key: cord-317455-6qx0v28w
authors: Brown, Paul A.; Courtillon, Céline; Weerts, Erik A. W. S.; Andraud, Mathieu; Allée, Chantal; Vendembeuche, Anthony; Amelot, Michel; Rose, Nicolas; Verheije, Monique H.; Eterradossi, Nicolas
title: Transmission Kinetics and histopathology induced by European Turkey Coronavirus during experimental infection of specific pathogen free turkeys
date: 2018-09-10
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13006
sha: 
doc_id: 317455
cord_uid: 6qx0v28w

Numerous viruses, mostly in mixed infections, have been associated worldwide with poult enteritis complex (PEC). In 2008 a coronavirus (Fr‐TCoV 080385d) was isolated in France from turkey poults exhibiting clinical signs compatible with this syndrome. In the present study, the median infectious dose (ID (50))(,) transmission kinetics and pathogenicity of Fr‐TCoV were investigated in 10‐day‐old SPF turkeys. Results revealed a titre of 10(4.88) ID (50)/ml with 1 ID (50)/ml being beyond the limit of genome detection using a well‐characterized qRT‐PCR for avian coronaviruses. Horizontal transmission of the virus via the airborne route was not observed however, via the oro‐faecal route this proved to be extremely rapid (one infectious individual infecting another every 2.5 hr) and infectious virus was excreted for at least 6 weeks in several birds. Histological examination of different zones of the intestinal tract of the Fr‐TCoV‐infected turkeys showed that the virus had a preference for the lower part of the intestinal tract with an abundance of viral antigen being present in epithelial cells of the ileum, caecum and bursa of Fabricius. Viral antigen was also detected in dendritic cells, monocytes and macrophages in these areas, which may indicate a potential for Fr‐TCoV to replicate in antigen‐presenting cells. Together these results highlight the importance of good sanitary practices in turkey farms to avoid introducing minute amounts of virus that could suffice to initiate an outbreak, and the need to consider that infected individuals may still be infectious long after a clinical episode, to avoid virus dissemination through the movements of apparently recovered birds.

Infectious bronchitis virus is a highly contagious virus transmitted very quickly among naive birds in the field. It is responsible worldwide for respiratory diseases, egg drop with poor eggshell quality, reduced hatchability, nephritis and sometimes, in early infection of future breeders, genital atrophy responsible for the syndrome of "false laying" in chicken breeders or layers (Jackwood, & Wit, 2013) .

Turkey coronavirus, originally identified in the USA in the 1970s as one of the agents responsible for an acute enteritis named bluecomb (Panigrahy, Naqi, & Hall, 1973; Ritchie, Deshmukh, Larsen, & Pomeroy, 1973) and since with a multifactorial disease known as poult enteritis complex of turkeys (PEC) , has now been detected in most areas where turkeys are farmed Cavanagh et al., 2001; Dea & Tijssen, 1988; Domańska-Blicharz, Seroka, Lisowska, Tomczyk, & Minta, 2010; Martin, Vinco, Cordioli, & Lavazza, 2002; Maurel et al., 2009; Teixeira et al., 2007) , although TCoVs isolated in Europe have been shown to have a different genetic lineage to those isolated in the USA (Brown et al., 2016; Maurel et al., 2011) . PEC includes several intestinal disorders that occur in turkeys mostly within the first three weeks of life (Guy, 2013) and its clinical signs often include diarrhea, stunting, anorexia, dehydration, weight loss, and immune dysfunction (atrophy of the thymus and the bursa of Fabricius) that promotes secondary infections. The wide distribution of both IBV and TCoV and their highly contagious nature have considerable economic repercussions.

The contagious nature of a disease can be measured by the "reproduction number" (R0) defined as "the expected number of secondary cases produced by a single (typical) infection in a totally susceptible population" (Masters & Perlman, 2013) . The parameters necessary to calculate R0 are (a) the speed of transmission and (b) the shedding duration of the infectious viruses. Generally, a virus with an R0 less than 1 will disappear quickly because an infected individual will have a low ability to infect another. A virus with an R0 greater than one will spread in the susceptible population. For IBV, an R0 of 19.95 has been estimated (de Wit, de Jong, Pijpers, & Verheijden, 1998) , which is a figure comparable to the R0 of highly contagious human viruses such as measles virus (R0 12-18) (Masters & Perlman, 2013) . For TCoV, R0 has not yet been fully calculated; however, a study with an American TCoV isolate demonstrated that infectious virus particles can be shed up to six weeks post-infection in experimentally infected turkeys .

The current study focused on strain Fr-TCoV 080385d that was detected in France in 2008 in turkeys with clinical signs compatible with PEC. Fr-TCoV is the only European TCoV strain isolated to date, although coronaviruses have been detected in turkeys in Poland, Great Britain and Italy (Cavanagh, 2001; Domańska-Blicharz et al., 2010; Martin et al., 2002) . The aim of this study was to determine the transmission properties of the virus by evaluating its ID 50 and reproduction number (R0) under experimental conditions in 10day-old SPF turkeys, in order to better understand the diffusion of the disease. Histopathological examination and in-situ detection of TCoV antigen at the sites of replication in the intestinal tract were also performed.

Three animal experiments (Exp 1, 2 and 3) were performed in agreement with the national regulations of the French Ministry for higher education and research on animal welfare and after approval from the French Agency for Food, Environmental and Occupational Health & Safety's (ANSES) ethical committee.

Virus Fr-TCoV 080385d isolated from duodenal contents of 42-dayold turkeys affected by PEC in November 2008 was propagated by inoculating embryonated SPF turkey eggs (Anses, Ploufragan, France) via the intra-amniotic route, as previously described (Guionie et al., 2013) . Because Fr-TCoV 080385d does not induce clinical lesions in the embryo, the intestines of inoculated embryos were screened 4 days post-inoculation by qRT-PCR (Maurel et al., 2011) , and the intestines of positive embryos were collected and pooled to prepare a virus stock (22). Five-fold serial dilutions of this stock were inoculated into seven eggs per dilution, and a titre of 10 4.01 EID 50 /ml was calculated according to Reed & Muench (20) .

One hundred microliters of intestinal or cloacal swab material was lysed with 300 μl of Buffer RLT (Qiagen, France) by mixing and incubating at room temperature for 15 min. RNA was extracted using MagAttract RNA Tissue Mini M48 kit or MagAttract Virus Mini M48 kit for BioRobot M48 (Qiagen, France) and eluted in 100 μl of buffer AVE following the manufacturer's instructions. The presence of TCoV genome was detected using a qRT-PCR specific for Avian Coronaviruses (Maurel et al., 2011) . The limit of detection (LoD) and the linear phase of this qRT-PCR were described as 2 log10 and from 3 to 9 log10 copies per microliter of extracted RNA, respectively. In this study, samples were considered positive with a result higher than 2 log10 copies per microliter of extracted RNA. All results are given as copy number (cp)/μl of extracted RNA expressed in log10 together with the SD 2.4 | Exp 1. Titration of Fr-TCoV in 10-day-old SPF

Thirty 10-day-old SPF turkeys were separated in 5 groups of 6 birds, and housed for 3 days in negative pressure isolators allowing ad lib feeding and drinking. Each isolator had a cardboard floor with a metal grid platform underneath and a surface area of 1.4 m 2 . Groups 1, 2, 3 and 4 were inoculated via the oral route with 0.25 ml of BROWN ET AL.

strain Fr-TCoV 080385d diluted to 10 −1.5 , 10 −3.0 , 10 −4.5 and 10 −6.0 respectively in MEM Hepes (Gibco, France) supplemented with penicillin (200 μ/ml final concentration) and streptomycin (0.2 mg/ml final concentration). Control group 5 was inoculated with MEMH plus antibiotics alone via the same route. At 1-day post-inoculation (dpi), two SPF turkey contacts were introduced into groups 1-4 as sentinels to demonstrate horizontal transmission of infectious virus. From 1 to 3 dpi, cloacal swabs were collected from all subjects, sampling the contacts first, followed by those that had been inoculated.

RNA was extracted from these samples for molecular analysis as described above. The 50% endpoint was calculated using the method of Reed and Muench (Reed & Muench, 1938) .

Thirty-two 10-day-old SPF turkeys were separated into groups, one containing 29 subjects and a second containing 3. Each group was housed in a separate negative pressure room at a density of seven birds per m² and floors were covered with wood chippings (reproducing common commercial rearing conditions in France). The group of three subjects was inoculated with 0.25 ml of strain Fr-TCoV 080385d diluted at 10 −4.5 in the same media as used in Exp. 1, via the oral route. At 1 dpi, cloacal swabs were collected to confirm their Fr-TCoV 080385d positive status by qRT-PCR. At 2 dpi, one positive subject was placed as a seeder infected bird among the group of 29 SPF subjects (contacts). Cloacal swabs were collected from all subjects every 2 hr until 16 hr post-contact (hpc), at 24 hpc and 2 days post-contact (dpc) then weekly until 41 dpc.

During the 2-hr-sampling regime, the order in which the subjects were taken was respected throughout. This ensured that each subject was sampled precisely every two hours. Sampling staff wore a new pair of sterile gloves for each sampled bird, so as not to transfer the virus through bird-handling. RNA was extracted from these samples to perform qRT-PCR, to determine infection and the excretion period for each subject. One representative positive sample selected at 6 dpc of Exp 2.

(codified T6) was diluted (same media as Exp. 1) so as to inoculate via the oral route 10 5.7 RNA copies in three 10-day-old SPF turkeys. They were housed in a negative pressure room, under the same rearing conditions as in Exp 2, with three 11-day-old SPF turkeys introduced as contact-birds at 1 dpi to demonstrate horizontal transmission. Cloacal swabs were collected daily for qRT-PCR analyses from all birds until 3 dpi, when the birds were humanely euthanized and duodenum, jejunum, ileocaecal junction and bursa of Fabricius were collected. These samples were fixed for 24 hr in 4% formaldehyde then transferred to 70% ethanol and finally embedded in paraffin wax for histopathology and anti-TCoV immunohistochemistry (see section Histopathology).

This process was repeated using one representative positive sample from 13, 21, 27, 34 and 41 dpc of Exp 2. (codified T13, T21, T27, T34 and T41, respectively) to make a total of six experiments. Airborne transmission was evaluated in each of these experiments by using six 10-day-old SPF turkeys housed in a park in the same containment cell but separated from the other animals, at a distance of 3 meters. The sampling programme was as described above. Housing, circulation of personal, change of boots, clothes and gloves was organized to minimize physical contamination. 

Fr-TCoV was detected with qRT-PCR at 1 dpi in all six inoculated subjects of group 1 (dilution 10 −1.5 , mean ± SD 5.19 ± 0.94 log 10 cp/μl), in 5 out of 6 subjects of group 2 (10 −3 , 4.46 ± 1.81 log 10 cp/ μl) and in 3 out of 6 subjects in group 3 (10 −4.5 , 3.59 ± 1.37 log 10 cp/μl). At 2 and 3 dpi, all subjects of these groups, including contactbirds, were positive, demonstrating horizontal transmission. No viral RNA was detected throughout the experiment in groups 4 (10 −6 ) and 5 (MEMH). The result obtained at 1 dpi (before horizontal transmission) gave a virus titre of 10 4,88 ID 50 /ml.

The following data are shown graphically in Figure 1 .

An inoculated subject with a viral RNA load of 5.28 log 10 cp/μl at 1 dpi that had been placed among 29 contacts, transmitted the virus to one contact between 8 and 10 hpc, though the level of viral RNA detected at 10 hpc in this newly infected bird (2.05 log 10 cp/μl) was almost at the LoD. However, between 10 and 12 hpc the level of viral RNA detected in the same bird increased to 3.39 log 10 cp/μl and a second contact was positive at 2.19 log 10 cp/μl. 

The data are shown graphically in Figure 2 . In three out of six Exp 2.

samples (T6, T27 and T41), the number of positive inoculated birds and the level of viral RNA detection increased over time during the sampling period, culminating at 3 dpi with RNA detected in all birds including contacts (mean ± SD = 4.89 ± 0.69, 5.75 ± 0.32 and 4.55 ± 0.70 cp/μl, respectively). No viral RNA was detected throughout the period, neither in inoculated or contact subjects exposed to T13, T21 and T34, nor in subjects assigned to the assessment of airborne transmission.

Intestinal samples taken from infected subjects at 3 dpi from Exp. 3

showed well-preserved characteristic architectural features. Except for some very mild hyperemia and rare epithelial desquamation, no clear histopathological changes were seen in any of the samples (Figure 3a) . Immunohistochemical staining showed an abundance of viral protein expressed in the ileum, caeca and bursa of all inoculated or contact subjects exposed to T6, T27 and T41 (expression in the caecum and bursa is shown for T41 in Figure 3 ). As shown in Figure 4 histograms, antigen detection in the other regions of the intestine (duodenum or jejunum) was inconsistent in both the inoculated and contact-birds exposed to the same samples, as illustrated by the fact that no viral protein was detected in the duodenum of any contact subject exposed to T6, T27 and T41. No viral protein expression was seen in any of the intestines taken from the inoculated and contact subjects exposed to T13, T21 and T34. to be taken into consideration. Similarly, at one ID 50 /ml, viral RNA levels were also beyond the limit of detection of a well characterized qRT-PCR (Maurel et al., 2011) , so that 10-day-old turkeys might be also more sensitive than qRT-PCR to detect infectious TCoV. Such a high susceptibility of young hosts was also reported in a recent paper where an enteric coronavirus of pigs (porcine epidemic diarrhea virus, PEDV) was shown to infect more efficiently 5-day-old piglets than tissue culture (minimal infectious dose = 0.056 TCID50) (Thomas et al., 2015) . RNA levels at the PEDV MID have also been reported to be beyond the limit of detection by qRT-PCR (Goyal, 2014) . The effects of turkey age on the ID 50 of Fr-TCoVwere not investigated in the current study however, the fact that TCoV associated enteric disorders such as PEC or poult enteritis mortality syndrome (PEMS) are predominantly diseases of younger subjects lends support to more resistance in older birds. was also difficult to justify on an ethical level in respect to the principals of the 3 Rs (Russell & Burch, 1959) .

Although the "duration of excretion" for every individual could not be obtained, the experiments performed in the current study (in which one sample from each date was re-inoculated) revealed that some subjects continued to shed infectious virus for at least six weeks when others ceased at two. At this time the authors have no data on why some subjects stopped excreting infectious virus four weeks in advance of others or if, in fact, excretion detected at six weeks was representative of subjects with intermittent excretion profiles as has been observed in cats experimentally infected with FECV (Kipar, Meli, Baptiste, Bowker, & Lutz, 2010) . In cats, this intermittent excretion has been suggested to be linked with persistent infection in the colon (lower intestine) from which viruses then have the potential to re-infect the small intestine at any time. The presence of virus in both regions of the gut can then result in renewed excretion (Kipar et al., 2010) . Concerning Fr-TCoV these questions should be assigned to specifically designed trials and histopathological examination, however, the present study seems to indicate a clear tropism of Fr-TCoV for lower intestine, as described for FECV (Kipar et al., 2010) . Indeed, in the current study, Fr-TCoV's ability to infect the turkey intestinal tract was successfully demon- (Reddy et al., 2016) . However, although the stellate morphology and the localization of the depicted cell (see inset Figure 3c ) suggest that it likely belongs to one of the mentioned APC families, additional assays characterizing these cell types (for example double immunohistochemical staining for both viral protein expression and cell-characterizing protein epitopes) are needed to confirm a APC tropism for TCoV.

Fr-TCoV viral protein expression was not correlated with histopathologic changes in the sampled tissues collected here, contrary to previous observations following inoculation with a TCoV of US lineage that did induce lesions, albeit without associated clinical signs . This discrepancy could be explained by the F I G U R E 4 Detection of viral antigen in intestinal tissues. Immunohistochemistry of: intestinal tissues D = duodenum, J = jejunum, I = ileum, C = caeca B = bursa of Fabricius, taken 3 dpi from subjects inoculated with samples T6, T13, T21, T27, T34 orT41 of Exp 2 (a) and from their corresponding contacts (b) difference in age of the birds inoculated as in the US TCoV study, birds had been inoculated at 6 days of age; alternatively, like the distribution pattern discussed above, this discrepancy might be a time-dependent feature, with 3 dpi in our study being too early for morphologic changesresulting from epithelial damage, local tissue reactions and influx of immune cells to manifest. Furthermore the specific date postinoculation when microscopic lesions were observed in the US TCoV study was not given . It is equally possible that under experimental conditions the European lineage of TCoV simply has a different pathogenic profile to those of the US lineage. Infection studies for EU and US TCoVs in turkeys of the same age and under the same controlled conditions are required for comparative analysis into the pathological profiles of these different lineages.

In conclusion an extremely low dose of European isolate Fr-TCoV strain 080385d is required for infection of 10-day-old turkey poults under experimental conditions. The virus spreads very quickly via the oro-faecal route among susceptible subjects (a new subject at least every 2.5 hr) and infectious virus may continue to be excreted for at least six weeks after the initial infection which may be linked to a preferential tropism for the lower intestines. These results stress the importance of good sanitary practices at the entrance to livestock buildings and the need to consider that infected individuals may still be infectious long after the clinical episode.

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The authors wish to thank the Département des Côtes d'Armor/Conseil Régional de Bretagne/Conseil Régional des Pays de Loire/France Agrimer/Office de l'élevage/Comité Interprofessionnel de la Dinde Française for their financial support. This research was part of the EPICOREM ANR programme: Eco-epidemiology of Coronaviruses:From wildlife to human & emergence threat assessment.

The authors declare that they have no conflict of interest.

http://orcid.org/0000-0002-6697-7688