Prevalence of Middle East Respiratory Syndrome Coronavirus in Dromedary Camels, Tunisia

Free-roaming camels, especially those crossing national borders, pose a high risk for spreading Middle East respiratory syndrome coronavirus (MERS-CoV). To prevent outbreaks, active surveillance is necessary. We found that a high percentage of dromedaries in Tunisia are MERS-CoV seropositive (80.4%) or actively infected (19.8%), indicating extensive MERS-CoV circulation in Northern Africa.

MERS cases is highly probable because of the broad range of manifestations, from asymptomatic infection to acute pneumonia.
Furthermore, epidemiologic surveillance of MERS-CoV is limited in Tunisia, and no respective data for neighboring countries is publicly available. Two studies analyzing MERS-CoV prevalence in dromedaries in Tunisia reported high seropositivity of the sampled animals (49.0% and 87.3%) (8,9). However, those studies analyzed dromedary camels from livestock markets, slaughterhouses, and meat farms, without representing natural habitats. This limitation complicates drawing realistic conclusions about geographic and age-dependent distributions. We investigated the prevalence of MERS-CoV in dromedary camels in Tunisia primarily by sampling animals that roam freely through the desert during summer to determine an authentic representation of the distribution pattern.

The Study
Winter is mating and birthing season for dromedary camels. Therefore, animals kept for milk and meat production gather in areas that provide access to salty plants and other minerals. This environment provides an optimal opportunity to catch and examine large numbers of animals from different herds and origins, given that the camels that roam freely through the desert the rest of the year congregate simultaneously.
In January 2020, we collected serum samples and nasal swabs of 382 gathered animals in Tunisia. We sampled an additional 119 camels used for transport or patrol purposes, all of which were males and kept enclosed. The specimens were collected from 20 different locations within the Kebili Governorate (Table; Figure 1, panel A). Furthermore, serum samples of 22 camel keepers and 2 veterinarians were obtained. We Free-roaming camels, especially those crossing national borders, pose a high risk for spreading Middle East respiratory syndrome coronavirus (MERS-CoV). To prevent outbreaks, active surveillance is necessary. We found that a high percentage of dromedaries in Tunisia are MERS-CoV seropositive (80.4%) or actively infected (19.8%), indicating extensive MERS-CoV circulation in Northern Africa.
We analyzed all 501 dromedary serum samples for MERS-CoV-specific antibodies by ELISA and found 80.4% to be seropositive for MERS-CoV IgG. At 85.7%, MERS-CoV seropositivity was higher in female than in male camels (65.6%) (Table; Figure 1, panel A).
Although none of the calves (0-6 months of age) and 4.9% of the juvenile camels (6-24 months of age) were seropositive for antibodies against MERS-CoV, relative seropositivity increased with age (Table). None of the camel keepers or veterinarians was seropositive, indicating no previous MERS-CoV infection.
Screening the dromedary nasal swab specimens for active virus infections with real-time reverse tran-scription PCR revealed MERS-CoV RNA in 19.8%. However, cycle thresholds >30 (for all but 6 samples) indicated low virus concentrations. Female animals (23.0%) actively shed MERS-CoV RNA, whereas only 10.7% of the male camel specimens were PCRpositive. In contrast to the immunologic findings, a high percentage (40%) of juvenile camels (<2 years of age) shed MERS-CoV RNA, compared with 17.8% of the adult camels that tested positive (Table, Figure 1, In summary, 433 of 501 dromedaries tested positive for MERS-CoV. A total of 334 animals were seropositive for MERS-CoV IgG but did not shed MERS-CoV RNA. Of these, 30 dromedary swab specimens contained MERS-CoV RNA, but no specific antibodies were found in the respective serum samples. Sixty-nine PCR-positive dromedary camels also had MERS-CoV antibodies, indicating reinfection ( Figure  1, panel B).
Attempts to cultivate the virus from all respective PCR-positive swab specimens were unsuccessful, most likely because of the low virus concentrations in the samples. Presumably, whole-genome sequencing did not work for the same reasons. However, we performed Sanger sequencing of cDNAs obtained from PCR-positive samples with the highest viral concentrations and subsequently conducted phylogenetic analysis with a 720-bp fragment of the spike receptorbinding protein. The analyzed nucleotide sequences from the dromedaries in Tunisia differ from previously published MERS-CoV sequences and therefore form a separate group distinct from strains found in Arabia. Two MERS-CoV isolates in Egypt, however, cluster in the same clade ( Figure 2).

Conclusions
On the continent of Africa, active surveillance, longitudinal studies, and epidemiologic monitoring are scarce, and little is known about the prevalence and circulation of MERS-CoV in many regions. Whether MERS-CoV lineages in Africa have a lower tendency to cross the species barrier and infect humans is not fully understood. Therefore, closing the gaps in surveillance and virus prevalence data remains a focus for all regions with dromedary camel populations.
However, most studies focus on locations where large numbers of camels congregate (e.g., abattoirs, large-scale farms, harbors, or livestock markets). At these locations, dromedaries are kept at a substantially increased population density compared with their normal habitats. This practice, referred to as crowding, increases stress for individual animals (11). Under these circumstances, increased intensive animal contact can lead to higher transmission rates of various microorganisms. Crowding, in combination with animal transport, is known to promote infections of the upper and lower respiratory tract, especially in cattle (12). In contrast, the camels investigated in our study represent a rare example of MERS-CoV prevalence in animal groups with a natural herd structure in northern Africa.

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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 7, July 2021 We found extensive MERS-CoV IgG seropositivity (80.4%) and high ratios of MERS-CoV RNA (19.8%) among dromedaries in Tunisia. Compared with adult animals, juvenile camels were more likely to have active MERS-CoV infections and less MERS-CoV IgG in their serum samples. Furthermore, some dromedaries appeared to have MERS-CoV reinfections, explained by the fact that coronaviruses tend to establish endemic infection patterns with high seroprevalence and low but continuous viral shedding in their natural host (13). Waning antibodies in combination with antigenic drift of the virus fosters reinfection events (14). A limitation of our study is that the low sample size of humans tested, comprising 22 camel keepers and 2 veterinarians (data not shown), precludes drawing generalized conclusions. Furthermore, no nasal swab specimens were collected from camel keepers to check for active MERS infections. Also, no phenotypic or whole-genome analysis of MERS-CoV strains from the dromedary camels was possible because virus growth and next-generation sequencing were not successful because of low viral concentrations.
In conclusion, the high seroprevalence of MERS-CoV antibodies and the active shed of MERS-CoV RNA indicate the widespread nature of the virus in dromedaries in Tunisia. However, more extensive studies in the human and dromedary camel populations and in-depth whole-genome sequence analysis of circulating MERS-CoV strains are required to increase epidemiologic understanding of the disease and its infection dynamics.

Immunoserological testing
Seroprevalence of anti-MERS-CoV IgG in dromedary camels as well as camel keepers was investigated by performing indirect ELISA assays (Euroimmun AG, Germany) according to the manufacturer's protocol.

Molecular testing
Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (QIAGEN, Netherlands). Subsequently, real-time reverse transcription PCR (rtRT-PCR) was performed targeting the genetic region upstream of the E gene (upE) (for screening) and ORF1a (for confirmation), respectively, as described elsewhere (1,2).

Statistical analysis
Associations between MERS-CoV prevalence in dromedary camels and the study parameters (sex, age and sampling site) were analyzed by Pearson Chi-square test for comparison of categorical data. At a p-value of less than 0.05, statistical significance was considered.

Virus isolation in cell culture
Isolation of live virus was attempted on Vero E6 cells under BSL3 conditions. Briefly, 250 µl of virus transport medium of MERS-CoV rtRT-PCR-positive nasal swabs was used as inoculum for cell cultures in T25 flasks. Cells were washed with serum free medium, inoculated with sample fluid and incubated for one hour. Afterwards, the inoculum was removed and the cellular layer was washed three times. The final culture medium contained MEM with 2% FBS and 5x antibiotic-antimycotic additive (GIBCO, USA). Cells were incubated for 7 days with one subsequent subpassage of supernatant on fresh cells. Cultures were regularly screened for cytopathic effects and supernatant was screened for an increase in MERS-CoV RNA.

Genomic spike RBD amplification
For phylogenetic analysis a partial 774-bp fragment of the spike (s) gene containing the RBD was amplified by running a rtRT-PCR (SSIII qRT-Kit, Invitrogen, United States) using the primer pair pre-RBD fwd (GAATCTGGAGTTTATTCAGTTTCGT) and pre-RBD rev (ACGGCCCGAAACACCATAG) (3). Subsequently, spike RBD (720-bp) was amplified by performing a nested PCR with the primers RBD fwd (GAAGCAAAACCTTCTGGCT) + RBD rev (ATATTCCACGCA) using Q5 HotStart MasterMix Kit (New England Biolabs, USA) with the respective pre-fragment as DNA template. The resulting PCR product was gel-purified (Mini kit for gel extraction and PCR clean up, Macherey-Nagel, Germany) and sequenced with the latter primers (Eurofins Genomics Germany GmbH, Germany).

Sequence analysis
The phylogenetic tree was constructed based on 13 s RBD (GenBank AcNo: MW322770-MW322782) sequences, obtained from rtRT-PCR-positive dromedary specimens with the highest viral amounts and representative MERS-CoV sequences from different countries using Geneious Prime (Version 2020 1.2) by applying the neighbor-joining method with Tamura-Nei's genetic distance model and 1,000 bootstrap replicates.

Ethics Statement
The use of the biologic material described in the underlying study was approved by the Ethics Committee of the Military Hospital in Tunis (Decision N° 57/2020/CLPP) and written consent forms were signed by all included participants.