key: cord-0885645-vo01vbxy
authors: Shehata, Awad A.; Basiouni, Shereen; Sting, Reinhard; Akimkin, Valerij; Hoferer, Marc; Hafez, Hafez M.
title: Poult Enteritis and Mortality Syndrome in Turkey Poults: Causes, Diagnosis and Preventive Measures
date: 2021-07-10
journal: Animals (Basel)
DOI: 10.3390/ani11072063
sha: 3f25ceb8e59ed2ed561c0a3c844358b8a27af743
doc_id: 885645
cord_uid: vo01vbxy

SIMPLE SUMMARY: The poult enteritis and mortality syndrome (PEMS) causes severe economic losses in turkeys. Several agents were described to be associated with the PEMS; however, a specific etiological agent(s) has not been identified. The diagnosis of PEMS is still a huge challenge for several reasons: (1) no specific clinical signs or pathognomonic lesions, (2) isolation of some enteric viruses still difficult, (3) the pathogenicity of several enteric viruses in turkeys is not fully understood, (4) PEMS is an interaction between several known and might be unknown agents and (5) opportunistic microorganisms also have a role in the pathogenesis of PEMS. Both electron microscopy and molecular techniques can be used for diagnosis of PEMS and might help to discover unknown causes. Until now, no specific vaccines against enteric viruses associated with PEMS. However, biosecurity, maintaining a healthy gut and strengthening the immune system of turkey poults using probiotics, prebiotics and/or phytogenic substances are crucial factors to prevent and/or reduce losses of PEMS in turkeys. This review is a call for scientists to perform further research to investigate the real cause(s) of PEMS and to develop a preventive strategy against it. ABSTRACT: Poult enteritis and mortality syndrome (PEMS) is one of the most significant problem affecting turkeys and continues to cause severe economic losses worldwide. Although the specific causes of PEMS remains unknown, this syndrome might involve an interaction between several causative agents such as enteropathogenic viruses (coronaviruses, rotavirus, astroviruses and adenoviruses) and bacteria and protozoa. Non-infectious causes such as feed and management are also interconnected factors. However, it is difficult to determine the specific cause of enteric disorders under field conditions. Additionally, similarities of clinical signs and lesions hamper the accurate diagnosis. The purpose of the present review is to discuss in detail the main viral possible causative agents of PEMS and challenges in diagnosis and control.

Several challenges such as intense global competition between producing countries, permanent changes in social, political and consumer perceptions regarding food safety, animal welfare and environmental protection are influencing turkey production and health [1] . 

TCoV is known for about 70 years; it was first isolated in 1951 in the USA by Peterson and Hymas [18] . Later on, the virus was reported in several countries worldwide including Australia, Brazil, Italy, UK, France and Poland [19] [20] [21] [22] [23] [24] . In Europe, TCoV was isolated for the first time in 2008 from turkey poults suffering from enteritis [21] . TCoV infections remain a leading cause of massive economic losses in young turkeys in many countries [25] . The virus belongs to the family Coronaviridae, which is classified into two subfamilies, namely, Letovirinae and Orthocoronavirinae. While the subfamily Letovirinae includes the genus Alphaletovirus, the subfamily Orthocoronaviridae contains four genera based on the phylogenetic analysis and genome structure: Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Gammacoronavirus (γCoV) and Deltacoronavirus (δCoV), [26] . Both γCoV and δCoV infect birds, but some can also infect mammals [27, 28] . The γCoV contains three subgenera, namely, Igacovirus and Brangacovirus, both identified in birds, and Cegacovirus, reported in mammals (beluga whale, SW1 virus) [26] . TCoV belongs to the genus γCoV and subgenus Igacovirus, which contains other avian coronaviruses (ACoVs) such as infectious bronchitis virus (IBV) and guinea fowl coronavirus (GfCoV). The virus is enveloped, containing single-stranded, positive-sense, non-segmented RNA of 28-kb [29, 30] .

Like other ACoVs, the genome of ACoV consists of 15 non-structural proteins, encoded by open reading frame (ORF) 1a/b at the 5'-end, and four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N), encoded by other ORF at the 3'-end [21, [31] [32] [33] . Generally, ACoVs have similar phylogenetic relationships and genomic structures and close nucleotide identities. The IBV, TCoV and GfCoV exhibited nucleotide identities of 90% for the replicase, E, M and N genes [28, 29, 34] . However, the S gene of ACoVs shares at most 36% identity [31, 35] . Three distinct genetic groups of TCoV isolates in USA were identified, namely, in North Carolina isolates formed group I, Texas isolates formed group II, and Minnesota isolates formed group III, suggesting the endemic circulation of distinct TCoV genotypes in different geographic states [36] . Recombination in coronaviruses is common. Wang and others documented a recombination event between a chicken coronavirus and TCoV in China using viral metagenomic analysis [37] . Additionally, an atypical TCoV strain was isolated from duodenum of 5-week-old turkey poults suffering from acute enteritis in Poland. Molecular analysis revealed recombination between different γCoV genomic backbones, suggested potential transmission of coronaviruses between different bird species [32] .

The TCoV is involved in the economically devastating PEMS, a multifactorial syndrome [10] . The natural host of TCoV is turkeys, as this virus did not cause a disease in chickens under experimental conditions [30] . TCoV causes high morbidity rates that may reach 100% and a sudden increase in mortality of 10-50% in turkeys during the first 4 weeks of age. Although TCoV is more common in young poults, exposure of older ages results in stunting with low mortality rates [38] . The natural route of infection of TCoV is orally by ingestion of contaminated fecal materials. It replicates in enterocytes at the apical portion of the intestinal villi in the jejunum and ileum and in the immune organs such as the bursa of Fabricius [39] . The virus was also detected in dendritic cells, monocytes and macrophages, highlighting its potential replication in antigen-presenting cells [40] . Movement of contaminated equipment, personal or vehicles and other birds probably spread the virus. The disease is more common and severe during summer months (May to August) with sporadic occurrence in autumn. Once turkeys are infected with the virus, they remain life-long shedders [11] .

Post-mortem lesions are mainly found in the GIT and bursa of Fabricius. Pale duodenum and jejunum that are distended watery with gaseous contents were reported. In addition, ceca were distended and filled with watery contents. Atrophy of the bursa of Fabricius may also be observed. Microscopic lesions include villus atrophy, infiltration with mononuclear inflammatory cells in the lamina propria and decreased numbers of goblet cells on villous tips. Lymphoid atrophy of follicular cells of bursa of Fabricius and heterophilic infiltration are also reported [10, 40, 41] . Experimentally, TCoV shedding persists until 14-weeks post inoculation [42] . However, inoculation with the TCoV NC95 isolate was shed up to 7-weeks [43] . Additionally, latent infection with TCoV without clinical signs is reported [44] .

The TAstV was first reported in 1980 in turkey poults suffering from diarrhea and increased mortality in UK [45] . Since then, it has been documented worldwide [14, [46] [47] [48] . TAstV infections are common in 4-week-old turkey poults as a coinfection in enteric disease. TAstVs, belonging to the family Astroviridiae, are non-enveloped, single stranded positivesense RNA viruses with a genome size of 6.5-7.5 kb long. It contains three ORFs: ORF1a encodes the non-structural proteins serine-protease, ORFab encodes RNA-dependent RNA-polymerase and ORF2 encodes the structural proteins of the viral capsid [49] . Three astrovirus types, namely, TAstV-1 (7003 nt), TAstV-2 (7325 nt) and avian nephritis virus (ANV, 6927 nt) have been detected in commercial turkey flocks, with a prevalence of 100, 15.4 and 12.5%, respectively. The TAstV-2 has frequently been associated with PEC, PEMS and PES [5, 6, 50, 51] . Additionally, TAstV-2 has been detected in apparently healthy flocks of turkeys [50, 51] . The ANV is associated with nephritis and RSS in chicken and turkey flocks and other avian species [52] .

The TAstVs replicates in the basal portion of the lamina and rarely in the crypts [53, 54] . Oral inoculation of TAstV in one-day-old specific pathogen free (SPF) turkey poults decreased the absorption of D-xylose [55] , resulting in maldigestion of disaccharides, malabsorption and consequent osmotic diarrhea [56] . It was also found that 24 h after experimental infections of one-day-old turkey poults, birds showed signs of intestinal infection including yellowish brown watery to foamy diarrhea, followed by emaciation and stunting growth [57, 58] . Significant reduction of body weights as a result of decreased absorption of nutrients was also found [58] . The main pathological changes are mainly located also in the digestive tract and usually non-specific including dilated ceca with yellowish frothy contents, fluid distention and inflammation of intestines [57] . It was also suggested that TAstV virus might cause immunosuppression, hence the virus was detected in bursa and thymus [59] .

Adenoviruses, family Adenoviridae, are DNA viruses with an icosahedral capsid and a double-stranded, linear genome. Adenoviruses are described in many species of vertebrate animals, including mammals, birds, reptiles, amphibians and fish [60] [61] [62] . Three different genera namely, Aviadenovirus, Siadenovirus and Atadenovirus can infect poultry [63] . The Aviadenovirus comprises the fowl aviadenovirus (FAdV) and the turkey aviadenovirus (TAdV) species [63] . FAdVs are grouped into the five species (FAdV-A to FAdV-E), 12 serotypes (FAdV-1 to FAdV-8a and FAdV-8b to FAdV-11) and 12 genotypes. Three TAdV species, namely TAdV-B (type TAdV-1), TAdV-C (type TAdV-4) and TAdV-D (type TAdV-5) can infect turkeys [64] [65] [66] , isolated from respiratory disease and PEMS. These viruses also cause inclusion body hepatitis in turkey poults and may be responsible for lower hatchability rates in breeder flocks [67, 68] . Generally, further studies are required to understand the pathogenicity of aviadenoviruses in turkeys, hence all aviadenoviruses were identified within diseased turkey flocks in Germany, however, no apparent link between case history and type of isolate were identified [65] .

Hemorrhagic enteritis (HE) has been reported worldwide, i.e., Canada, England, Germany, Australia, India, Japan, Israel and the USA [69] . Surprisingly, the HE virus does not replicate in intestinal epithelium; however, it replicates in the endothelial cells that causes vascular damage and ischemic necrosis of intestinal villi. HE virus causes severe immunosuppression in turkeys, which subsequently stimulate opportunistic bacteria [70] . The HE infection is common in 4-12-week-old turkey poults [69] . The main signs include depression, bloody droppings and sudden death [71] . The main post-mortem lesions are enlarged mottled spleen and distended and congested intestines, more prominent in the proximal small intestine. The intestine might be filled with bloody exudate [69, 71] . Microscopic examination revealed characteristic lesions including hyperplasia of white pulp and lymphoid necrosis in the spleen at death [71] . Moreover, severely congested mucosa, degeneration and sloughing of the villus epithelium and hemorrhage at the villus tips were reported.

Rotaviruses, genus Rotavirus, family Reoviridae, have been associated with enteritis in mammalian and poultry species [72] [73] [74] . Rotaviruses are non-enveloped icosahedral particles and contain double stranded-RNA of 11 segments, which encode structural (VP1 to VP4, VP6 and VP7) and non-structural proteins (NSP1 to NSP6). According to VP6, rotaviruses are classified into 10 groups (RVA-RVJ) [75] . Turkey rotaviruses belong to group A rotaviruses [45] , however, rotaviruses that are antigenically distinct (referred as rotaviruslike particles) from group A turkey rotaviruses were also detected in turkey poults [76] . The pathogenicity of rotaviruses in turkeys depends on several factors including the virulence of involved strains, coinfections with other pathogens and management [77] . The main clinical signs of rotaviruses infections are diarrhea, depression, high mortality rates, chronic runting and stunting.

Avian reoviruses have been associated with enteric disease, arthritis/tenosynovitis, respiratory distress, immunosuppression, poor feed conversion and malabsorption syndrome in poultry [78] . Avian reoviruses belong to family Orthoreoviridae, genus Orthoreovirus. These viruses are non-enveloped and have linear double stranded RNA with 10 segments. Fusogenic reovirus strains are characterized by the ability to fuse with infected cells and form multinucleated syncytia, affecting mammals, birds and reptiles, while non-fusogenic viruses are mainly infecting mammals [79] . Based on the molecular differences between avian reoviruses, species-specific reovirus types are being described, namely turkey reovirus for turkey, duck reovirus for duck, goose reovirus for goose and avian reovirus chickens [80] . Generally, young birds without maternal antibodies can be infected with reoviruses [81] . However, the course of infection depends on the age of birds and their sensitivity, pathogenicity of the reovirus strain, infectious dose, route of infection, presence of maternal antibodies and immune status.

Turkey reoviruses are associated with arthritis and PEMS in turkeys [82] . Turkey arthritis reovirus causes tenosynovitis in turkeys, leading to the reduction of performance due to mortality and low feed conversion ratio. Additionally, although reoviruses have been isolated from turkey poults suffering from enteritis disease, they have been isolated from apparently healthy birds [83] . Experimentally infected SPF poults showed mild clinical signs and exhibited no post-mortem lesions, highlighting that turkey reovirus might not be the primary cause of PEMS [5] . There are conflicts about the replication of reoviruses in the intestine of turkeys [5] . It is suggested that turkey reoviruses cause severe bursa atrophy in poults at a young age that probably lead to a permanent immunosuppression [5] , which in turns cause enteric disease by stimulating the opportunistic microorganisms. Moreover, experimentally infect young poults with turkey reovirus induced subclinical tenosynovitis [84] . Sharafaldin et al. investigated the pathogenesis of turkey reoviruses. The virus was detected in cloacal swabs at 1-2 dpi and peaked at 14 dpi. Additionally, cytokines were elevated in intestines (at 7-14 dpi) and in gastrocnemius tendons (at 14 dpi), suggesting a possible correlation between viral replication and cytokine response in the early infection. Still, there is limited information about the pathogenesis of reovirus in turkeys and development of its diagnosis and control [85] .

The diagnosis of PEMS based on the clinical picture and gross lesions is difficult because there are no specific clinical signs or pathognomonic lesions. Generally, cultivation of some enteric viruses is also a challenge. Therefore, negative contrast electron microscopy (EM), PCR and serology can help in the diagnosis of viruses causing enteric complex in turkeys. The combined use of TEM and PCR in the diagnosis of PEMS is possible to potentiate the advantages of both methods. While the strengths of the TEM lie in the detection of the entire spectrum of virus groups, the PCR can be used for a more sensitive and differentiated diagnosis after narrowing down the spectrum to certain viruses.

The negative contrast EM enables the discovery and morphological assessment of all microorganisms present in a sample and thus offers great advantages compared to many other used methods, Although the visual representation of viruses using EM requires special knowledge regarding sample preparation and evaluation, this method enables the detection of the entire spectrum of viruses in just one preparation approach with reasonable effort. EM can be particularly successful in virus infections with a high presence of pathogens, for example during an acute infection phase [86] . Enteric viral infections stand out through the excretion of large amounts of pathogen in the feces and thus offer good requirements for the visual detection and differentiation of the virus particles based on specific morphological features [87, 88] . In addition, with samples with a lower virus concentration, significant virus enrichment can be achieved by means of ultracentrifugation [89] . In addition to the identification of known viruses that can be recognized in the context of mono or mixed infections, it is also possible to discover new and previously unknown viruses [69, 90] . The negative contrasting electron microscope is an ideal instrument for both individual bird diagnostics and for flock monitoring studies [87, 91] . As a rule, 1-2% solutions of the heavy metal salts of molybdenum, tungsten or uranium are used as contrast media [92] . However, phosphotungstic acid (PTA) can be also used for contrasting because of its low toxicity [87] .

Molecular-based methods are used also in the diagnosis of enteric viruses of turkeys [16, 43, 49, [93] [94] [95] [96] [97] [98] . Sellers and his colleagues developed a multiplex RT-PCR for simultaneous detection of enteric viruses in turkeys. Later on [79] developed a multiplex RT-PCR test for the detection and differentiation of turkey astrovirus-1 (TAstV-1), TAstV-2, ANV, chicken astrovirus (CAstV) and rotavirus in turkey and chicken samples [46] . El-Adawy and others developed and validated a simple, sensitive, specific and cost-effective multiplex PCR (mPCR) assay as a molecular screening approach for the detection of six enteric avian pathogens; Campylobacter spp., Salmonella spp, Clostridium perfringens, Escherichia coli, Histomonas meleagridis and Tetratrichomonas gallinarum for use in the daily practice of a clinical microbiology laboratory. The sensitivity and specificity of multiplex polymerase chain reaction (mPCR) was tested and evaluated. The mPCR is advantageous when compared with conventional detection methods because it allows detecting and distinguishing multiple pathogenic agents through the use of one test. It is cost effective, time saving, specific and sensitive [99] .

The diagnosis of TCoV depends on EM, PCR and serology. EM was used for identification of TCoV in turkey poults suffering from PEMS. TCoV was detected in turkey poults located in Germany using EM. When identifying TCoV particles, it must be considered that fragments of cell membranes often look very similar to these virus particles (Figure 1 ).

This often makes it difficult to clearly assess electron microscopic specimens. Immunoelectron microscopy can also help in these cases. However, a virus-specific antiserum is needed for this method of preparation. The detection of TCoV using PCR is described in several studies. Due to the great genetic homology between the IBV and TCoV [10, 100] , the first attempts to detect TCoV using PCR were based on the genetic analysis of already sequenced IBV strains [43, 46] . A highly conserved non-coding region (3'UTR) at the 3'-end of the RNA strand is particularly suitable for the design of PCR primers [101, 102] . The 3'UTR sequence fragments showed high homologies between the TCoVs strains and IBV [103] . Based on this fact, IBV (e.g., IBV vaccine strain H120) can be used as a positive control for [20, 23] . Additionally, based on the fact that N and M genes are highly conserved, there were attempts to identify TCoV using PCR targeting these genes [43, 46, 103] . The high sensitivity of this diagnostic method makes it possible to detect even very small amounts of virus particles. It was found that TCoV can be detected in cloacal swabs just 24 h after an oral infection of turkey poults [5, 42] . The use of the 3'UTR primers and the N-gene primers had identical results. Despite the fact that no coronavirus genome was found in the of Bursa Fabricii samples, detection was successful in at least 27% of the cloacal swabs. These results highlighting that feces and intestinal samples are the best samples suited for PCR detection of TCoV [22] . This often makes it difficult to clearly assess electron microscopic specimen noelectron microscopy can also help in these cases. However, a virus-specific a is needed for this method of preparation. The detection of TCoV using PCR is d in several studies. Due to the great genetic homology between the IBV and TCoV the first attempts to detect TCoV using PCR were based on the genetic analysis o sequenced IBV strains [43, 46] . A highly conserved non-coding region (3'UTR) end of the RNA strand is particularly suitable for the design of PCR primers [101, 3'UTR sequence fragments showed high homologies between the TCoVs strains [103] . Based on this fact, IBV (e.g., IBV vaccine strain H120) can be used as a posi trol for the identification of TCoV based on 3'UTR-PCR [20, 23] . Additionally, bas fact that N and M genes are highly conserved, there were attempts to identify TC PCR targeting these genes [43, 46, 103] . The high sensitivity of this diagnostic makes it possible to detect even very small amounts of virus particles. It was fo TCoV can be detected in cloacal swabs just 24 hours after an oral infection of turk [5, 42] . The use of the 3`UTR primers and the N-gene primers had identical results the fact that no coronavirus genome was found in the of Bursa Fabricii samples, d was successful in at least 27% of the cloacal swabs. These results highlighting t and intestinal samples are the best samples suited for PCR detection of TCoV [22 The ELISA and immunofluorescent assay (IFA) can be used for the detectio bodies in sera collected from birds at 10-15 days after the onset of clinical signs t the diagnosis of PEMS. Commercially available ELISA plates that are coated antigens could be successfully used for the detection of antibodies to TCoV in a capture ELISA [104] . The recombinant S1 spike polypeptide was also used to d TCoV-specific antibody ELISA [42] . Abdelwahab and others developed a reco ELISA based on the N protein of TCoV expressed in a prokaryotic system for the d of antibody of TCoV. The relative sensitivity and specificity of the recombinan The ELISA and immunofluorescent assay (IFA) can be used for the detection of antibodies in sera collected from birds at 10-15 days after the onset of clinical signs to help in the diagnosis of PEMS. Commercially available ELISA plates that are coated with IBV antigens could be successfully used for the detection of antibodies to TCoV in antibodycapture ELISA [104] . The recombinant S1 spike polypeptide was also used to develop a TCoV-specific antibody ELISA [42] . Abdelwahab and others developed a recombinant ELISA based on the N protein of TCoV expressed in a prokaryotic system for the detection of antibody of TCoV. The relative sensitivity and specificity of the recombinant ELISA compared with IFA were 86% and 96%, respectively [105] .

In contrast, TCoV could not be detected in turkey stocks suffering from PEMS [50, 51] , highlighting the fact that PEMS is a multifactorial syndrome and other potential causative agents should also be investigated.

The diagnosis of astroviruses can be done based on EM and RT-PCR. The TAstV was recognized as five or six-rayed star-shaped particles by negative contrast EM from samples collected from poults suffering from PEMS in Germany (Figure 2 ). However, this morphology only applies to about 10% of all the particles shown, while the rest have a smooth surface [4, 58, 106, 107] . In some cases, it is difficult to distinguish between astrovirus particles and other enteral viruses such as picorna and enteroviruses, so that such particles are often referred to as small round viruses (SRV) [58] . The average size of the particles is 29.6 nm [106] . The RT-PCR can be used also for the diagnosis of TAstV using primers specific to the polymerase or capsid genes [48, 49, 108] . Mixed infections with TAstV and TCoV have been reported in turkeys causing a severe negative impact on intestinal absorptive functions as causative factors of PEMS [55] . Additionally, coinfection of turkeys with TAstV and rotavirus was also reported in the US [65] . TAstV and TCoV have been reported in turkeys causing a severe negative impact on intestinal absorptive functions as causative factors of PEMS [55] . Additionally, coinfection of turkeys with TAstV and rotavirus was also reported in the US [65] . The diagnosis of adenoviruses depends on virus isolation and molecular identification. Virus isolation can be done using cell cultures derived from the homologous species [94] . However, adenoviruses could be successfully derived from turkeys using chicken embryo liver (CEL) cells isolated from SPF chickens. The main cytopathic effects on CEL cells are rounded cell degeneration after the 1st and 4th passages [65] . Adenovirus could be also detected in turkey poults using EM (Figure 3 ). Molecular typing can be done based conventional PCR targeting the L1 region of the hexon gene [65, 109] . Amplification and sequence analysis of the polymerase gene can be used to distinguish between TAdV-B, TAdV-C and TAdV-D [65, 110] . The diagnosis of adenoviruses depends on virus isolation and molecular identification. Virus isolation can be done using cell cultures derived from the homologous species [94] . However, adenoviruses could be successfully derived from turkeys using chicken embryo liver (CEL) cells isolated from SPF chickens. The main cytopathic effects on CEL cells are rounded cell degeneration after the 1st and 4th passages [65] . Adenovirus could be also detected in turkey poults using EM (Figure 3 ). Molecular typing can be done based conventional PCR targeting the L1 region of the hexon gene [65, 109] . Amplification and sequence analysis of the polymerase gene can be used to distinguish between TAdV-B, TAdV-C and TAdV-D [65, 110] .

TAstV and TCoV have been reported in turkeys causing a severe negative impact on in testinal absorptive functions as causative factors of PEMS [55] . Additionally, coinfectio of turkeys with TAstV and rotavirus was also reported in the US [65] . The diagnosis of adenoviruses depends on virus isolation and molecular identifica tion. Virus isolation can be done using cell cultures derived from the homologous specie [94] . However, adenoviruses could be successfully derived from turkeys using chicke embryo liver (CEL) cells isolated from SPF chickens. The main cytopathic effects on CE cells are rounded cell degeneration after the 1st and 4th passages [65] . Adenovirus coul be also detected in turkey poults using EM (Figure 3 ). Molecular typing can be done base conventional PCR targeting the L1 region of the hexon gene [65, 109] . Amplification an sequence analysis of the polymerase gene can be used to distinguish between TAdV-B TAdV-C and TAdV-D [65, 110] . Due to the high sensitivity of PCR in determining the genotype of rotaviruses, it is a good alternative to EM or virus antigen ELISA [111] . In human medicine, PCR methods are established that amplify sections of gene 4 (VP4), gene 9 (VP7) or gene 9 (NSP4) [108, 112, 113] . To detect avian rotaviruses using PCR, highly conserved primers of the NSP4 gene can be used [51, 79] . The NSP4 gene sequences of the rotaviruses detected by these authors were 96.1%-97.5% identical. Rotavirus particles could also be detected in turkey poults using EM (Figure 4 ). Due to the high sensitivity of PCR in determining the genotype of rotaviruses, it is a good alternative to EM or virus antigen ELISA [111] . In human medicine, PCR methods are established that amplify sections of gene 4 (VP4), gene 9 (VP7) or gene 9 (NSP4) [108, 112, 113] . To detect avian rotaviruses using PCR, highly conserved primers of the NSP4 gene can be used [51, 79] . The NSP4 gene sequences of the rotaviruses detected by these authors were 96.1%-97.5% identical. Rotavirus particles could also be detected in turkey poults using EM (Figure 4 ). 

PEMS is an interaction between enteric pathogens and opportunistic infections in young turkeys. The main role of enteric viruses as primary agents in this syndrome is not fully understood. However, it is obvious that the interaction between enteric viruses and opportunistic bacteria/parasites and management increased the pathological effects. The development of PEMS depends on the virus-host interaction, virus pathotype, age of birds, immune status, biosecurity, and healthy conditions of the GIT. Therefore, several measures should be taken to control PEMS in turkeys, (1) reduction of the pathogenic load using antibiotic alternatives, (2) maintaining gut healthy and strengthening the immune system, (3) hygienic measures and (4) vaccinations.

There is an increasing trend to use alternatives to antibiotics including probiotics, prebiotics, organic acids, essential oils and botanical extracts for turkey [114] in the aim of reducing the pathogenic load. Several studies highlighted the benefits of these products in the improvement of animal performance and reduction infections in turkeys [115] [116] [117] [118] [119] [120] . Lactic acid bacteria (LAB) proved to be an efficient antibiotic alternative to control Salmonella in turkeys by the reduction of intestinal colonization of Salmonella Typhimurium [121] and Salmonella Enteritidis [122] . Higgins and others found also that supplementation of turkey poults with LAB following antibiotic treatment improved significantly animal performance, compared with non-treated or probiotic-treated poults [123] . Additionally, Leyva-Diaz found that combinations between curcumin and copper acetate reduced the colonization of Salmonella Typhimurium in turkey poults and maintained a better intestinal homeostasis [124] . It was also found that Propionibacterium freudenreichii subsp. freudenreichii modulated the beneficial microbiota and reduced the multidrug-resistant Salmonella Heidelberg colonization in turkey poults [125] . 

PEMS is an interaction between enteric pathogens and opportunistic infections in young turkeys. The main role of enteric viruses as primary agents in this syndrome is not fully understood. However, it is obvious that the interaction between enteric viruses and opportunistic bacteria/parasites and management increased the pathological effects. The development of PEMS depends on the virus-host interaction, virus pathotype, age of birds, immune status, biosecurity, and healthy conditions of the GIT. Therefore, several measures should be taken to control PEMS in turkeys, (1) reduction of the pathogenic load using antibiotic alternatives, (2) maintaining gut healthy and strengthening the immune system,

There is an increasing trend to use alternatives to antibiotics including probiotics, prebiotics, organic acids, essential oils and botanical extracts for turkey [114] in the aim of reducing the pathogenic load. Several studies highlighted the benefits of these products in the improvement of animal performance and reduction infections in turkeys [115] [116] [117] [118] [119] [120] . Lactic acid bacteria (LAB) proved to be an efficient antibiotic alternative to control Salmonella in turkeys by the reduction of intestinal colonization of Salmonella Typhimurium [121] and Salmonella Enteritidis [122] . Higgins and others found also that supplementation of turkey poults with LAB following antibiotic treatment improved significantly animal performance, compared with non-treated or probiotic-treated poults [123] . Additionally, Leyva-Diaz found that combinations between curcumin and copper acetate reduced the colonization of Salmonella Typhimurium in turkey poults and maintained a better intestinal homeostasis [124] . It was also found that Propionibacterium freudenreichii subsp. freudenreichii modulated the beneficial microbiota and reduced the multidrug-resistant Salmonella Heidelberg colonization in turkey poults [125] .

Several medical plants such as rosemary, sage, thyme and oregano exhibited a broadspectrum of antimicrobial properties and antioxidative effects [126, 127] and improved animal performance in turkeys [128] . Essential oils have also a broad spectrum of antibac-terial and antiparasitic effects [129, 130] by increasing the permeability of the cell wall of microorganisms and/or inactivation cellular enzymes [130] . It was found that benzoic acid and essential oils improved performance, increased lactic acid bacteria populations and decreased coliform bacteria in the caeca of turkey poults [131] . Additionally, thymol and essential oils improved the antioxidant status of turkeys. Similar results were described in broiler chickens in which essential oils improved the intestinal microbial balance through reduction of coliform bacteria and increasing the Lactobacillus spp. of commercial broiler chickens [132] .

Generally, protozoan and immunosuppressive diseases such as Marek's disease in turkeys should be taken in to consideration [133] . Improvement of the immune system of turkey poults has a role to resist diseases. The β-glucans have an immunomodulatory effect due to increasing the activity of immune cells such as macrophages and neutrophils [134] [135] [136] . It also decreases Salmonella Enteritidis invasion and stimulates phagocytosis, bacterial killing, and oxidative burst in heterophils isolated from 4-d-old male Leghorn chickens 24 h after the oral challenge [137] . Supplementation of turkeys with probiotics (mannan-oligosaccharide) in combination with probiotics enhanced the immunoglobulin levels and improved performance [138] . Due to the negative impacts of protozoan such as Histomonas meleagridis in turkeys on the health and welfare, preventative management measures should be strictly applied to prevent the infection [2] . Additionally, litter management requires also thoughtful consideration and active management [139] .

Although there is no specific treatment, the prevention of PEMS includes vaccination against potential pathogens in the case of available vaccines and hygienic measures. Until now, there are no available vaccines against viruses causing PEMS. In addition, there is no specific treatment. Although several efforts were done to develop effective vaccine against TCoV using classical methods (attenuated and inactivated vaccines) and molecular based (DNA and vector) vaccines, early and protective humoral and cellular immune responses could not be obtained by the developed vaccines [25] . Further improvements and optimization of vaccination regimes against TCoV are urgently needed.

Although no etiological agent has been identified as a specific cause of PEMS condition, several potential infectious agents and non-infectious predisposing factors are associated with this condition. The fact that the pathogenicity of several enteric viruses in turkeys remains unclear and it cannot be excluded that PEMS initiated by an unidentified virus. Opportunistic microorganisms such as Salmonella, E. coli, Clostridium and parasitic infections complicate the disease, leading to severe economic losses. Although isolation of the enteric viral agents is a challenge, EM and molecular identification can be used for diagnosis. No vaccine against viruses associated with this condition are available. Biosecurity including disposal of dead birds, litter management is very important in preventing the spread of any infectious agent between farms and between birds of different ages within the farm. Additionally, general management measures such as raising the temperature, use of antibiotic alternatives to combat secondary bacterial infections and supportive treatment might minimize the economic losses. Maintaining healthy gut and strengthening the immune system of turkey poults are crucial factors to prevent enteritis in turkeys. This can be achieved by supplementation of birds with probiotics, prebiotics and/or phytogenic substances.

Turkey production and health: Current challenges

A case study of histomoniasis in fattening turkeys identified in histopathological investigations

Overview about currently ongoing viral diseases of turkeys

Multiplex real-time reverse transcription-polymerase chain reaction for the detection of three viruses associated with poult enteritis complex: Turkey astrovirus, turkey coronavirus, and turkey reovirus

A retrospective study on poult enteritis syndrome in minnesota

The occurrence of enteric viruses in light turkey syndrome

Mortality patterns associated with poult enteritis mortality syndrome (PEMS) and coronaviral enteritis in turkey flocks raised in pems-affected regions

Metagenomic analysis of the turkey gut RNA virus community

High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus

Partial characterization of a turkey enterovirus-like virus

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome

Electron microscopic identification of viruses associated with poult enteritis in turkeys grown in california 1993-2003

Viral agents associated with poult enteritis and mortality syndrome: The role of a small round virus and a turkey coronavirus

Astrovirus, reovirus, and rotavirus concomitant infection causes decreased weight gain in broad-breasted white poults

Partial genome sequence analysis of parvoviruses associated with enteric disease in poultry

Cross-sectional survey of selected enteric viruses in polish turkey flocks between

Antibiotics in the treatment of an unfamiliar turkey disease

Coronavirus associated with an enteric syndrome on a quail farm

Poult enteritis and mortality syndrome in turkeys in great britain

First full-length sequences of the s gene of european isolates reveal further diversity among turkey coronaviruses

Detection of turkey coronavirus in commercial turkey poults in brazil

Identification of turkey astrovirus and turkey coronavirus in an outbreak of poult enteritis and mortality syndrome

A coronavirus outbreak in northwest arkansas

Infection with the turkey coronavirus: A recurring problem in turkeys

Animals 2021

26. International Committee on Taxonomy of Viruses (ICTV) Master Species List

Origin and evolution of pathogenic coronaviruses

Spotlight on avian coronaviruses

Complete genomic sequence of turkey coronavirus

Emergence of a group 3 coronavirus through recombination

First complete genome sequence of european turkey coronavirus suggests complex recombination history related with us turkey and guinea fowl coronaviruses

Recombinant turkey coronavirus: Are some s gene structures of gammacoronaviruses especially prone to exchange?

Full genome sequence of guinea fowl coronavirus associated with fulminating disease

Coronaviruses in avian species-review with focus on epidemiology and diagnosis in wild birds

Review of infectious bronchitis virus around the world

Genotyping of turkey coronavirus field isolates from various geographic locations in the unites states based on the spike gene

A recombinant infectious bronchitis virus from a chicken with a spike gene closely related to that of a turkey coronavirus

Turkey coronavirus. In Diseases of Poultry

Development of a competitive enzyme-linked immunosorbent assay for detection of turkey coronavirus antibodies

Transmission kinetics and histopathology induced by European turkey coronavirus during experimental infection of specific pathogen free turkeys

Use of recombinant s1 spike polypeptide to develop a tcov-specific antibody ELISA

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3 untranslated region identifies the virus as a close relative of infectious bronchitis virus

Cleanup and prevention of TCV enteritis

Detection of astroviruses in turkey faeces by direct electron microscopy

Development of a multiplex reverse transcriptionpolymerase chain reaction diagnostic test specific for turkey astrovirus and coronavirus

Detection and characterization of chicken astrovirus associated with hatchery disease in commercial day-old turkeys in Southwestern Nigeria

Astrovirus: A cause of an enteric disease in turkey poults

Development of an RT-PCR diagnostic test for an avian astrovirus

Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys

Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms

The first whole genome sequence and characterisation of avian nephritis virus genotype 3

Localization of astrovirus in experimentally infected turkeys as determined by in situ hybridization

Veterinary Virology

Pathogenicity of turkey coronavirus in turkeys and chickens

Astrovirus infection in hatchling turkeys: Alterations in intestinal maltase activity

Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the united states between

Astrovirus induces diarrhea in the absence of inflammation and cell death

Identifying agent(s) associated with poult enteritis mortality syndrome: Importance of the thymus

Unconventional gene arrangement and content revealed by full genome analysis of the white sturgeon adenovirus, the single member of the genus Ichtadenovirus

Identification of two novel adenoviruses in smooth-billed ani and tropical screech Owl

Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses

Virus Taxonomy. IXth Report of the International Committee on Taxonomy of Viruses

The first complete genome sequence of a non-chicken aviadenovirus, proposed to be turkey adenovirus 1

Investigations on aviadenoviruses isolated from turkey flocks in Germany

Whole-genome sequences of two turkey adenovirus types reveal the existence of two unknown lineages that merit the establishment of novel species within the genus Aviadenovirus

Inclusion-body hepatitis in day-old turkeys

Group I avian adenovirus and avian adeno-Associated virus in turkey poults with inclusion body hepatitis

Virus infections of the gastrointestinal tract of poultry

The immune response of young turkeys to haemorrhagic enteritis virus infection at different levels and sources of methionine in the diet

Hemorrhagic enteritis and related infections

Fields Virology

Zoonotic aspects of rotaviruses

Genotype constellation and evolution of group a rotaviruses infecting humans

Candidate new rotavirus species in schreiber's bats, Serbia

Comparison of immune electron microscopy and genome electropherotyping techniques for detection of turkey rotaviruses and rotaviruslike viruses in intestinal contents

Detection of viruses in avian faeces by direct electron microscopy

Retrospective analysis of turkey arthritis reovirus diagnostic submissions in minnesota

A multiplex RT-PCR test for the differential identification of turkey astrovirus type 1, turkey astrovirus type 2, chicken astrovirus, avian nephritis virus, and avian rotavirus

Early pathogenesis of experimental reovirus infection in chickens

The history of avian reovirus

Enteric viruses associated with mid-growth turkey enteritis

Infectious enteritis of turkeys: Characterization of two reoviruses isolated by sucrose density gradient centrifugation from turkeys with infectious enteritis

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus

A newly emergent turkey arthritis reovirus shows dominant enteric tropism and induces significantly elevated innate antiviral and t helper-1 cytokine responses

Electron Microscopy in Viral Diagnosis

Electron microscopy in diagnostic virology; A practical guide and atlas

Virus morphology

Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related microorganisms

Development of antigen-capture enzyme-linked immunosorbent assay and RT-PCR for detection of turkey astroviruses

Salmonella infections. In Diseases of Poultry

Specimen staining and contrast methods for transmission electron microscopy

Enteric viruses in Brazilian turkey flocks: Single and multiple virus infection frequency according to age and clinical signs of intestinal disease

PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus

Molecular characterization of avian reoviruses using nested PCR and nucleotide sequence analysis

Detection of avian adenovirus by polymerase chain reaction. Avian Dis

Detection and molecular characterization of gene 3 and 5 of turkey coronavirus from turkeys with severe enteritis in Brazil

Viral agents related to enteric disease in commercial chicken flocks, with special reference to latin america

Detection of some turkey enteric pathogens by single multiplex polymerase chain reaction

Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay

Coronaviruses in poultry and other birds

Molecular identification and characterization of novel coronaviruses infecting graylag geese (Anser anser), feral pigeons (Columbia livia) and mallards (Anas platyrhynchos)

Detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus

Astrovirus infections

Letter: 28 nm particles in faeces in infantile gastroenteritis

Diversity within the VP4 gene of rotavirus p[8] strains: Implications for reverse transcription-pcr genotyping

Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses

Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

PCR for Clinical Microbiology: An Australian and International Perspective; Schuller

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens

Design of a multiplex nested PCR for genotyping of the nsp4 from group a rotavirus

Alternative antimicrobials in the turkey industry-Challenges and perspectives

The influence of probiotic bacteria (Bacillus Toyoi) on livability and performance of young meat-type turkeys

Effects of a direct-fed microbial (Primalac) on turkey poult performance and susceptibility to oral Salmonella challenge

Growth performance and gastrointestinal responses in heavy tom turkeys fed antibiotic free corn−soybean meal diets supplemented with multiple doses of a single strain Bacillus subtilis probiotic (DSM29784)

The Effect of a direct-fed microbial (primalac) on turkey live performance

Performance and condemnation rate analysis of commercial turkey flocks treated with a Lactobacillus spp.-based probiotic

Developing probiotics, prebiotics, and organic acids to control Salmonella spp. in commercial turkeys at the University of Arkansas USA

Characterization and evaluation of lactic acid bacteria candidates for intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults

Isolation and identification of lactic acid bacteria probiotic culture candidates for the treatment of Salmonella enterica serovar Enteritidis in neonatal turkey poults

Evaluation of intervention strategies for idiopathic diarrhea in commercial turkey brooding houses

Evaluation of curcumin and copper acetate against Salmonella Typhimurium infection, intestinal permeability, and cecal microbiota composition in broiler chickens

Effect of supplementation of a dairy-originated probiotic bacterium, Propionibacterium freudenreichii subsp. freudenreichii, on the cecal microbiome of turkeys challenged with multidrug-resistant Salmonella Heidelberg

Bioactive natural compounds and antioxidant activity of essential oils from spice plants: New findings and potential applications

A review on applications and uses of thymus in the food industry

Effect of dietary dried oregano leaves on growth performance, carcase characteristics and serum cholesterol of female early maturing turkeys

Efficacy of a herbal product against Histomonas Meleagridis after experimental infection of turkey poults

The dietary combination of essential oils and organic acids reduces Salmonella Enteritidis in challenged chicks

Dietary supplementation of benzoic acid and essential oil compounds affects buffering capacit y of the feeds, performance of turkey poults and their antioxidant status, ph in the digestive tract, intestinal microbiota and morphology

The effect of volatile oil mixtures on the performance and ilio-caecal microflora of broiler chickens

History and current status of Marek's disease in turkeys

Glucan-induced enhancement of host resistance to selected infectious diseases

The effect of molecular weight and β-1,6-linkages on priming of macrophage function in mice by (1,3)-β-D-Glucan

Polysaccharide immunomodulators as therapeutic agents: Structural aspects and biologic function

Purified β-Glucan as an abiotic feed additive up-regulates the innate immune response in immature chickens against Salmonella enterica serovar Enteritidis

The effects of probiotic and mannanoligosaccharide on some haematological and immunological parameters in turkeys

Considerations in selecting turkey bedding materials