Association Between Wolbachia Infection and Susceptibility to Deltamethrin Insecticide in Phlebotomus papatasi (Diptera: Psychodidae), the Main Vector of Zoonotic Cutaneous Leishmaniasis.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups. Result: The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi. Conclusion: Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management.

Evaluation of Repellency and Lethal Effects of Ultrasonic Waves on the Blattella germanica (Blattodea: Blattellidae).

Background: The German cockroach, Blattella germanica (Insecta: Blattodea, Blattellidae), which occurs widely in human buildings, is a small cockroach species. Cockroach control chemical pesticides are toxic to the environment, and it is sometimes impossible to prevent them. Controlling Blattella germanica through ultrasonic waves can be efficient and less dangerous for the environment. Methods: In this study, the repellency and lethal effect of ultrasonic waves on male and female German cockroaches was tested in a twin glass cubic chamber at laboratory condition. The wave frequencies tested ranged from 20 to 100kHz with 5kHz steps. A signal generator generated these frequencies, and the piezoelectric transmitter of these ultrasonic waves was positioned in the chamber's center on the upper side. Results: Fisher's test showed that there was the greatest repellency effect in both male and female at frequencies of 35 and 40kHz. According to the results of the regression test, the most lethal effect is at the frequencies of 40 and 75kHz. Conclusion: The operating ultrasonic frequencies investigated in this study can be used to repel and kill German cockroaches as pests endangering human health and environment.

Wolbachia infection in native populations of Blattella germanica and Periplaneta americana.

Cockroaches are significant pests worldwide, being important in medical, veterinary, and public health fields. Control of cockroaches is difficult because they have robust reproductive ability and high adaptability and are resistant to many insecticides. Wolbachia is an endosymbiont bacterium that infects the reproductive organs of approximately 70% of insect species and has become a promising biological agent for controlling insect pests. However, limited data on the presence or strain typing of Wolbachia in cockroaches are available. PCR amplification and sequencing of the wsp and gltA genes were used to study the presence, prevalence and molecular typing of Wolbachia in two main cockroach species, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), from different geographical locations of Iran. The Wolbachia endosymbiont was found only in 20.6% of German cockroaches while it was absent in American cockroach samples. Blast search and phylogenetic analysis revealed that the Wolbachia strain found in the German cockroach belongs to Wolbachia supergroup F. Further studies should investigate the symbiotic role of Wolbachia in cockroaches and determine whether lack of Wolbachia infection may increase this insect's ability to tolerate or acquire various pathogens. Results of our study provide a foundation for continued work on interactions between cockroaches, bacterial endosymbionts, and pathogens.

Molecular Detection of Dipylidium caninum in Isolated Fleas of Dogs in Meshkinshahr County, Northwest of Iran.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as humans, livestock, dog, cat, rabbit, squirrels, rats, and mice. The desire for blood feeding from warm-blooded animals has led to becoming an intermediate host for some tapeworms like Dipylidium. The aim of this study was to detect D. caninum larval infection in fleas of dogs living in Mesh-kinshahr County, northwest of Iran. Methods: Fleas were collected from 42 dogs using brushing the hair in Meshkinshahr for one year (2014-2015). After the morphological study, fleas were preserved in 90% ethanol for molecular identification. After DNA extraction, the 28S ribosomal RNA gene (∼670 bp) of D. caninum was amplified using specific primers. Finally, the PCR products were sequenced. Results: Overs, 974 fleas were collected from the dogs. In the morphological study, three species Ctenocephalides canis, Ct. felis, and Pulex irritans were identified. PCR and sequence analysis results showed that 4 isolates Ct. Canis were infected with D. caninum. Also, no positive specimens were isolated from the other two species. Conclusion: Ct. canis is reported as the most important species of fleas in transferring D. caninum in that region.

SARS-CoV-2 in domestic cats (Felis catus) in the northwest of Iran: Evidence for SARS-CoV-2 circulating between human and cats.

This study aimed to investigate the prevalence of COVID-19 in domestic cats, focusing on the disease in the northwest of Iran and then showing the natural transmission of SARS-COV-2 circulating between domestic cats and humans. After receiving ethic codes from Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1399.303) and confirmed by the Center of Communicable Diseases Control (CDC) of Iran, 124 domestic cats were collected from the homes and only one hospital of Meshkin -Shahr district from northwestern Iran where SARS-CoV-2 patients were hospitalized and quarantined during 2020. Samples were prepared from fluid materials of oropharynx and nasopharynx. All samples were tested by real-time PCR (RT-PCR) using specific genes N and ORF1ab in Pasteur Institute of Iran, and then partial sequence analyses of S gene were performed. All collected cats were kept in separated cages until SARS-COV-2 infection was confirmed with the RT-PCR. RT- PCR Ct values of 123 collected cats were ≥40; thus, all of them showed negative results, but one of the collected cats with close contact with its owner, whom confirmed SARS-CoV-2 showed positive results with gene N(Ct=30) and gene ORF1ab (Ct=32). Furthermore, the positive pet cat showed respiratory and gastro-intestinal clinical manifestations, and its owner was infected with SARS-CoV-2 two weeks ago. Cats are susceptible animals to SARS-CoV-2 infection. Epidemiological evidence showed that SARS-COV-2 is able to transmit to healthy cats due to having close contact with its owner as a reverse zoonosis.

PCR Positivity of Gerbils and Their Ectoparasites for Leishmania Spp. in a Hyperendemic Focus of Zoonotic Cutaneous Leishmaniasis in Central Iran.

Background: Various arthropods, including Rhipicephalus sanguineus and Ctenocephalides felis felis have been suggested as secondary vectors of Leishmania spp. many years ago. This study was conducted to determine zoonotic cutaneous leishmaniasis (ZCL) PCR positivity of reservoir hosts and their ectoparasites for Leishmania spp. in Segzi plain in Esfahan Province from October 2016 to October 2017. Methods: Microscopic examination and nested PCR were used to detect and identify Leishmania spp. isolated from rodents' ears and ectoparasites, and then, the results were confirmed by two methods, PCR-restriction fragment length polymorphism and sequencing. Results: Totally, 93 rodents (92 Rhombomys opimus and one Nesokia indica) and nine different species of ectoparasites (n=527) including fleas, mites, and ticks were collected during different seasons in the study area. Fourteen R. opimus were positive for Leishmania spp. by microscopic examination while one N. indica and 77 R. opimus were positive by nested PCR. The infection rate of rodents with Leishmania major and Leishmania turanica was 39.79% (n=37) and 15.05% (n=14), respectively. Mixed natural infections with L. major and L. turanica were seen in rodents. Moreover, 72.22% of fleas (39/54), 75.0% of mites (5/8), and 100% of tick nymph (1/1) were PCR positive for Leishmania parasites. Conclusions: The highest rate of infection with L. major and L. turanica in R. opimus populations was observed in summer and spring, respectively. It is suggested that the role of L. turanica and the probable role of ectoparasites in the epidemiology of disease should be investigated. A Xenodiagnostic test is recommended for future study.

Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mechanism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. Methods: A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and preserved in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infantum using a semi-nested polymerase chain reaction (PCR) assay. Results: The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) collected from infected dogs. No leishmanial infection was observed in P. irritans. Conclusion: It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined.

Pulex irritans on Dogs and Cats: Morphological and Molecular Approach.

Background: The painful bite of Pulex irritans; causes wound on the host body and is a vector for Bartonella bacteria species, which can cause trench fever, Rickettsia species, Rocky Mountain spotted fever and Mediterranean spotted fever. We conducted a study to find out the hosts, abundance, as well as the molecular characteristics of this flea species in Meshkin-Shahr County, Ardabil Province, northwest of Iran. Methods: After collecting fleas from different reservoir hosts and transferring them to the laboratory, identification was done using different morphological characters as well as the internal transcribed spacer1 (ITS1) molecular marker. Results: This morphological study indicated that from the 1053 fleas, which were collected from 162 different animals, including cats and dogs, 74 specimens belonged to human flea, Pulex irritans. In addition, in molecular analysis showed a high sequence similarity (99.5%) with the P. irritans counterparts from Spain country and Zanjan of Iran available in GenBank. Conclusion: Pulex irritans species is an obligatory hematophagous ectoparasite of human and animals. Therefore, considering the relatively high frequency of this species on the body of cats and dogs, it is suggested to conduct more studies on its distribution and the possibility of being a vector of pathogens among these animals and human. The results of these studies will be used to compile and review the control programs of this vector.

Insecticide Resistance in the West Nile Encephalitis, Japanese Encephalitis, Avian Malaria and Lymphatic Elephantiasis Vector, Culex pipiens complex (Diptera: Culicidae) in Iran.

Background: Culex pipiens complex is considered as a vector of some important diseases such as West Nile fever, equine encephalitis, Rift valley fever, St. Louis encephalitis, Elephanthiasis and avian malaria in the world. The main measure for vector control is using insecticides. High use of insecticides caused resistance in the populations. The aim of this study was to review the status of insecticide resistance in the vector. Methods: Insecticide resistance in this species was found by the available papers and map of the data for carbamates, organochlorine, organophosphates, pyrethroids, microbial and insect growth regulator insecticides were done. An intensive search of scientific literature was done in "PubMed", "Web of Knowledge", "Scopus", "Google Scholar", "SID", and related resources. Results: Results showed that a wide variety of resistance to different insecticides in the country. Due to importance of this species in transmission of diseases. Discussion: resistance management strategies should be further considered to prevent from in secticide resistance and replacement of novel approach for vector control.

Molecular Identification of Phlebotomus caucasicus and Phlebotomus mongolensis (Diptera: Psychodidae) in a Hyperendemic Area of Zoonotic Cutaneous Leishmaniasis in Iran.

Phlebotomus caucasicus Marzinovsky and Phlebotomus mongolensis Sinton are morphologically similar sand fly species. Finding a reliable, fast, and simple method to differentiate these two sand flies is important in understanding their role in the transmission of Leishmania parasite. In our study, 20 specimens of male P. caucasicus, 4 specimens of male P. mongolensis, and 16 specimens of female of both species (Caucasicus group) were examined by polymerase chain reaction (PCR). The result shows identical patterns with a visible fragment of about 500 bp in size. In restriction fragment length polymorphism (RFLP), we observed identical patterns with TasI used as the restriction enzyme. After alignment with sequences of the ITS2 partial gene in GenBank, a perfect match was obtained for the P. mongolensis, but not for P. caucasicus whose sequence was not present in the GenBank. Based on the results of our study, the RFLP-PCR method with nucleotide gene fragment ITS2 was a rapid and reliable method for differentiating these sand fly species.

Wild Rodent Ectoparasites Collected from Northwestern Iran.

BACKGROUND: Rodents play an important role as reservoir of some pathogens, and the host of some ectoparasites as well. These ectoparasites can transmit rodents' pathogens to human or animals. The aim of this study was to assess the distribution and infestation load of ectoparasites on rodents in Meshkin-Shahr District, northwestern Iran. METHODS: Rodents were captured using baited live traps in spring 2014 from Meshkin-Shahr District and were transferred to the laboratory for identification to the species level. Their ectoparasites were collected, mounted and identified. RESULTS: Three rodent species including Meriones persicus (74%), Mus musculus (16.9%) and Cricetulus migratorius (9%) were identified. Among all rodents, 185 specimens (90.69%) were infested with a total of 521 ectoparasites. Overall, 10 arthropods species were collected, including fleas (97.6%), one mite (1.6%) and one louse species (0.6%) as follows: Xenopsylla nubica, X. astia, X. buxtoni, X. cheopis, Nosopsyllus fasciatus, N. iranus, Ctenocephalides felis, Ctenophthalmus rettigismiti, Ornithonyssus sp and one species of genus Polyplax. The most prevalent ectoparasites species was X. nubica (89%). CONCLUSION: Nearly all rodent species were infested with Xenopsylla species. Monitoring of ectoparasites on infested rodents is very important for awareness and early warning towards control of arthropod-borne diseases.

Feline leishmaniosis due to Leishmania infantum in Northwest Iran: The role of cats in endemic areas of visceral leishmaniosis.

Visceral leishmaniosis caused by Leishmania infantum (L. infantum) is an important zoonotic disease endemic in the Mediterranean region. Domestic dogs and other wild canines are the main reservoir hosts for the parasite, while domestic cats (Felis catus) may be carriers of L. infantum, and play a role as secondary reservoirs for the parasite. In the present study, serological (DAT), parasitological (microscopic smears and culture) and molecular methods (nested PCR) were used to evaluate infection with L. infantum in 103 stray cats collected from villages of Meshkin Shahr district, located in Ardabil province which is a well-known endemic region of human and canine visceral leishmaniosis in Iran. Overall, 25 out of 103 cats (24.27%) displayed anti-Leishmania antibodies with different titers. Amastigote forms of the parasite were detected in microscopic smears of the spleen of a cat with high anti-Leishmania antibodies using DAT. L. infantum was identified on microscopic slides by nested PCR, and the results were confirmed by sequence analysis. Based on the high rate of seropositive cats in this study, we conclude that cats may have an important role in the maintenance of L. infantum in the endemic areas of zoonotic visceral leishmaniosis in Iran.

Transmission of Leishmania infantum by Rhipicephalus sanguineus (Acari: Ixodidae) in Dogs.

BACKGROUND: Rhipicephalus sanguineus is the most widely distributed tick in the world, which is partly due to its biological flexibility and the global distribution of its major host, the domestic dog. In Mediterranean region it could be principal reservoir host for Leishmania infantum, usually transmitted by the phlebotomine sand flies. In this study, we evaluated the vector potential of R. sanguineus in transmitting L. infantum to uninfected dogs. METHODS: During 2014, five dogs with clinical manifestations of canine visceral leishmaniasis (CVL), high anti-Leishmania antibody titers and tick infestation, were selected from CVL endemic areas (Tehran and Alborz provinces). At least, twenty live ticks were removed from each infected dog. After morphological identification, the ticks were divided into two groups; ticks belonging to the first group were dissected for parasitological examinations and semi-nested PCR assay, and those of the second group were selected for the transmission of CVL caused by L. infantum to uninfected dogs. Following tick infestation, all uninfected dogs were kept for 9 months and examined monthly for clinical and serological tests. RESULTS: Nearly, 67% of ticks were infected by L. infantum using the semi-nested PCR. All other parasitological tests of ticks were negative. Clinical examinations and serological tests of the investigated dogs revealed negative results. Nested-PCR test results performed on splenic biopsy samples of dogs were also negative. CONCLUSION: L. infantum-positive R. sanguineus ticks were unable to transfer L. infantum from infected dogs to healthy ones. The detection of L. infantum DNA in ticks collected from naturally infected dogs by semi-nested PCR does not prove their vectorial competence.

Feline Dirofilariosis Due to Dirofilaria immitis in Meshkin Shahr District, Northwestern Iran.

BACKGROUND: Dirofilaria immitis is a common nematode of the cardiovascular system, which infects carnivores all over the world. The infection is prevalent in dogs, but in cats and human is rare. Dirofilariosis is transmitted by mosquitoes. Cats are accidental hosts and are naturally resistant to the infection, compared to the dogs. Mild infection can cause severe illness in cats and may lead to death, so it is clinically important to diagnose the disease. METHODS: In the present study, 103 stray cats were collected from Meshkin Shahr district, Aradabil Province, northwestern Iran that is an endemic area for canine dirofilariosis. Blood samples were prepared from the saphenous vein of each cat and were examined for the presence of microfilariae by the modified Knott test. RESULTS: A 2 yr old male cat (0.97%) was microfilaremic. The cat was subjected to necropsy and 4 adult D. immitis (2 male and 2 female worms) were found in the right ventricle of the heart. PCR was then carried out and D. immitis was confirmed. CONCLUSION: Cats and other carnivores in Meshkin Shahr district are at risk of infection due to the high activity of vectors in this climate and it is important to follow up the infection in this area.