Comparison of breeder/layer coccidiosis vaccines: Part 1 -precocity and pathogenicity

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1 C The Author Published by Oxford University Press on behalf of Poultry Science Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Comparison of breeder/layer coccidiosis vaccines: Part 1 -precocity and pathogenicity Greg F. Mathis, Linnea J. Newman,,1 Steve Fitz-Coy, Brett Lumpkins, Robert Charette, and Lorraine Fuller Southern Poultry Research, 2011 Brock Road, Athens, GA 30607, USA; Merck Animal Health, 2 Giralda Farms, Madison, NJ USA; and Peccary Recherches, 839 Gault, Mont Saint Hilaire, Quebec, Canada J3H 3Y5 Primary Audience: Breeder/Layer Pullet Producers, Veterinarians, Biologics Producers SUMMARY Coccidiosis control for breeding and laying chickens requires the development of immunity against multiple Eimeria species. Vaccines approved for use in Europe are precocious (attenuated) strains. Only one precocious vaccine (Paracox R 8) was widely available to breeder and layer producers in the European Union (EU) from 1991 until 2015, with registration in all EU member states except Luxembourg. Recently, 2 new products have been introduced to the market: Hipra Evalon R and a 2 part product originally designated as Huveguard R Start (now designated Mmat) and Huveguard R Plus (now designated NB). Nonattenuated (nonprecocious) vaccines, by contrast, are used in other parts of the world, but are not available in the EU. Three precocious vaccines (Paracox R 8, Evalon R and the combined Huveguard vaccines) were compared to each other and to a nonattenuated vaccine from North America (Coccivac R D2) with respect to precocity and pathogenicity. All 3 precocious vaccines demonstrated significantly reduced oocyst output compared to the nonattenuated breeder/layer coccidiosis vaccine. One vaccine (Paracox R 8) demonstrated oocyst output for all species at 96 h (more precocious), while the other 2 vaccines did not have output until 24 or even 48 h later for individual species (less precocious). When tested at 40X the manufacturer s recommended dose (attempting to simulate the field effect of uneven application), all 3 precocious vaccines demonstrated lower lesion scores and better weight gain over the 7 d post challenge compared to the nonattenuated vaccine. Key words: Eimeria spp., coccidiosis, vaccination, precocity, pathogenicity 2017 J. Appl. Poult. Res. 0:1 5 DESCRIPTION OF PROBLEM Coccidiosis, caused by various species of the genus Eimeria, is a ubiquitous problem of floor-reared chickens, including replacement broiler breeders and replacement layer pullets. 1 Corresponding author: linnea.newman1@merck.com The common pathogenic species, E. acervulina, E. maxima, E. necatrix, E. tenella, and E. brunetti cause intestinal lesions within the host that can result in reduced weight gain of individual birds, reduced uniformity of flock weights, and mortality [1]. While coccidiosis is often controlled with chemoprophylaxis in broilers, the poultry

2 2 JAPR: Research Report industry depends upon acquired immunity to protect long-lived breeder and layer flocks against these parasites [2]. Immunity is speciesand sometimes strain-specific [3, 4] and is developed via exposure to coccidia, including repeated exposure to low numbers of oocysts [5] created either by vaccination with live sporulated oocysts (either attenuated or nonattenuated) [6], or by controlled natural exposure with step-down doses of an anticoccidial drug to control field infection until immunity develops [7]. One method of stimulating immunity is to use an attenuated, live, sporulated oocyst vaccine developed with precocious Eimeria species. Precocious strains are missing one or more secondary schizogony stages compared to the normal life cycle of the parent Eimeria strain, and thus have a shortened pre-patent period and a significantly reduced oocyst output. The development of precocious Eimeria isolates through serial selection of the earliest oocysts shed by infected birds has been described by researchers Jeffers, McDonald, Shirley, and Long in the mid-1970s through mid-1980s [8 15]. The more precocious an isolate is, the less pathology is induced by asexual stages, and the fewer oocysts are shed. The precocious strains are still capable of inducing a protective immune response to challenge with the parent strains under laboratory and field conditions. The immune response to vaccination with live coccidiosis vaccines under field conditions depends upon repeated reinfection through ingestion of sporulated oocysts shed through the feces of the vaccinates into the poultry house environment [5]. Excessive reduction of oocyst output through precocity or reduced sporulation due to dry environmental conditions may hinder this process and delay the onset of immunity [16, 17]. Uneven hatchery or field application or uneven uptake of the fecal oocysts in the field environment can occasionally result in naïve or partially immune birds that can become exposed to wild Eimeria spp. in the house, or to higher doses of oocysts shed by properly vaccinated hatchmates after the first Eimeria life cycle has been completed [18]. Until 2015, most of the European Union had only one live Eimeria vaccine for the vaccination of replacement birds (Paracox R,MSD Animal Health, Madison, NJ, USA). Paracox R (P) contains precocious strains of Eimeria acervulina, E. maxima (2 strains, CP and MFP), E. mitis, E. praecox, E. tenella, E. necatrix, and E. brunetti, for a total of 8 strains [19]. Two biological companies have recently introduced new live Eimeria vaccines to Europe. Evalon R (E) (Hipra, Amer, Girona, Spain) contains precocious strains of E. acervulina, E. maxima, E. tenella, E. necatrix, and E. brunetti for a total of 5 strains [20]. Huveguard R Start (Huvepharma, Sophia, Bulgaria) contains 4 precocious strains, E. acervulina, E. maxima, E. mitis, ande. tenella for administration at 1 d of age [21], while Huveguard R Plus is a separate product containing 2 precocious strains E. necatrix and E. brunetti, to be given at 14 d of age [22]. At the time of the study, the Huveguard trade names were not fully established. They have since been registered in some countries as Huveguard R Mmat and Huveguard R NB, respectively. Together the Huveguard R products provide protection for the common challenge species of breeder and layer replacements. In this study, both Huveguard products are given simultaneously (HSP) to inoculate birds with the full complement of Eimeria species for comparison with the other vaccines. The aim of this study was to compare Paracox R (P), Evalon R (E) and Huveguard R Start plus Huveguard R Plus (HSP) with respect to precocity and pathogenicity with a nonattenuated vaccine Coccivac R -D2 (Merck Animal Health, Madison, NJ, USA). Coccivac R -D2 contains nonattenuated strains of E. acervulina, E. mivati, E. maxima, E. tenella, E. necatrix, and E. brunetti for a total of 6 strains [23]. Vaccines P, E, and D2 are all approved for application at 1dofage. MATERIALS AND METHODS In each of the studies, the guidelines presented in the Guide for the Care and Use of Agricultural Animals in Research and Teaching, 3rd Edition, 2010 [24] were followed. The guidelines are approved by the institutional animal care and use committee (IACUC Southern Poultry Research, Athens, GA, USA).

3 MATHIS ET AL.: BREEDER/LAYER COCCIDIOSIS VACCINES PART 1 3 Part A: Precocity Fifty male Cobb 500 broiler chicks were placed into 5 groups of 10 birds in isolation units. At 3 d of age, the birds in each group were inoculated via oral gavage with 5 times (5 ) the manufacturer s recommended dose of one of the 4 test vaccines, P, E, HSP, or D2. One group remained un-inoculated, as negative controls. The authors chose 5 dose to increase the oocyst output and the opportunity to capture oocysts at their earliest shedding point. Pooled fecal samples from each group were collected via hand collection of fresh feces, with clean gloves used between each sample, at 5 24-h intervals beginning at 72 h (72, 96, 120, 144, and 168 h). Fecal samples were subjected to a salt solution for flotation and samples were enumerated using a McMaster counting chamber using techniques described in Holdsworth et al. (2004) [25]. Morphological identification of oocysts was based upon the descriptions by Reid [1], but for vaccine D2, E. acervulina and E. mivati were counted together as one species. Part B: Pathogenicity Five hundred one-day-old male Cobb 500 broilers were separated into 10 birds per cage 10 cages per treatment group. Five treatment groups were included: 3 attenuated vaccines (P, E, HSP), one nonattenuated vaccine group (D2), as well as uninoculated controls. At 7 d of age, a start weight (birds weighed as a group per cage) was determined, and then each vaccine group received 40 of the manufacturer s recommended dose of the respective vaccine via gavage, while uninoculated controls were housed in a separate location to avoid cross-contamination. The 40 level was selected to induce pathology without mortality in the nonattenuated vaccine group based upon a prior dose titration. At 7 d postinoculation, the birds from all treatment groups and the negative controls were weighed by cage. One bird per cage was humanely sacrificed by cervical dislocation for lesion scoring. Lesion scoring was done for E. acervulina, E. maxima, E. tenella, E. necatrix, and E. brunetti as described in Johnson and Reid [26], with E acervulina and E. mivati scored together for vaccine D2. Data was analyzed as described [27, 28]. All statements of statistical significance are based upon probability RESULTS AND DISCUSSION Part A: Precocity None of the vaccines had detectable oocyst output prior to 96 h postinoculation. Vaccine P was the most precocious: it yielded oocysts at 96 h for all species, and produced higher counts for each species at that time point compared to the other vaccines. Vaccine E yielded 96-h oocyst output for 4 of the 5 species at lower numbers, and vaccine HSP yielded low-level 96-h oocyst output for 2 of the 5 species. The nonattenuated vaccine D2 had low oocyst output at 96 h for E. acervulina and E. maxima, and no oocysts could be detected at 96 h for E. tenella, E. necatrix, or E. brunetti. Vaccine D2 induced the highest overall oocyst output from 96 h through 192 h at a magnitude ranging from 4 (E. necatrix) to 115 (E. tenella) compared to the attenuated vaccines. Vaccine P produced the highest oocyst output of the attenuated vaccines over the same time period. (Table 1) Part 2: Pathogenicity The authors chose a 40 manufacturer s recommended dose in an attempt to simulate what can happen to naïve birds when they are missed by initial vaccination, and then become exposed to oocysts shed following initial vaccination, or subsequent oocyst shedding by vaccinated hatchmates. The results for lesion scores and average daily gain are presented in Table 2. Ata40 dose, the lesion scores of the nonattenuated vaccine (D2) were significantly higher compared to the controls for all species. Vaccine HSP was not significantly different from vaccine D2 with respect to E. acervulina and E. maxima. Vaccine P and vaccine E lesion scores did not differ significantly from uninoculated controls for any of the 5 Eimeria species. The average daily weight gain during the 7 d postinoculation was significantly less in all of the vaccine groups compared to uninoculated controls. Average daily weight gains for the 3 attenuated vaccines were significantly better than the nonattenuated vaccine D2

4 4 JAPR: Research Report Table 1. Precocity and Fecundity: Oocyst output 1 at 96 h and total oocyst output 2 from 96 to 192 h. Eimeria species Vaccine P Vaccine E Vaccine HSP Vaccine D2 Oocyst output at 96 h (precocity) E. acervulina E. maxima E. tenella E. necatrix E. brunetti Total oocyst output from 96 to 192 h (fecundity) E. acervulina ,155 3 E. maxima E. tenella ,914 E. necatrix E. brunetti ,540 1 Oocysts per gram of feces (OPG). 2 OPG sum of collections at h. 3 The authors counted E. acervulina and E. mivati together as E. acervulina due to the difficulty of morphological distinction. Table 2. Pathogenicity: Average lesion Scores 1 and average daily gain (g) following 40X inoculation. 2 Uninoculated Eimeria species Control Vaccine P Vaccine E Vaccine HSP Vaccine D2 3 E. acervulina 0.0 b 0.2 b 0.3 b 0.6 a,b 2.6 a E. maxima 0.0 b 0.5 b 0.9 b 1.0 a,b 2.1 a E. tenella 0.0 b 0.1 b 0.0 b 0.0 b 1.7 a E. necatrix 0.0 b 0.0 b 0.0 b 0.0 b 0.6 a E. brunetti 0.0 b 0.1 b 0.1 b 0.1 b 0.9 a Weight gain (g) a b b b c 1 Johnson and Reid Lesion Score 2 Differing superscripts indicate significant difference between treatment groups (P < 0.01) 3 Lesion score represents combined E. acervulina and E. mivati due to the similarity of the lesions in location and appearance. group. It is important to note, however, that even at a 40 manufacturer recommended dose, the nonattenuated D2 vaccine produced an average lesion score of 1.7 for E. tenella, with only one bird out of 10 exhibiting a score of 3 on the Johnson and Reid scale [26]. Despite a lack of attenuation, the vaccine is not highly pathogenic. CONCLUSION AND APPLICATIONS 1. The results of the study show that there are marked differences between the attenuated vaccines and the nonattenuated vaccine with respect to precocity, fecundity and pathogenicity. The nonattenuated vaccine induced higher lesion scores and induced greater oocyst output compared to the attenuated vaccines. 2. Higher oocyst output increases the opportunity under field conditions for every bird in a flock to find and recycle sporulated vaccinal oocysts, which contributes to a rapid development of immunity [29]. Higher oocyst output, however, can also result in greater pathogenicity when a nonattenuated vaccine completes successive life cycles in a flock, particularly if some of the birds remain naïve following the initial vaccination. 3. The attenuated vaccines themselves demonstrated numerical differences in precocity and fecundity. Of the attenuated strains, vaccine P demonstrated the highest level of precocity, vaccine HSP the lowest level of precocity and vaccine E was intermediate. Greater precocity implies a reduction in pathogenicity due to loss of more secondary schizogony stages. The pathogenicity study demonstrated significantly better weight gain in the face of 40 manufacturer s recommended dose for the attenuated

5 MATHIS ET AL.: BREEDER/LAYER COCCIDIOSIS VACCINES PART 1 5 vaccines compared to the nonattenuated D2; however, with respect to lesion scores, vaccine HSP did not differ from vaccine D2. 4. The lower oocyst output of the attenuated strains implies that the onset of immunity under field conditions may be slower, with implications on flock uniformity as well as protection against challenge. Further investigation into the comparative onset of immunity of the 3 attenuated vaccines is needed. REFERENCES AND NOTES 1. McDougald, L. R., and S. H. Fitz-Coy, Coccidiosis. Pages in Diseases of Poultry, Swayne, D. E., J. R. Glisson, L. R. McDougald, L. K. Nolan, D. L. Suarez, and V. L. Nair eds. Wiley-Blackwell, Hoboken, NJ. 2. Shirley, M. W., A. C. Bushell, V. McDonald, and B. Roberts, A live attenuated vaccine for the control of avian coccidiosis: trials in broiler breeders and replacement layer flocks in the United Kingdom. Vet. Record. 137: Martin, A. G., H. D. Danforth, J. R. Barta, and M. 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Merck Animal Health, Coccivac D2 Product Label (NAC No.: ). Coccivac D2 Product Label, Merck Animal Health, Madison, NJ. 24. Federation of Animal Science Societies Guide for the care and use of agricultural animals in research and teaching, 3rd edition. Online PDF document. Guide 3rd ed.pdf. 25. Holdsworth, P. A., D. P. Conway, M. E. McKenzie, A. D. Dayton, H. D. Chapman, G. F. Mathis, J. T. Skinner, H. C. Mundt, and R. B. Williams, World Association for the advancement of veterinary parasitol (WAAVP). Guidelines for evaluating the efficiency of anticoccidial drugs in chickens and turkeys. Vet. Parasitol. 121: Johnson, J., and W. M. Reid, Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28: All analysis was performed using the R language and environment for statistical computing and graphics [28]. Homogeneity of variances was tested for average daily gain using the bartlet.test function of the stats package. The assumption of equal variance was violated (p = ), thus the data was analyzed using Welch s ANOVA using the oneway.test function of the stats package. Post hoc test on the means was performed with the Games-Howell post hoc test using the oneway function of the userfriendlyscience package. Discontinuous variables were analyses by nonparametric ANOVA using the Kruskal-Wallace rank sum test one way analysis of variance using the kruskal.test from the stats package. A pairwise multiple comparison of mean rank post hoc test after Nemeyi was performed using the function posthoc.kruskal.nemenyi.test of the PMCMR package. 28. R Core Team R: A language and environment for statistical computing. R Foundation for Statistical Computing. 29. Severins, M., D. Klinkenberg, and H. Heesterbeek, Effects of heterogeneity in infection-exposure history and immunity on the dynamics of a protozoan parasite. J. Roy. Soc. Interface. 4: