Molecular-based survey of Rickettsia spp. and Coxiella burnetii in mosquitoes (Diptera: Culicidae) from Fars province, southern Iran, during 2017-18 CURRENT STATUS: POSTED

Background Mosquito-borne diseases are main problems of public health worldwide, especially in tropical and subtropical regions. Mosquitoes can transmit human filariasis, arboviruses, malaria, and dirofilariasis. It has been recently found that Anopheles gambiae is a vector of Rickettsia felis. Both cells of Anopheles gambiae and Aedes albopictus help developing of R. felis. These studies indicated that mosquitoes could be vector of Rickettsia spp., especially R. Felis. Since there was no study on roles of the Iranian mosquitoes in transmission of Rickettsia spp. and Coxiella burnetii, the present study for the first time investigated roles of mosquitoes in the transmission of these pathogens using the PCR technique in Iran. Methods The present study was conducted in Fars province, and mosquitoes were manually caught (hand-catch, total catch etc.) in Qir and Karzin Counties from four different geographical regions during the activity seasons of mosquitoes in 2017-18. The primers design were done to investigate the probability of mosquito’s contamination with Rickettsia spp. and Coxiella burnetii from gltA genes (Rickettsia sp. citrate synthase kinase) for Rickettsia spp. and IS111 A Transposase gene for Coxiella burnetii. The conventional PCR was used after the extraction of DNA from mosquitoes to study the contamination. Results A total of 1103 adult mosquitoes were collected and identified from four regions of the County. Among them, 3 genera and 11 species were identified including Anopheles (25.74%) (An. dthali, An. stephensi, An. superpictus), Culex (51.84%) (Cx pipiens, Cx sinaiticus, Cx bitaeniorhynchus, Cx theilers, Cx laticinctus, Cx tritaeniorhynchus, and Cx torrentium) and Culiseta (22.39%) (Cu. longiareolata) genera. All tested mosquitoes were negative in terms of contamination to Rickettsia spp. and Coxiella burnetii.

. Rickettsia spp. are transmitted through infected arthropods such as ticks, mites, fleas and lice.
Coxiella burnetii is also a gram-negative small bacterium that cannot grow in the medium (18,19).
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, act 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)(33)(34). The research indicates that mosquitoes can play roles in transmitting Rickettsia, especially R. felis. Since there was no study on roles of the Iranian mosquitoes in transmitting Rickettsia and Coxiella burnetii, the present study for the first time investigated roles of mosquitoes in transmission of these pathogens using the PCR (polymerase chain reaction) technique in an endemic area of south Iran ( near Persian gulf with subtropical weather).
2 Methods with rainy winter and hot and dry summer. The maximum temperature is 46 °C in the summer and the minimum temperature is 25.1 °C in the winter. The mean precipitation of this city is 270 mm per year. Qir and Karzin County has borders with Firuzabad, Jahrom, Khonj and Farashband counties.
Sampling was carried out in four regions (including Qir, Efzar, Karzin and Emam Shahr) in the mosquito activity season every 2 months ( Figure 1).

Mosquitoes collecting and identifying
Mosquitoes were collected by hand-catch, total catch from indoor and outdoor places (human and animal) (7,35,36). All needed data like date, location and hour of collecting were recorded along with mosquito species. Adult specimens of mosquitoes were pinned and kept at the Museum of Medical Entomology (Shiraz University of Medical Sciences, Department of Medical Entomology). In this study another innovative and effective method was used for catching mosquitos. In this method, wasted tires were placed in the target places and filled with water to attract mosquitoes for resting to these moist environments.
Two to three days after using tires, they were covered with nets with small pores, and shake them, so that mosquitoes, which were resting flied upwards and got caught by the net. The aspirator was then inserted slowly from the corner of net and mosquitoes were collected and transported into the cups.
The caught mosquitoes were transferred alive to the Medical Entomology Laboratory of Shiraz University of Medical Sciences for further studies. After pinning, the mosquitoes were identified using a valid identification key (37).
Adult mosquitoes were collected by simple random sampling. Since there was no research on this field, and the prevalence was not known in Iran, sample size was calculated according to the statistical consultant's opinion based on similar studies using the sample size formula; and 600 mosquitoes were necessary for this project. For this study, about 1103 specimens were caught and then Anophelinae and Culicinae mosquitoes were identified using valid identification keys (37,38).
Molecular tests were performed on both Anophelinae and Culicinae mosquitoes.

Designing primer for gene identification of Rickettsia spp. and Coxiella burnetii
The primers were designed to identify Rickettsia spp. and Coxiella burnetii in mosquitoes. Therefore, the DNA sequences of Coxiella burnetii (MH920311.1 and CP018150.1) and various Rickettsia species (CP002912.1, CP002912.1, CP002912.1 and MG906669.1) were obtained from the NCBI website; and the alignment was done using Mega6 software, and then the primer was designed based on Conserve points using Oligo7.0 and Generunner4.0 software according to gltA (Rickettsia spp. citrate synthase kinase) and IS111 A Transposase genes to identify Rickettsia and Coxiella burnetii species (Table 1).

Conventional PCR
Materials with listed concentrations in Table 2 with a final volume of 20μl were used do the conventional PCR. Variables of conventional PCR during the research were obtained from using specific forward and reverse primers of gene as well as the PCR at the specific binding temperature(Annealing) relating to the same primers. All PCRs were done using the Analytikjena machine made in Germany. The following program was used to do the conventional PCR (Table 3).     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)(43)(44)(45)(46)(47)   In a study by Doosti et al. in 2014, they studied the prevalence of C. burnetii in camels of Iran. In this study, blood samples were taken from 130 camels; and the PCR method was used for their study.
10% of samples were positive (63). In recent years, studies have indicated that mosquitoes can be potential vectors for Rickettsiae.
According to studies, the importance of presence of mosquitoes has multiplied in regions where there are many vector-borne diseases; and mosquitoes can be also considered as vectors of Rickettsiae.
Some studies on the potential of mosquitoes for transmitting Rickettsial diseases are as follows: 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).

Conclusion
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 replication within their bodies and having different geographic conditions. 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 done in other endemic areas of Iran in order to define the prevalence of Rickettsial infection rate 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. Figure 1 Map of Qir and Karzin County and sampling locations