Molecular characterization of Leishmania species from stray dogs and human patients in Saudi Arabia.

Leishmania major and Leishmania tropica cause cutaneous leishmaniasis in humans and dogs in several parts of the world, with a large number of cases recorded in the Middle East. However, when they occur in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. To assess the frequency and to identify the species of Leishmania that infect humans and stray dogs in Riyadh and Al-Qaseem (Saudi Arabia), 311 stray dogs and 27 human patients who were suspected for Leishmania infection were examined for CL by a nested polymerase chain reaction (nPCR). Seven (25.9%) out of 27 human patients scored positive for Leishmania spp. (i.e., L. major in five patients from Riyadh and L. tropica in two patients from Al-Qaseem). Out of 311 dogs, five (1.6%) were infected by L. tropica. Data herein presented demonstrate the occurrence of L. tropica in dogs and humans in Saudi Arabia, as well as the occurrence of L. major in humans.

Molecular epidemiology of certain vector-borne bacterial microorganisms in domestic animals and their ectoparasites in Egypt.

Vector-borne bacterial diseases (VBBD) are a diverse group of tropical and subtropical zoonotic diseases. This study investigated the possibility of domestic animals to carry certain vector-borne bacterial microorganisms (VBBMs), as well as the presence of these targeted DNAs in their ectoparasites in different localities of Egypt using molecular analyses. For this study, 234 animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) in addition to 115 ectoparasites (95 ticks and 20 lice) were investigated for the molecular detection of Bartonella spp., Borrelia spp., and Rickettsia spp., targeting 16S-23S rRNAITS, 16S rRNA, and gltA genes, respectively. The results indicated that the overall prevalence of VBBD was observed in 17 animals (7.26%), of which 16 (6.84%) were positive for Bartonella spp. and one (0.43%) was positive for Borrelia theileri. All blood samples were negative for the DNA of Rickettsia spp. In addition, the results demonstrated that all ectoparasites were free from VBBDNA. Furthermore, of the animals examined for ectoparasite infestation, 28 (11.97%) and 5 (2.14%) represented Rhipicephalus annulatus ticks and Haematopinus tuberculatus lice, respectively, which infested animals. Analysis of epidemiological factors revealed that gender, age, and ectoparasitic infestation of animals had a significant effect on Bartonella infection, whereas no significant difference between animal species was observed. Hence, we report a potential novel Bartonella sp. from cattle and buffaloes, including a new genotype of Bo. theileri from cattle, in Egypt.

Morphological and molecular description of immature stages of Ornithodoros savignyi (Acari: Argasidae).

This study was designed to provide more details about larva, first nymph, and second nymph of Ornithodoros savignyi using a combination of light microscopy (LM), scanning electron microscopy (SEM), and partial sequence of mitochondrial 16S ribosomal ribonucleic acid (rRNA). The main characteristics of larva are wrinkled integument with many grooves, gnathosoma without camerostome cheeks, hypostome with a pair of large teeth apically, and tarsus without humps. The comparisons between the first and the second nymphs are different shape and distribution of dorsal grooves; a few spots without mammilla on the dorsal surface of the second nymph; 27 and 63-65 pairs of setae on the dorsal surface of the first and second nymphs, respectively; small holes on mammillae that are more dense in the second nymph; basis capitulum with two pairs of small setae in the second nymph; and one pair of sate in the first nymph, hypostome with dental formula 2/2 in the first nymph, and 3/3 in the second nymph. The partial 16S rRNA sequence of the second nymph that was determined as O. savignyi (450 bp) was deposited in GenBank under the accession number KU163242.

Molecular characterization of some equine vector-borne diseases and associated arthropods in Egypt.

Equine vector-borne diseases (EVBDs) are emerging and re-emerging diseases, and most of them are zoonotic. This study aimed to investigate EVBDs in equines and associated arthropods (ticks and flies) from Egypt using molecular analyses, in addition to a preliminary characterization of associated ticks and flies by the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and molecular techniques. In this study, 335 blood samples were obtained from equines that appeared to be in good health (320 horses and 15 donkeys) in Cairo and Beni Suef provinces, Egypt. From the same animals, 166 arthropods (105 sucking flies and 61 ticks) were collected. Ticks and flies were preliminary characterized by the MALDI-TOF and molecular tools. Quantitative PCR (qPCR) and standard PCR coupled with sequencing were performed on the DNA of equines, ticks, and flies to screen multiple pathogens. The MALDI-TOF and molecular characterization of arthropods revealed that louse fly (Hippobosca equina) and cattle tick (Rhipicephalus annulatus) infesting equines. Anaplasma platys-like (1.6%), Anaplasma marginale (1.6%), Candidatus Ehrlichia rustica (6.6%), a new Ehrlichia sp. (4.9%), and Borrelia theileri (3.3%) were identified in R. annulatus. Anaplasma sp. and Borrelia sp. DNAs were only detected in H. equina by qPCR. A. marginale, Anaplasma ovis, and Theileria ovis recorded the same low infection rate (0.6%) in donkeys, while horses were found to be infected with Theileria equi and a new Theileria sp. Africa with recorded prevalence rates of 1.2% and 2.7%, respectively. In conclusion, different pathogens were first detected such as A. platys-like, Candidatus E. rustica, and a new Ehrlichia sp. in R. annulatus; A. marginale, A. ovis, and T. ovis in donkeys; and a new Theileria sp. "Africa" in horses.

Evaluation of a vaccine candidate isolated from Cryptosporidium parvum oocyst in mice.

Background and Aim: Cryptosporidiosis is a leading cause of diarrheal disease worldwide and is an animal and public health burden. This study aimed to evaluate the protective potential of affinity-purified Cryptosporidium parvum oocyst antigen as a vaccine candidate according to fecal oocyst shedding, humoral and cellular immune responses, histopathological changes, and the number of parasite developmental stages in ileal and hepatic tissues. Materials and Methods: We isolated oocysts from naturally infected buffalo calves and identified them molecularly as C. parvum isolates (GenBank: ON730707 and ON730708) by targeting the Cryptosporidium oocyst wall protein gene. We propagated the C. parvum oocysts in mice. In addition, we prepared crude antigen from the isolated oocysts by purification using cyanogen bromide-activated Sepharose-4B affinity chromatography coupled with rabbit hyperimmune serum. Then, we divided 81 parasite-free mice into three groups: (1) non-vaccinated non-infected mice, (2) mice orally infected with 1 × 105 C. parvum oocysts on week 4 of the experiment, and (3) mice immunized twice with 40 µg/kg of the purified fraction at 2-week intervals. Then, we challenged the vaccinated group with C. parvum oocysts after 2 weeks, and the positive control group was infected at the same time. Results: We observed a prolonged prepatent period and decreased oocyst shedding in the vaccinated infected mice compared with the non-vaccinated infected mice (t < 0.001). The vaccinated mice had significantly higher immunoglobulin G levels than those in the other two groups at all examined weeks. In addition, the production of cytokines interferon-gamma, interleukin (IL)-10, IL-12, and IL-15 was activated post-vaccination. After the challenge, all tested cytokines were significantly increased (p < 0.001) in the two infected groups compared with the non-vaccinated non-infected group, with the highest levels in the vaccinated infected group. Vaccinated infected mice exhibited significantly fewer pathological lesions in the ileum and liver than non-vaccinated infected mice, which showed prominent histopathological lesions. Endogenous developmental stages of C. parvum indicated that the ileum was more parasitized than the liver and that vaccination resulted in a lower number of oocysts in ileal and hepatic tissues (p < 0.05). Conclusion: Our prepared affinity-purified vaccine candidate could be promising in protecting against cryptosporidiosis.

Emergent and Neglected Equine Filariosis in Egypt: Species Diversity and Host Immune Response.

Equine filariosis (EF) is a neglected vector-borne disease caused by nematode species belonging to the Onchocercidae and Setariidae families. Aside from their zoonotic potential, some species are responsible for serious health problems in equids worldwide, leading to significant economic difficulties. Here, we molecularly investigated equine blood samples (320 horses and 109 donkeys from Egypt) and four adult worms isolated from the peritoneal cavity of 5 out of the 94 slaughtered donkeys. In addition, quantitative enzyme-linked immunoassays (ELISAs) targeting circulating cytokines were used to identify whether the immunological profile of the infected animals is a Th1 (i.e., INF-gamma as indicator) or Th2 (i.e., IL-5 and IL-10 as indicators) response type. Overall, 13.8% and 0.3% of the donkeys and horses, respectively, were scored as positive for filaroid DNA. The 18S phylogeny revealed the occurrence of three different filaroid species, identified here as Mansonella (Tetrapetalonema) sp., Setaria digitata and Dirofilaria repens. Th1 (INF-gamma and IL-5) and Th2 (IL-10) immune response types were identified in equines infected with S. digitata and Mansonella (T.) sp., respectively. These results provide new data on the species diversity of EF in Egypt and extend knowledge of the downregulation of the protective immune response by the potentially zoonotic Mansonella (T) sp. There is an urgent need to implement control measures to preserve equine health and limit the propagation of these vector-borne filaroids in Egypt.

Molecular Epidemiological Investigation of Piroplasms and Anaplasmataceae Bacteria in Egyptian Domestic Animals and Associated Ticks.

Piroplasmosis and anaplasmosis are serious tick-borne diseases (TBDs) that are concerning for the public and animal health. This study aimed to detect the molecular prevalence and epidemiological risk factors of Piroplasma and Anaplasma species in animal hosts and their associated ticks in Egypt. A total of 234 blood samples and 95 adult ticks were collected from animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) from six provinces of Egypt (AL-Faiyum, AL-Giza, Beni-Suef, Al-Minufia, Al-Beheira, and Matruh). Blood and tick samples were investigated by polymerase chain reaction coupled with sequencing targeting 18S and 16S RNA genes for Piroplasma and anaplasmataceae, respectively. Statistical analysis was conducted on the potential epidemiological factors. Of the 234 animals examined, 54 (23.08%) were positive for pathogens DNA distributed among the six provinces, where 10 (4.27%) were positive for Piroplasma, 44 (18.80%) for anaplasmataceae, and 5 (2.14%) were co-infected. Co-infections were observed only in cattle as Theileria annulata and Anaplasma marginale plus Babesia bigemina, A. marginale plus B. bigemina, and T. annulata plus B. bigemina. Piroplasmosis was recorded in cattle, with significant differences between their prevalence in their tick infestation factors. Animal species, age, and tick infestation were the potential risk factors for anaplasmosis. All ticks were free from piroplasms, but they revealed high prevalence rates of 72.63% (69/95) with anaplasmataceae. We identified T. annulata, B. bigemina, and A. marginale in cattle; A. platys in buffaloes; A. marginale and A. ovis in sheep; for the first time, A. ovis in goats; and Ehrlichia sp. in Rhipicephalus annulatus ticks. Our findings confirm the significant prevalence of piroplasmosis and anaplasmosis among subclinical and carrier animals in Egypt, highlighting the importance of the government developing policies to improve animal and public health security.

Multiple vector-borne pathogens of domestic animals in Egypt.

Vector Borne Diseases (VBDs) are considered emerging and re-emerging diseases that represent a global burden. The aim of this study was to explore and characterize vector-borne pathogens in different domestic animal hosts in Egypt. A total of 557 blood samples were collected from different animals using a convenience sampling strategy (203 dogs, 149 camels, 88 cattle, 26 buffaloes, 58 sheep and 33 goats). All samples were tested for multiple pathogens using quantitative PCR and standard PCR coupled with sequencing. We identified Theileria annulata and Babesia bigemina in cattle (15.9 and 1.1%, respectively), T. ovis in sheep and buffaloes (8.6 and 7.7%, respectively) and Ba. canis in dogs (0.5%) as well as Anaplasma marginale in cattle, sheep and camels (20.4, 3.4 and 0.7%, respectively) and Coxiella burnetii in sheep and goats (1.7 and 3%; respectively). New genotypes of An. centrale, An. ovis, An. platys-like and Borrelia theileri were found in cattle (1.1,3.4, 3.4 and 3.4%, respectively), An. platys-like in buffaloes (7.7%), An. marginale, An. ovis, An. platys-like and Bo. theileri in sheep (3.4, 1.7, 1.7 and 3.4%, respectively), An. platys, An. platys-like and Setaria digitata in camels (0.7, 5.4 and 0.7%, respectively) and Rickettsia africae-like, An. platys, Dirofilaria repens and Acanthocheilonema reconditum in dogs (1.5, 3.4, 1 and 0.5%, respectively). Co-infections were found in cattle, sheep and dogs (5.7, 1.7, 0.5%, respectively). For the first time, we have demonstrated the presence of several vector-borne zoonoses in the blood of domestic animals in Egypt. Dogs and ruminants seem to play a significant role in the epidemiological cycle of VBDs.

A report on tick burden and molecular detection of tick-borne pathogens in cattle blood samples collected from four regions in Saudi Arabia.

Babesiosis, theileriosis and anaplasmosis are among the most commonly reported tick-borne diseases in cattle and are associated with significant economic losses. Through the present study the researchers aimed to report the presence of various pathogens that cause babesiosis, theileriosis and anaplasmosis in cattle collected from different provinces in Saudi Arabia and to report their phylogenetic relationship. A total of 362 blood samples of cattle along with ticks that were present on the cattle were collected from four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia. Blood samples were screened by polymerase chain reaction (PCR) for the presence of various Babesia, Theileria and Anaplasma species by amplification of their 18S rRNA and/or 23S rRNA genes. A total of 541 ticks were collected and identified from the cattle. These included Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma excavatum, Rhipicephalus annulatus and Rhipicephalus turanicus. Regarding tick-borne pathogens, the overall prevalence was 1.9 % (7/362) for Theileria annulata, (2/362) 0.6 % for Theileria and (21/362) 5.8 % for Anaplasma ovis. Four of the cattle were found to be co-infected with more than one pathogen (1.1 %). We did not detect any Babesia species in the blood of the studied cattle. Prevalence of the Theileria and Anaplasma species was highest in cattle that resided in Riyadh, followed by cattle from Al-Hasa and Al-Qassim. Representative amplified partial-gene sequences of T. annulata (GenBank accession numbers MK826137-39) and A. ovis (GenBank acc. no. MK 880224) were submitted to GenBank. The presence of ticks on cattle was found to be associated with a high prevalence of Theileria spp. (P = 0.02) and Anaplasma ovis (P < 0.001). We report novel genotypes of T. annulata and A. ovis from cattle in Saudi Arabia and we recommend that molecular surveys are undertaken throughout the country to address the prevalence and geographical distribution of tick-borne infections for their effective diagnosis and treatment.

Molecular Survey of Vector-Borne Pathogens of Dogs and Cats in Two Regions of Saudi Arabia.

Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.

Q fever: A neglected disease of camels in Giza and Cairo Provinces, Egypt.

BACKGROUND AND AIM: Q fever is a zoonotic disease caused by Coxiella burnetii. Cattle, sheep, and goat are the main reservoir of C. burnetii. In Egypt, the epidemiological data about C. burnetii in camels are limited. Therefore, the current study was conducted to identify C. burnetii infection in camels by different molecular tools and to estimate its seropositivity through the detection of anti-C. burnetii antibodies in camel sera. MATERIALS AND METHODS: Blood samples were collected 112 from camels in Giza and Cairo Provinces, Egypt. All blood samples were screened by trans-quantitative polymerase chain reaction (trans-qPCR) for C. burnetii and positive samples subjected to standard PCR using the superoxide dismutase enzyme coding gene of C. burnetii. Sera of studied camels were examined for the presence of antibodies against C. burnetii using enzyme-linked immunosorbent assay. RESULTS: Out of 112 camels, 19 were positive for C. burnetii by qPCR with an overall prevalence of 16.9% (18.6% in Giza and 15.1% in Cairo Provinces, respectively). The seroprevalence of anti-C. burnetii IgG antibodies in the examined camels was 4.5% (5/112). CONCLUSIONS: Trans-qPCR assay is a rapid and sensitive tool for the detection of C. burnetii in acute stage. Camels should be considered one of the major reservoirs for C. burnetii in Egypt.

Molecular and immunological characterization of Hyalomma dromedarii and Hyalomma excavatum (Acari: Ixodidae) vectors of Q fever in camels.

BACKGROUND AND AIM: Q fever Coxiella burnetii is a worldwide zoonotic disease, and C. burnetii was detected in mammals and ticks. Ticks play an important role in the spread of C. burnetii in the environment. Therefore, the aims of this study were to detect Q fever C. burnetii in camels and ixodid ticks by molecular tools and identification of Hyalomma dromedarii and Hyalomma excavatum using molecular and immunological assays. MATERIALS AND METHODS: A total of 113 blood samples from camels and 190 adult ticks were investigated for the infection with C. burnetii by polymerase chain reaction (PCR) and sequencing the targeting IS30A spacer. The two tick species H. dromedarii and H. excavatum were characterized molecularly by PCR and sequencing of 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit-1 (CO1) genes and immunologically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. RESULTS: A total of 52 camels (46%) were positive for Q fever infection. Only 10 adult ticks of H. dromedarii were infected with C. burnetii. The IS30A sequence was around 200 bp in length for C. burnetii in H. dromedarii ticks with a similarity of 99% when compared with reference data in GenBank records. The length of 16S rDNA and CO1 was 440 and 850 bp, respectively, for both H. dromedarii and H. excavatum. The phylogenetic status of H. dromedarii was distant from that of H. excavatum. SDS-PAGE revealed seven different bands in the adult antigens of either H. dromedarii or H. excavatum with molecular weights ranged from 132.9 to 17.7 KDa. In western blot analyses, the sera obtained from either infested camel by H. dromedarii or infested cattle by H. excavatum recognized four immunogenic bands (100.7, 49.7, 43.9, and 39.6 kDa) in H. dromedarii antigen. However, the infested camel sera identified two immunogenic bands (117 and 61.4 kDa) in H. excavatum antigen. Furthermore, the sera collected from cattle infested by H. excavatum recognized three immunogenic bands (61.4, 47.3, and 35 kDa) in H. excavatum antigen. CONCLUSION: Molecular analyses indicated that both camels and ticks could be sources for infection of animals and humans with Q fever. Furthermore, the molecular analyses are more accurate tools for discriminating H. dromedarii and H. excavatum than immunological tools.

Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt.

AIM: Rickettsioses have an epidemiological importance that includes pathogens, vectors, and hosts. The dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii play important roles as vectors and reservoirs of Rickettsiae. The aim of this study was to determine the prevalence of Rickettsiae in ixodid ticks species infesting dogs and camels in Egypt, in addition to, the morphological and molecular identification of R. sanguineus and H. dromedarii. MATERIALS AND METHODS: A total of 601 and 104 of ticks' specimens were collected from dogs and camels, respectively, in Cairo, Giza and Sinai provinces. Hemolymph staining technique and OmpA and gltA genes amplification were performed to estimate the prevalence rate of Rickettsiae in ticks. For morphological identification of tick species, light microscope (LM) and scanning electron microscope (SEM) were used. In addition to the phylogenetic analyses of 18S rDNA, Second internal transcript spacer, 12S rDNA, cytochrome c oxidase subunit-1, and 16S rDNA were performed for molecular identification of two tick species. RESULTS: The prevalence rate of Rickettsiae in ticks was 11.6% using hemolymph staining technique and 6.17% by OmpA and gltA genes amplification. Morphological identification revealed that 100% of dogs were infested by R. sanguineus while 91.9% of camels had been infested by H. dromedarii. The phylogenetic analyses of five DNA markers confirmed morphological identification by LM and SEM. The two tick species sequences analyses proved 96-100% sequences identities when compared with the reference data in Genbank records. CONCLUSION: The present studies confirm the suitability of mitochondrial DNA markers for reliable identification of ticks at both intra- and inter-species level over the nuclear ones. In addition to, the detection of Rickettsiae in both ticks' species and establishment of the phylogenetic status of R. sanguineus and H. dromedarii would be useful in understanding the epidemiology of ticks and tick borne rickettsioses in Egypt.