Responses Of Chickens Vaccinated With A Live Attenuated

Veterinary Parasitology 129 (2005) 179–186 www.elsevier.com/locate/vetpar

Responses of chickens vaccinated with a live attenuated multi-valent ionophore-tolerant Eimeria vaccine G.Q. Li *, S. Kanu, S.M. Xiao, F.Y. Xiang Department of Veterinary Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, PR China Received 11 June 2004; received in revised form 25 August 2004; accepted 7 September 2004

Abstract Coccidiosis, caused by Eimeria species, is a serious economic disease of chickens (Gallus gallus) and the search for vaccines to control the disease is intensifying especially with the increasing threat of drug resistance. A live attenuated multi-valent ionophore-tolerant Eimeria vaccine has been developed that contains three ionophore-resistant Eimeria species, E. tenella, E. maxima and E. acervulina. The attenuated lines were derived from virulent field strains resistant to monensin ionophore by selection for early development in chicks. The vaccine was administered by gavage and through drinking water to broiler chickens, Chinese Yellow strain, reared in wire cages. Vaccinated medicated birds performed better than vaccinated unmedicated and medicated unvaccinated groups. The final mean weights of vaccinated medicated birds were significantly higher (P < 0.05), and a better vaccine protection index, using both vaccinating methods, was achieved. Results indicated that concomitant use of ionophores and vaccines could be a useful adjunct to planned immunization in the control of coccidiosis. # 2005 Elsevier B.V. All rights reserved. Keywords: Coccidiosis; Chicken; Ionophore-tolerant; Eimeria spp.; Vaccinated medicated; Vaccine protection index

1. Introduction Coccidiosis is a disease of fowl caused by an obligate microscopic protozoan parasite, which belongs to the genus Eimeria (phylum Apicomplexa). The disease is characterized by loss in weight gain, increased feed conversion ratio, loss of skin pigment and decreased egg production (Ruff, 1991). * Corresponding author. Tel.: +86 20 85280241; fax: +86 20 85282693. E-mail address: [email protected] (G.Q. Li).

Coccidiosis is an economically important parasitic disease of poultry resulting in losses exceeding between US$ 1.5 and 2 billion annually to the poultry industry worldwide (Stevens, 1998). In the UK alone, the total cost of coccidiosis infections has been estimated to be at least £42 millions per annum, of which 74% is due to sub-clinical effects on weight gain and feed conversions and 24% is the cost of prophylaxis and therapy of commercial birds (Williams, 1998). As the world’s poultry industry continues to grow, so do concerns about coccidiosis which remains one of the most commonly reported diseases of chickens. Coccidiosis is easily

0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.09.034

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transmitted by direct or indirect contact with droppings of infected birds especially in intensively managed poultry hence causing an inevitable choice of protecting the birds against the disease. Although coccidiosis can be controlled by prophylactic chemotherapy, use of broad spectrum anticoccidial agents and good management practices and sanitary procedures, the emergency of rapid drug resistance coccidia coupled with difficulties and high costs of developing new drugs for effective treatment and prevention has lead to a pressing need to search for new approaches such as immunological, biotechnological and genetic methods as a more promising alternatives (Grag et al., 1999). A variety of anticoccidial drugs have been developed in the past with the ionophorous antibiotics being the most extensively used groups, but resistance has developed to almost all of them (Chapman, 1997), and there are concerns about drug residues in poultry products and strong desires of consumers to ban drugs from animal feeds. The development of coccidial resistance to drugs is probably the greatest single factor causing demise of the effectiveness of drugs. There is, therefore, a pressing need to move away from chemotherapeutic control of coccidiosis towards vaccination. Many anticoccidial vaccines, including live virulent vaccines (e.g. CoccivacTM and ImmucoxTM) and live attenuated vaccines (e.g. ParacoxTM and LivacoxTM) are currently in use. The recent uptake of attenuated coccidiosis vaccines worldwide has given the poultry industry greater confidence that vaccines may be used without any subsequent disease problems that require attendant medication. The major obstacle of these live vaccines (with either virulent or attenuated parasites) is that they are usually administered to chickens in the first week of age resulting in low-level infection and depends on litter for the recycling of vaccine parasites. During this period, use of therapeutics or feed additives that interfere with Eimeria development is not permitted, thus increasing the risk of contracting the coccidiosis at an early (1–3 weeks) age and decreases as immunity has developed (Vermeulen et al., 2001). Furthermore, Chapman (1999) noted that acquisition of immunity in medicated birds requires that the drug in question permits some parasite development in the host and that oocysts be present in the environment in sufficient numbers to stimulate an immune response.

Recently, a new development has been made in the use of live Eimeria strains, which are relatively tolerant to ionophores (Danforth, 2000). The advantage of these vaccines is that they allow the use of ionophores during the first 3–4 weeks when the immunity of the birds is not complete. This limits the increase of infection pressure due to expanding field strains during the development of immunity, hence reducing the overall risk of contracting coccidiosis (Vermeulen et al., 2001). In this experiment, we investigate the immunological responses of broiler chickens vaccinated with a live attenuated ionophore-tolerant multivalent Eimeria precocious line for protection against avian coccidiosis.

2. Materials and methods 2.1. Chickens Chinese Yellow disease-free chickens were purchased from the South China Agricultural University poultry farm, and used in the present experiment. They were initially kept in a coccidian-free house and transferred to wire cages for the experiments. 2.2. Coccidia vaccine The vaccine is composed of a live attenuated ionophore-tolerant stabilized suspension of multivalent sporulated oocysts of E. tenella, E. maxima and E. acervulina (Li et al., 2004). The parasites were isolated from chicken feces in a farm in Nanhai city, Guangdong province, Southern China, by established methods (Long, 1972). The three Eimeria species isolated were then tested for drug sensitivity. Drug resistant oocysts were sporulated and blended aseptically into a suspension of 0.1% Carboxymethyl cellulose sodium, which ensures the homogeneity of oocysts and maintenance as suspension in drinking water. 2.3. Challenge inocula The material used to challenge the vaccinated chickens were virulent strains of the same Eimeria species in the vaccine. Each virulent strain had

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previously been titrated using 55 susceptible Chinese Yellow chickens in the pathogenecity test. The number of sporulated oocysts needed in each inoculum, was given by gavage, to produce a statistically significant (P  0.05) reduction in weight gain during 1 week after infection. 2.4. Vaccinations Two vaccination trials were conducted using two vaccination methods namely, by gavage and through drinking water. 2.4.1. Vaccination by gavage This test was carried out on 300 broiler chickens of the Chinese strain (150 of each sex). Birds were divided into five groups, with three replication of 20 birds (10 of each sex) kept on disinfected wire cages at a stocking rate of 1 ft2 per bird. The first group was vaccinated by gavage when the birds were 3 days of age and a booster dose repeated a week later and fed with rations containing 5% Monensin ionophore vaccinated and medicated (VM). The second group was vaccinated by similar method with a similar dose as in group one but was not given feed containing any therapeutic drug vaccinated unmedicated (VUM). The third group was not vaccinated but was fed with rations similar to those given to group 1 medicated unvaccinated (MUV). The fourth and fifth groups were maintained as positive and negative controls, respectively (unvaccinated unmedicated challenged control (UVUM-C), and unvaccinated unmedicated non-challenge control (UVUM-NC)). All groups, except group 5, were challenged 3 weeks later with the virulent strains of the three Eimeria species contained in the vaccinal material. Lesion scores were determined 6 days after challenge according to the method of Johnson and Reid (1970). In addition to average weight gains, feed conversion ratio (FCR), and oocysts per gram of feces (OPG) were also determined and lesion score protection index (LSPI) calculated according to the method of Danforth (1998). 2.4.2. Vaccination by drinking water A total of 360 Chinese Yellow chickens (180 of each sex) were randomly divided, by equal weights, into five groups of 12 birds each with three replications. Three

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best vaccine batches (VB1, VB2 and VB3), determined in the gavage vaccination trials were suspended in 0.1% Carboxymethyl cellulose sodium solution for even distribution of oocysts. The vaccine combinations of three Eimeria species, E. tenella:E. maxima:E. acervulina were 5000:2500:5000 in VB1, 2500:1250:2500 in VB2 and 1250:750:1250 in VB3 and the challenged dose was 50,000:100,000:500,000. The vaccinal material was then administered to groups 1–3 at 3 days of age and a week later and fed with rations containing 100 mg/kg monensin anticoccidial drug. Groups 4 and 5 were maintained as unvaccinated unmedicated challenge and unchallenged control, groups, respectively. All groups, except group 5, were later challenged 3 weeks later with a predetermined dose of the virulent parent strain of the Eimeria species contained in the vaccine. Lesion scores were again determined 6 days post-challenge according to Johnson and Reid (1970) and oocysts production per gram of feces determined to calculate vaccine protection index (Rose and Mockett, 1983). Average live weight gains and FCR were also determined at the end of the experiment. 2.5. Statistical analysis The statistical analysis of weight gain, including the various doses of vaccine combination used, was based on Duncan’s multiple range test of analysis of variance (ANOVA) using the SAS system (Version 8.1) statistical package. The factors included in the ANOVA model are the mean weight gains and the treatment comprising of VM, VUM, MUV, UVUMC and UVUM-NC in vaccination by gavage and vaccine batch, VB1, VB2, VB3, UVUM-C and UVUM-NC in vaccination by drinking water. The group and animal effects are random, meaning that they are a representative sample of all groups and animals. By statistically modeling in this way, the conclusions can be extended beyond the immediate experiments to future batches and animals. The factors sex and treatment are fixed effect factors. For each Eimeria species, the mean weight gains per batch of chickens in the five treatment groups (VM, VUM, MUV, UVUM-C and UVUM-NC) were compared. The statistical significance necessary to demonstrate that the vaccinated birds were immune is that the mean weight gain of the unvaccinated unmedicated non-challenge groups need to be

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statistically significantly (P  0.05) lower than that of the VM groups. Protective immunity of the vaccinated birds was established when the mean weight gain of the VM groups was statistically significantly (P  0.05) greater than that of the UVUM-C groups. In addition to the primary criterion of weight gain, FCRs and lesion scores were compared numerically when the same trend were sought and used as a supportive secondary criterion to weight gains.

male and female birds, respectively. There was no significant differences between VUM and MUV groups. The males of the VUM group had VPI slightly higher (78%) than the females (76%). However, the female birds of the MUV group had higher VPI (72.61%) than the male birds (70%). Considering the average oocysts produced per bird after challenge, VM birds produced less oocysts 3 weeks after challenge. The VUM birds produced 3% more oocysts than the MUV group, while the MUV group produced about 1% more oocysts than the VUM group. The plausible explanation is that VM birds were well protected by deterring the establishment of the parent oocysts, which were expelled much earlier. As a result, few oocysts were able to establish themselves without any adverse consequences on the bird’s performances. Compared to the UVUM challenge control group, VM produced 88% oocysts, while VUM and MUV produced 85% and 86%, respectively, less oocysts post-challenge at the end of the first week. There was, however, a dramatic fall in oocyst production in the subsequent 2 weeks that

3. Results 3.1. Vaccination by gavage Table 1 show the summary of chickens vaccinated by gavage with live attenuated ionophore-tolerant multi-valent vaccine and/or medicated with anticoccidial ionophore (monensin), and is illustrated in Fig. 1. The vaccine protection index (VPI) was highest for VM birds with indices of 86.93% and 88.07% for

Table 1 Summary of performance of Chinese Yellow broiler chickens immunized by gavage with live ionophore-tolerant multivalent Eimeria vaccine and/or medicated with anticoccidial drugs Group

Treatment

Sex

FCR

Lesion score

Total oocyst production (Av/(bird day))

Post-challenge mean weight gain (g)

UI

MI

C

7a

7d

14e

21f

28g

Mean

14b

21c

VPI (%)

1

VM

M F

1.98 2.26

0.2 0.3

0.2 0.3

0.4 0.3

36600 33300

20000 25800

6600 8300

216.39 207.92

287.64 281.08

463.81 459.24

612.59 611.11

395.11A 389.84A

86.93 88.07

2

VUM

M F

1.96 2.42

0.6 0.6

0.5 0.7

0.6 0.7

105100 105000

47500 51600

9900 10900

216.25 212.64

258.06 254.31

441.19 419.76

497.62 459.28

353.28B 336.50C

78.00 76.00

3

MUV

M F

1.92 2.48

0.8 0.8

0.7 0.8

0.8 0.8

142500 134100

70000 66600

16600 27300

217.50 213.75

259.17 255.69

408.09 410.00

451.43 434.52

334.05C 328.49C

70.00 72.61

4

UVUM-C

M F

4.66 4.97

3.0 3.0

3.0 3.0

3.0 2.8

268300 281800

134100 185800

68300 78300

216.53 215.14

229.72 224.31

366.90 388.81

395.18 398.81

302.08D 306.77D

N/A N/A

5

UVUM-NC

M F

1.85 1.83

0.0 0.0

0.0 0.0

0.0 0.0

– –

– –

– –

219.58 217.50

261.25 260.42

468.57 466.43

543.57 535.71

373.24B 370.02B

N/A N/A

VM: vaccinated and medicated; VUM: vaccinated unmedicated; MUV: medicated unvaccinated; UVUM-C: unvaccinated unmedicated challenged control; UVUM-NC: unvaccinated unmedicated non-challenged control; VPI: vaccine protection index; UI: upper intestine; MI: middle intestine; C: caeca; N/A: not applicable. Means with the same letters (A–D) are not significantly different at a = 0.05 (DMRT). a Total oocyst produced (7 days) after challenge. b Oocysts produced (14 days) after challenge. c Oocysts produced (21 days) after challenge. d Mean weight gain (7 days post-challenged). e Mean weight gain (14 days post-challenged). f Mean weight gain (21days post-challenged). g Mean weight gain (28 days post-challenged).

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followed. The FCR for VM groups was 1.98 for male birds and 2.26 for females (n = 10). This was 7.23% higher for VM male birds and 6.10% for VUM and 3.75% for MUV compared to that of the UVUM female challenge control groups. 3.2. Vaccination by drinking water Overall performance of chickens medicated and vaccinated with different vaccine batches (doses) by drinking water is summarized in Table 2 and illustrated in Fig. 2. After a week post-challenge, there were no significant differences between unvaccinated unmedicated unchallenged control group and birds vaccinated with vaccine batch 2. The performances of birds vaccinated with vaccine batches 1 and 3 were significantly (P < 0.0001) less than the unvaccinated unmedicated challenged control group, but no apparent differences were observed between sexes in each treatment group.

Fig. 1. Post-challenge mean weights of chickens vaccinated by gavage with ionophore-tolerant multi-valent Eimeria vaccine and/ or medicated with ionophore (monensin).

Table 2 Summary of performance of Chinese Yellow broilers chickens vaccinated by drinking water with live ionophore-tolerant multi-valent Eimeria vaccine and/or medicated with anticoccidial drug (monensin) Vaccine batch

Treatment

Sex

FCR

Lesion score

Total oocyst production (Av/(bird day))

Post-challenged mean weight gains (g)

UI

MI

C

7a

17c

7d

14e

21f

28g

Mean

12b

VPI (%)

1

VB1

M F

3.96 4.06

1.2 1.5

2.5 1.0

1.5 1.6

44167 39000

22500 24167

27583 8667

239.68 229.17

333.67 337.50

379.17 383.00

401.33 402.17

338.46B 337.96B

62.00 60.00

2

VB2

M F

1.96 2.22

0.9 0.6

1.5 0.7

0.8 1.0

19000 18333

10333 12167

3167 4333

243.11 239.89

409.00 400.17

451.33 434.00

519.50 525.00

405.74A 399.77A

80.30 77.00

3

VB3

M F

3.92 4.48

0.8 0.8

0.7 0.8

1.7 1.6

31667 35000

15167 17250

10500 9500

204.89 207.89

329.50 344.17

398.33 404.17

436.83 438.51

342.39B 348.69B

57.00 59.00

–

UVUM-C

M F

4.66 4.97

3.0 3.0

3.0 3.0

3.9 3.9

301000 304667

164000 165000

91500 91917

241.17 245.67

368.00 369.75

388.50 390.75

400.75 405.50

349.61B 352.92B

N/A N/A

–

UVUM-NC

M F

1.85 1.83

0.0 0.0

0.0 0.0

0.0 0.0

– –

– –

– –

258.67 260.17

411.00 411.50

456.00 455.00

489.50 488.50

403.79A 403.79A

N/A N/A

VB1: vaccine batch 1; VB2: vaccine batch 2; VB3: vaccine batch 3; UVUM-C: unvaccinated unmedicated challenged control; UVUM-NC: unvaccinated unmedicated non-challenged control; VPI: vaccine protection index; UI: upper intestine; MI: middle intestine; C: caeca; N/A: not applicable. Means with the same letters (A–D) are not significantly different at a = 0.05 (DMRT). a Total oocyst produced (7 days) after challenge. b Oocysts produced (12 days) after challenge. c Oocysts produced (17 days) after challenge. d Mean weight gain (7 days post-challenged). e Mean weight gain (14 days post-challenged). f Mean weight gain (21days post-challenged). g Mean weight gain (28 days post-challenged).

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Fig. 2. Post-challenge mean weights of chickens vaccinated by drinking water with ionophore-tolerant multi-valent Eimeria vaccine and/or medicated with ionophore (monensin).

However, the unvaccinated unmedicated unchallenged control group still outweighed 411.00 g and 411.50 g for males and females, respectively, compared with birds vaccinated with vaccine batch 2 with mean weights of 409 g and 400.17 g for males and females, respectively. Comparing the three vaccine batches, birds vaccinated with vaccine batch 2 had a better performance with FCRs of 1.96 and 2.22, and a vaccine protection index of 80.3% and 77%, for males and females, respectively. This was followed by birds vaccinated with vaccine batch 1 with FCRs of 3.96 and 4.06 and vaccine protection index of 63% and 60% for males and females, respectively. In summary, vaccine batch 2 with an Eimeria combination of 2500 oocysts of E. tenella, 1250 oocysts of E. maxima and 2500 oocysts of E. acervulina provided the best protection index (80.30%) for birds, hence considered superior to the other vaccine combinations. It can therefore be asserted, based on results obtained, that the best vaccination dose of the vaccine for oral administration by drinking water is 2500:1250:2500 oocysts of E. tenella, E. maxima and E. acervulina, respectively.

4. Discussion Previous laboratory and experimental studies (Norton and Joyner, 1986; Shirley and Millard, 1986; Shirley, 1989) have demonstrated that a multivalent vaccine containing precocious lines of coccidia induces in chickens a strong immunity to

challenge with virulent homologous or heterologous strains. Drug resistance has been documented against almost all anticoccidial drugs used so far. Resistance to polyether ionophorous anticoccidial has become a major problem and there is growing evidence for a significant shift in the effectiveness of these drugs. In raising broiler chickens with a relative short life span (40–45 days), the problem of drug residue has forced farmers to comply with certain withdrawal periods for the safety of consumers. In this research, a live multivalent ionophore-tolerant anticoccidial vaccine was developed and tested to evaluate the efficacy of the vaccine during the growing period of broilers (Chinese Yellow chickens strains) reared on wire cages. The present study suggests that the concomitant use of anticoccidial ionophores and live attenuated ionophore-tolerant anticoccidial vaccines has the potential to improve live weight gains and minimise the spread of virulent field strain when the bird’s immunity is not well developed, and subsequently allow the gradual development of the bird’s local immunity at a later date of the bird’s life span. This dual approach strategy (chemotherapy and immunoprophylaxis) will certainly encourage the use of live ionophore-tolerant anticoccidial in intensively broiler production industries. It is on this basis that this research was designed with the intent of addressing drug resistant problems and providing adequate protection against avian coccidiosis. Comparison between the performances of broilers either vaccinated or treated with anticoccidial drugs

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reveal few consistent differences. These observations tend to confirm the expectation that there is unlikely to be any consistent improvement of bird’s weight gains or FCRs by anticoccidial vaccines, unless drug resistance has been a problem on a farm. However, for some yet unexplained reasons, vaccinated birds tend to have lower overall mortality than drug-treated ones (Williams, 2002). Although live anticoccidial vaccines may not offer any basic advantages in performances when compared with drugs, there are other benefits. A major advantage of vaccines is that they cannot induce coccidial drug resistance. Indeed, if resistant coccidia already exist on a farm, their adverse effects may be ameliorated by the use of either non-attenuated or attenuated vaccines. With respect to studies on the performance of live anticoccidial vaccines in broilers, there is no reason to expect that a vaccine will perform better, under identical conditions, than any non-ionophore anticoccidial drug, unless the resident coccidia are drug resistant (Williams, 2002). On the other hand, the performance of vaccinated birds should be no worse than drugtreated birds if the anticoccidial drug has no additional therapeutic properties. However, in comparison with ionophorous anticoccidial drugs, it is possible that vaccinated birds might have poorer feed conversion ratios (FCRs). This is possible because, even in disease free chickens, ionophores used at recommended concentration tend to improve FCRs (Wheelhouse et al., 1985). The performance of birds vaccinated by gavage or through drinking water and given monensin anticoccidial drug and/or medicated unvaccinated or vaccinated unmedicated were compared using the following primary criteria: protection from clinical coccidiosis, weekly bird weights, mean bird weights after challenge, percentages of bird losses (culled or found dead), total number of oocysts in feces, lesion score protection index and feed conversion ratio. Many scientists involved in coccidiosis research have often questioned the criteria that is best to assess chickens performance and appropriately evaluate drug resistance. All criteria in this experiment have proved to be of value under certain circumstances. Weight gains measured the effects of coccidiosis upon growth of the birds, lesion score estimated the pathology caused by infection, and FCR measures the amount of feed intake and conversion into animal tissues. Weight

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gain has proven to be the most useful criterion for evaluating the efficacy of anticoccidial drugs during the acute phase of infection, and these may be compared directly with unmedicated infected and unmedicated uninfected controls. In this research work, weight gains were similarly compared with unvaccinated unmedicated challenged and unchallenged controls. Lesion scores were also used but this procedure is inherently subjective since it requires different regions of the intestine, depending on the Eimeria species involved, to ascertain pathology of the parasite. It should also be remembered that, unlike other criteria, lesion scores do not increase linearly with number of oocysts inoculated (Chapman, 1994). In this experiment, lesion scores were very much erratic especially for species such as E. maxima and E. acervulina. Lesion scores were, therefore, used as a secondary criterion and as supportive evidence to weight gains. A criticism of the use of lesion scores for assessing coccidial infections is that under some circumstances lesion scores do not correlate with weight gain and those lesions may be present even though weight gain is not depressed (Long and Johnson, 1988). An interesting aspect of this broiler vaccine is the ‘‘reaction’’ (the presence of coccidial lesions and slowing of growth rate) that occurred with some vaccine doses during the 2 weeks following vaccination. However, the birds later exhibited a compensatory weight gain that brought them up to almost the same weight as unvaccinated unmedicated unchallenged control groups by 5–6 weeks of age. Moreover, because the birds may have acquired some immunity, the reaction to challenge with homologous virulent strains was minimal compared to unvaccinated unmedicated challenged controls. The live anticoccidial vaccine developed and tested in this experiment proved to be efficacious and provided satisfactory protection for the chickens. The vaccine, if administered by gavage, requires a less dosage of attenuated oocysts but a slightly higher dosage is needed when administered through drinking water.

Acknowledgements This research was partially financed by the Council of State Education Committee of the People’s Republic of China and by Natural Science Fund of

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Guangdong province (No. 010354). We would like to thank Prof. Zhu Xingquan for his critical review of this manuscript.

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