. However, in the two vaccinates which had a temperature reaction, adhesions were present in the thoracic cavity, but Mccp was not isolated from their tissues. This finding may be related to individual variation or to other unknown causes. Similar observations have been reported before (Rurangirwa et al 1987e).
In conclusion, one dose of Iyophilised Mccp vaccine induced an immunity in goats that protected totally against mortality and was 95% efficacious against clinical disease. In the two vaccinated goats that had pyrexia, no detectable carrier state resulted. Therefore, this vaccine is suitable for use in control programmes for CCPP.
The vaccine has not yet been Iyophilised for general use in the field although the same formulation in liquid form is in use. Plans are now well advanced to freeze-dry the vaccine.
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