الفهرس 
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Abstract
Flea-borne pathogens are diseases transmitted through fleas and act as agents that cause disease in a host. This study was conducted in Egypt’s Northern West Coast (NWC) and South Sinai Governorate (SSG). Five big cities from both areas were selected (Mersa Matruh, Sidi Berrani, and Maryout from the NWC area, and El Tur and Ras Sudr from the SSG area).
The present study aimed to characterize those pathogens molecularly using samples from both sexes and flea subspecies to identify helminths, protozoa, rickettsia, bacteria, and viruses in flea-infested species of small ruminants in the study areas. Also, it extended to the geographical distribution of different pathogens and their association with subspecies of fleas.
The study was performed in two steps, primarily with a survey of flea-infested sheep and goats, and ended with the genotyping of flea-borne pathogens present.
For the survey:
A total of 765 animals, including 460 sheep and 305 goats, were selected for flea investigations from April 2020 to September 2021. There were 471 from NWC and 294 from SSG. Their ages ranged from 9 months to 5 years. It was divided into two groups (less than three years and more than three years).
Of those animals, 265 goats and 390 sheep were female, whereas 40 goats and 70 sheep were male. Also, 1009 fleas were collected from infested animals in the two areas to identify parasitic, rickettsial, and viral infections.
Furthermore, 223 fleas were randomly collected from 249 sheep in the NWC area to investigate some bacterial infections that needed to be cultured in suitable media before PCR.
Microscopical identification of fleas-infesting small ruminants in the two studied areas was performed using a stereoscopic microscope SZ-PT-40 (Olympus, Japan), according to Skuratowicz key.
Data statistical analysis of the obtained data was analyzed by SAS, 2004.
Results of the present survey showed that:
Fleas were found in animals with highly significant differences (P < 0.0001) throughout the year. The infestation rate in the rainy season in December and January was 13.8%, while it increased during the dry season from March to October (86.2%).
Hosts and place of collection on the animal have no statistically significant differences (P > 0.5).
Location and season of flea collection were highly significant risk factors for the infection (P = 0.0001).
Sheep were more preferable hosts for flea infestations (58.9%) than goats (41.1%) in the two studied areas.
Most fleas were collected signicantly around the sexual organs and on the head (54.6%), compared to the neck and ventral of the animal (25.7%) and, to a lesser extent, the legs and tail (19.7%), and with no significant difference (P> 0.05) between the two studied areas.
The infestation rates are not significantly different (P > 0.05) between both sexes of animals. Of the 511 infested animals, 66.3% of females and 70.0% of males’ harbored fleas. Whereas female sheep recorded the highest rate in NWC and the lowest rate in SSG, male goats recorded the highest infestation values compared to male sheep in the two areas.
The older animals have higher infestation rates than the younger ones in both areas, except older sheep in the SSG area have the lowest infestation rate (31.25%).
Ctenocephalides were the only ones collected from sheep and goats in the two different localities under microscopical examination.
Data analysis showed a highly significant difference between C. felis and C. canis infestations in the two areas (P = 0.00024).
Totally, the cat flea C. felis was more abundant (52.8%) than the dog flea C. canis (47.18%). Whereas, C. felis was more prevalent in the NWC area (57.7%) than C. canis (42.3%), C. canis was more common (54.4%) compared to C. felis (45.6%) in the SSG area.
Males of the two flea species were more prevalent than females in the two areas, with a significant difference among the sexes of both flea species in the NWC area only (P = 0.004).
For molecular characterization of flea-borne pathogen:
Molecular identification of flea-borne pathogens in infested small ruminants was performed by different PCR-based assays using different species-specific primers.
Gene sequencing and phylogenetic analysis of the identified pathogens were carried compared to that maintained in the GenBank database.
The obtained accession numbers were recorded in the GenBank database.
Results of molecular characterization of pathogens
A- Flea-borne helminths
Four DNAs extracted from four pooled samples collected from two various geographic locations in the NWC and SSG, Egypt, were used to determine the ITS-2 and 18S rDNA sequences.
For trematodes, four generated bands of 539 bp were identified from all specimens as Fasciola hepatica; their sequences had identities ranging from 83.9 to 100% and genetic distances of 0.0 to 14.5.
The alignment of all ITS-2 sequences with the reference sequences of Fasciola spp. in the GenBank database revealed four different nucleotide substitutions at the nucleotide positions.
For nematodes, four bands of 900 bp were generated from all specimens, and the alignment of all 18S rDNA sequences with the reference sequences showed 45 specific nucleotides for two distinct pathogen genotypes, Haemonchus contortus, and Trichostrongylus colubriformis.
For T. colubriformis, the sequence identities ranged from 78.6 to 85.2%, and the genetic divergence was between 0.0 and 14.8.
The three H. contortus isolates are typically 100% similar to each other. The genetic similarity between those isolates and others in GenBank ranged from 81.3 to 100%, and the genetic divergence was 0 to 1.9.
Two trees were constructed using sequences of nematodes publicly available under accession numbers ON113484, ON113485, and ON113486 (for H. contortus), and ON113498 (for T. colubriformis). Whereas ITS-2 sequences for trematodes under accession numbers ON123996, ON123994, ON123995, and ON123993 (for F. hepatica).
B- Flea-borne protozoa
Eight protozoon parasite DNAs were amplified for their presence in four samples containing flea-extracted DNA. Those protozoon parasites were: Babesia, Theileria, Cryptosporidium, Sarcocystis, Neosporum caninum, Giardia intestinalis, Haemoproteus, Plasmodium, and Leucocytozoon.
The results revealed one generated product for Cryptosporidium and the absence of the others. It amplified specific bands at 553 bp in the flea-extracted DNA that infested sheep in the two areas, whereas it was absent in the flea-infested goats. It was detected in both sexes of C. felis and C. canis.
The sequence analysis of the PCR products identified three typical C. parvum strains in flea-infested sheep at both locations. It exhibited a genetic similarity of 100% with the reference sequences in GenBank, and divergence ranged from 0 to 0.2. Their identities range from 77.2% to 100% and their genetic distances range from 0.0 to 27.4 compared to other GenBank strains.
Analysis of the three generated fragments showed sequences with accession numbers: ON081033 isolated from both sexes of C. felis and ON081034 and ON081035 isolated from both sexes of C. canis.
It clustered with some Egyptian isolates from Sakha in Kafr el-Sheikh Governorate (AB514059, AB514061, AB514046, and AB514045) and others maintained in GenBank [(KY706489 (Italy), DG388390 (The Netherlands), AB0892292 (Japan), BX538351 (United Kingdom), and Z22537 (Italy)].
C- Flea-borne Rickettsia
Gram-negative bacteria called rickettsia require arthropods as either reservoirs or vectors since they can only replicate inside their host cells. We amplified and sequenced the 16S rRNA of every Rickettsia bacterium that was available using the polymerase chain reaction and DNA sequence analysis.
Rickettsia sp. was detected only in two samples of male C. canis collected from sheep in the two studied areas by PCR (one for Rickettsia and one for Anaplasma).
Molecular analysis identified the gene band of 533 bp of Rickettsia as R. massiliae under accession number ON076427. It shared an identity of 100% with two accession numbers (CP000683 and NR025919) submitted from France.
The similarity between R. massiliae in the present study and the preserved isolates in GenBank ranged from 97.8% to 100%, whereas the genetic distance drifted from 0 to 3.6.
Anaplasma was identified as A. marginale under accession number ON081028. It displays high homogeneity (100%) with three reference strains (M93392, CP001079, and CP000030) isolated from cattle in the USA, but with identities ranging from 93.8% to 100% with the other known strains reported to date, with genetic distances from 0.0 to 11.
D- Flea-borne viruses
Until now, there has been no proof that fleas handle this way of transmission. Therefore, unless proven otherwise, they may serve as virus vectors, even though the data showed that PCR amplification had not occurred or had not created bands at the predicted level.
PCR-based assays targeting the ORF103, NS3, and Rdrp genes were used for identifying Capripox, Bluetongue, and Coronaviruses, respectively.
The findings showed that the Bluetongue, Corona viruses, and Lumpy Skin Disease virus were absent and generated zero bands at 670 bp and 251 bp, respectively. It also supported the hypothesis that Ctenocephalides play an adverse role in the spread of coronavirus.
Capripox virus (CPV) was only detected in four isolates from all sexes of the two Ctenocephalides species that produced four bands at 570 bp after PCR amplification.
Phylogenetic analysis of our isolates of Sheeppox virus revealed that samples from sheep collected in China, Saudi Arabia, and Egypt and kept in the GenBank database were 100% identical. Their sequences were recorded under accession numbers ON081029, ON081030, and ON081031.
The Goat pox virus isolate is 100% inappreciable to a different Egyptian reference strain recovered only from goats and registered in GenBank under accession number MW546999. It also shared a close relationship with AY077836 from goats in Kazakhstan, MN072622 from Turkey, and KX576657 from a lamb in Italy.
E- Flea-borne bacteria
Arthropod-borne transmission of bacterial infections is still uncommon, although Yersinia pestis, gram-negative bacteria, and other bacterial diseases have developed in phylogenetically distinct families. This part was carried only in flea-infested sheep in NWC, and some Rickettsia and bacterial genera may have been removed through sample preparation or not in the present study.
Helicobacter felis, Staphylococcus, Enterococcus, Yersinia pestis, and Campylobacter sp. were detected negatively in both flea species, whereas others were amplified by specific bands targeting their genes, such as Mycobacterium, Mycoplasma, and Pseudomonas.
Flea-infested sheep harbored Mycobacterium bovis and mycoplasmas, with sequence-significant data identities of >90%-100% and >82%-100%, respectively, compared to archived genes.
Phylogenetically, the present isolation of Mycobacterium bovis (ON076426) was grouped in one clade with seven preserved strains in GenBank with 100% identity. Of those, CP039850 and CP039851 (Belgium), CP040832 (Brazil), and CP033311 (South Africa) were isolated from Tapirus terrestris, Homo sapiens, animal organs, and fleas.
The present study also showed genetic diversity within Mycoplasma species clustered in three clades. One contained the first sequence of 16S rRNA under accession number ON076423 (M. agalactiae), which is 100% identical to that kept in GenBank; AF332751, AF332748 (Sweden), and NR114847 (USA).
The second cluster contained the accession number of 16S rRNA, ON076424 (M. arginini), which was 100% identical to LR215044 (United Kingdom), NR041743 (Sweden), and JN935883 (USA).
The third sequence of 16S rRNA under accession number ON076425 (M. ovipneumoniae) in a separate cluster is 100% identical to LR215028 (United Kingdom), KJ433280 (Norway), NR025989 (Sweden), and EU265779 (western North America) isolated from different sheep.
Our research reveals the ability of the 16S rRNA gene-based microbiome method to identify Pseudomonas, even at low concentrations. However, PCR amplification of Pseudomonas infections in the DNA of pooled cultured samples from infested sheep showed it was present in all isolates and all linked to environmental pollution.
Gene sequencing and phylogenetic analysis revealed two genotypes from the submitted isolates in two distinct clusters: one contained two Pseudomonas aeruginosa isolates, and the second included Pseudomonas fragi isolates.
The two isolates of Pseudomonas aeruginosa fall into one cluster with those of identity 100% under accession numbers: CP045002, CP034908, CP021380, and MN911373, isolated from ciliated protozoa in China. They also clustered with two previously identified P. mosselii strains with the accession numbers JF904871 and MK026781.
The identified P. fragi expressed in GenBank under accession number ON100875 and grouped with another five identical strains under accession numbers: MT631986, MN062088 from China, MH463556, MH463554 from India, and MH012195 isolated from the Ficus carica plant in Iran.