Mosquito-borne diseases are the main public health problems worldwide, especially in tropical and subtropical regions [1-5]. Despite the fact that their bites are widely annoying, they are probably the most dangerous vectors of disease or cause of mortality in humans [1]. Their infection issues are related to their ability to transmit various types of pathogens, such as filarial nematodes, Plasmodiums and Arboviruses. Among them, malaria, dirofilariasis, dengue fever, west nile virus (WNV), yellow fever and some other viral diseases are the most pathogenic [1, 6]. Due to climatic conditions in tropical and subtropical regions, the invasion of mosquitoes, which bite the human, may be high in urban and rural places leading to the increase of mosquito-borne diseases [7]. Among international travelers, malaria, dengue, and Rickettsial diseases are the most common causes of febrile illnesses; and the exposure to mosquitoes is reported as the most common source of fever [2, 8]. Most vector mosquitoes, which can cause human diseases, are divided into three genera, namely Anopheles, Aedes and Culex. These genera can transmit human filariasis, arboviruses and dirofilariasis; however, only Anopheles mosquitoes can transmit five known Plasmodium parasites that cause human malaria [7]. Despite a great number of researches on mosquito-borne infections, there is no study on the probable transmission of Rickettsia spp. and Coxiella burnetii by mosquitoes to humans in Iran, and there are a few studies worldwide.

These are bacteria of the Rickettsiales order and are referred to as short, gram-negative and rod-shaped bacteria and need eukaryotic cells for growth and proliferation [9]. In terms of history, Rickettsiales order is divided into three families, namely Rickettsiaceae, Bartonellaceae and Anaplasmataceae. Rickettsiaceae Family consists of three tribes, namely Rickettsieae, Ehrlichieae and Wolbachieae; and rickettsieae consists of three genera, namely Coxiella, Rickettsia, and Rochalimaea. Based on morphological, antigenic and metabolic characteristics, Rickettsia genus is divided into three groups: the typhus group (TG) consisting of R. typhi, R. prowazekii, and R. Canada members [10-15].

The spotted fever group (SFG) contains 20 different species of Rickettsia spp. (R. felis) [11, 13, 14, 16]. The scrub typhus group includes Orientia tsutsugamushi (Rickettsia tsutsugamushi until 1995) [12-15, 17]. Rickettsia spp. are transmitted through infected arthropods such as ticks, mites, fleas and lice.

Coxiella burnetii is also a small gram-negative bacterium that cannot grow in the medium [18, 19]. This bacterium is the cause of Q fever disease that is common worldwide. Rickettsial diseases of Iran include endemic typhus (murine typhus) [20-24], epidemic typhus (louse-borne typhus) [21, 24, 25] and Q fever [26-29].

In 1924, the presence of intracellular microorganisms of Rickettsia was reported in ovarian and testicle cells in Culex pipiens [30]. Later, this bacterium was introduced as Wolbachia pipientis, a member of Anaplasmataceae family in Rickettsiales order. In addition, Yen et al. introduced another type of transovarially transmitted Rickettsia through the electron microscope in 1975 [31]. Therefore, we assumed that there are probably other unknown species of Rickettsia in mosquitoes. In sub-Saharan Africa, R. felis is the cause of 15% of the unknown fever [32]. It has been recently found that Anopheles gambiae, the main vector of malaria in sub-Saharan Africa, acts as a vector of R. felis [2, 30, 31]. Interestingly, both cells of Anopheles gambiae and Aedes albopictus help to develop R. felis [7, 32-34]. The research indicates that mosquitoes can play roles in transmitting Rickettsia, especially R. felis. Since there have not been any studies on the roles of the Iranian mosquitoes in transmitting Rickettsia and Coxiella burnetii, the present study investigates the roles of mosquitoes in the transmission of these pathogens using the PCR (polymerase chain reaction) technique in an endemic area in south of Iran (near Persian Gulf with subtropical weather) for the first time.

In general, there are few studies on Rickettsial diseases in Iran. A study was recently conducted by Ghavami et al. who investigated an ITS1 part of gltA gene and Rickettsia contamination in human flea (Pulex irritans) in Iran. Their results indicated that 4.9% out of 182 samples of human flea were contaminated with Rickettsia sp [41]. Azizi et al. conducted an overview of epidemic Typhus in the world in the 19th and 20th centuries and stated that typhus was a major Rickettsial disease in Iran [24]. Historically, typhus (Rickettsial disease) was a major health problem with an economic impact on Iran; however, there is limited published information on the prevalence of typhus over the centuries. Typhus has been an endemic disease in Iran, but its prevalence has been rare. In the Qajar period (1796-1925), infectious diseases, including typhus were common. In the twentieth century, the prevalence of typhus occurred in Iran [24].

Spotted Fever Group (SFG) Rickettsioses occur worldwide and may cause serious illnesses in humans. They are transmitted to humans by vector arthropods such as ticks, fleas and lice [42-47]. In the Middle East, a number of known species of Rickettsia genus have been reported, but some studies are suspicious [48]. Al-Deeb et al. reported the first case of spotted fever group of Rickettsia sp. and Theileria annulata in Hyalomma dromedarii tick that was collected from camels in the United Arab Emirates [49]. Furthermore, Rickettsia sibrica mongolitimonae was diagnosed in a male patient in Turkey in 2016 [50]. Mediterranean Spotted Fever (MSF) is caused by Rickettsia conorii subsp. Israelensis is the main cause of fever and chills from spotted fever group rickettsiosis in collected ticks from mice, deers, goats, red foxes and wild boars [48, 51].

According to conducted studies in the Middle East, the presence of different types of ticks, which can transmit Rickettsia in several countries including Yemen, Saudi Arabia, and Oman throughout the Middle East, can cause Rickettsial diseases in the Middle East [48, 52, 53]. Despite the evidence of the presence of Rickettsial SFG pathogens in the Middle East, there is no study on this disease in Iran. Therefore, it is necessary to study the SFG Rickettsiae to have a better understanding of the epidemiology of this disease in Iran [48]. There is only a paper on the spotted-fever group rickettsioses, and it investigates 40 samples of human serum collected from 4 countries, including Iran, for the presence of spotted fever group rickettsia antibodies among other pathogens by Kovacova et al. From 40 human laboratory samples, 45% were positive in terms of SFG rickettsia by the ELISA test, and 27.5% were positive using the IFA test [48, 54].

In the study of Idris et al. [2000], 347 human serum samples for Rickettsia infections were obtained in Dhofar, Oman, and their results indicated that 59% of samples were positive [55].

Studies indicate that there are different genera of the Rickettsia order, including Coxiella, Anaplasma, Ehrlichia, and Wolbachia in different arthropods in Iran. Coxiellosis, erlichiosis, anaplasmosis and SFG rickettsioses, which are zoonotic, increase the presence of their agents in the environment and the risk of human-animal transmission. The insufficient information about SFG Rickettsioses in Iran indicates the absence of pathogens in the population. However, there is a need for studies on the SFG Rickettsioses among a wide range of organisms across the country as it is difficult to determine the range of hosting, the zoonotic potential and actual prevalence of SFG Rickettsioses in Iran. Several studies indicate the presence of Rickettsia species in Iran; some of them are as follows:

Spitalska et al. [2004] examined tick-borne diseases in small domestic ruminants in Iran. Collected ticks were infected with Ehrlichia ovina, Theileria lestoquardi and Theileria spp [56].

Jalali et al. [2013] investigated the Anaplasma infection in blood samples of sheep in Ahvaz by the PCR-RLFP method. In 80% of samples, there was an infection caused by A. ovis and A. marginale [57]. Noaman et al. (2017) conducted an overview of Anaplasmosis in the Iranian ruminants [58].

Yousefi et al. [2017] investigated the molecular evidence of the presence of Anaplasma phagocytophilum as a tick-borne pathogen in small domestic ruminants in Iran. One percent of blood samples were positive. The research was the first report of this pathogen in Iran [59]. Parvizi et al., (2013) isolated Wolbachia pipientis from Phlebotomus kandelakii and Phlebotomus perfiliewi sandflies [60]. Karimian F., et al., [2018] studied wsp-based analysis of Wolbachia strains associated with Phlebotomus papatasi and P. sergenti (Diptera: Psychodidae) main cutaneous leishmaniasis vectors, the introduction of a new subgroup wSerg [61].

Q fever is caused by a small and intercellular gram-negative bacterium, called Coxiella burnetii. Domestic ruminants, including cow, sheep and goats, are considered the main reservoirs of C. burnetii to cause human infection. Infected ticks can play important roles in the transmission of bacteria among mammals, rodents and other mammals as well as wild and domestic birds. Q fever is domestic in Iran [23, 29, 62]. It has been reported that rodents have been infected by named infectious agents in Iran during the 1970s; however, there has been no report of rodent infections and transmission to humans in recent decades [62]. There are many reports about the infection of other animals and their products with C. burnetii in different provinces of Iran like Fars, Khuzestan, Yazd, Chaharmahal and Bakhtiari, Isfahan, Gilan and Mazandaran [23, 26, 27, 29].

In research by Rahimi in 2010, it was investigated that the prevalence of C. burnetii in cow's milk samples from dairy cattle of Fars, Kerman, Qom, Yazd and Khuzestan provinces. Eighteen percent of collected samples from Fars were infected [27].

In a study by Doosti et al. in 2014, the prevalence of C. burnetii in camels of Iran was studied. In this study, blood samples were taken from 130 camels; and the PCR method was used for their study. Ten percent of samples were positive [63].

A research was conducted by Parhizgari et al. in 2017, and they reviewed Emerging and re-emerging infectious diseases in Iran and declared that Q fever was an endemic disease caused by C. burnetii in Iran [64].

Rahmdel et al. [2018] investigated C. Burnetii in cow's milk samples in Shiraz (capital of Fars province) using the PCR method. Their results indicated that 3% of milk samples were contaminated, and cows' raw milk could be a potential source of C. burnetii in Shiraz in southern Iran [65]. Ghavami et al. (2018) investigated ITS1 spacer and Rickettsia infection rate in human fleas (Pulex irritans) using the molecular method. Their results indicated that 4.9% of 182 samples of human fleas were infected with Rickettsia spp [41]. Traub et al. (1974) studied the ecology of scrub typhus. In their study, they predicted that the infection could be present in the northern regions of Iran despite no evidence of infection [66].

In recent years, studies have indicated that mosquitoes can be potential vectors for Rickettsiae. According to studies, the importance of the presence of mosquitoes has multiplied in regions where there are many vector-borne diseases; and mosquitoes can also be considered as the vectors of Rickettsiae. Some studies on the potential of mosquitoes for transmitting Rickettsial diseases are as follows:

Among the first studies on the presence of Rickettsiae in mosquitoes, research reported the presence of intracellular microorganisms of Rickettsia in ovarian and testicle cells of Culex pipiens mosquito in 1924 [30]. Later, this bacterium was known as Wolbachia pipientis, a member of Anaplasmataceae family in Rickettsiales order. In addition, Yen introduced another type of transovarially transmitted Rickettsia using electron microscopy in 1975 [31].

In 2006, Horta et al. could develop R. Felis in the c6/36 cell of Aedes albopictus mosquito. Their results indicated that mosquito cells could be used to cultivate this rickettsia. In the study, 90 to 100% of cells were infected after adding pathogen to these cells after 15 days of incubation at 25 °C [33].

In a similar study in 2007, they examined the proliferation of transmitted Rickettsiae by arthropods in two types of mosquito cells. In this study, they used two cells, Aedes albopictus Aa23 and Anopheles gambiae Sua5B. They proliferated three species, namely Rickettsia felis, R. montanensis and R. peacockii in the cells. They found that the cells were also suitable for the cultivation of difficult - cultivable Rickettsiae such as R. peacockii [67].

Another study in 2011, examined the impact of Wolbachia on Culex quinquefasciatus. It was found that this kind of Rickettsia affected the ovary and reproduction of mosquito. The infected females are laying eggs before the usual time, and these eggs spend more time than healthy eggs. Healthy females produce more eggs than infected ones [68].

In a study in 2011, the R. Felis infection was investigated as a pathogen that was transmitted by mosquitoes in the future. They argued that due to the separation of this Rickettsia from people with fever in tropical regions, where there were numerous Ae.albopictus and Ae.aegypti mosquitoes, and they easily bit people, Aedes spp. mosquitoes could spread infection in the future [32]. In a study in the same year, they investigated the role of Wolbachia in controlling mosquito-borne diseases. The results indicated that Fruit fly Wolbachia strains could infect mosquitoes and affected the longevity, reproduction and pathogen entry to the mosquito's body [69].

Research in 2012 provided an examination of Rickettsia species in Anopheles of Africa. The real-time PCR and conventional PCR method were used by sequencing gltA, ompB, ompA and sca4 genes. DNA of Rickettsia was not separated from An.arabiensis, An.funestus and An.gambiae (molecular form M). R. felis was separated from An.gambiae molecular form S as the most important vector of malaria in Africa [7].

In a study in 2015, the researchers investigated a potential transmission of R.felis by An.gambiae mosquito. In this test, An.gambiae were infected with Rickettsia in the laboratory, and the infected mosquitoes were exposed to balb/c rats. The results indicated that the mosquitoes were able to transmit R. felis to rats; hence, mosquitoes could be potential vectors of this Rickettsia [70].

In another study in 2017, the researchers examined molecular characteristics of the species of mosquito-borne Rickettsiae in collected sample mosquitoes from a Korean civilian region. In this study, Rickettsiae were detected using real-time PCR and sequencing rss, gltA, 17kDa, ompB, and sca4 genes. Three new Rickettsia genotypes were detected from Mansonia uniformis, Culex pipiens, and Aedes esoensis [71].

In a research in Africa in 2017, a new species of Wolbachia was detected in malaria vectors. They could isolate this bacterium from 5 Anopheles species [72].

In one of the latest studies in this field in 2019, the researchers studied R. felis in mosquitoes in China. They investigated three species, including Culex pipiens, C. tritaeniorhynchus, and Aedes albopictus in terms of Rickettsia; and all three species were positive [73].

All these studies indicate the higher importance of mosquitoes in regions where there are Rickettsia infections. There are few studies on the presence of Rickettsial diseases in Iran.

According to the results of this study, mosquitoes in this part of the country are not considered as vectors of Rickettsia spp. and Coxiella burnetii currently. We can attribute the negative outcomes to several main factors, such as the inability of mosquitoes to support pathogens in order to establish, grow and replicate within their bodies and to have different geographic conditions.

In general, limited studies have been conducted on Rickettsial diseases in Iran, and since Rickettsia spp. has been reported from different vectors in Iran and other neighboring countries, there is a need for further studies on this field in tropical areas of Iran, especially in mosquitoes as a possible vector with high abundance and mobility.

Given the fact that there are numerous travels to different regions of the world with Rickettsia infection such as Southeast Asia, and Middle Eastern countries; and people enter Iran from neighboring countries such as Pakistan with the possibility of infection, there is a need for more comprehensive and accurate studies on this field, especially in Southern provinces of Iran such as Sistan and Baluchestan, Fars, khozestan, Bushehr, and Hormozgan.

Rickettsial disease should be considered important and travelers, who are returning from neighboring countries especially Saudi Arabia travelers, for the Hajj ritual with symptoms of fever and headache, should be screened. The importance of these diseases and their causes should be taken seriously into consideration before they create serious problems for the society.

It is suggested that a comprehensive study at a wider level should be conducted in other endemic areas of Iran in order to define the prevalence of Rickettsial infection rates in mosquitoes. In this case, we will be able to understand the exact role of mosquitoes in the cycle of transmission of these pathogens to humans in Iran.