Another approach to control is vaccination. This tool started to be developed in the 1 960s. Early reports showed that subcutaneous injection of a suspension of Cfv cells produced rapid recovery of fertility in heifers (Te Punga, 1 962). Work done in Australia at the same time focused on intravaginal inoculation of live and dead Cfv cells suspensions in the hope of generating good immune responses. Immunity was only achieved by inoculation of live bacteria (Newsam, 1 965b, c). Some time later, Clark et al. ( 1 968a; 1 968b) reported that after subcutaneous injection of phenol-killed whole Cfv cell suspensions, had curative effects and prevented re-infection of cured females and males. Thereafter, many researchers have focused on different methods of vaccine preparation (Cl ark et al., 1 972a), age of vaccination (Clark et al. , 1 972b ), dosages and the effects of single or double vaccination (Clark, 1 975), the best timing for vaccination (Berg and Firehammer, 1 978) and the use of annual or biannual boosters (Clark and Dufty, 1 982; Hoerlein and Carrol, 1 970). The use of Cff cells for the formulation of vaccines against Cfv was also evaluated since they share many antigens ( Bouters et al. , 1 973; Clark et al. , 1 975). The results were encouraging, as no significant differences were found between vaccines made of different Cfv strains (Eaglesome et al., 1 986), vaccines made from Cfv biovar intermedius (Johns et al., 1 977), or even dual vaccines prepared from strains of Cfv and Cfv biovar intermedius (Clark et al. , 1 977). Cell-free vaccines have also been evaluated for their effectiveness to prevent or cure BVC with encouraging results (Schurig et al., 1 978). Finally, passive immunisation of animals was evaluated. This also appeared to have a curative effect (Berg et al. , 1 979).
The pattern of immunoglobulin secretion promoted by these vaccines was also studied. In females, IgG and IgA increased in both serum and vaginal mucous after vaccination, although no consistent relationship between the two 35
was observed (Schurig et al. , 1 975). In males diffusion of IgG and IgM into the prepuce along with local secretion of IgA occurs after vaccination (Van Aert et al., 1 976). Border et al. ( 1 980) reported that it was the capsular antigens (K) of Cfv that elicited immunity. Further investigation showed that antibodies against K antigens were secreted 4 weeks after vaccination and antibodies against somatic antigen (0) were secreted within 6 week of vaccination (Vasquez et al. ,
1 983).
Hjerpe ( 1 990) published a comprehensive rev1ew on the subject of vaccination against Cfv. According to this material, vaccines develop opsonins of the IgG class against superficial heat-labile K antigens of Cfv cells. The IgG diffuses into the vagina and uterus. The presence of IgG in the vagina does not last for a long period and bacterial invasion may still occur although fertility is not lost (vaginal carrier stage). In males, immunization results in the presence of IgG, IgM and IgA in the preputia! cavity. Hence, vaccination will prevent infection of bulls and transmission of the disease to susceptible cows. Vaccines are based on oil or aluminium hydroxide adjuvants. The oil adjuvant promotes longer lasting immunity although granulomatosis may develop at the injection site. If the vaccine is oil based, then injection should be subcutaneous with a dose given 1 to 7 months before mating plus an annual boost. Males require a double vaccination (1 and 8 weeks before mating) plus an annual booster. If the vaccine is aluminium hydroxide based it may be administered subcutaneously or intra muscularly with the first dose 6 weeks before mating, a booster 1 0 days before mating and an annual booster thereafter at least 1 0 days before mating. For bulls a double vaccination is recommended (2 and 4 weeks before mating) plus an annual booster 4 weeks before mating.
Some authors have had problems cunng BVC in bulls usmg only vaccination. The complementary use of an intra muscular injection of 6.25g of procaine penicillin plus dihydrostreptomycin sulphate was proposed as an support of vaccination (Hum et al. , 1 993) .
Some commercial laboratories have also developed polyvalent vaccines against as spectrum of common reproductive diseases. The effectiveness in preventing BVC of one vaccine containing Leptospira interrogans serovar
pomona, Haemophilus somnus, Cif, Cfv, ffiR and BVD antigens in aluminium 3 6
hydroxide adjuvant and another vaccine containing the same antigens except Haemophilus somnus was evaluated (Cobo et al. , 2003 ). It was found that these vaccines produced no immunity against Cfv. This shows that it is better to have proper knowledge of the disease status of the herd than trying to prevent all of the possible diseases blindly.
1.9.3 Prevention
If the herd IS free of BVC and no preventive vaccme IS available,
introduction of only virgin bulls should be done in case of need. If an older non virgin bull is to be used, treatment with antibiotics (locally and systemically) should be implemented an at least 4 consecutive samplings should deliver negative results before allowing mating (Hartley, 1 952). If new female stock is needed, the farmers must have certainty about their health status and the history of the provider in that matter. A sexual rest of at least 6 months should be allowed before mating.
If vaccination is available, the previously explained vaccination plans should be applied (Section 1 .9.2.2).
In all cases, the application of the reproductive management rules explained in Section 1 .9.2. 1 are useful to prevent spread of the disease in case of introduction into a herd.
1. 10 Bovine genital campylobacteriosis in New Zealand
The disease was well known in Australasia in the decades between 1 950- 1 980 and many studies on campylobacteriosis have been published in New Zealand and Australian Journals (Clark, 1 97 1 ; Hartley, 1 952; Hemmisen, 1 98 1 ; Newsam, 1 960; Newsam and Peterson, 1 964; Te Punga, 1 958, 1 962; Te Punga and Boyes, 1 95 8 ; Te Punga et al., 1 964; Te Punga and Moyle, 1 96 1 ).
The implementation of AI together with reproductive management rules (Section 9.2. 1 .) decreased the prevalence of the disease in New Zealand, until it was thought that it no longer existed in the country. The organism was last isolated in 1 993 in New Zealand (Loveridge and Gardner, 1 993), although it was suspected again in 1 996 (Black and Orr, 1 996).
In the early 2 1 st century, the presence of Cfv in some herds in the Taihape 37
area was suspected (Hughes, 200 1 ). The reproductive history of these herds resembled that of endemic campylobacteriosis, whilst tests for other potential causes of low fertility had failed to identify any other source of the problem. The IgA ELIS A was used in an attempt to demonstrate the presence of Cfv in those herds. According to the rules of interpretation of the test (Hum et al., 1 994) it was concluded that the disease was present. However, the organism was never isolated from any of preputia! washes taken from bulls on farm and at an abattoir. One year later, the IgA ELISA test was used to evaluate the presence of Cfv in a dairy herd in the Gisborne area (Tattersfield, 2002). It was found that, according to the tests results the infection with Cfv was in the herd, although, again, the organism was not isolated from any of the preputia! washes obtained from the bulls.
Results from research by McFadden et al . (2004a) at Massey University showed that there was no relationship between the fertility of beef herds and the proportion of positive results to the IgA ELISA test in those herds. Moreover, when the standard of interpretation given with the test was used, positive animals were widespread throughout the country. Again the agent was not isolated from the 54 samples collected from bulls as part of the survey, although both Cff and C. jejuni were commonly present.
The reproductive histories of the researched herds in 200 1 and 2002 do agree with bovine genital campylobacteriosis. Natural mating is still used in both beef and dairy productions (Anonymous, 2005 ; McFadden et al. , 2004b). The country counts with the right geography and management to promote this disease to be maintained in some animals (hill country where no control over the animals is exercised other than joining the bulls to the cow herd and taking them out once the mating season ends; cows grazing off farm or in hill country where no control over possible abortions is done). Only a few carrier animals (females or males) in a herd that undergoes natural mating for some time during the year are enough to maintain a certain proportion of the bovine population as constant reservoirs of the organism. These animals will change from on year to the next but if the disease is present in New Zealand, it will surely be quartered in a few animals per herd.
1 . 1 1 Conclusions
In the light of all the above, it may be said that BVC presents many challenges to the clinician, the microbiologist and the epidemiologist in New Zealand.
It is a disease that if endemically present in a herd, the symptoms will be subtle enough for the farmer to need time to recognize that a problem is present and call the veterinarian (Hughes, 200 1 ). Once the clinician is involved, the fact that the disease has not been diagnosed in New Zealand for so long means that it will probably be one of the last causes of infertility to be considered. When Cfv is finally suspected, the challenge is now to try and diagnose it. Microbiological isolation should always be sought but the extremely fastidious characteristics of this organism renders isolation a very erratic means of undoubtful diagnosis.
If other means of diagnosis are used, such as the IgA ELISA test, there is no certainty about the results delivered by this test under New Zealand conditions. In the light of the evidence shown in previous sections it appears that there are major problems in the use or interpretation of the ELISA test in New Zealand herds, which need to be observed before it can be widely used in the field.
None of the diagnostic method developed until now are sensitive and/or specific enough to be used alone, while culture should always be the goal. However, isolation of the organism had not proved possible in any samples taken from bulls and cows in New Zealand during 200 1 /2002. This raises the question if the isolation methods used in New Zealand are adequate for the agent to be isolated. Perhaps, since none of the diagnostic methods has yet proven effective, evaluation of the newly developed techniques is needed. For example, preliminary evaluation of PCR as a diagnostic technique to be applied to clinical samples from bulls has been done and encouraging results were obtained (Pannachio et al., 1 993).
Every effort should be made for the diagnosis of this disease if present in New Zealand. Once confirmed, its characteristics are such that in two or three years of proper reproductive management, application of artificial insemination and maybe vaccination is enough to control it and possibly eradicate it. B ut until some proper attempt of diagnosis is made nation wide, no sure statement about 39
its presence or absence can be made.
1. 12 Hypothesis
In the light of the above evidence it appears that there are some difficulties in using the IgA ELISA test in New Zealand herds, which need to be understood before it can be widely used on the field. Moreover, it may also be that the cultural methods used in New Zealand are not adequate for the agent to be isolated either at sampling level or at media level. Furthermore, exploration of other means of diagnosis needs to be researched.
It is the aim of this work to:
1 ) Evaluate possible cross-reactions of the IgA ELISA test with other Campylobacter subspecies
2) Evaluate the sampling methods used in New Zealand
3) Evaluate the use of the PCR technique (Hum et al., 1 997; Pannachio et al., 1 993) on clinical samples from bulls.