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INFECTIOUS ANEMIA IN CHICKENS -

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					                      INFECTIOUS ANEMIA IN CHICKENS -
                   PREV ENTION BY A LIV E ATTENUATED VACCI NE

                                        M. Voss
                       Lohmann Animal Health, Cuxhaven, Germany


Already 2 years before the first isolation of chicken anemia virus by Yuasa in 1979, two
outbreaks of gangrenous dermatitis and aplastic anemia were seen in Northern
Germany. The disease was characterised by severe anemia in the first weeks of life
followed by high losses up to 15 % caused by gangrenous dermatitis at an age of 18-
35 days.
The chicken anemia virus is now spread world-wide, often not recognised. The virus is
present in all chicken producing countries. The disease becomes apparent mainly
when vertical transmission via the hatching egg takes place. Disease outbreaks have
increased during the last years due to more intensive biosecurity measures in the
rearing of breeder flocks to avoid infections with salmonella. By this, the horizontal
infection of breeder populations in the rearing period was prevented. However, due to
the contact with hatcheries at the beginning of production these breeder flocks were
infected by CAV. For a period of 5 to 6 weeks these flocks vertically transmit the virus
to the progeny.

Based on this observation that always young parent flocks may produce symptomatic
offspring it seemed to be necessary to immunise the parent birds during rearing.
Vaccination experiments with an embryo propagated CAV vaccine were started in
Germany in 1985. This vaccine is licensed since April 1990 in Germany. The aim of
vaccination is to provide breeder flocks with high and uniform antibody levels. This will
avoid vertical transmission of the virus and provide the progeny with maternal
antibodies. Depending on the spreading capacity of the virus used for immunisation of
breeder flocks, either mass application by drinking water, individual vaccination by
injection or wing web method are necessary to produce protective titer levels in all birds
of a given flock.
As vaccination programs, specially in broiler breeders, are getting more and more
intensive with several handlings of the birds in the rearing, further vaccinations by
individual handling of the bird should be avoided. Therefore the big advantage of the
vaccine is the high spreading capacity in a vaccinated flock which allows mass
application by drinking water at an age of 12 to 16 weeks.

The spreading capacity of the vaccine is demonstrated in figure 1. In this experiment,
one week old orally vaccinated and contact SPF birds were placed in isolators. Neither
clinical and patho-morphological signs nor depression of the hematocrit occurred. The
ELISA serology 4 and 6 weeks p.v. proved the development of antibodies in the
vaccinates and in the contacts as well, whereas the antibody level of the contact birds
tends to be lower as compared to the vaccinates 4 weeks p.v., titer levels have
equalised after 6 weeks.
              28th day p.v.                                                 42nd day p.v.

      1                                                         1

  0,8                                                          0,8

  0,6                                                          0,6

  0,4                                                          0,4

  0,2                                                          0,2

      0                                                         0
              vaccinated         contact                    vaccinated                   contact
                                      Guildhay ELISA S/P titer
                            cage 1                                                  cage 2
Fig. 1: Safety and spreading of a CAV vaccine virus in young chicks

Yet, the vaccine should only be given in breeder flocks at an age of 10 to 16
weeks. Figure 2 demonstrates the safety of the live vaccine in day old chicks.
Ten doses of vaccine were individually applied by crop instillation. Only in birds
without maternal antibodies the residual vaccinal pathogenicity became evident
by the development of clinical signs: reduced haematocrit and microscopic
lesions 14 days post infection. None of these signs were detectable in CAV
antibody positive day old chicks.


100                          100                         100                        30                            30
 90                           90
 80                                                                                 25                            25
                              80                          80
 70                           70                                                    20                            20
 60                           60                          60
 50                           50                                                    15                            15
 40                           40                          40
 30                           30                                                    10                            10
 20                           20                          20                        5                              5
 10                           10
  0                            0                           0                        0                              0
          1    2        3           1      2       3             1      2       3         1      2       3             1       2       3
                                                                                                             hct (%)

          no clinical                   survival                     microsc.                 infected                      controls
      symptoms (%)                      rate (%)                pathology (%)


                    SPF (n=30)                         Broiler breeder (n=30)                        Layer breeder (n=30)


Fig. 2: Safety test of a commercial CAV vaccine (10-fold overdosage) in day old chicks

A further experiment demonstrates the spreading capacity of the vaccine virus under
laboratory conditions (fig 3). Groups of orally vaccinated and contact birds were placed
in cages and the antibody development was quantified four weeks p.v. by CAV-SN-test
and IDEXX-ELISA. All birds became highly positive, even if less than one dose per bird
had been administered.
                                              CAV-SN                                     S / N IDEXX
                                                                        0,6
                               12

                               10                                       0,5

     vaccinated orally         8                                        0,4

                               6                                        0,3
     contact vaccination
                               4                                        0,2

                               2                                        0,1

                               0                                            0
                                         0,1x         1x      10x                     0,1x         1x           10x
                                                    Dosage                                     Dosage

Fig. 3: Horizontal spreading of vaccine virus and induction of antibodies

In order to assess potential immunosuppressive effects of an early CAV-
vaccination, the Newcastle Disease (ND)-seroresponse of chicks which were
CAV vaccinated at day-old (fig. 4) was tested.

35                         5                                 9000                            100
                         4,5                                 8000                             90
33                         4                                                                  80
                                                             7000
                         3,5                                                                  70
                                                             6000
31                         3                                                                  60
                                                             5000
                         2,5                                                                  50
                           2                                 4000
29                                                                                            40
                         1,5                                 3000                             30
27                         1                                 2000                             20
                         0,5                                 1000                             10
25                         0                                    0                              0
      1     2       3          1     2    3     4                   1   2     3   4                1    2   3   4   5
          hct (%)                  ND: HI-log2                      ND-ELISA                       Mortality after
                                                                                                   challenge (%)
     SPF, CAV + ND vaccinated                                                         SPF, ND vaccinated
     Layer breeder, CAV + ND vaccinated                                               SPF, negative control
     Layer breeder, CAV contact vaccinated, ND vaccinated                                    (n = 30/group)



Fig. 4: ND-Immunoresponse of chicks vaccinated against CAV on day 1

Chicks with (groups 2 and 3) and without (group 1) CAV-antibodies were vaccinated by
crop instillation or by contact (group 3). 14 days p.v., 10 birds per group were screened
for lesions and the haematocrit was determined. As expected, only the CAV-antibody
free SPF chicks developed signs of anemia. The remaining 20 birds per group were
then vaccinated against ND. 14 days later, they were bled and challenged. All ND-
vaccinated birds were protected against the challenge. No depression of ND-antibody
formation was evident in CAV-vaccinates as compared to controls which were
vaccinated against ND only.
As field conditions differ from a laboratory environment, data from flocks maintained
under conventional conditions were obtained. The aim of these experiments was to
verify the efficacy of the vaccine to induce high and uniform levels in all birds of orally
immunised commercial flocks.

Fig. 5 and 6 demonstrate the CAV-titer development in a broiler breeder flock
measured by SN-test and two commercial ELISA kits, respectively. Still in progress
three weeks p.v., the seroconversion is completed within five weeks. According to
published data on the protective CAV-antibody level, all tested individuals possessed
sufficient antibodies to block CAV-shedding after intramuscular challenge (threshold
titer 210, according to Malo et al., 1995). All sera reacted positive in the IDEXX-ELISA
already 4 weeks p.v. (the manufacturer considers all S/N ratios lower than 0,6 as
positive).

          mean S/N IDEXX mean S/P Guildhay                        Titer SN

   1,2                        0,8                      12
      1                       0,7                      10
                              0,6
   0,8                                                 8
                              0,5
   0,6                        0,4                      6
   0,4                        0,3                      4
                              0,2
   0,2                        0,1                      2
      0                        0                       0
          0 1 3 4 5                 0 1 3 4 5                   0 1 3 4 5
                                                                weeks p.v.
Fig. 5: CAV Titer development in a broiler breeder flock after vaccination by drinking
        water, tested by ELISA and SN-test
            neg. IDEXX                    neg. SN

 12
          10 10               12    10   10
 10
                              10
  8                            8
  6                            6
  4                            4
                                                   2
  2               1            2              1
                      0   0                                0
  0                            0
          0 1 3 4 5                 0    1    3    4        5
Fig. 6: Number of individuals to be considered as CAV-negative by IDEXX-ELISA and
        SN, respectively

Within four randomly selected, orally vaccinated broiler breeder flocks at the start of
production, only one serum out of 80 had a titer below the suggested threshold value of
210 in the SN-test (fig. 7). No tested individual remained negative for CAV-antibodies.


                                                                 No. of birds

                                                                        20


                                                                        15


                                                                        10


                                                                        5
      4
            3
 flock no.                                                              0
                2
                       1                                  11      >12
                                       8         9   10
                                 7
                                       titer (SN-test)
Fig. 7: CAV SN-titers of broiler breeder flocks at 21 weeks of life
The efficacy and persistence of maternal protection during the production period
of a layer breeder flock was assayed by a challenge experiment (table 1). At the
beginning and close to the end of the laying period, respectively, eggs were set
and day-old chicks were either challenged or tested for CAV-antibodies (one full
sib each). 14 day post challenge, lesions were scored and haematocrit values
were determined. It turned out that no chick hatched from the vaccinated flock
developed any lesions or reduction of the packed cell volume. Because of the
fact that all birds were protected, no minimal protective titer could be established
by the aid of the corresponding antibody levels of the siblings.

Table 1: Protection of chicks derived from a layer breeder flock vaccinated against
         CAV
                            lesion score

                     thymus          bone marrow          hct           S/N IDEXX   S/P Guildhay

 Production week 5
          vaccinated        0               0             30,7              0,19        0,68
          control          0,6             0,6            26,7                -           -

Production week 33
        vaccinated         0               0              30,7              0,47        0,40
          control          1,1             2,1            25,1               -            -



The importance of complete seropositivity of all parent flocks is not only
important in meat type breeders but also in layers. A Marek´s Disease Virus from
a German white layer pullet flock with excessive Marek´s mortality was isolated.
It soon became evident that the MDV-isolate also contained CAV which had
circulated in the pullets. A pathogenicity test was carried out with this CAV-
contaminated isolate in not MD-vaccinated day-old chicks. No CAV-related
mortality occurred, but the extent of typical Marek´s tumor formation over 70
days of life was markedly lower in chicks which had CAV-antibodies at the time
of infection.

 mortality (%)
   100
    90
    80
    70
    60
    50
    40
    30
    20
    10
     0
              white           white        brown layer
             leghorn         leghorn        (CAV ab+)
              (SPF)         (CAV ab+)
Fig. 8: Marek´s mortality up to day 70 after infection with CAV / MHV - isolate
This finding suggests that the well-described negative influence of CAV on
Marek´s protection is still a field issue.

Conclusions:

 CAV can be efficiently controlled by oral vaccination of breeder flocks with the live,
  attenuated vaccine.
 The vaccine is safe and potent. No undesired side effects have been reported after
  more than seven years of extensive field use.
 The vaccine has a marked spreading capacity. Seroconversion in orally vaccinated
  flocks is completed within about five weeks. Individual application of the vaccine is
  not necessary
 Broiler and layer breeder flocks should be CAV antibody-positive before the first
  hatching eggs are set. Screening for antibodies is easily done by means of
  commercially available ELISA kits which provide reliable results for field work.


References

Coombes, A. L., and G. R. Crawford(1996): Chicken anemia virus: a short review.
World´s Poultry Science Journal 52, 267-277

Malo, A., and M. Weingarten (1995): Determination of the minimum protective
neutralizing antibody titre in adult chickens. Intervet, VSD Newsletter 11, November
1995: 1-5
Otaki Y, Nunoya T, Tajima M, Kato A, and Nomura Y (1988): Depression of vaccinal
immunity to Marek´s disease by infection with chicken anemia agent, Avian Pathol. 17,
33

Vielitz, E., and M. Voß (1994): Experiences with a commercial CAV-Vaccine. In:
Proceedings of the international symposium on Infectious Bursal Disease and
Chicken Anaemia, Rauischolzhausen, Germany, June 21-24. Giessen, Institut für
Geflügelkrankheiten 1994: 465-481

Yuasa, N., Taniguchi, T., and I. Yoshida (1979): Isolation and some characteristics of
an agent inducing Anaemia in chicks. Avian Diseases 23, 366-385

				
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