Development of immunization trials against Eimeria spp.

Trials in Vaccinology 5 (2016) 38–47

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Trials in Vaccinology journal homepage: www.elsevier.com/locate/trivac

Review Article

Development of immunization trials against Eimeria spp. Tarek A. Ahmad a,b,⇑, Bassant A. El-Sayed c, Laila H. El-Sayed b a

Scientific Support and Projects, Bibliotheca Alexandrina, Alexandria, Egypt SeptivaK Research Group, Immunology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt c Zoology Department, Faculty of Science, Alexandria University, Alexandria, Egypt b

a r t i c l e

i n f o

Article history: Received 12 August 2015 Revised 28 January 2016 Accepted 5 February 2016

Keywords: Avian coccidiosis Eimeria Vaccine Immunotherapy Poultry

a b s t r a c t Coccidiosis is a major intestinal disease affecting economically valuable livestock animals such as chickens and turkeys. Economic losses are associated with decreased productivity in afflicted animals. The different Eimeria spp. are the main etiologic agents for that virulent disease. The usefulness of prophylactic and therapeutic anticoccidial compounds has decreased in recent years due to the emergence of drug resistance in Eimeria, together with their possible toxic effect to the human consumers. Despite that, biosecurity and disinfection measures are the cornerstone to control the emergence of the pathogen, the immunization methods proved to be more practical and promising to prevent outbreaks due to coccidia. Since the early 1950s, several attempts were followed to formulate commercial immunotherapies, but up till now none proved to be sufficient. This review summarizes, classifies, and evaluates the trials performed to prevent avian coccidiosis, thereafter introduces an out of frame scientific strategy to find a solution for that emerging parasite. Ó 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Contents 1. 2. 3. 4. 5. 6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment and drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of Eimeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Passive immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Active immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Live virulent vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Live attenuated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Live-tolerant to ionophores vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Generations of subunit vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Animals as humans, suffer from diseases. Coccidiosis is a disease that affects a variety of wild type and domesticated ⇑ Corresponding author at: Scientific Support and Projects, Bibliotheca Alexandrina, Alexandria, Egypt. E-mail addresses: [email protected], [email protected] (T.A. Ahmad).

38 40 40 40 41 41 41 41 43 43 44 45 45

vertebrates including chickens, turkeys, rabbits, cattle, sheep, goats, pigs, fish, and reptiles. The most virulent and economically important types of that disease are those infecting poultry and mammalian livestock. In poultry, the disease usually provokes severe intestinal disorders, diarrhea, dehydration, loss of weight, anorexia, and weakness. Therefore, obviously affects the animals husbandry, due to the reduced production it causes and the remarked mortality rate [1]. The main etiologic agents of coccidiosis in poultry are members of the genus Eimeria; which

http://dx.doi.org/10.1016/j.trivac.2016.02.001 1879-4378/Ó 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47 Table 1 The most important farm animal hosts of Eimeria species. Host

Eimeria spp.

References

Chickens

E. acervulina, E. brunette, E. necatrix, E. tenella, E. maxima, E. mitis, E. praecox E. adenoids, E. meleagrimitis, E. dispersa, E. meleagridis, E. gallopavonis, E. innocua, E. subrotunda E. stiedae, E. flavescens, E. intestinalis E. bovis, E. zuernii, E. alabamensis, E. auburnensis, E. brasiliensis, E. bukidnonensis, E. canadensis, E. cylindrica, E. ellipsoidalis, E. pellita, E. subspherica, E. wyomingensis E. crandallis, E. ovinoidalis, E. faurei, E. granulosa, E. intricata, E. pallida, E. parva, E. weybridgensis E. arloingi, E. christenseni, E. caprina, E. hirci, E. ninakohlyakimovae, E. alijevi, E. aspheronica, E. caprovina, E. hirci E. debliecki, E. polita, E. scabra, E. spinosa, E. porci, E. neodebliecki, E. perminuta, E. suis

[2,3]

Turkeys Rabbits Cattle

Sheep Goats

Pigs

[2] [6] [7]

[7] [7]

[7]

are obligatory intracellular apicomplexan protozoan parasites that belong to family Eimeriidae [2]. The clinical disease depends on the ingested dose of the sporulated oocysts of Eimeria by susceptible fowl [3]. The different in-host stages of Eimeria spp. invade the cells of the intestine and duodenum (enterocytes), and replicate resulting in variable pathological changes. These changes range from local destruction of the mucosal barrier and underlying tissue, to systemic effects such as blood loss, and death [4]. Collectively those symptoms are associated with drop in egg production, impaired growth rate due to nutrients malabsorption in adults, necrotic enteritis due to Clostridium perfringens, and high mortality

39

rates especially in young’s [5]. Table 1 clusters the different species of Eimeria in groups according to their domesticated hosts. The table orders each group from the most virulent to least to that specific host. The infection of poultry with Eimeria spp. (Fig. 1) begins when the host swallows the sporulated oocysts that excyst in the intestine and each releases 8 sporozoites (SZ). Once free within the intestine, the sporozoites penetrate the host’s enterocyte in very short time, encapsulate themselves safely within a parasitophorous vacuole, and replicate. Merozoites (MZ) are then released in tremendous numbers, they rupture the host cell, and invade new ones. It has been proven that each Eimeria sp. is programmed genetically for a specific number of merogonic divisions. As much the parasite reinvades the cell, as much it damages the intestine and causes severe manifestations. After the last speciesspecific merogonic generation, merozoites invade the enterocytes to differentiate into male and female gametocytes (GAM). After fertilization the oocyst ruptures from the host’s enterocytes and leaves the host with feces [2,8,9]. Experienced farmers noticed that lactating or previously infected animals show increased survival against the parasite for several months [10,11]. Despite the loss of an adequate full perception of immunity against coccidiosis, many studies underlined the immunological mechanisms that are active during primary and secondary infection of Eimeria spp. [12]. Hosts usually develop both immunological specific and nonspecific responses against the intracellular developmental stages of Eimeria spp. Specific responses include mucosal and circulating humoral response that initiates against the sporozoite, the merozoite, and the late sexual stages [4,13]. On the other hand, the gut-associated lymphoid

Fig. 1. (A) Diagrammatic life cycle of Eimeria and (B) simplified structure of Eimeria spp. sporozoite.

40

T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47

Table 2 Overview of the most widely used preventive methods to control coccidiosis. Strategy

Preventive method

Effect

References

Management and biosecurity

Disinfection of the materials, the people and equipments entering the farm

Reduce infection

[3]

Oral phytotherapy and feed additives

Arteminsin and citric extracts Oregano, Echinacea, mushrooms (lectin), turmeric, and betain T. violacea antioxidant-rich plant extracts Lectin (FFrL) extracted from the mushroom Fomitella fraxinea Garlic extract Mixture of castor and cashew oils

Inhibit the development of Eimeria spp. Indirectly affect the development of coccidia Decreased oocyst production Reduction in oocyst shedding

[35–40] [3,11,41– 46] [47] [48]

Attenuate inflammation and injury due to liver coccidiosis Increase livability of infected animals and reduce intestinal lesions

[49] [50]

Prebiotics

Mannanoligosaccharides (MOS) ‘BioMosÒ, SAF-MannenÒ, Y-MosÒ, and CelmanaÒ’

Block the binding of the pathogens to the mucosal surface Stimulate the immune response, enhances the development of Bifidobacteria spp. and Lactobacillus spp., and suppresses the number of enterobacteriacea

[51,52]

Probiotics

MitoGrowÒ and MitoMaxÒ

Lowers intestinal invasion, development of coccidian and oocyst shedding Increase antibody response

[3,53]

Other products

Ibuprofen (non-steroidal anti-inflammatory drug-NSAID) Mucolytic enzyme (protease)

Reduces the inflammation due to coccidiosis

[54]

Impair the attachment of Eimeria to the mucus layer

[3]

tissue involves intraepithelial and lamina propria CD8+ cd T and CD4+ ab T cell lymphocytes in the cell mediated responses. Moreover, natural killer (NK) cells appeared to play a main role in non-specific responses [8,12,14,15]. 2. Treatment and drug resistance Since the 1940s and up till now, more than 30 sulfanilamide drugs were used for the prophylactic control and treatment of coccidiosis. These anticoccidial products either affect cofactor synthesis, mitochondrial functions, or the cell membrane function of Eimeria spp. [3]. However, the worldwide use of such drugs led to the development of lowered efficacy, cross-resistance, and multi-drug resistance against them all [16]. It was proposed that the genetic recombination is the major reason underlying the selection of resistant phenotypes during the development of the parasite [17]. Meanwhile, the attempt of the rotation of various anticoccidial drugs in single and/or shuttle programs are followed to diminish the time for the development of resistance to an extent [18]. In parallel, solutions for finding new drug targets were studied. In 1998, researchers identified a gene (ets3a) that may be important in controlling the life cycle of E. tenella [19]. More recently, the identification of many drug possible targets such as, the enzymes of the sporozoite mannitol cycle [15,20], the trophozoite histone deacetylase, the E. tenella CDC2-related kinase 2 (EtCRK2), and the protozoal cyclic GMP dependent protein kinases, together with the genes encoding them from Eimeria maxima, paved the way for the introduction of new anticoccidial products [21]. Simultaneously, the use of nano-particles was introduced to replace the available coccidiostats. Although nano-silver did not show a success [22], nano-zinc was promising against mice’s coccidia [23]. However, all those solutions are still under trials and Eimeria outbreaks are still causing harm and economic loss to the animal husbandry.

infections has been estimated to be 2.4–3 billion USD, of which 76% is caused by clinical or sub-clinical coccidiosis and 24% by drug-related costs [2,10,24]. Therefore, the attempts to study and control Eimeria through epidemiological and environmental factors flourished. It was found that the severity of the coccidiosis disease depends on several factors, such as the facts that some species of Eimeria showed to be more ‘antigenic’ than others [25]. Although a large dose of Eimeria spp. oocysts (more than 103) are required to generate protective immune response, E. maxima requires only a small number of oocysts to induce almost full immunity. In other species such as E. tenella, high doses between 15  103 and 45  103 sporozoites, induce complete mortality in chick embryos [26,27]. The second factor that affects the epidemiology of Eimeria is a cluster of host related ones. Moreover, the clinical signs resulting from Eimeria infection are significantly influenced by the host’s genetic factors, since genetically divergent strains show different levels of susceptibility to coccidiosis [28,29]. Simultaneously, animal husbandry related factors affect the occurrence of coccidiosis. Poor hygiene of people and equipment, presence of disease vectors such as rodents and insects in the farm, and the occurrence of other infectious agents such as viruses and enteric bacteria collectively affect the spread of the disease and the severity of the infection [3]. Moreover, it was proven that the presence of immunized chicks in the litter plays a role in reducing infection [30,31]. Finally, some environmental factors were shown to enhance the infection. Although wet litter decreases the sporulated oocyst formation rate for Eimeria, its use to control the spread was non-practical since the increased humidity was found to cause footpad lesions and skin burns in animals [3]. In the practical farm’s life, sanitization as a part of prevention showed to play a main role in reducing the dissemination of the parasite. However, new preventive approaches appeared to show some hope to diminish the emergence of Eimeria outbreaks [2].

3. Epidemiology of Eimeria 4. Prevention The spread of coccidiosis disease is a recurrent matter, since oocysts are carried by both infected and recovered hosts [3]. Over 20 billion broilers are produced globally each year. The US produces 9 billion broilers alone while Brazil produces around the same number, with a prevalence of clinical and sub-clinical coccidiosis that attains 5% and 20%; respectively. The annual cost sustained by the global poultry industry from the Eimeria

The elimination of the source of Eimeria oocysts and continuous disinfection by anti-oocysts preparation such as ammonium hydroxide [32], together with the prophylactic chemotherapy are the corner stone strategies used by most farmers to prevent the parasite outbreaks. However, substitutes for coccidiosis control and alternative anticoccidial strategies (Table 2), such as

T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47

biosecurity, management, phytotherapy, pre- and probiotics, and vaccination developed [3,33,34]. Although the management and biosecurity measures could interrupt the introduction of Eimeria spp. to a farm, in practice they could not stand alone to prevent coccidiosis outbreaks. Moreover, phytotherapy, aromatherapy, and pre- and pro-biotics show confusing results and have therefore not been applied at large scale in the field till now [11]. Therefore, practice proved that the most solid and successful prevention and control strategy for coccidiosis is immunization [55]. 5. Passive immunization The first attempts to passively immunize chickens was in 1988, when a monoclonal antibody (mAb) raised against the surface antigen (1073.10) of E. tenella sporozoite produced in mice was able to agglutinate sporozoites in vitro, to lyse the parasite in the presence of complement, and that the passive transfer of the ammonium sulfate-precipitated peritoneal ascites fluid was able to protect against challenge with E. tenella. It was demonstrated as well that the antiserum raised against the different purified or recombinant antigens of the different stages of the parasite showed to provide partial protection [56]. However, studies on recovered chicken sera taken at IgG peak demonstrated to provide 97% immunity in naive chicks against homologous challenge [57]. Additionally, it was found that maternal immunity induced by live infection with E. maxima can provide partial cross-protection against E. tenella [58]. Monoclonal antibody raised against the 56-kDa gametocyte antigen of E. maxima, showed to reduce oocyst shedding after challenge by 40% in chicks [59]. Simultaneously, monoclonal antibody raised against the 12-kDa oocyst wall protein of E. tenella was also found to be capable of providing passive protection [60]. Meanwhile, sonicated gametocytes of E. maxima or its affinity-purified antigens have demonstrated to confer partial maternal protection against E. tenella and Eimeria acervulina [13,61]. 6. Active immunization Observations in various avian and murine models proved that even a single infection with oocysts from a given Eimeria sp. can provoke homologous immunity against reinfection. Therefore, several research groups together with the pharmaceutical bodies introduced to the market a variety of commercial vaccines especially for poultry [62]. Since killed parasite given by different routes of vaccination failed to induce protective immunity, the majority of the commercial ones were live vaccines [4]. Thus, up till now there are more than 25 commercial anticoccidial vaccines for use in poultry (Table 3). The following paragraphs classify those products, together with the research trials in the field. The effectiveness of live vaccines lies in the oral introduction of very low doses of the Eimerian oocysts. The ingested living antigens develop and initiate both humoral and cellular responses against the parasite’s different stages. The robust immune response against the pathogen protects the host from further infections [15]. The in ovo administration of living oocysts was practiced as a result of a finding that E. tenella is able to develop in the chick embryo [63] and allows the 18–19 days old embryo to develop an immunity against the antigenic determinants of the pathogen. However, the use of asexual subunit vaccines usually enable the immune system to control a specific stage from the life cycle of Eimeria [15]. Although immunoglobulins production is a considerable mechanism to limit the propagation of several pathogens [13], T-cell mediated response is the major criterion for the control of intracellular parasites such as Eimeria [8,64,65]. Therefore, the potency of all vaccines against Eimeria were measured in term of

41

T-cell lymphocytes activity and by challenge in term of lesion score (LS), body weight gain (BWG), relative growth rate (RGR), oocysts decrease rate (ODR), survival rate (SR), and anti-coccidial index (ACI). 6.1. Live virulent vaccines Live vaccines group was the first to be studied, due to the fact that live parasites reduce further reinfection. They comprise parasites derived from laboratory or field strains, without any modification that changes their natural virulence [63]. The first commercial live anticoccidial vaccine, CocciVacÒ, was introduced to the US market in 1952. It comprised a mixture of wild-type strains of E. tenella oocysts, and conferred a homologous protection against those strains included in the mixture. Therefore, the vaccine went through a number of reformulations over the past 6 decades and variants of the original product, CocciVac-BÒ, CocciVacDÒ and ImmucoxÒ, are still in use today in more than 40 countries [63,66]. In parallel live oocysts vaccines proved to be efficient in turkeys [67]. Recent research focused on the delivery of Eimeria’s oocysts in gel-beads in day-old chicks [68]. Although vaccination with wild-type parasites may be very successful, immunovariant infections within a flock can lead to the emergence of uncovered strain. This may lead to the ingestion of large numbers of oocysts by susceptible animals and increases the risk of disease outbreaks [69]. Therefore, the attenuation of the parasite appeared to overcome that defect. 6.2. Live attenuated vaccines These vaccines consist of parasites of artificially reduced virulence as active ingredients. At first, heat treatment and Xirradiation were used to attenuate Eimeria parasite, but both were unsuccessful to induce potent preparations. Though, vaccine attenuated by irradiation (E. tenella radiovaccine) reported to be a promising Immunoprophylactic trend against coccidiosis [70,71]. Later on, various methods for the attenuation of the Eimeria parasite evolved, such as de-routed vaccines of sporozoites and high doses of merozoites inoculated intra-rectally [72]. However, only two of these methods have been used to attenuate commercial vaccines; either by passing parasites through embryonated hens’ eggs (embryo-adapted line) such as LivacoxÒ vaccines of E. tenella, or by selection for precocity (i.e.; the production of parasite strains that complete their endogenous life cycle in their host faster than the parent (wild) strains (Table 3). Therefore, they induce immunity without damaging the intestine) such as LivacoxÒ and ParacoxÒ vaccines [63,73]. Practically, embryo-adapted line was difficult to obtain with a satisfactory combination of immunogenicity and attenuation of virulence. Moreover, another obstacle that limited embryoadaptation as a mean of producing potential vaccination parasites was the total lack of success in obtaining complete development of the Eimeria spp. in the embryonic chorio-allantoic membrane. On the other hand, the method of selection for precocity introduced by Jeffers in 1975 was the most widely used for the attenuation of Eimeria parasite in chickens [74]. The unique feature of the attenuated parasites obtained from the precocious type is the reduced virulence and pathogenicity of the parasite compared to the parent strain. Although the production of precocious lines of all Eimeria species of fowl has been already accomplished, the produced parasites have lower pathogenicity than the wild ones [63,75,76]. Therefore, they failed to provide a satisfactory degree of protective immunity against a heavy challenge. Currently, adjuvants that consist of different cytokines are paired with the attenuated vaccines in order to improve the immunogenicity. However, economic concerns arose as a limitation [3].

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T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47

Table 3 First generation vaccines against coccidiosis.* Vaccine

Targeta Speciesb

Oocyst typec

Form Routed

Age

Concurrent conditions

Protection Application Last manufacturer

DMÒ coccidiosis

Bo

WT

Aq.

3d

Sulfa drugs

NAe

Sulfa drugs Sulfa drugs Sulfa drugs Trithiadol Trithiadol Trithiadol Trithiadol Trithiadol Trithiadol

e

NA NAe NAe NAe NAe NAe NAe NAe NAe

(Amprol)

18 week

–

18 week

(Amprol)

18 week

–

NAe

–

NAe

–

NAe

–

36 week

Ò

Et

Coxine NObiCOXÒ CoccivacÒ MF CoccivacÒ-t/A MF-CoccivacÒ-t/3 MF-CoccivacÒ-t/4 MF-CoccivacÒ-t/B MF-CoccivacÒ-t/C MF-CoccivacÒ-t/D

Bo Bo Bo L Bo Bo Bd L L

CoccivacÒ-T

T

CoccivacÒ-D2 Coccivac -B52

Bo/Bd/ Eac, Eb, Ema, Emi, En, Et L Bo Eac, Ema, Emi, Et

ImmunocoxÒ-C1/I

WT WT WT WT WT WT WT WT WT

Aq. Aq. Aq. Aq. Aq. Aq. Aq. Aq. Aq.

Mf Mf Mf W/Mf W/Mf W/Or W/Or W W

WT

Aq.

WT

Aq.

WT

Aq.

Bo/R/T Eac, EmaX2, En, Et

WT

Gel

ImmunocoxÒ-C2/II

Bd/L

Eac, Eb, Ema, En, Et

WT

Gel

ImmunocoxÒ-T

T

Ead, Emel, Eg, Ed

WT

Gel

ParacoxÒ

Aq.

ParacoxÒ-5

Bo/Bd/ Eac, Eb, EmaX2, Emi, En, Ep, A (P) L Et Bo Eac, EmaX2, Emi, Ep, Et A (P)

W/F/Oc/ 1– H 14 d F/Oc 1– 14 d F/Oc/H 1– 14 d H/W/Or 1– 14 d H/W/Or 1– 14 d H/W/Or 1– 14 d H/W/F 1–9 d

Aq.

H/W/F

1–3 d –

36 week

ParacoxÒ-8

Bd/L

Eac, EmaX2, Emi, En, Ep, Et A (P)

Aq.

W/F

1–9 d –

36 week

Livacox -D

C

Eac, Et

A (P, Ea)

Aq.

W

–

NAe

LivacoxÒ-T

Bo

Eac, Ema, Et

A (P, Ea)

Aq.

H/W/F

–

NAe

LivacoxÒ-Q

Bd/L

Eac, Ema, En, Et

A (P, Ea)

Aq.

H/W/F

–

NAe

Supercox

Bo

Eac, Ema, Et(A(P))

WT, A (P)

Aq.

W

1– 10 d 1– 10 d 1– 10 d 1d

–

NAe

Ò

Ò

Eac, Eh, En, Et Eac, Ema, En, Et Eac, Ema, En, Et Eac, Eh, Ema, En, Et Eac, Eh, Et Eac, Ema, En, Et Eac, Eb, Eh, Ema, En, Et Eac, Eb, Eh, Ema, En, Ep, Et Eac, Eb, Eh, Ema, Emi, En, Ep, Et Ead, Emel, Eg, Ed

F

1d 3d 3d – – – – 10 d 10 d

Ò

Nobilis -Cox ATM

Bo

Eac, EmaX2, Et

WT/T

Aq.

H/W/F

1–5 d Ionophores

NA

AdventÒ

Bo

Eac, Ema, Et

WT

Aq.

H/W/F

1d

Sulfa drugs

NAe

HatchpakÒCocci III Vac MÒ

Bo

Eac, Ema, Et Ema (ionopore resistant)

WT WT/T

Aq. Aq.

H/W F/W

1d 1d

NAe Ionophores

NAe NAe

ViracoxÒ500 EimerivaxÒ 4 m

Bo Bd / L

Eac, Ema, Ep, Et Eac, Ema, En, Et

WT A (P)

Aq. Aq.

H/W/F Oc

1–5 d NAe 1d –

NAe 8 weeks

EimerivaxÒ 3 m

Bo

Eac, Ema, Et

A (P)

Aq.

Oc

1d

8 Weeks

Immuner Gel-CocÒ

Bo/Bd/ Eac, Eb, Ema, Et L Bo Eac, Ema, Emi, Ep, Et

A (P)

Gel

Or

1–5 d –

A (P)

Aq.

W/Or

1d

–

NA

InovocoxÒ/TM

Bo

Eac, EmaX2, Et

WT

Aq.

In ovo

–

–

NAe

InovocoxÒ-EM1

Bo

Eac, Ema, Et

WT

Aq.

In ovo

–

–

NAe

EimerivacÒ Plus

Bd/L

Eac, Ema, Emi, En, Et

A (P)

Aq.

W/F

5– 25 d 5– 25 d

–

NAe

–

e

Hipracox

Ò

Ò

Eimerivac Plus

Bo

Eac, Ema, Emi, Et

A (P)

Aq.

W/F

–

e

NAe e

NA

1952–1954 Dorn & Mitchell (USA) 1954–1959 Gland-O-Lac (USA) 1955–? Nobilis (Netherland) 1955–? Nobilis (Netherland) 1959–? Dorn & Mitchell 1959–1966 (USA) 1960–1984 1960–1984 1964–? 1964–1984 1984– present 1985– present 1989– present 1985– present 1985– present 1992– present 1989– present 1989– present 1989– present 1992–? 1992– present 1992– present 1996– present 2001– present 2002– present ?–present 1989– present 2001–? 2003– present 2003– present 2005– present 2006– present 2006– present 2006– present 2013– present 2013– present

MSD (UK)

Vetech (Canada)

MSD (UK)

Biopharm (Czeck)

Qilu Pharm. (China) Intervet/MSD (UK) Novus Int. (USA) Merial/Sanofi (USA) Elanco (USA) Stallen’s (Swiztz.) Bioproperties (Australia)

Immuner (Argentina) Hipra (Spain) Zoetis/Pfizer (USA)

Guandong Acad. (China)

NB. Coccidiosis usually attach 21–28 days old chicks. The vaccines are always applied for healthy chicks or embryonated-eggs (18–19 days old embryo). Aqueous preparation of the vaccines are formulated with xanthan gum to ensure oocyst homogenous suspension. The immunity is conferred 10–14 days post single dose of vaccination. Chicks are safe to consume after 21 days for vaccination with WT, while no safety time is necessary for attenuated vaccines. Chicks vaccinated with attenuated vaccines are favored to be left on the litter to recycle the oocysts. The protection was conferred to chickens along the whole production cycle, unless otherwise specified. a Target animals: Bd = breeders; Bo = broilers; C = caged chickens; L = layers; R = roasters; T = turkey. b Eimeria species: Eac = E. acervulina; Ead = E. adenoides; Eb = E. brunetti; Ed = E. dispersa; Eg = E. gallopavonis; Eh = E. hagani; Ema = E. maxima (Ema X2 = two different serotypes); Emel = E. meleagrimitis; Emi = E. mitis; En = E. necatrix; Ep = E. praecox; Et = E. tenella. c Oocyte treatment: A = attenuated; Ea = egg adapted; P = attenuated by selection for precocity; WT = wild type virulent living oocyte; WT/T = Live oocyte tolerant to ionophores. d Route of administration: F = feed spray; H = colored-spray cabinet on hatchery; Mf = moist feed; Oc = ocular drops; Or = oral; W = water suspension. e NA = no available data, but it usually refers that the protection is not necessary along the entire broiler cycle. * Sources of data: ([63,2]; manufacturers’ technical bulletins and internet websites).

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T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47 Table 4 Overview of the different antigens used to prepare the subunit and DNA vaccines generations. Stage

Location

Eimeria spp.

Antigen name

Type of vaccine, route, adjuvant & %ODR (ACI) Protein

Recombinant

DNA

SZ, MZ

SA Subcellular enzyme

Et Eac

Mzp5-7 LDH

– Sc/Quil A/65

Eac Et

IM/DC/90 –

MIC

Et

Profilin (3-1E) EtMIC1 (Etp100 & TFP100) EtMIC2

– IM/IFNc, IL2/57 (169) IM –

[81] [18,82,83]

SA TRAP/CSP

Sc, IM/Vaccinia Virus Sc/Amphigen/90 OR IM/IB/53 (159) Sc/CFA Sc/IFA/61

IM/70

TRAP Subcellular structural protein Soluble MIC – SA

Ema Eac

MIC4 (TFP250)

a-Tubulin

– –

In ovo/CpG/60-70 OR IM/CFA/85 (180) IM/CFA,IFA Sc/CFA/36

– –

[88–90] [91] [87] [92]

Et Eac Et

EtMIC3 cSZ-JN1 IMP-1

Sc/TG-IFA/54 – –

– Sc/CFA/74.34(161) Sc/FliC, IFA, & TLR-5/88

IM/48.5 IM/77.4(169) IM/59.7

[93] [94] [95,96]

SZ

RB GPI-linked SAG SA SA Rhomboid proteases

Et Et Et Et Et

SO7 (RB1 and GX3262) EtSAG1 (TA4) EtCDPK (pEtk2) AMA-1 ETRHO1

– Sc/CFA – – –

IM Oral in S. typhimurium OR Sc/CFA – TG-IFA/Sc IM/CFA,IFA/77.3

IM/76 (160) IM/IL2/75(192) IM/IL2/70-80 IM/16 –

[97–100] [101–105] [106] [95] [79]

MZ

SA-EAMZp35 LDH SA

Eac Eac Eac

p250 EaSC2 Eam45 and Eam20

IM/CFA OR oral in E. coli/82 – –

– – –

[107,108] [18] [18]

Sc/CFA

Refs.

[84,85] [86,87]

Subcellular enzyme

Et

GAPDH

– Sc/Quil A/64 Sc/Quil A/ 65,64 –

Sc/CFA

–

[109]

GAM

WFp WFp

Ema Ema

GAM56 GAM82

– –

IM/CFA IM/CFA

IM/53.7 –

[110,111] [110]

MZ/ GAM

Cathepsein-L-like protease

Et

EtCalL

–

Sc/CFA/86

–

[112]

Key: ACI: anti-coccidial index; AMA-1: apical membrane antigen 1; CDPK: calmodulin-domain kinase; CFA: Complete Freund’s Adjuvant; CpG: short oligonucleotide containing CpG; DC: dendritic cells and heir exposomes; Eac: E. acervulina; EFliC: truncated flagellin; Ema: E. maxima; Et: E. tenella; ETRHO1: rhomboid-like protein; IMP: immune mapped protein; GAM: gametocyte; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPI-linked SAGs: glycosylphosphatidylinositol (GPI)-linked surface antigens (SAGs); IB: inclusion bodies; IFA: Incomplete Freund’s Adjuvant; IM: intra muscular; LDH: lactate dehydrogenase; MIC: microneme (protein); MZ: merozoite; Mzp: Merozoite protein; NA: not available; ODR: Oocyst Decrease Rate; RB: Refractile Body; SA: surface antigen; Sc: subcutaneous; SZ: sporozoite; TG: Titermax Gold; TLR: toll-like receptor; TRAP/CSP: thrombospondin-related anonymous protein (TRAP)/circum-sporozoite protein (CSP); WFp: wall forming protein; –: not determined. Gene bank accesses for the different antigens are provided in their references.

6.3. Live-tolerant to ionophores vaccines This strategy depends on co-administering anticoccidial drugs while the animals are vaccinated by live vaccine strain that tolerate that drug [73]. Ionophores are the most widely used anticoccidial drugs for that purpose. They disrupt the trans-membrane ion concentration gradients required for the proper functioning and survival of the parasite. Ionophores act against the susceptible field parasites prior to the evolvement of the vaccine action, they also protect against coccidiosis due to species not present in the vaccine, and they control necrotic enteritis due to C. perfringens [3,77]. In 1985, Edgar and Fitz-Coy [27] successfully immunized broilers with CocciVac-DÒ during in-feed medication with monensin ionophore antibiotic. Similarly, NobilisÒ COX ATM and Vac MÒ vaccines (Table 3) depend on other ionophores to enhance the immunity [78]. Unfortunately, the risk of oocysts reverting to a more pathogenic state, or the variable susceptibility of the host’s genetic back ground or the resistance toward ionophores chemotherapeutics were safety measures that directed recent researches toward the different generations of the subunit vaccines to overcome the risk of the live ones [79]. 6.4. Generations of subunit vaccines These are anticoccidial vaccines composed of distinct protective antigens, such as micronemes, rhoptries, refractile bodies, merozoites, or gametocytes (Fig. 1B) of Eimeria parasite [1]. They are either native or recombinant (vectored) subunit second generation (2nd GN) extracts or DNA (3rd GN) vaccines. CoxAbicÒ was the first

commercial subunit vaccine against coccidiosis, which contained a purified native protein isolated from the gametocytes of E. maxima (Affinity Purified Gametocyte Antigen; APGA). The vaccine blocks the transmission of the parasites through inhibiting the development of oocysts. However the difficulties associated with antigen production and the fact that the vaccine only provides an unreliable protection (53%) against challenge with Eimeria infections, limited the advancement of CoxAbicÒ vaccine [59,80]. Likewise, protein antigens of the different asexual stages (Fig. 1) showed to be more promising as subunit vaccines [113]. These proteins are localized either on the surface of sporozoite or merozoite stages, or they are secreted by the rhoptries and micronemes during the process of invasion (Fig. 1). Among those antigens, dense granule (DG) proteins are thought to be the key players during the invasion process. DG antigens are believed to be promising targets for vaccination since; the contents of DG are deposited in the parasitophorous vacuole membrane. This means that they are present at the actual interface between the parasite and the host’s proteins in the context of major histocompatibility complex molecules [18,114]. Table 4 summarizes the various antigens that were tested as subunit recombinant and DNA vaccine candidates. Simultaneously, it clarifies the formulation of each preparation, its route of administration, and potency. It has been reported that the delivery of recombinant Eimeria antigens using live vectors such as Salmonella typhimurium, Fowlpox virus (FPV), or Herpes virus of turkeys (HVT) expressing Eimeria DNA sequences is a considerable approach to protect against coccidiosis [1]. In 2012, a novel approach using exosome derived from dendritic cells loaded with Eimeria antigen was tested for vaccination. However, partial

44

T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47

Table 5 Anti-coccidial index of the most promising homologous multi and monovalent T-cell epitopes of Eimeria applied in pVax DNA vaccine preparations [116]. Vaccine preparation

Target stage

Epitope fragment

Eimeria spp.

ACI

Quadrivalent/adjuvant Quadrivalent Trivalent Quadrivalent/adjuvant Divalent Quadrivalent Monovalent Trivalent Quadrivalent/adjuvant Quadrivalent/adjuvant Quadrivalent Monovalent Divalent Monovalent Monovalent Quadrivalent Trivalent Monovalent Monovalent Monovalent Trivalent Monovalent Divalent Monovalent Monovalent Divalent Monovalent Monovalent Monovalent Monovalent

SZ/SZ/MZ SZ/SZ/MZ SZ/SZ/MZ SZ/SZ/MZ SZ/SZ SZ/SZ/MZ SZ SZ/SZ/MZ SZ/SZ/MZ SZ/SZ/MZ SZ/SZ/MZ SZ SZ/SZ SZ SZ SZ/SZ/MZ SZ/SZ/MZ MZ SZ SZ/MZ SZ/SZ/MZ SZ/MZ SZ/SZ SZ SZ SZ/SZ SZ/MZ SZ/MZ MZ SZ/MZ

NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 NA4-1-TA4-1-LDH-2-EMCDPK-1 NA4-1-TA4-1-LDH-2 NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 NA4-1-TA4-1 NA4-1-TA4-1-LDH-2-EMCDPK-1 NA4-1 NA4-1-TA4-1-LDH-2 NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 NA4-1-TA4-1-LDH-2-EMCDPK-1 EMCDPK-1 NA4-1-TA4-1 NA4-2 TA4-1 NA4-1-TA4-1-LDH-2-EMCDPK-1 NA4-1-TA4-1-LDH-2 LDH-2 pEtK2-1 MIC4-2 NA4-1-TA4-1-LDH-2 MIC4-1 NA4-1-TA4-1 EMCDPK-1 pEtK2-2 NA4-1-TA4-1 3-1E-2 5401-1 LDH-1 3-1E-1

En En En Et En Et En Et Eac Ema Ema Ema Et En Et Eac Eac Eac Et Et Ema Et Eac Ema Et Ema Eac Et Eac Eac

180.96 179.17 176.92 175.91 175.11 174.91 174.77 172.15 170.4 170.18 169.23 167.18 165.91 165.58 165.48 164.34 164.2 163.72 163.52 162.83 160.72 158.34 156.7 155.72 155.28 154.07 151.8 151 145.79 141.32

Key: 5401: Gene 5401 encoding for a part in EtMIC4; ACI: anti-coccidial index; Eac: E. acervulina; EMCDPK: E. maxima calmodulin-domain protein kinase; Ema: E. maxima; Et: E. tenella; IL-2: interleukin-2; LDH: lactate dehydrogenase; MIC: microneme protein; MZ: merozoite; NA: sporulated oocyst antigen; pEtK2:; pVax: eukaryotic expression vector; SZ: sporozoite; TA4: TA4 surface antigen. Gene bank codes are provided in the corresponding reference.

protection against coccidiosis was achieved by this type of vaccines [115]. Table 4 proposed the importance to use many antigens such as IMP-1, SO7, ETROH1, p250, EaSC2, and Eam. It highlighted the special potency of LDH, profiling (3-1E), EtMIC2, MIC4, cSZ-JN1, EtSAG1 (TA4), EtCDPK (pEtK2), and the EtCalL. Epitopes mapping of T-cell mediated antigenic determinants was adopted by Nanjing Agricultural University. In 2015 Song et al. [116], applied the in silico (DNAStar software) and binding techniques to investigate several promising epitopes from the sporozoite and merozoite stages of Eimeria [83,103], and as concluded from previous research (Table 4). The efficacy of the epitopes in monovalent and multivalent cloned preparation in eukaryotic expression vector (pVax) was estimated (Table 5). The Chinese trials confirmed the remarkable superiority of the sporozoite’s and merozoite’s antigens over the gametocytes’ to induce an anticoccidial effect against the pathogen. The research revealed that the quadri-valent adjuvanted DNA vaccine could offer an excellent protection of more than ACI-170 for all tested strains [116]. The multivalent epitope DNA adjuvant-supplied vaccine harbored in one plasmid was able to confer a protection against the major emerging Eimeria species.

7. Comment Eimeria spp. are the major etiologic agents for coccidiosis in livestock. The disease causes economic loss in farm animals due to the drop in milk, egg, and meat production, weakness, and the increment of the chance to induce mortality. The dissemination of the parasite’s oocysts is feasible, especially in highly productive crowded farms and readily introduces outbreaks. The routine infection-control includes the introduction of oocysts-free feed to the animals, the biosecurity precautions to access the farms,

continuous disinfection by potent anti-oocysts preparations, and the introduction of prophylactic anticoccidial drugs in the animals feed and drinking water. Despite these strict procedures, coccidiosis still affects livestock. Moreover, the concerns regarding drug residues in animal products increased consumer and governmental demands to prohibit their use and limited it to control active infections only [34]. Although anticoccidial chemotherapeutics showed to be potent for some time, Eimeria spp. developed resistances. This may render the infection uncontrollable and causes remarkable economic losses, paving the way toward applying new control methods [2,3]. Several studies investigated the factors that may reduce the severity of coccidiosis, but none was practical. Simultaneously, alternative anticoccidial strategies were evaluated and demonstrated to be either insufficient or not promising to be used at a large scale. Until now, all alternatives to prevent coccidiosis infections were not applicable or outstanding to control outbreaks. Therefore, since initial infections were able to induce extended homologous protective immunity, the idea of vaccination against coccidiosis arose to offer the last promising alternative to win the battle against that pathogen. Trials for passive immunization confirmed that antisera raised against purified antigens only confer partial homologous protections, while full homologous protection is secured by antisera raised against the whole stages of the parasite. Additionally they proved that the antisera raised against gametocytes induce heterologous maternal immunity. Although these studies highlighted the unique advantages of the gametocytes proteins, anti-gametocyte preparations only prevent infected animals from producing infective oocysts. Therefore, they play a role in limiting the dissemination of the parasite, but neither treat the infected animal, nor protect it from further infection. Moreover, the antiserum preparations are still expensive to be used in the veterinary field.

T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 38–47

Unlike bacteria, the life cycle of protozoa undergoes several developmental stages (Fig. 1A). Therefore, the type of vaccines produced against those pathogens does not only depend on their method of preparation, but also on the stage and on the antigen used to produce that vaccine. The fact that the killed Eimeria spp. vaccines do not provide protection confirmed that oocysts do not contain powerful antigenic determinants for the immune system. However, the observation that the infected animals poses a homologous protection that lasts for several months, directed the majority of anticoccidial vaccines toward the live ones. Although, the first vaccine toward Eimeria spp. was composed of live virulent oocyst preparation, being homologous obliged the vaccinologists to formulate tri- to heptavalent preparations to cover the most common strains (Table 2). Although the vaccine showed to be potent and cheap, the idea of being unable to protect against the non-included strains, and the emergence of asynchronous outbreaks was always considered. Since the vaccines were administered orally in sub-lethal doses, worries were also attributed to use it for immuno-compromised animals or those with increased genetic susceptibility. The use of attenuated live vaccines, showed to be the solution for that disquiet and many commercial products are already in the market for that type. However, the attenuation of the parasite by heat, X-irradiation or embryo-adapted lines was unsatisfying on the level of safety and immunogenicity, and therefore paved the way to the attenuation by precocious protocols. However, the last one failed to provide a sufficient protection against heavy challenges, unless formulated with expensive cytokines to improve its immunogenicity. Moreover, the preparations were unable to protect from active infections and were not safe enough to use. Therefore, live vaccines formulated with potent tolerable anticoccidial drugs, evolved to produce safer therapeutic vaccines. Those vaccines profited from the privilege of controlling the coccidia species not included in the preparation and the anaerobic bacterial infections, that deteriorate the cases of infection. However, the short-time effect and the toxicity of the chemotherapeutics were concerned as limitations. Although several commercial brands of anticoccidial live vaccines were produced, concerns about their safety in respect to the host immune state and the parasite were always considered. In the mid-90’s, the idea of preparing native second generation subunit vaccines from APGA protein of gametocytes was adopted by a commercial company in Israel. The aim was to produce a fully safe maternal heterologous vaccine against E. maxima. Although the product was commercialized, difficulties for preparing the antigen increased its price. However in 2004, the efficiency of a DNA vaccine based on the wall forming protein (WFp) antigen of the gametocyte was proved. Despite that GAM-based vaccines prevent oocysts’ formation and dissemination; they also have no effect on protecting the vaccinated chicks, as has been previously noted. Moreover, the encapsulated stages showed to be non-immunogenic. For those reasons the scope was switched toward the use of the merozoites and sporozoites surface or secreted proteins as vaccine candidates. Those proteins were produced either by direct extraction, loaded in delivery vectors, cloned to produce recombinants, or produced by DNA vaccines in their hosts. The majority of those effective protein antigens were shared by the merozoites and the sporozoites stages, while fewer were restricted to one of them. Simultaneously, the fact that sporozoites presence in the life cycle of the pathogen is very short for less than 20 min sometimes, rendered it an elusive target for vaccines and paved the way to the merozoites-based vaccines. However until recently, none of the investigated or produced vaccines was enough to confer a complete safe protection against the pathogen. Unfortunately, only recent studies started to map the epitopes of some stages of Eimeria spp. [117,118] to detect the most obvious antigenic

45

determinants to the immune system and use them as vaccine and diagnostic candidates [119]. This was clearly followed by researchers at the Nanjing Agricultural University in China [94]. They applied in silico and binding epitope mapping techniques to reveal the most potent heterologous antigenic determinants of Eimeria [116]. This fact renders more investigation of the most potent epitope of the Eimeria different stages a target of merit (Table 4), such as the EtMIC2, cSZ-JN-1, and ETCalL antigens. Those candidates may be administered in a complex, by a novel immunization regimen. In ovo administration seems to be a promising route of immunization to ensure the even distribution of the vaccine in labor-less method, and ensures a very early protection for the chicks. This complex should be fully safe, independent on toxic chemotherapeutics, it should provide a therapeutic effect, reduce oocyst formation, with a wide spectrum heterologous, maternal, cheap in production, and should be potent against heavy infections and outbreaks. The candidates are believed to be used in the form of DNA vaccines composed of merozoites’ proteins as being immunologically active immunogens, together with the gametocytes’ proteins that play a main role in reducing the production and dissemination of oocysts. Obviously till now all the control products were unable to confer full protection from heavy infection or/and outbreaks. They all lacked a corner that rendered them ineffective to a certain extent. This is confirmed by the continuous use of prophylactic anticoccidial drugs till now, regardless of their toxic effect on human consumers. Therefore, the control of coccidiosis in farm animals is truly an open topic for futuristic investigations. Ideas are open from the introduction of new control regimens, to researches on producing genetically resistant hosts. Furthermore, obviously the agriculture and animal husbandry is a cultural practice of a specific group of people in a particular region. Therefore, the experience coming from the trial and error is affected by the environmental conditions of that area. Since then, it shall be overlooked case by case. Our observation for the practice of the Egyptian farmers showed that in case of severe diarrhea for farm animals, that may be due to coccidia, farmers isolate the animals and restrict their food to only dry straw while they orally introduce garlic sap to the infected animals. Farmers also noted that lactating animals never attain infection at any age, regardless the mothers previously attained infection or not. This may highlight the biochemical composition of the mammalian’s milk and the physiological conditions it induces. Therefore, further scientific evaluation on those safe environment-attributed cultural practices should be performed to integrate them to the biosecurity, disinfection, and vaccination strategies. Acknowledgement The authors would like to thank Miss. Aya-Allah Saleh, Mr. Nader Zakhary and Mr. Karim Hisham volunteers at the Bibliotheca Alexandrina for their support and efforts to edit this review. References [1] M.C. Jenkins, Advances and prospects for subunit vaccines against protozoa of veterinary importance, Vet. Parasitol. 101 (2001) 291–310. [2] M.W. Shirley, A.L. Smith, F.M. Tomley, The biology of avian Eimeria with an emphasis on their control by vaccination, Adv. Parasitol. 60 (2005) 285–330. [3] H. Peek, Resistance to anticoccidial drugs: alternative strategies to control coccidiosis in broilers. PhD Diss., Univ. Utrecht, Utrecht, the Netherlands, 2010. [4] C. Yun, H. Lillehoj, E. Lillehoj, Intestinal immune responses to coccidiosis, Dev. Comp. Immunol. 24 (2000) 303–324. [5] A. Tewari, B. Maharana, Control of poultry coccidiosis: changing trends, J. Parasit. Dis. 35 (2011) 10–17. [6] M. Pakandl, Coccidia of rabbit: a review, Folia Parasitol. 56 (2009) 153–166. [7] C. Chartier, C. Paraud, Coccidiosis due to Eimeria in sheep and goats, a review, Small Ruminant Res. 103 (2012) 84–92.

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