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.
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.
Vector Borne Diseases (VBDs) are emerging and re-emerging infectious diseases, that pose a health threat to humans, livestock, companion animals and wildlife [1]. VBDs are a global burden and cause severe economic losses through high mortality rates and production declines in the livestock industry, as well as impacts on human and animal health [2,3]. Moreover, about a quarter of vertebrate pathogens of veterinary importance are VBDs [4]. The World Organization for Animal Health (OIE) list includes many VBDs such as piroplasmoses, anaplasmoses and Q fever. The epidemiology and spread of VBDs are influenced by various factors such as globalization and increasing international trade, urbanization, climate change, travel and mobility of animals which pose unprecedented challenges to clinicians and veterinarians [5-6].Piroplasmoses are tick-borne infectious diseases caused by apicomplexans of the order Piroplasmida, which includes three genera namely: Theileria, Babesia and Cytauxzoon [7]. Theileria annulata, T. ovis and Babesia bigemina are etiological agents of tropical theilerioses and babesiosis in ruminants especially cattle, buffalo and sheep [8]. Similarly, Ba. canis and Ba. vogeli are the main causative agents of canine babesiosis [9]. Piroplasmoses are common in Asia, Southern Europe and Africa [10]. The main clinical signs of piroplasmoses are fever and hemolytic anemia and deaths of up to 50% in the case of acute infection in susceptible herds [11,12]. Recovered animals may become asymptomatic carriers with long-term persistent infection [13,14]. Piroplasmoses have been detected in several provinces of Egypt and are widespread [15-18].Anaplasmataceae include many tick-borne bacteria that infect mammals and consist of at least five genera: Anaplasma, Ehrlichia, Neoehrlichia Neorickettsia, and Aegyptianella [19-20]. Bovine anaplasmosis caused by Anaplasma marginale and An. centrale mainly in tropical and subtropical regions cause mild to severe anemia in ruminants [20,21]. Ovine anaplasmosis is a neglected mild disease in sheep, goats and wild ruminants caused by An. ovis and is common in different areas of the world [22,23]. In addition, there are many Anaplasmataceae bacteria pathogenic to dogs, such as An. platys and Ehrlichia canis [24,25]. Overall, these bacteria could cause persistent infection in mammals making them reservoir, which has lasting effect on the spread and new outbreaks of anaplasmosis [26,27]. In Egypt, anaplasmosis has been reported in cattle, water buffaloes and camels in different provinces [16,28-34].Rickettsioses are bacterial infectious diseases that cause health problems in humans and animals worldwide [35,36]. Rickettsiae are divided into spotted fever group (SFG; mainly transmitted by ticks), typhus group (TG; transmitted by lice and fleas), Rickettsia belli group and Rickettsia (R.) candensis group [37]. R. africae is the most common rickettsial species in Africa that causes African tick-borne fever in humans [38]. Other rickettsiae such as R. aeschlimannii, R. conorii and R. sibirica mongolitimonae, R. massiliae have been detected in ticks and animals in Africa [39-43]. In Egypt, SFG have been identified in vectors, animals and humans since 1989 [44-48]. SFG rickettsiae were found in ticks (Hyalomma sp. and Rhipicephalus sanguineus) collected in Sinai province [49-51]. Moreover, R. siberica mongolitimonae was detected in a French traveler returning from Egypt [52]. Finally, R. africae was detected by molecular biology in Hyalomma sp. and camels [53-55].Borrelioses are zoonotic infectious diseases and are divided into two groups: Lyme disease group (caused by Borrelia burgdorferi and related species) and relapsing fever group [56]. Relapsing fever borrelioses are arthropod-borne spirochetal diseases, usually transmitted by soft ticks; they are common in subtropical regions worldwide [57]. In Africa, relapsing fever is most common in the northern hemisphere and is caused by various Borrelia spp. such as Bo. hispanica, Bo. duttonii, and Bo. crocidurae [57-60]. Bo. theileri is the etiological agent of bovine borreliosis in ruminants, which causes anemia and fever and, unlike other members of the relapsing fever spirochetes, is transmitted by hard ticks [58]. In Egypt, data on borrelioses in animal hosts are sparse. Only the few studies have detected Bo. burgdorferi [61,62] and Bo. theileri in hard ticks [62].Q fever is a zoonosis that infects humans and animals through direct contact or a tick bite [63]. Coxiella burnetii is the causative agent of Q fever that may be severe in humans [64]. Infection in animals it is usually subclinical except that reproductive diminution and abortions may occur [65]. Coxiella burnetii infects a wide range of animals, especially sheep, goats, cattle and camels, which serve as reservoirs [64,66]. In Egypt, the seroprevalence of C. burnetii was estimated in buffaloes, sheep, cattle and camels [67-70]. In addition, C. burnetii has been detected molecularly in goats, camels and ticks (H. dromedarii) [70-72].Filarial nematodes are vector-borne helminths belonging to the order Spiruridae, suborder Spirurina and families Filariidae and Onchocercidae and pose a serious threat to humans and livestock [73,74]. Dirofilaria repens and D. immitis, followed by Acanthocheilonema sp. are the most important etiological agents of filarial infections in dogs [9,73,75]. Setaria digitata is a filarial nematode of cattle and buffaloes and is not pathogenic to these natural hosts, but when transmitted by mosquitoes to accidental hosts such as camels and horses, it can have serious pathological effects [76,77]. In Egypt, information on filarial infections in ruminants and dogs are scarce. In Africa, there are some reports of filarial infections in different places of the continent [78-80].Diagnosis of all these diseases is challenging due to the non-specific febrile illness, difficulty in isolation and cross reactivity of serological methods [35,59]. Therefore, the advanced molecular techniques have been used to increase the sensitivity and specificity of diagnosis, to detect previously unknown pathogens and distinguish closely related species [5]. In Egypt, the epidemiology and prevalence of these diseases remain neglected and poorly understood. To date, few studies have been conducted on individual VBDs in vectors or animal hosts. Here, we provide the first data for molecular screening and characterization of multiple vector-borne pathogens in different animal hosts to better understand the epidemiological approach of VBDs in Egypt.
Materials and methods
Ethical approval
This study was approved by the Medical Research Ethics Committee at the National Research Centre, Egypt with the number 19058.
Study area and samples collection
We conducted a cross-sectional observational study with a total of 557 apparently healthy domestic animals (203 dogs, 149 camels, 88 cattle, 26 buffaloes, 58 sheep and 33 goats) using a convenience sampling strategy [81]. Animal blood samples were randomly collected from different provinces in Egypt between 2016 and 2018. The details of the sample locations were presented in Fig 1 and Table 1. For each animal host, 5 ml of blood was collected in a sterile EDTA tube using a sterile syringe and stored at -20°C for molecular purposes. The prevalence of infection of different pathogens by different animal hosts was calculated according to Thrusfield et al. [81].
Fig 1
Map of Egypt showing the different provinces where the blood samples from different animal hosts were collected for our study.
https://en.wikipedia.org/wiki/Governorates_of_Egypt and the picture has CC BY-SA 3.0.
Table 1
The information data of collected samples.
Provinces
Geographic coordinates
Animal Hosts
Locations
Numbers of Animals
Cairo
30° 03’ 45.47" N, 31° 14’ 58.81" E
Dog
Police Academy (El-Abbasia)
75
Police Academy (El-Tagamoa)
67
Police Academy (El-Dowaika)
61
Camel
Police Academy (Gasr-El Swiss)
52
Giza
29° 58’ 27.00" N, 31° 08’ 2.21" E
Camel
Police Academy (El-Haram)
96
sheep
households
5
Goat
households
6
Beni-Suef
29° 03’ 60.00" N, 31° 04’ 60.00" E
Cattle
households
63
Sheep
households
48
Goat
households
20
Buffalo
households
20
Qalyubia
30.41°N, 31.21°E
Cattle
households
2
Buffalo
households
6
Goat
households
2
Sinai
28° 32’ 13.79" N, 33° 58’ 14.39" E
Sheep
households
5
Goat
households
5
Camel
Free rearing
1
El-Wady El-Geded
24°32′44″N, 27°10′24″E
Cattle
households
11
Qena
26° 09’ 60.00" N, 32° 42’ 59.99" E
Cattle
households
10
Beheira
30.61°N, 30.43°E
Cattle
households
2
Map of Egypt showing the different provinces where the blood samples from different animal hosts were collected for our study.
https://en.wikipedia.org/wiki/Governorates_of_Egypt and the picture has CC BY-SA 3.0.
DNA extraction
DNA was extracted from 200 μl of each blood sample using EZ1 DNA Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at -20°C until use for molecular screening.
Screening of multiple pathogen DNA by qPCR
All samples were first screened for pathogen DNA by qPCR using genus-specific primers and probes targeting the 5.8S rRNA gene of piroplasms, the 23S rRNA gene of Anaplasmataceae, the gltA gene of Rickettsia sp., the 16S rRNA gene of Borrelia sp., the IS1111 of C. burnettii, BartoITS3 of Bartonella sp. and the pan-fil 28S rRNA gene of Filariidae. For positive Filariidae in dog samples, a triplex qPCR targeting Cox1 was used to detect D. immitis, D. repenes and Ac. reconditum. The sequence of primers and probes used in this study is showed in Table 2. The qPCR was preformed using a CFX 96 Real Time System (Bio-Rad Laboratories, Foster City, CA, USA). The total reaction volume of 20 μl included 10 μl of Eurogentec Master Mix Roche, 0.5 μl of each primer, 0.5 μl of FAM-labeled probe, 0.5 μl of UDG, 5 μl of DNA template, and 3 μl of DNAse- and RNAse-free water. Thermal cycling was performed according to the instructions provided by the manufacturer of the Master Mix PCR kit. To evaluate the PCR reaction, a positive control (pathogen DNA) and a negative control were added to each reaction. The sample was considered positive if the cycle threshold (Ct) was less than 35 Ct [82].
Table 2
Primers and probes used for qPCR, Standard PCR and sequencing in this study.
All samples considered positive by qPCR were subjected to standard PCR and sequencing. Primers targeting 969 bp and 1200 bp region of the 16S rRNA gene, respectively, were used to identify Piroplasma and Borrelia. For the identification of Anaplasmataceae, standard PCR were performed with primers targeting a 520 bp fragment of the 23S rRNA gene. The positive samples with 23S rRNA gene were confirmed with Anaplasma genus-specific primers targeting the 525 bp fragment of the rpoB gene. Rickettsia genus-specific primers targeting the gltA gene were used and the positive samples were confirmed by the ompB gene. Moreover, multi-spacer typing (MST) for C. burnetii was performed by amplifying of three intergenic spacers (Cox2, Cox5 and Cox18). Identification of Filariidae was performed using 18S rRNA primers targeting 1155 bp. All primer sequences used in standard PCR and sequencing are listed in Table 2. All PCR reactions were performed in an Applied Biosystems 2720 Thermal Cycler model (Thermo Fisher Scientific Courtaboef, France) using AmpliTaq 360 Master Mix (Thermo Fisher Scientific Courtaboef, France) according to the manufacturer’s recommendations. Negative and positive controls were included in each reaction. PCR products were visualized by electrophoresis on a 1.5% agarose gel stained with Syper Safe stain (Invitrogen, USA) and analyzed using Lab Image software (BioRad, Marnes-La-Coquette, France).PCR products were purified using NucleoFast 96 PCR plates (Macherey Nagel, EURL, Hoerdt, France), according to the manufacturer’s recommendation. The purified PCR products were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems). The sequences obtained were assembled and edited using ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia), and the corrected sequences were compared with the sequences available in GenBank by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Phylogenetic analyses
Multiple sequence alignments were performed between the obtained sequences and other reference sequences in GenBank using CLASTAL W in MEGA software version X [83]. Phylogenetic trees were inferred using the Maximum-Likelihood method and Tamura-Nei model with 500 bootstrap replicates in MEGA X software [83,84].
Results
In this study, all samples (557) were screened by qPCR. None of the animals were positive for Bartonella sp., while different animal hosts were positive for piroplasms, Anaplasma sp., Rickettsia sp., Borrelia sp., C. burnettii and Filaria sp (Table 3).
Table 3
The prevalence of pathogens in animals by PCR.
Animal Hosts
No. of examined Animals(Total = 557)
Pathogens amplified
No. of infected Animals (%)
Cattle
88
PiroplasmidaT. annulataBa. bigeminaAnaplasmataceaeAn. marginaleAn. centraleAn. ovisAn. platys-likeBorrelia sp.Bo. theileriCo-infection :An. marginale + T. annulataAn. marginale + Bo. theileriAn. centrale + T. annulataAn. platys-like + Ba. bigemina
Fifty of 557 (8.9%) animal hosts were positive for piroplasms based on 5.8S rRNA qPCR system. Standard PCR and sequencing based on 18S rRNA gene succeeded in amplifying and identifying two Theileria sp.; T. annulata in cattle (14/88), T. ovis in sheep and buffaloes (5/58 and 2/26, respectively) and two Babesia sp.; Ba. bigemina in cattle (1/88) and Ba. canis in dogs (1/203). However, camels and goats were free of Piroplasmida DNA. The overall prevalence of piroplasmoses in different animal hosts was 23/557 (4.1%) as it was 17% in cattle, 8.6% in sheep, 7.7% in buffaloes and 0.5% in dogs. In our study, BLAST analysis revealed that cattle were positive for T. annulata and Ba. bigemina, including two genotypes of T. annulata, one genotype in 13 cattle with 100% (910/910) similarity to those of T. annulata detected in donkey blood in Turkey (GenBank: MG569892), a new genotype in one cattle with 99% (908/910) identity to the same reference dataset, and a new genotype of Ba. bigemina in one cattle with 99% (865/866) identity to those of Ba. bigemina detected in cattle blood from Switzerland (GenBank: KM046917). Similarly, we found that 5 sheep and 2 buffaloes were positive for a genotype of T. ovis with 100% (897/897) identity to T. ovis detected in wild sheep from Turkey (GenBank: KT851427). Finally, we identified Ba. canis in a dog with 100% (884/884) similarity to those of Ba. canis vogeli detected in a dog from Egypt (GenBank: AY371197). The phylogenetic tree of these genotypes was illustrated in Fig 2.
Fig 2
18S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Theileria sp. and Babesia sp. in the present study (Bold) related to other Theileria sp. and Babesia sp. available in GenBank. The sequence of 18S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 1066 positions in the final dataset. The scale bar represents a 2% nucleotide sequence divergence.
18S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Theileria sp. and Babesia sp. in the present study (Bold) related to other Theileria sp. and Babesia sp. available in GenBank. The sequence of 18S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 1066 positions in the final dataset. The scale bar represents a 2% nucleotide sequence divergence.For Anaplasmataceae, 172 out of 557 (30.9%) animal hosts were positive for anaplasmoses by 23S rRNA qPCR system. Based on the 23S rRNA gene, only 87 out of 557 animal hosts were successfully amplified by standard PCR, consequently, sequencing identified only 48 out of 557. The overall prevalence of anaplasmoses in different animals was 8.6%, with 28.4% in cattle (25/88), 6.9% in buffaloes (4/58), 7.7% in sheep (2/26), 6.7% in camels (10/149) and 3.4% in dogs (7/203), while goats were free of Anaplasma DNA. BLAST analysis revealed that cattle, sheep and camel were positive An. marginale, including two different genotypes of An. marginale, the first originated from sixteen cattle, two sheep and one camel with 100% (455/455) similarity to those of An. marginale detected in Rh. bursa collected from cattle in France (GenBank KY498335), and another new genotype was detected in two cattle with 99% (454/455) identity to the same reference dataset (GenBank KY498335). Moreover, one case of cattle was positive for An. centrale with 100% identical to An. centrale strain Israel (GenBank NR076686). From cattle and sheep, a genotype of An. ovis was identified with 100% (454/454) similarity to An. ovis in sheep blood from Niger (GenBank KY644694). We found that dogs and camels were positive for An. platys, including two different genotypes of An. platys, one genotype from six dogs and one camel with 100% (458/458) identity to An. platys in dog blood from France (GenBank KM021425) and another genotype from one dog with 100% (458/458) homology to An. platys in dog blood from France (GenBank KM021414). Finally, from cattle, buffaloes, sheep and camels, a new potential Anaplasma sp. was identified including, four different genotypes of this Anaplasma sp., the first genotype from six camels, the second from two camels, the third from one cattle and one sheep and the last from two cattle and two buffaloes with 98% (450/458), 98% (448/458), 98% (447/458) and 97% (446/458) similarity, respectively, to An. platys in dog blood from France (GenBank KM021414). Sequence analysis of this Anaplasma species revealed that this species has a homology score below 99% (more than 10 nucleotides different) and are closely related to An. platys, that means these sequences could be considered as potential new species of Anaplasma and can be called as An. platys-like. The phylogenetic tree showed that the new potential Anaplasma sp. in two separates and well-supported branches (bootstraps 99 and 96) belong to the cluster of An. platys (Fig 3).
Fig 3
23S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Anaplasma sp. in the present study (Bold) related to other Anaplasma sp. and Ehrlichia sp. available in GenBank. The sequence of 23S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 432 positions in the final dataset. The scale bar represents a 5% nucleotide sequence divergence.
23S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Anaplasma sp. in the present study (Bold) related to other Anaplasma sp. and Ehrlichia sp. available in GenBank. The sequence of 23S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 432 positions in the final dataset. The scale bar represents a 5% nucleotide sequence divergence.To better characterize different Anaplasma genotypes, rpoB genus-specific PCR primers were applied and 23 good quality sequences were identified. The result revealed that, 12 cattle and one sheep were positive for a genotype of An. marginale with 100% (487/487) homology with An. marginale in Rhipicephalus bursa from France (KY498345), and another genotype of An. marginale from one cattle with 99% (486/487) similarity with the same reference dataset. We also identified that cattle and sheep were positive for An. ovis, one genotype was found in two cattle and another in a sheep with 100% (489/489) and 99% (487/489) identical to those of An. ovis in sheep blood from Niger (GenBank KY644695), respectively. From dogs, we identified a new genotype of An. platys obtained from two dogs with 99% (488/489) homology to An. platys in dog blood from France (GenBank KX155493). Finally, from cattle, buffaloes and sheep, a new potential species of Anaplasma. was identified, its sequences had a homology score of less than 90%, confirming that these sequences are likely to be a new potential species of Anaplasma (like 23S rRNA gene). The only two different genotypes (one from two buffaloes and another from a cattle and a sheep) showed a low identity of 89% (432/486) and 88% (431/486), respectively, with An. platys in dog blood from France (GenBank KX155493), while identification of the genotype derived from camels failed. Phylogenetic analysis revealed a new potential Anaplasma sp. (An. platys-like) in a separate and well-supported branch (bootstraps 99) with the same clade belonging to An. platys (Fig 4).
Fig 4
rpoB gene based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Anaplasma sp. in the present study (Bold) related to other Anaplasma sp. and Ehrichia sp. available in GenBank. The sequence of rpoB gene were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 534 positions in the final dataset. The scale bar represents a 10% nucleotide sequence divergence.
rpoB gene based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Anaplasma sp. in the present study (Bold) related to other Anaplasma sp. and Ehrichia sp. available in GenBank. The sequence of rpoB gene were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 534 positions in the final dataset. The scale bar represents a 10% nucleotide sequence divergence.Rickettsial infection was detected by qPCR targeting gltA gene in dogs (3/557; 0.54%); the other animal hosts were free of rickettsiosis. To identify Rickettsia sp., standard PCR and sequencing were performed using gltA gene, and it was possible to amplify a 728 bp fragment of this gene from these three positive samples. A BLAST search of the obtained sequences with those in GenBank revealed that two different genotypes, one genotype was 100% (728/728) identical with R. africae previously detected in H. dromedarii from Egypt (GenBank: HQ335126), and the other sequence had 99% (726/728) identity with the same reference. Moreover, ompB gene was used to confirm the detection of R. africae-like infection in dogs. Based on the BLAST search, the sequences obtained from dogs were identified as R. africae (GenBank: MN629894) and showed (757/758) 99% similarity with the reference stain of R. africae detected in a traveler returning from Tanzania (GenBank: KU721071). The phylogenetic tree of these R. africae-like in dogs based on gltA was shown in Fig 5.
Fig 5
gltA gene based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Rickettsia sp. in the present study (Bold) related to other Rickettsia sp. available in GenBank. The sequence of gltA gene were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 728 positions in the final dataset. The scale bar represents a 2% nucleotide sequence divergence.
gltA gene based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Rickettsia sp. in the present study (Bold) related to other Rickettsia sp. available in GenBank. The sequence of gltA gene were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 728 positions in the final dataset. The scale bar represents a 2% nucleotide sequence divergence.Screening of Borrelia sp. in all animal hosts we found that 3 cattle and 2 sheep were positive for Borrelia sp. (5/557; 0.9%). Standard PCR and sequencing using 16S rRNA gene identified it as Bo. theileri. Alignment of five obtained sequences of Borrelia sp. from our samples revealed that all sequences were identical to each other. Furthermore, comparison of the obtained sequences with sequences from the GenBank database showed that 1139/1143 (99%) identity with Bo. theileri detected in Rh. geigyi in Mali (GenBank: KF569941). The phylogenetic position of this new Bo. theileri genotype was shown in Fig 6.
Fig 6
16S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Borrelia sp. in the present study (Bold) related to other Borrelia sp. available in GenBank. The sequence of 16S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 1142 positions in the final dataset. The scale bar represents a 5% nucleotide sequence divergence.
16S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Borrelia sp. in the present study (Bold) related to other Borrelia sp. available in GenBank. The sequence of 16S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 1142 positions in the final dataset. The scale bar represents a 5% nucleotide sequence divergence.Two out of 557 (0.36%) blood samples from one sheep and one goat tested positive for C. burnetii DNA by qPCR targeting IS1111. MST genotyping was performed using Cox2, Cox5 and Cox18, with only Cox2 successfully identified and the other spacers failing amplification. A BLAST search for the two sequences obtained showed that (351/351) 100% identity with the reference sequences of C. burnetii recorded in GenBank.Concerning Filariidae, four out of 557 (0.7%) animal hosts collected from three dogs and one camel tested positive for Filaria sp. DNA. By BLAST analyses, two dogs were found to have D. repens with 100% identity to those of D. repens previously detected in a Japanese woman returned from Europe (GenBank AB973229), and another sequence obtained from one dog showed 99% (1114/1119) similarity to Ac. viteae (GenBank: DQ094171). Moreover, S. digitata with (1107/1111) 99% identity to S. digitata from UK (GenBank: DQ094175) was found in a camel. The phylogenetic analysis of these Filaria sp. was constructed and presented in Fig 7.
Fig 7
18S rRNA based phylogenetic analysis of genotypes identified in this study.
Phylogenetic tree highlighting the position of Filaria sp. in the present study (Bold) related to other Filaria sp. available in GenBank. The sequence of 18S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 1110 positions in the final dataset. The scale bar represents a 10% nucleotide sequence divergence.
Phylogenetic tree highlighting the position of Filaria sp. in the present study (Bold) related to other Filaria sp. available in GenBank. The sequence of 18S rRNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA X using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There was a total of 1110 positions in the final dataset. The scale bar represents a 10% nucleotide sequence divergence.Finally, seven of different animal hosts were positive for more than one vector-borne pathogen (co-infections; 7/557; 1.3%). In cattle, five co-infections were observed (5/88; 5.7%) as An. marginale plus T. annulata (2/88; 2.3%), An. marginale plus Bo. theilerii (1/88; 1.1%), An. centrale plus T. annulata (1/88; 1.1%) and An. platys-like with Ba. bigemina (1/88; 1.1%). Moreover, one co-infection in sheep was recorded as An. platys-like plus Bo. theilerii (1/58; 1.7%) and one case in dogs R. africae-like with Anaplasma (1/203; 0.5%) (Table 3).
Discussion
The sustainable and economic progress of developing countries depends mainly on domestic animal resources, as they provide vital food, draught power and manure for crop production, and generate income [85]. However, animal-associated diseases, especially, VBDs are a global burden [2]. Recently, the spectrum of VBDs affecting animals has expanded and the attention of clinicians and veterinarians is growing. Therefore, the diagnosis of VBDs is crucial to develop the epidemiological mapping of these diseases and this can be achieved through the advances in molecular biology [86].Concerning piroplasmoses, the overall prevalence among animal hosts was 4.1%, including the highest prevalence among cattle 17%, then sheep 8.6%, buffaloes 7.7% and dogs 0.5%. Based on the 18S rRNA gene, two genotypes of T. annulata was detected in cattle from different provinces (El-Wady El-Geded, Beni-Suef, Qena and Beheira) and one case of Ba. bigemina was detected in cattle from Beni-Suef. In accordance to our results, many studies reported the high prevalence of T. annulata compared to other piroplasms in cattle from different provinces in Egypt [87-89]. In the current study, we observed that the majority of cases (10 out of 15) were detected in cattle from El-Wady El-Geded province that in accordance with Al-Hosary et al. [89], who stated that the prevalence of T. annulata in cattle from El-Wady El-Geded province was 63.6%. This finding might be due to the climate in this province, which is dry and sunny throughout the year, which is conducive to tick activity [89]. Likewise, we identified T. ovis in sheep from Giza and Beni-Suef and buffaloes from Beni-Suef. In Egypt, there are few studies reporting T. ovis in sheep [90] and buffaloes [91]. In parallel, a recent study reported that T. ovis was detected in sheep from Menoufia and El-Wady El-Geded province [92], implying that this pathogen is widespread in sheep throughout Egypt. Finally, we detected one case of Ba. canis in a dog from Cairo province with 100% identity with Ba. canis vogeli detected in a dog from Egypt (GenBank: AY371197). Canine babesiosis is distributed worldwide and was later detected in Egypt by Passos et al. [93] and Salem and Farag [94]. In Africa, Ba. canis vogeli has been detected in different regions such as South Africa [95], Tunisia [96] and Côte d’Ivoire [80].Family Anaplasmataceae was known to cause human and animal diseases, is transmitted by ticks and has a worldwide distribution [26,97]. In the current study, the overall prevalence of anaplasmosis was 30.9% (172/557) by qPCR, while we obtained only 48 samples with good quality sequences, possible due to the higher sensitivity of qPCR compared to standard PCR or due to the co-infection with family Anaplasmataceae. The overall infection rate of An. marginale was 3.8% (21/557) in cattle, sheep and camels from different localities (Beni-Suef, Qena, El-Wady El-Geded and Cairo). In Egypt, An. marginale was first mentioned in the national report in 1966, after which the disease was reported in numerous provinces [32-34,98]. Several studies reported endemicity of An. marginale in cattle [16,28,31-34], buffaloes [30] and camels [29]. However, An. marginale was detected for the first time in sheep. To our knowledge, An. marginale has not yet been described in sheep. For the first time, An. centrale was detected in a bovine from El-Wady El-Geded province, Egypt. Anaplasma centrale is closely related to An. marginale but less pathogenic, so it has been used as a live vaccine to protect against bovine anaplasmosis [99,100]. We also found that sheep and cattle from Beni-Suef province (upper Egypt) were positive for An. ovis with a prevalence rate of 0.7% (4/557). To the best of our knowledge, An. ovis has never been detected in cattle and sheep in Egypt. In parallel, a recent study reported that An. ovis was detected in sheep in Menoufia province (one of Delta provinces) [34], implying that this pathogen is widespread in cattle and sheep throughout Egypt. Anaplasma ovis is the etiological agent of ovine anaplasmosis in small ruminants and causes mild and subclinical infections [23]. In Africa, some studies reported An. ovis in sheep from Tunisia [101], Senegal [25] and Algeria [102,103], and in cattle from Algeria [103]. In addition, we found that dogs from Cairo and a camel from Giza province were positive for two genotypes of An. platys, with an infection rate of 1.4% (8/557). In Egypt, An. platys was never molecularly identified in dogs and camels. Later, Loftis et al, [51] detected An. platys in ticks collected from dogs. Anaplasma platys is the causative agent of canine anaplasmosis, which causes severe thrombocytopenia in dogs [104]. Interestingly, we detected that cattle, buffaloes and sheep from Beni-Suef province and camels from Giza and Cairo provinces were positive for a new potential Anaplasma sp. with a prevalence rate of 2.5% (14/557). This probably new species was genetically related to canine An. platys, which is why it was commonly referred to as An. platys-like. This An. platys-like genotype has never been detected in Egypt, except in a recent study where An. platys-like bacterium was detected only in cattle in Menoufia province [34], implying that this new potential pathogen circulates between different animal hosts (excluding dogs that seem to be susceptible for a type An. platys only) and different provinces in Egypt. Later, An. platys-like was detected in various animal hosts such as cattle in Italy [105], Algeria [106] and Tunisia [107], camels in Tunisia [108,109] and sheep and goats in South Africa [110] and Senegal [25]. Various Anaplasma sp. were identified by the 23S RNA gene and which further confirmed by the rpoB gene.Rickettsioses are VBDs of humans and animals and are mainly transmitted by ticks [35]. In Africa, the human pathogens R. africae, R. aeschlimannii, R. conorii and R. massiliae have been identified in ticks and animals [39-41]. In our study, rickettsial DNA was detected in dogs from Capital Cairo with a prevalence of 1.5% (3/203) in dogs. Phylogenetic analysis showed that our genotypes (R. africae-like) clustered in a separate and well-supported branch (bootstraps 94) with R. africae previously detected in Egypt (Fig 5) [53]. To the best of our knowledge, R. africae has not been previously detected in dogs anywhere in the world. Thus, this is the first detection of R. africae-like pathogens in dog anywhere in the world. African tick-bite fever, a benign disease with severe complications in elderly populations, and transmitted mainly in the south and West Africa by Amblyomma variegatum [35,111]. Likewise, R. africae was identified in other tick genera as Hyalomma sp. [42,53,54,112] and in Rh. sanguineus
(the most common tick parasitizing dogs) [113].Relapsing fever borrelioses caused by group of the spirochete group Borrelia sp. and is transmitted by soft and hard ticks [57]. In the present study, we identified Bo. theileri in bovine and ovine blood for the first time in Beni Suef province, Egypt, with an overall prevalence of 0.9% (5/557). Alignment of five sequences obtained revealed that there is a new potential genotype of Bo. theileri circulating between cattle and sheep in Beni-Suef province, which is 99% identical to Bo. theileri found in Rh. geigyi in Mali [58]. Borrelia theileri is considered one of the relapsing fever borreliae and the etiological agent of bovine borreliosis in cattle, transmitted by hard ticks, mainly Rhipicephalus sp. [114]. In Egypt, Bo. theileri was reported in Rh. annulata collected from donkeys in the same province [115]. Later, Bo. theileri was also detected in Rh. annulata in Egypt [62]. Recently, some studies have detected Bo. theileri in cattle such as Argentina [116] and Cameroon [117]. Similarly, Bo. theileri has been detected in the blood of sheep in Algeria [102]. It appears that, Bo. theilerii is not exclusively pathogenic to cattle.Q fever is a tick-borne disease that is a major public health concern [65]. The infection in human manifests as acute or chronic febrile disease often associated with endocarditis and abortion [65]. In Egypt, Q fever was first detected in a high-risk group of cattle farmers [118]. Later, many reports demonstrated the prevalence of the disease in goats, sheep, cattle and camels [67-72,119,120]. In this study, the overall prevalence of Q fever in sheep and goats from Sinai province is 0.3% (3% in goats and 1.7% in sheep). This result was in accordance with Abdel-Moein and Hamza [71] who reported an overall prevalence of Q fever of 0.9% and 3.4% in goats. PCR and sequencing amplified only Cox2 with a 100% match with the C. burnetii reference recorded in GenBank, while genotyping and sequencing of the positive samples with other spacers (Cox5 & Cox18) failed. This result can be explained by the fact that the high sensitivity of qPCR can detect low DNA concentrations and the lower prevalence of C. burnetii in blood is lower than feces and urine [121,122].For filarial infections, we detected four cases of filarial infection with an overall prevalence of 0.7%, 1.5% (3/203) in dogs and 0.7% (1/148) in camels. In dogs from the capital Cairo, we identified two different species of Filariidae as D. repens and Acanthocheilonema sp. Acanthocheilonema viteae is the filarial nematode of rodents, while Ac. reconditum is the etiological agent of filariasis in dogs. Also, there is no sequence of Ac. reconditum for the 18S rRNA gene in GenBank. Therefore, we suspect that the identified species is, however, Ac. reconditum. Therefore, this is the first report of D. repens and Ac. reconditum in dogs in Egypt. Subcutaneous dirofilariasis of domestic dogs is caused by D. repens and is common in Africa, Asia and Europe [123]. It is a mosquito-borne nematode that is a public health problem [124]. Acanthocheilonema reconditum colonizes the peritoneal cavity and adipose tissue and can cause skin lesions with allergy and is transmitted by fleas and biting lice [78,125,126]. In Africa, some studies reported microfilariae of Ac. reconditum in dogs in South Africa [127], Côte d’Ivoire [80]. Moreover, a camel from Giza province was positive for filarial nematodes, and was identified as S. digitata. To our knowledge, S. digitata has not been previously detected in camels. Setaria digitata is the natural filarial nematode of the Bovidae and the adult worm is resident in the peritoneal cavity [128,129]. Accidental transmission of S. digitata to unnatural hosts such as horses, donkeys, sheep and goats causes worrisome pathological problems such as corneal opacity and blindness [74,130,131-133].Finally, we reported 1.3% (7/557) co-infections in animals, with the highest percentage in cattle 5.7% (5/557). Co-infection in cattle is common and has been reported in many studies [33,34,117,134]. We observed that all cases of co-infections including Anaplasma sp. with another pathogen such as piroplasms, Borrelia or even Rickettsia. Regarding the endemicity of VBDs, we observed the most infected region in Beni-Suef province, where the same genotypes or even new potential pathogens circulated between different animal hosts with a risk of transmission to other adjacent provinces and to humans. Furthermore, we observed that the highest prevalence among animal hosts was anaplasmoses (48/557; 8.6%), followed by piroplasmoses (23/557; 4.1%). Molecular analysis revealed an interesting diversity of these VB pathogens in ruminants and dogs. Therefore, further studies are needed for a better understanding of the epidemiological mapping of pathogen-host-vector in this region or even in the whole Egypt.In conclusion, the current study is the first large-scale epidemiological observational study that performed molecular screening and characterization of multiple vector-borne pathogens in different animal hosts for better understanding of the endemicity of VBDs in Egypt. We identified for the first time An. centrale, An. ovis, a new An. platys-like and Bo. theileri in cattle, a new An. platys-like in buffaloes, An. marginale, An. ovis, a new An. platys-like and Bo. theileri in sheep, An. platys, a new An. platys-like and S. digitata in camels and R. africae-like, An. platys, D. repens and Ac. reconditum in dogs in Egypt. Therefore, ruminants and dogs in Egypt are reservoirs for multiple neglected, emerging and re-emerging vector-borne pathogens, especially new potential pathogens. Our observational study aimed to describe the repertory of possible vector-borne zoonotic pathogens in Egypt. However, convenient sampling approach did not permit us to evaluate the association of identified pathogens with host characteristics and to describe the geographic distribution of pathogens that limited our study. Further studies are needed to determine the pathogen-host-vector connections and other epidemiological factors of VBDs throughout Egypt, as well as to decipher the zoonotic potential of newly identified genotypes and their animals and public health significance.19 Mar 2021Dear Dr. Mediannikov,Thank you very much for submitting your manuscript "Multiple vectors borne diseases in Domestic Animals in Egypt" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. Please pay careful attention to each of the Reviewers' comments, as many relate to the clarity of presentation of the data and the validity of the conclusions. Correction of the english language errors will be important to the resubmission.One Reviewer stated that this work is not appropriate for this journal, so please strengthen the aspects of the work that focus on disease as well as on the presence of the pathogens, particularly since infection does not necessarily lead to disease.We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Jenifer Coburn, PhDAssociate EditorPLOS Neglected Tropical DiseasesAmanda BastosDeputy EditorPLOS Neglected Tropical Diseases***********************Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: AbstractLine 13: you can just say vector-borne pathogens.Line 21: please replace ‘proved’ with found.Line 26: the presence of different….Line 28: ruminants seem to play….Author summaryLine 34: please remove ‘was’ after that.Line 39: these findings suggest that….Line 40: please rephrase the end part of the sentence to ‘emerging and re-emerging potentially new vector borne pathogens that have significant implications in human health’ or something along those lines.IntroductionLine 46: humans, livestock, companion animals and…Line 46: vbds are a worldwide burden….Line 50: such as.Line 51: spread of VBDs…Line 51: such as globalization…Line 53: pose.Line 54 – 112: these paragraphs can be deleted or significantly condensed.Line 118: this sentence needs to be rephrased.Line 119: provide.Materials and MethodsThis section is fairly straightforward and concise but the other sections are heavily wordy and can be greatly condensed.Line 128: I’m assuming all these animals were domesticated? Please mention it explicitly.Line 161: ‘performed’ instead of applied.Reviewer #2: The objectives of the study are articulated with the hypothesis. It is not clear what are the criteria for inclusion of animals in the research and how the sample size calculation was performed to obtain the prevalence. In addition, they mention that information such as gender, breed, age, health status and vector infestation were collected, but it is not described or analyzed in the manuscript. There are no concerns about complying with ethical or regulatory requirements.Reviewer #3: The objectives of the study were clearly stated.The study design did not appropriately address the stated objectiveThe population were not described and hence cannot be measured with the study hypothesisThe sample size is not sufficient in ensuring adequate power to address the aim off the studyMore analysis needs to be carried out. Authors should mention exactly where sampling was done in the provinces whether households, kraals, abattoir and their respective confounders to the study.--------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures (Tables, Images) of sufficient quality for clarity?Reviewer #1: I think the whole results section needs to be reworked. As it is written, it is very difficult to read and grasp the main points of the research. The emphasis is heavily placed on the pathogens and not on the animals, but I think that it should be the other way around (this is not a microbiology journal). For example, instead of saying “No Bartonella sp. DNA was detected in all animal hosts, while, piroplasms, Anaplasma sp., Rickettsia sp., Borrelia sp., C. burnetii and Filaria sp. DNA were detected in different animal hosts (Table 3)” please write it as ‘None of the animals were positive for Bartonella sp. while different animal species were positive for……..’ or something along those lines.In line 133: You mention that you also recorded data about each animal’s gender, breed, age, vector infestation and health status but you do not report whether any of these factors had an effect on the detection of pathogens.Reviewer #2: The results of the analysis are adequate and clearly described. The figures and tables were well chosen and have quality. I suggest the inclusion of a map of Egypt showing the origin of the animals.Reviewer #3: The analysis matches the analysis plan, but more analysis needs to be done.The results are clearly presented but not complete.The figures (Tables, Images) are of sufficient quality for clarity--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: Needs to be rewritten. I could not make sense of the results section. Therefore, I don’t think any of my specific comments on the discussion/conclusions section would be helpful. I would request the authors to please rewrite the results and discussion sections from mainly the host animals’ perspective and not from the pathogens’ perspective. Also, please try to be concise as the way it is written is very wordy.I would like to see table 3 with the rows and columns switched.Reviewer #2: The conclusion is adequate, but the authors do not describe the limitations of the analysis. The relevance of the results for advancing the discussion of the subject and the importance of the findings for public health are highlighted.Reviewer #3: The conclusion are not supported by the data presented.The limitations of the study were not mentioned--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.Reviewer #1: This manuscript can be greatly condensed to better explain the results and their importance in the discussion sections.Reviewer #2: Line 21: replace “Dirofilariarepens” by “Dirofilaria repens”Line 55: replace “Piroplamida” by “Piroplasmida”Lines 119-121: It is not the first data... There are at least two published works with objectives similar to the present manuscript:Tumwebaze MA, et al. Parasitol Int. 2020 Oct;78:102150. doi: 10.1016/j.parint.2020.102150AL‑Hosary et al. Parasites Vectors (2020) 13:495 https://doi.org/10.1186/s13071-020-04372-zLines 128-131: It is not clear what are the criteria for inclusion of animals in the research and how the sample size calculation was performed to obtain the prevalence. I suggest the inclusion of a map of Egypt showing the origin of the animals.Lines 132-133: The authors mention that information such as gender, breed, age, health status and vector infestation were collected, but these data are not described or analyzed in the manuscript.Lines 135-136: Why was the blood extraction kit not used?Lines 149-151, and line 166: Was a reagent blanck control used?Line 190: Was a reagent blanck control used?Line 191: How was the sample size determined for the calculation of prevalence? What is the justification for using the term "Prevalence" throughout the text?Lines 285-290: It is important to describe the presence of co-infections also in the abstractLines 318-319: Replace the term “E. canis” by “Ba. canis”. In addition, citation number 89 refers to the first molecular description of Babesia vogeli in Brazil, so it does not seem appropriate to use this reference here.Lines 338-339, 356-357, 427-431: This study reports the first detection of A. ovis in sheep and A. platys-like strains in cattle in Menoufia and Egypt:Tumwebaze MA, Lee SH, Adjou Moumouni PF, Mohammed-Geba K, Sheir SK, Galal-Khallaf A, Abd El Latif HM, Morsi DS, Bishr NM, Galon EM, Byamukama B, Liu M, Li J, Li Y, Ji S, Ringo AE, Rizk MA, Suzuki H, Ibrahim HM, Xuan X. First detection of Anaplasma ovis in sheep and Anaplasma platys-like variants from cattle in Menoufia governorate, Egypt. Parasitol Int. 2020 Oct;78:102150. doi: 10.1016/j.parint.2020.102150Lines 383-384: Borrelia theileri description in Brazil was from ticks, not from bovine samplesLine 397: Replace “faces” by “feces”Lines 425-433: It is importante to describe the limitations of the analysis in conclusion.Reviewer #3: (No Response)--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: Overall, I think that the manuscript needs to be improved a lot more to be considered for reevaluation.Reviewer #2: The present work demonstrates important data about agents transmitted by vectors in animals in Egypt, contributing to reduce an existing gap on this knowledge.There are some recent works describing some pathogens that the authors thought were unpublished data. However, this does not detract from the research that was really extensive, covering various genera of pathogens transmitted by vectors to farm and companion animals.Reviewer #3: TITLE‘Multiple vectors borne diseases in domestic animals in Egypt’... Title is misleading it was not the disease that was investigated but the presence of pathogens.ABSTRACTAbstract needs to be re-written. Certain sentences do not read well and need restructuring. Authors should check how capitals and small letters are used and rectify all anomalies. The type of study should be stated in the abstract. Line 26 – 27 states that ‘For the first time, we detected the presence different zoonotic, mostly vector-borne pathogens in the blood of domestic animals in Egypt…. The question is by the use of mostly is the author implying that some of the pathogens were not VBD? If this is so which ones are those and why include them in the write up since the aim of the study was on VBD. All the new genotype discovered during the study should be mentioned under the abstract section.INTRODUCTIONSome of the diseases mentioned such as piroplasmosis presents itself as asymptomatic in livestock, some pathogens like Theileria present itself in the asymptomatic and is not zoonotic therefore what is its economic importance to merit it being studied. In inference the mortality and morbidity of these diseases should be highlighted clearly so that the relevance of its pathogens being studied is put in proper perspective.On line 46 what is the meaning of companion in that context.METHODSThe section was not well written. Authors should rewrite it by arranging it systematically following the order of; study area or study setting description-make sure each province is discussed in detail including their population, seasons and topography etc, sampling design and procedure-should be as detailed as possible including how each animal was sampled per province, where e etc, exclusion criteria, statistical analysis, ethical approval and consent.The convenient sampling that was carried out does not allow for authors to generalize the study to the whole country. The authors should be clear on the distribution of the animals in the provinces in the country and why. Authors should stop beginning sentences with acronym. Describe how field and laboratory processes were carried out.OVERALLFor substantial portion of the article it is not sequentially written out to allow for flow of thought.RESULTSThere should be another table that shows the demographics, vector infestation and health status of the animals with respective analysis. Reconcile from line 187 to that of the breakdown of the various VBD pathogens mentioned at the abstract section as well as with the various tables.DISCUSSIONLine 312 and 319 should state the authors whose studies this research is referring to before the references are written in parenthesis.Check tenses, missing words and misuse of words and correct them.Authors should also include in the discussion dynamics of all the VBD as present in the provinces, as per their proximity to each other and the potential beneficial or detrimental effect they might pose on the people of living in and around the study provinces and then the nation as a whole.It should also be discussed the new strain found their potential virulence or pathogenecity.How all pathogens identified economically impact the general populace should be further discussed.CONCLUSIONBased on the sampling technique the conclusion is farfetched, the convenient sampling done in this study cannot be used in projecting what pertains in the whole country--------------------PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods27 May 2021Submitted filename: Revision Letter-HA.docxClick here for additional data file.23 Jun 2021Dear Dr. Mediannikov,Thank you very much for submitting your manuscript "Multiple Vectors-borne Pathogens in Domestic Animals in Egypt" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. Please pay careful attention to the points raised by Reviewer 3, and please make every effort to have your manuscript proofread for correct usage of the English language. For example, there is a grammatical error in the Title.Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Jenifer Coburn, PhDAssociate EditorPLOS Neglected Tropical DiseasesAmanda BastosDeputy EditorPLOS Neglected Tropical Diseases***********************Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #2: The objectives of the study are related to the hypothesis. The authors clarified the criteria for inclusion of animals (convenience sampling) in the research, how the prevalence was calculated and added, as suggested, a figure containing the location of the samples obtained on the map of the country.The authors, when asked about not presenting results from data mentioned in the material and methods (about sex, race, age, health status and vector infestation), justified that these data will be presented on another article. Therefore, I suggest that this part is not mentioned as part of this manuscript.There are no concerns about complying with ethical or regulatory requirements.Reviewer #3: Authors clearly articulated the objectives of the study with the right study design and sufficient sample size but the animal population was not clearly described with respect to their sex, breed, age, vector infestation and health as stated in line 135.--------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures (Tables, Images) of sufficient quality for clarity?Reviewer #2: Data results are adequate and clearly described. The figures and tables were well chosen and of good quality. The authors accepted the suggestion to include a map of Egypt with the location of the samples collected.Reviewer #3: The analysis presented does match the analysis plan with the figures (Tables, Images) being of sufficient quality for clarity however authors on several occasions at the results section restated their methods adding up to the bulkiness of the manuscript.In line 209 how was 172 out of 557 arrived at for Anaplasmataceae in the animal hosts?The table 3 shows An. platys in 7 dogs why is line 226 reporting 6?Reconcile line 230 and 231 to table 3.Include the An. platys-like pathogen obtained from two dogs in table 3.--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #2: The conclusion is adequate and the authors pointed out the limitations of the analysis. The relevance of the results for advancing the discussion of the subject and the importance of the findings for public health are highlighted.Reviewer #3: yes the conclusions are supported by the data presented with limitations adequately described--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.Reviewer #2: The required changes were made, with no further changes suggested by my review.Reviewer #3: Minor editsReword line 67, and lines 69 to 70 to convey appropriately the message it wishes to convey.Authors should check spacing and punctuations through out the manuscript.Abbreviations of all scientific names should be synchronized through out the manuscript eg A. ovis and An. ovis, and A. platy and An. platy.The prevalence of piroplasmoses have been stated on line 193 and 194, it needn't be repeated on line 197.Sentence on lines 214 to 216 should be rewritten to convey the information it is intended to send to readers.On line 424 replace ...' and there is'... with ...' with'...--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #2: (No Response)Reviewer #3: Authors should not repeat most of the results in the discussion section, just discuss.Discussion on piroplasmoses with respect to T. ovis should be more detailed.Are all the organisms transmitted by ticks? If not kindly mention the other vectors and discuss them with their corresponding pathogens.Again all zoonotic pathogens should be mentioned as well as their devastating effect.--------------------PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.23 Aug 2021Submitted filename: Revision letter-HA-OM.docxClick here for additional data file.26 Aug 2021Dear Dr. Mediannikov,We are pleased to inform you that your manuscript 'Multiple Vector-borne Pathogens of Domestic Animals in Egypt' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. 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All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.Best regards,Jenifer Coburn, PhDAssociate EditorPLOS Neglected Tropical DiseasesArmanda BastosDeputy EditorPLOS Neglected Tropical Diseases***********************************************************14 Sep 2021Dear Dr. Mediannikov,We are delighted to inform you that your manuscript, "Multiple Vector-borne Pathogens of Domestic Animals in Egypt," has been formally accepted for publication in PLOS Neglected Tropical Diseases.We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. 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Authors: N Boudebouch; M Sarih; C Socolovschi; H Amarouch; M Hassar; D Raoult; P Parola Journal: Clin Microbiol Infect Date: 2009-05-18 Impact factor: 8.067
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