Link of a ubiquitous human coronavirus to dromedary camels.

The four human coronaviruses (HCoVs) are globally endemic respiratory pathogens. The Middle East respiratory syndrome (MERS) coronavirus (CoV) is an emerging CoV with a known zoonotic source in dromedary camels. Little is known about the origins of endemic HCoVs. Studying these viruses' evolutionary history could provide important insight into CoV emergence. In tests of MERS-CoV-infected dromedaries, we found viruses related to an HCoV, known as HCoV-229E, in 5.6% of 1,033 animals. Human- and dromedary-derived viruses are each monophyletic, suggesting ecological isolation. One gene of dromedary viruses exists in two versions in camels, full length and deleted, whereas only the deleted version exists in humans. The deletion increased in size over a succession starting from camelid viruses via old human viruses to contemporary human viruses. Live isolates of dromedary 229E viruses were obtained and studied to assess human infection risks. The viruses used the human entry receptor aminopeptidase N and replicated in human hepatoma cells, suggesting a principal ability to cause human infections. However, inefficient replication in several mucosa-derived cell lines and airway epithelial cultures suggested lack of adaptation to the human host. Dromedary viruses were as sensitive to the human type I interferon response as HCoV-229E. Antibodies in human sera neutralized dromedary-derived viruses, suggesting population immunity against dromedary viruses. Although no current epidemic risk seems to emanate from these viruses, evolutionary inference suggests that the endemic human virus HCoV-229E may constitute a descendant of camelid-associated viruses. HCoV-229E evolution provides a scenario for MERS-CoV emergence.

Hepatitis E Virus Infection in Dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983-2015.

A new hepatitis E virus (HEV-7) was recently found in dromedaries and 1 human from the United Arab Emirates. We screened 2,438 dromedary samples from Pakistan, the United Arab Emirates, and 4 African countries. HEV-7 is long established, diversified and geographically widespread. Dromedaries may constitute a neglected source of zoonotic HEV infections.

Molecular differentiation of Sarcocystis buffalonis and Sarcocystis levinei in water buffaloes (Bubalus bubalis) from Sarcocystis hirsuta and Sarcocystis cruzi in cattle (Bos taurus).

The purpose of the present study was to obtain sarcocysts of Sarcocystis buffalonis and Sarcocystis levinei from water buffaloes and characterize the isolates by molecular methods in order to determine whether the two species were genetically different from Sarcocystis hirsuta and Sarcocystis cruzi, respectively, from cattle, which had been characterized before. About 35 macroscopically visible (3-4 × 1-2 mm) and 20 barely visible (1-3 × 0.2 mm) sarcocysts were excised from the esophagus of 18 naturally infected and freshly slaughtered adult water buffaloes at three slaughterhouses in Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were first characterized at the mitochondrial cytochrome c oxidase subunit I gene (cox1) gene through PCR amplification and direct sequencing. Selected isolates were subsequently further characterized at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit by direct sequencing or cloning. Only six of the isolated macroscopic sarcocysts belonged to S. buffalonis, whereas the others belonged to Sarcocystis fusiformis. Twelve of the smaller cysts belonged to S. levinei and seven to Sarcocystis sinensis. The characterization of the sarcocysts of S. sinensis and some of the sarcocysts of S. fusiformis have been reported before. Fifteen additional sarcocyst isolates of S. fusiformis were characterized at cox1 in the present study and found to be identical or closely similar to previous isolates. At cox1, the sequence identity between the six isolates of S. buffalonis was 99.8-100 % (two haplotypes), whereas the identity between the 12 isolates of S. levinei was 99.0-100 % (10 haplotypes). The identity between cox1 sequences of S. buffalonis and S. hirsuta (n = 56) was 92.9-93.6 % (on average 93.4 %), and the identity between cox1 sequences of S. levinei and S. cruzi (n = 22) was 92.9-94.0 % (on average 93.5 %). The phylogenetic analyses placed with high support the cox1 sequences of S. buffalonis and S. hirsuta into two monophyletic sister groups, and the same was true for the cox1 sequences of S. levinei and S. cruzi. Hence, the study established that S. buffalonis and S. levinei are distinct species different from S. hirsuta and S. cruzi, respectively. Nucleotide sequences of S. buffalonis could be distinguished from those of S. hirsuta also at the 28S rRNA gene (clearly different) and the ITS1 region (small and uncertain difference) but not at the 18S rRNA gene. Sequences of S. levinei could be distinguished from those of S. cruzi both at the 18S and 28S rRNA genes (ITS1 region not examined). However, the cox1 gene was superior to the 18S and 28S rRNA genes as regards the ability to unambiguously delimit the species within each species pair, since at the latter markers, the number of consistent nucleotide differences between the species was low and there was a slight overlap between the intraspecific and interspecific sequence divergence. Comparison of the newly generated 18S rRNA gene sequences of S. levinei from water buffaloes with similar sequences deposited in GenBank suggested that S. levinei and S. cruzi are not strictly intermediate host specific but might occasionally infect cattle and water buffaloes, respectively.

Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt.

A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15 dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.

MERS coronavirus neutralizing antibodies in camels, Eastern Africa, 1983-1997.

To analyze the distribution of Middle East respiratory syndrome coronavirus (MERS-CoV)-seropositive dromedary camels in eastern Africa, we tested 189 archived serum samples accumulated during the past 30 years. We identified MERS-CoV neutralizing antibodies in 81.0% of samples from the main camel-exporting countries, Sudan and Somalia, suggesting long-term virus circulation in these animals.

Domestic cats (Felis catus) are definitive hosts for Sarcocystis sinensis from water buffaloes (Bubalus bubalis).

The definitive hosts of Sarcocystis sinensis in water buffaloes have hitherto been unknown, but the close similarity of this species to the cat-transmitted Sarcocystis bovifelis in cattle suggested they were felids. In a previous study, two domestic cats were fed macroscopic sarcocysts of Sarcocystis fusiformis contained within or dissected from the esophageal muscles of water buffaloes, while no microscopic sarcocysts of S. sinensis were noticed. Both cats started shedding small numbers of sporocysts 8-10 days post infection (dpi) and were euthanized 15 dpi. Using a PCR-based molecular assay targeting the mitochondrial cox1 gene of S. fusiformis, both cats were shown to act as definitive hosts for this species. In the present study, DNA samples derived from oocysts/sporocysts in the intestinal mucosa of both cats were further examined by PCR for the presence of S. sinensis using 2 newly designed primers selectively targeting the cox1 gene of this species. All 6 DNA samples examined from each cat tested positive for S. sinensis. A 1,038-bp-long portion of cox1 was amplified and sequenced as 2 overlapping fragments from 5 of these DNA samples. The 5 sequences shared 99.3-100% identity with 7 previous cox1 sequences of S. sinensis obtained from sarcocysts in water buffaloes. Additionally, amplification of the ITS1 region with primers targeting various Sarcocystis spp., yielded amplicons of 2 different lengths, corresponding to those obtained from sarcocyst isolates of S. sinensis and S. fusiformis, respectively. This is the first study to show that cats act as definitive hosts for S. sinensis.

Serological detection and epidemiology of Neospora caninum and Cryptosporidium parvum antibodies in cattle in southern Egypt.

Neospora caninum and Cryptosporidium parvum are intracellular protozoan parasites that are distributed worldwide and of major economical concern in cattle industry. N. caninum can cause abortion storms and high culling rates, whereas C. parvum has zoonotic implications and can cause diarrhea in calves. There are currently no data on the prevalence of neosporosis and cryptosporidiosis in humans or animals in southern Egypt. Prevalence of these two infections was determined in a sample of cattle from two different areas in southern Egypt, Sohag and Qena, using enzyme-linked immunosorbent assay. A total 301 cattle were sampled, of which 18.9% were positive for N. caninum, 35.9% were positive for C. parvum and 10.0% were positive for both. Geographical location and breeding system were considered as potential risk factors for C. parvum infection. A higher prevalence of infection was identified on small scale farms, compared with larger, intensive systems, with a prevalence of 50.2% compared with 37.8%, respectively. Animals in Sohag had a significantly higher prevalence compared with Qena, with a seroprevalence of 46.1% compared with 31.6%, respectively. In brief, marked seroprevalence recorded in this study indicates a high incidence of N. caninum and C. parvum infections in cattle, and this necessitates the application of more effective strategies for combating these types of infections on farms in Egypt.

Monthly variations of Rhinoestrus spp. (Diptera: Oestridae) larvae infesting donkeys in Egypt: Morphological and molecular identification of third stage larvae.

This study was carried out to investigate the prevalence and monthly intensity of Rhinoestrus (R) spp. among donkeys slaughtered at Giza Zoo abattoir, Egypt. A total of 144 donkeys were examined at postmortem through two visits per month from January 2010 to December 2010. All donkeys were infested with one or more larval stages during all months of the examination period (100%). The 1st and 2nd stage larvae (L1 and L2) were mostly observed in the turbinate bones and seldom in the nasal passages, whereas the 3rd stage larvae (L3) were observed mostly in ethmoid and lamina cribrosa and rarely in nasal passages and pharynx. The highest monthly intensity of infestation with the total number of larval stages was recorded in January and August, while the lowest occurred in September. L1 was observed during all months with two peaks in January and June. L2 occurred from February to April, July, and August. L3 was present from March to May, August, and September. The ranked size of infestation with the total number of the 3 larval stages of Rhinoestrus spp. showed that a total of 107 donkeys had 1-10 larvae; 34 had 11-30 larvae; and 3 harbored 31-50 larvae. The morphology and molecular characterization of the third stage larvae of Rhinoestrus spp. were investigated. Morphologically, two morphotypes (1 and 2) of Rhinoestrus spp. (R. usbekistanicus like and the other R. purpureus like) were reported. Whereas molecular sequencing of mitochondrial cytochrome-oxidase subunit I showed 99% homology with those of R. usbekistanicus. In conclusion, Rhinoestrus spp. present in Egypt is mainly R. usbekistanicus, which includes two morphotypes, R. usbekistanicus like and R. purpureus like.