Impact and diagnosis of poultry coccidiosis

Mild or sub-clinical Eimeria infections are often difficult to diagnose as infected birds do not display any obvious symptoms (Shahraki et al., Reference Shahraki, Shariati-Sharifi, Nabavi and Jamshidian2018). Clinical Eimeria infections take a great toll on infected birds, resulting in a sudden drop in feed intake and a reduction in essential vitamin and mineral absorption. Symptoms include bloody diarrhoea, fatigue, dehydration and eventually death (Eke et al., Reference Eke, Ibeh, Omalu, Otuu, Hassan and Ubanwa2016). Natural coccidial infections in chickens are caused by a single Eimeria species or a combination of different Eimeria species (Guven et al., Reference Guven, Beckstead, Kar, Vatansever and Karaer2013; Gadelhaq et al., Reference Gadelhaq, Arafa and Aboelhadid2015; Giles et al., Reference Giles, van Limbergen, Sakkas, Belkhiri, Maes, Kyriazakis and Foster2019). The severity or level of pathogenicity observed in an infection depends on the species of Eimeria causing the infection (Brown-Jordan et al., Reference Brown-Jordan, Blake, Beard, Beharry, Serrette, Soleyn and Oura2018). E. mitis and E. praecox (low pathogenicity) and E. acervulina and E. maxima (moderate pathogenicity) induce a mild coccidial infection, whereas E. brunetti, E. necatrix and E. tenella (high pathogenicity) bring about internal haemorrhaging, predisposing the birds to necrotic enteritis and, in rare instances, lead to death (Barbour et al., Reference Barbour, Bragg, Karrouf, Iyer, Azhar, Harakeh and Kumosani2015; Huang et al., Reference Huang, Tang, Bi, Hao, Han, Suo and Yu2018; Macdonald et al., Reference Macdonald, Van Diemen, Martineau, Stevens, Tomley, Stabler and Blake2019).

Understanding the pathogenicity, genetic diversity, prevalence and global distribution of these parasites is vital to develop robust, effective and long-term control measures (Brown-Jordan et al., Reference Brown-Jordan, Blake, Beard, Beharry, Serrette, Soleyn and Oura2018). Morphological (oocyst shape and size) and pathological (lesion site) analyses were used to successfully diagnose coccidial infections. However, the analysis only partially identified each Eimeria species (Arafat and Abbas, Reference Arafat and Abbas2018; Yunus et al., Reference Yunus, Suprihati and Wijaya2019). These detection methods require trained experts, and are labour-intensive and time-consuming (Ogedengbe et al., Reference Ogedengbe, Hanner and Barta2011). Molecular, biochemical and immunological techniques have been developed to overcome these limitations (Boulton et al., Reference Boulton, Nolan, Wu, Psifidi, Riggio, Harman and Watson2018). According to Clark et al. (Reference Clark, Tomley and Blake2017), DNA-based approaches, such as assessment of the iso-enzyme patterns of oocysts, rRNA and rDNA probes and species-specific PCR approaches have been established. DNA diagnostic approaches rely on the comparison of orthologous sequences to identify the species of a genus (Sharma et al., Reference Sharma, Singh, Singh, Joachim, Rath and Blake2018). Tang et al. (Reference Tang, Huang, Liu, El-Ashram, Tao, Lu and Suo2018a) postulated that Eimeria species have been effectively and accurately identified using the 18S rDNA region or ITS-1 and ITS-2 sequences. Recent studies have reported the identification of three new genetic variants of Eimeria, OTUs, which further complicate the identification, diagnosis and treatment of coccidiosis (Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016).

Eimeria operational taxonomic units (OTUs) in chickens

Advances in DNA sequencing technology enabled researchers to unveil and study microorganism communities at far greater depths in the past decade (Huang et al., Reference Huang, Tang, Bi, Hao, Han, Suo and Yu2018). These studies involve the amplification and sequencing of gene markers, such as the internal transcribed spacer regions (ITS-1/2) and 16S small ribosomal subunit RNA (rRNA). Amplified gene markers are then aligned to known sequences in genetic databases for identification (Hinsu et al., Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018; Stenzel et al., Reference Stenzel, Dziewulska, Michalczyk, Ławreszuk and Koncicki2018). To date, a large number of novel organisms has been discovered, including novel genetic variants of Eimeria, OTUx, OTUy and OTUz (Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016). The term ‘operational taxonomic unit’ was first coined by Sneath and Sokal (Reference Sneath and Sokal1962). OTUs were initially regarded as a group of individuals clustered together by similarity and equivalence to but not necessarily in line with evolutionary taxonomy (Callahan et al., Reference Callahan, Mcmurdie and Holmes2017). However, studies describe OTUs as a pragmatic proxy for different taxonomic levels of microbial species in the absence of traditional systems of biological classification (Vermeulen et al., Reference Vermeulen, Lott, Eldridge and Power2016; Huang et al., Reference Huang, Tang, Bi, Hao, Han, Suo and Yu2018). Therefore, OTUs are clusters of unknown or uncultivated organisms that are grouped according to a similarity in DNA sequence to a specific taxonomic gene marker (Callahan et al., Reference Callahan, Mcmurdie and Holmes2017).

Three Eimeria OTUs in chickens, namely OTUx, OTUy and OTUz have been reported to date (Cantacessi et al., Reference Cantacessi, Riddell, Morris, Doran, Woods, Otranto and Gasser2008; Fornace et al., Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013; Godwin and Morgan, Reference Godwin and Morgan2015; Clark et al., Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016). Clark et al. (Reference Clark, Tomley and Blake2017) attributed the emergence of OTUs to be a consequence of geographical isolation, genetic drift and mutations. According to Wang et al. (Reference Wang, Gong, Wang, Li, Ai and Zhang2019), the distribution OTU-infected broiler chickens worldwide disseminated novel OTUs. Eimeria OTU infections cannot be treated or controlled with traditional anticoccidial prophylaxis developed for known Eimeria species (Huang et al., Reference Huang, Tang, Bi, Hao, Han, Suo and Yu2018; Sakkas et al., Reference Sakkas, Oikeh, Blake, Nolan, Bailey, Oxley and Kyriazakis2018). Hence, it is fundamental to access the potential of OTUs as candidates for anticoccidial vaccines by addressing the impact of OTUs on the global poultry industry (Vermeulen et al., Reference Vermeulen, Lott, Eldridge and Power2016).

Impact of Eimeria OTUs in poultry production

The economic impact of Eimeria OTUs is yet to be accurately established, as the presence of the genetic variants were only recently reported (Morgan and Godwin, Reference Morgan and Godwin2017). Hence, in addition to a decrease in production, additional funds are now required for the development of new strategies to identify OTUs, coupled with the development of novel OTU antigen vaccines (Vermeulen et al., Reference Vermeulen, Lott, Eldridge and Power2016; Boulton et al., Reference Boulton, Nolan, Wu, Psifidi, Riggio, Harman and Watson2018). OTUx resulted in a gross margin of $0.1 per broiler bird and $3.4 per layer bird per annum in Tanzania; $0.1 per broiler bird and $−0.6 per layer bird per annum in Zambia; $1.3 per broiler bird and $−0.4 per layer bird per annum in Ghana. OTUz generated a gross margin of $−3.6 per broiler bird and $−2.6 per layer bird per annum in Tanzania; $−1.9 per layer bird per annum in Zambia; and $−0.3 per broiler bird and $−2 per layer bird per annum in Ghana. However, these figures included additional expenses of feed and daily operational costs (Fornace et al., Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013).

Although these genetic variants have only been noted in the southern hemisphere, human movement and trade add to the risk of the spread of OTUs (Fornace et al., Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013; Clark et al., Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016; Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016; Morgan and Godwin, Reference Morgan and Godwin2017). This in turn confers significant consequences for the development and application of novel anticoccidial vaccines, as OTU variants may evade the immune protection conferred by vaccines (Clark et al., Reference Clark, Tomley and Blake2017; Hinsu et al., Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018). Godwin and Morgan (Reference Godwin and Morgan2015) confirmed the lack of or little protection of developed vaccines against these genetic variants. The authors further stated the dearth of information regarding the biology and exact impact of OTUs on poultry production in terms of how each OTU negatively affects the birds. For example, Paracox 8 ( × 10) and HuveGuard MMAT anticoccidial vaccines effectively retarded infections with known Eimeria species. However, the vaccine showed little to no effect on coccidial infections with OTUx or OTUz. Jatau et al. (Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016) reported similar results, whereby Eimeriavax^® 4 M which effectively treats against E. tenella, E. necatrix, E. maxima, and E. acervulina could not prevent colonization by the OTU genetic variants (Chang et al., Reference Chang, Wang, Regev-Yochay, Lipsitch and Hanage2015). This is of growing concern especially in countries where antibiotics have been phased out, resulting in vaccines being utilized as the sole anticoccidial treatment measure.

The impact of OTUs has been undermined due to a lack of mortality reports and indiscrete lesions. However, OTUx was reported to be moderately pathogenic, resulting in malabsorption. On the other hand, OTUy and OTUz were said to be highly and very highly pathogenic, respectively, resulting in haemorrhage (Clark et al., Reference Clark, Tomley and Blake2017). The relationship and distinctiveness between OTUs and known Eimeria species affecting chickens aid in uncovering the genetic variability of the variants (Godwin and Morgan, Reference Godwin and Morgan2015; Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016; Hinsu et al., Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018).

Eimeria OTUS: divergent strains or distinct species?

One thousand eight hundred Eimeria species have been noted in a vast range of hosts, including domestic pets (cats and dogs), poultry (chicken, turkey, duck) and ruminants (cattle, goats and sheep) (Chapman, Reference Chapman2014; Blake, Reference Blake2015; Silva et al., Reference Silva, Chávez-Maya, Macdonald, Pegg, Blake, Taubert and Hermosilla2017). However, only seven pathogenic Eimeria species have been noted in chickens, including E. praecox, E. tenella, E. necatrix, E. mitis, E. maxima, E. brunetti and E. acervulina (Clark et al., Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016). Each of these species differ in prevalence, location of infection, pathogenicity and immunology depending on the magnitude of the infection (Barbour et al., Reference Barbour, Bragg, Karrouf, Iyer, Azhar, Harakeh and Kumosani2015; Huang et al., Reference Huang, Ruan, Li and Zeng2017; Brown-Jordan et al., Reference Brown-Jordan, Blake, Beard, Beharry, Serrette, Soleyn and Oura2018).

The bayesian species delimitation statistical analysis tool using a Markov chain Monte Carlo (reverse jump) method was utilized to access the relationships between OTUs and known Eimeria species (Hinsu et al., Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018). Clark et al. (Reference Clark, Tomley and Blake2017) and Morgan and Godwin (Reference Morgan and Godwin2017) described the similarity between the OTUx sequence and ITS2 sequence of E. maxima, whereby OTUx clustered alongside E. maxima in a phylogenetic Tamrua-Nei model. The authors also detected the close cluster of OTUy and E. brunetti sequences, whereas OTUz sequences formed a distinct clade by itself. Cantacessi et al. (Reference Cantacessi, Riddell, Morris, Doran, Woods, Otranto and Gasser2008) and Fornace et al. (Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013) reported similar results. However, Fornace et al. (Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013) defined OTUz as a genetic descendant of E. necatrix and E. tenella. The similarity detected between OTUz and known Eimeria species in chickens were further complicated when Godwin and Morgan (Reference Godwin and Morgan2015) observed significant similarity between OTUz and E. mitis.

The short distance between OTUx and E. maxima signifies a close relationship, indicating that the genetic variants are sister species. However, the genetic variant differs significantly from E. maxima in oocyst shape and size, pre-patent period and the site of infection. Similar differences were observed between OTUy and E. brunetti (Godwin and Morgan, Reference Godwin and Morgan2015; Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016; Hinsu et al., Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018). Clark et al. (Reference Clark, Tomley and Blake2017) suggested that the great divergence between OTUz and respective Eimeria species indicate that OTUz should be regarded as a novel species. While each of the OTUs was partitioned separately and were confidently taxonomically assigned, an analysis of additional loci with greater stability is required (Hinsu et al., Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018). Characterization and alignment of OTUs to global Eimeria isolates aid in the creation of genetic markers used to identify the genetic variants (Morgan and Godwin, Reference Morgan and Godwin2017). However, to determine whether OTUs have recently diverged from closely related Eimeria species the prevalence, characterization and geographical distribution of these OTUs need to be accessed (de Macedo et al., Reference de Macedo, Santos, da Silva, do Rêgo Barros, Alves, Giannelli, Ramos and de Carvalho2019).

Prevalence and geographical distribution of Eimeria OTUs

The occurrence and prevalence of Eimeria species depend on both intrinsic (host age and class) and extrinsic (farm and flock size; seasonal variation and environmental factors, coupled with management strategies) factors (Kadykalo et al., Reference Kadykalo, Roberts, Thompson, Wilson, Lang and Espeisse2017). Godwin and Morgan (Reference Godwin and Morgan2015) reported OTUy to be the most widespread and abundant genetic variant in Australia with more than 45% of flocks infected, whereas OTUz was the least common (8%) and OTUx (13%) was only moderately observed. The authors further illustrated the prevalence of OTUs to be equivalent to the prevalence of common Eimeria species; E. maxima, E. tenella and E. brunetti in commercial flocks globally. Hinsu et al. (Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018) reported similar results in India with OTUy being the most dominant genetic variant.

OTUx and OTUy were initially discovered in Victoria, Australia, in two vaccinated flocks of broiler chickens (Morris et al., Reference Morris, Woods, Richards and Gasser2007). Hinsu et al. (Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018) confirmed these results and concluded that OTU variants were more common in the Northern region of Australia with 188 OTUs identified out of 592 samples. However, all three OTUs (OTUx; OTUy and OTUz) were observed at an average occurrence of 33% in both commercial and backyard farms throughout Australia (Cantacessi et al., Reference Cantacessi, Riddell, Morris, Doran, Woods, Otranto and Gasser2008; Godwin and Morgan, Reference Godwin and Morgan2015; Morgan and Godwin, Reference Morgan and Godwin2017). North Western Nigeria had a similar prevalence, with all three OTUs detected (Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016). The presence of only two variants, OTUx and OTUz, were noted in commercial farms throughout Zambia, Tanzanian and Ghana (Fornace et al., Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013). Clark et al. (Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016) observed similar results in Venezuela, India, Zambia, Uganda, Tanzanian and Ghana. The authors further reported a dispersion of the two cryptic variants in quantities comparable to E. necatrix and E. maxima throughout South America, Sub-Saharan Africa and Southern Asia

Hinsu et al. (Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018) reported similar results, detecting low levels of OTUy in India using Next Generation Sequencing (NGS) technology, thus providing the most northern occurrence of OTUs reported to date. Tang et al. (Reference Tang, Suo, Li, Du, Wang, Hu, Duan, Lyu, Liu and Suo2018b) recently observed additional, unidentifiable OTUs in chickens immunized with transgenic Eimeria in China. The occurrence of OTUs is only observed in the Southern Hemisphere (30^°N latitude) (Fig. 1) and is yet to be discovered in South Africa, Egypt, USA and Europe (Clark et al., Reference Clark, Tomley and Blake2017). The spread of OTUs north of the 30^oN line of latitude is of growing concern. Hence, identifying the factors driving the global distribution of OTUs is fundamental for coccidial control.

Fig. 1. The geographical distribution of reported Eimeria genetic variants, OTUx OYUy and OTUz throughout the world (Fornace et al., Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013; Clark et al., Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016; Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016; Morgan and Godwin, Reference Morgan and Godwin2017).

Factors influencing the polarized distribution of Eimeria OTUs

The elucidation of factors driving the polarized distribution of OTUs globally was initially brought into question by Clark et al. (Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016). The authors suggest the polarized distribution of OTUs may result from the discovery of novel trade routes for the distribution of goods, including commercial chicken broilers. Commercial chicken genotypes have recently been introduced to much of the southern hemisphere. These genotypes interbreed with indigenous chicken breeds, introducing OTU genotypes into chicken populations (Suganthi, Reference Suganthi2014; Morgan and Godwin, Reference Morgan and Godwin2017). However, the lack of persistence and nature of infection within a host make transmission by this means highly unlikely (Jatau et al., Reference Jatau, Lawal, Kwaga, Tomley, Blake and Nok2016). Additionally, commercial poultry breeds were historically dispersed north to south, as determined by ancient mitochondrial DNA signatures (Storey et al., Reference Storey, Athens, Bryant, Carson, Emery, DeFrance, Higham, Huynen, Intoh, Jones and Kirch2012). OTUs are also said to migrate via transmission machinery; equipment and farm workers; genetic drift via geographical isolation and switching hosts from other galliforms with a regional distribution (Clark et al., Reference Clark, Macdonald, Thenmozhi, Kundu, Garg, Kumar and Nolan2016). Novel trade routes increase the likelihood of the parasite population mixing (Ogedengbe et al., Reference Ogedengbe, Hanner and Barta2011).

Conversely, Hinsu et al. (Reference Hinsu, Thakkar, Koringa, Vrba, Jakhesara, Psifidi and Joshi2018) reported a significant similarity in OTUy genotypes detected in indigenous and commercial chicken breeds in India. This indicated that the polarized global distribution of OTUs is highly unlikely to be a consequence of host genotype specificity. Clark et al. (Reference Clark, Tomley and Blake2017) and Prakashbabu et al. (Reference Prakashbabu, Thenmozhi, Limon, Kundu, Kumar, Garg, Clark, Rao, Raj, Raman and Banerjee2017) reported that OTUs are more dependent on geographical location as compared to the seven-known chicken Eimeria species. However, geographical location alone serves as an inadequate explanation for the polarized distribution of OTUs. The origin of OTUs is yet to be discovered due to a lack of historical samples. Therefore, additional studies should be carried out to determine the geographical origin of OTUs (Fornace et al., Reference Fornace, Clark, Macdonald, Namangala, Karimuribo, Awuni and Rushton2013; de Macedo et al. Reference de Macedo, Santos, da Silva, do Rêgo Barros, Alves, Giannelli, Ramos and de Carvalho2019). However, OTUs could be distinct species of Eimeria, which diverged a long time ago, together with the seven known Eimeria species as opposed to diverging recently from closely related species. Thus, OTUs may be ubiquitous, but due to limited methods of detection and discrimination, may not be detected. All gene markers used to detect specific Eimeria species, i.e. ITS-1, ITS-2, 18s ribosomal and SCAR markers, are located within a single locus. Genome-wide association studies (GWAS) should be carried out on OTU variants to identify more effective gene markers for identification.