Nanoliposomal Trachyspermum ammi (L) sprague essential oil for effective control of malaria mosquito larvae, Anopheles stephensi Liston.

Controlling mosquito vectors at immature stages using larvicides is a practical strategy to stave off mosquito-borne diseases such as malaria. Developing nanoliposomes bearing essential oil is a promising approach to improving the efficacy and stability of EOs-derived larvicides. The main aim of this investigation was to assess the efficacy of nanoliposome containing Trachyspermum ammi L. EO (TAEO-NL) as a new potential formulation to control Anopheles stephensi Liston (Diptera, Culicidae) mosquito larvae. The chemical constituents of T. ammi L. essential oil (TAEO) were first investigated using gas chromatography-mass spectrometry (GC-MS) analysis; its dominant component (48.22%) was thymol. TAEO-NL with a particle size of 54.6 ± 5 nm and zeta potential of -18 ± 0.5 mV were then prepared using the ethanol injection method. Besides, the successful loading of TAEO was confirmed using Attenuated Total Reflection-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy analysis. A significant difference (P < 0.05) was observed in the efficacy of TAEO-NL and TAEO with lethal concentration 50% (LC50) values of 14.09 and 59.47 µg/mL against An. stephensi larvae. However, free nanoliposomes show negligible larvicidal effects (<5%). This nano-formulation could thus be suggested as a green product against insects to impede transmission of deadly infectious diseases with possible field applicability scope.

Pyrethroid-linked resistance allelic mutations by molecular analysis in wild human head louse (Phthiraptera: Pediculidae) populations from schoolgirls of South Iran.

Background: Human head louse, Pediculus humanus capitis De Geer, 1767 (Anoplura: Pediculidae), is one of the most frequent ectoparasites infesting Homo sapiens worldwide. Reduced sensitivity to treatment due to genetic mutations, in particular knockdown resistance (kdr) (or target site insensitivity) allele, has led to this infestation prevalence. Molecular characterization of this resistance has a crucial impact on selecting appropriate treatment protocol. The aim of this study was to investigate kdr gene mutations on voltage-sensitive sodium channel (VSSC) among wild head lice samples from Fars province, southern Iran. Methods: Head lice were collected using plastic detection combs on girls enrolled in public schools from 10 counties in Fars province. The specimens were screened in 10 pools (each pool per county containing 35 specimens), with three pools (30%) being positive. Following species identification with valid entomological keys, 350 (68%) out of 514 randomly collected adult head lice were analyzed after their somatic genomic DNA extraction using Sinaclon kit. Samples were investigated by polymerase chain reactions (PCR), and the amplicon was subsequently sequenced. Results: Sequence analysis showed that the sodium channel genes in the pooled ectoparasites had two intron and three exon regions. Single (L840F), double (I836L, E837K), and triple novel point mutations (V875L, Q876P, S879V); the last involving two concomitant allelic substitutions; were discovered in the second and third exon regions of head louse DNA on chromosome II from three (30%) counties. Other exon or intron regions remained non-mutated from the remaining seven counties. Conclusions: The detection of six amino acid substitutions from 30% of examined head lice among infested schoolgirls reveal that mutants are minutely developing. These findings provide further incentive to recapitulate the legitimacy of current control measures and resolve dynamics of resistance in human head louse populations.

Saprinus planiusculus (Motschulsky' 1849) (Coleoptera: Histeridae), a beetle species of forensic importance in Khuzetan Province, Iran.

BACKGROUND: Medico legal forensic entomology is the science and study of cadaveric arthropods related to criminal investigations. The study of beetles is particularly important in forensic cases. This can be important in determining the time of death and also obtain qualitative information about the location of the crime. The aim of this study was to introduce the Saprinus planiusculus on a rat carrion as a beetle species of forensic importance in Khuzestan province. METHODS: This study was carried out using a laboratory bred rat (Wistar rat) as a model for human decomposition. The rat was killed by contusion and placed in a location adjacent to the Karun River. Observations and collections of beetles were made daily during May to July 2015. RESULTS: Decomposition time for rat carrion lasted 38 days and S. planiusculus was seen in the fresh to post decay stages of body decomposition and the largest number of this species caught in the decay stage. CONCLUSION: The species of beetle found in this case could be used in forensic investigations, particularly during the warm season in the future.

Molecular detection of Crimean-Congo haemorrhagic fever virus in ticks collected from infested livestock populations in a New Endemic Area, South of Iran.

OBJECTIVE: Crimean-Congo haemorrhagic fever (CCHF) is a viral zoonotic disease with potentially fatal systemic effects on man. We aimed to determine the presence of CCHF virus among collected ticks from domestic livestock from October 2012 to September 2013. METHODS: A total of 1245 hard and soft ticks were collected from naturally infested ruminants in Marvdasht County, Fars Province, south of Iran. Nine tick species and one unidentified species in four disparate genera were detected. A total of 200 ticks were randomly selected and analysed by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of CCHF virus genome. RESULTS: The viral genome was detected in 4.5% (9 samples) of the studied tick population. The infected ticks belonged to the species of Hyalomma marginatum' Hyalomma anatolicum and Rhipicephalus sanguineus. The viruses detected in these three tick species were clustered in the same lineage as Matin and SR3 strains in Pakistan and some other Iranian strains. These results indicate that the ticks were wildly infected with a genetically closely related CCHF virus in the region. CONCLUSION: Regular controls and monitoring of livestock to reduce the dispersion of ticks and providing information to those involved in high-risk occupations are urgently required.

Malaria elimination trend from a hypo-endemic unstable active focus in southern Iran: predisposing climatic factors.

Human malaria is the most important vector-borne infectious disease in Iran. It remains focally endemic being restricted to almost 20 counties in three oriental provinces. As a result of control measures applied since 1988, these counties appear to be on the verge of eliminating malaria. Malaria elimination strategy has thus become the new goal in Iran. Malaria due to Plasmodium vivax, whose transmission is particularly hard to interrupt, accounts for nearly 90% of the cases. This study was thus undertaken to show malaria elimination trend from a hypo-endemic unstable active focus in southern Iran and to examine the role of prevailing climatic factors. This focus is now under elimination phase (Stratum III or annual parasitic index <1/1000 person/year). All malaria-positive cases were identified with active and passive procedures from 2003 to 2011. Although all ages were infected, more than half of the patients were in the higher than 20-year age group and a clear majority (77%) of them identified in the warmer months (May-October) of the year were positive with P. vivax parasites. The rate of falciparum to mixed infections was highest (1%) in the penultimate year. Case finding was negatively related to precipitation rate both annually and over the 9-year period and positively to ambient temperature in each year. Despite progress in the scale-up of its elimination, transmission of malaria remains active. This should be tackled by proactive case detection in specific hotspots of the study focus.

First molecular detection of Leishmania major within naturally infected Phlebotomus salehi from a zoonotic cutaneous leishmaniasis focus in southern Iran.

Human cutaneous leishmaniasis (CL) is a major notifiable public health problem in many parts of Iran. It is often caused by the zooflagellate parasite Leishmania major which is mainly transmitted by the bites of female phlebotomine sandflies belonging to the genus Phlebotomus (Diptera: Psychodidae). The annual incidence of CL in Fars province, southern Iran, was about 108-144 in 2007. The leishmanial infections of wild sandflies that may act as vectors were thus investigated at an endemic focus in this province. In all 330 female Phlebotomus sandflies were screened for the detection of Leishmania-specific kinetoplast DNA (kDNA) by polymerase chain reaction (PCR) methods. A two stage nested PCR protocol was used to establish the identity of Leishmania major species in naturally infected sandflies. The L. major kDNA was detected in 18 (5.5%) individual sandflies which belonged to four different Phlebotomus species (Phlebotomus papatasi, Phlebotomus salehi, Phlebotomus sergenti and P. major group). For the first time, one naturally infected P. salehi specimen contained the kDNA of the protozoan parasite, L. major, with a main band of 560 base pairs identified using the nested PCR method. It seems most likely therefore that P. salehi is potentially a rare sylvatic vector of L. major parasites in parts of this province. This is the first combined morphological and molecular studies of P. salehi in Iran.

Treatment outcome of Paederus dermatitis due to rove beetles (Coleoptera: Staphylinidae) on guinea pigs.

Linear dermatitis (or dermatitis linearis, DL) is a skin blistering inflammatory lesion caused by exposure to the pederin toxin from rove beetles. Although it is prevalent in many countries of the Middle East region, this is not a notifiable disease. In recent years, a number of clinical symptoms outbreaks of DL has been reported from a few neighboring countries of Iran, but no report of experimental treatment among small laboratory rodents is known. This is a prerequisite to ascertain the nature of the best treatment strategy in cases of infestation with these beetles, as it occurs among local settlers during hot seasons in certain parts of the southern Iranian province of Fars. Live Paederus beetles were collected, identified to species level, sexed apart and partly processed to obtain their hemolymph toxin pederin in ethanol for dermal application on guinea pigs. Two Paederus species were found. Paederus ilsae (Bernhauer) (Coleoptera: Staphylinidae) was more abundant than P. iliensis (Coiffait). Recovery from DL due to live P. ilsae beetles was quicker and less complex than that of pederin in ethanol on guinea pigs. The application of potassium permanganate with calamine to heal DL was also more effective than fluocinolone treatment. This topical corticosteroid is thus considered less able to avert the cytotoxic action of pederin on the skin of guinea pigs than the antipruritic and cleansing agents. It seems likely that fluocinolone has certain effects which delays the recovery period for the treated skin.

Gerbillus nanus (Rodentia: Muridae): a new reservoir host of Leishmania major.

Gerbillus nanus Blanford, 1875 known as Baluchistan gerbil, is a granivorous solitary naked-footed species. No evidence of its natural infection with the protozoan parasite, Leishmania, has so far been provided. Cutaneous leishmaniasis (CL) is a major public health problem in many parts of the world, including Iran. The annual nationwide incidence of human CL due to Leishmania major (CLM) in endemic rural areas was above 18,000 cases in 2008. The detection of L. major in rodents is of fundamental importance for incriminating them as potential reservoirs of CLM infection. Between April 2007 and April 2008, following detection of 245 clinical cases in Jask region of south-east Iran, wild rodents were captured and checked by the microscopic slide smears for leishmanial infections. Overall, 106 gerbilline rodents were captured from which 17 were identified as Gerbillus nanus. Females of Meriones hurrianae, Tatera indica and G. nanus were found to be naturally infected with L. MAJOR. The presence of these parasites in G. nanus has never been reported before. All the amastigote-infected rodents came from the eastern plain of this region, except one T. indica from the western plain which was found to be smear-positive or kinetoplast DNA-positive by PCR. The highest (11·8%) prevalence of infection among rodents confirmed by PCR to be infected with L. major was attributed to Baluchistan gerbil, G. nanus, which is thus incriminated as a potential reservoir host of L. major in Iran.

Post-earthquake outbreak of cutaneous leishmaniasis in a rural region of southern Iran.

Human cutaneous leishmaniasis (CL) is of increasing public-health importance in Iran. On 10 July 2003, two mild earthquakes struck the rural town of Zarindasht in the southern Iranian province of Fars. The results of passive detection of CL cases in this town (in which patients with any skin lesions were evaluated) from April 2002 to April 2004 indicated that the earthquakes may have led to an outbreak of the disease; annual incidence increased from 58·6 detected cases/100,000 in the 12 months before the earthquakes to (an outbreak peak of) 864 detected cases/100,000 in the following 12 months. In addition, the incidence of detected CL in the town that was struck by the earthquakes in 2003 was significantly higher in the 12 months after the earthquakes than that recorded, over the same 12 months, for Fars province as a whole (P<0·05). Most (70%) of the cases detected in the town were aged ≤10 years, about half (50·4%) of the detected skin lesions were on the face, and most (89·7%) of the skin lesions were caused by Leishmania major. Incidence over the study period showed marked seasonality, with most (79·5%) of the detected cases occurring between November and February. In areas where the disease is endemic, CL may need to be considered among the health threats posed by natural disasters such as earthquakes, and increased surveillance for CL after future earthquakes may be justified.

Cockroaches (Periplaneta americana and Blattella germanica) as potential vectors of the pathogenic bacteria found in nosocomial infections.

Although it has been difficult to prove the direct involvement of cockroaches (i.e. insects of the order Blattaria) in the transmission of pathogenic agents to humans, such insects often carry microorganisms that are important in nosocomial infections, and their medical importance in the spread of bacteria cannot be ruled out. In houses and institutions with poor standards of hygiene, heavy infestations with cockroaches, such as the peridomestic American cockroach (Periplaneta americana L.) and the domestic German cockroach (Blattella germanica L.), can occur. In the present study, cockroaches (126 B. germanica and 69 P. americana) were collected from four buildings (three public training hospitals and one house) in central Tehran, Iran. Each insect was processed, under sterile conditions, so that the bacteria on its external surfaces and in its alimentary tract and faecal pellets could be isolated and identified. The oldest and largest of the three hospitals sampled (a 1400-bed unit built 80 years ago) appeared to be the one most heavily infested with cockroaches, and cockroaches from this hospital accounted for most (65.4%) of the isolates of medically important bacteria made during the study. No significant difference was found between the percentages of P. americana and B. germanica carrying medically important bacteria (96.8% v. 93.6%; P>0.05). At least 25 different species of medically important bacteria were isolated and identified, and at least 22 were Gramnegative. The genus of enteric bacteria most frequently isolated from both cockroach species, at all four collection sites, was Klebsiella. The cockroaches from each hospital were much more likely to be found contaminated with medically important bacteria than those from the house. The hospital cockroaches were also more likely to be carrying medically important bacteria internally than externally (84.3% v. 64.1%; P<0.05). The implications of these and other recent results, for the control of cockroaches and nosocomial infections, are discussed.

Tick-borne relapsing fever in a new highland endemic focus of western Iran.

Tick-borne relapsing fever (TBRF) is a neglected zoonotic disease caused by infection with spirochaetes of the genus Borrelia. Humans usually contract it from the bite of infected soft ticks of the genus Ornithodoros. In Iran, where the disease is endemic in the mountainous north-western provinces, reports of over 200 cases annually probably under-estimate the true incidence. The species, distribution and infection of ticks that are potential vectors of Borrelia and the clinical and epidemiological characteristics of the local TBRF cases were recently investigated in the villages in and around the county town of Bijar, in north-western Iran. A blood sample from each suspected case of TBRF was checked for B. persica by dark-field microscopy, data were collected on the demographics and clinical manifestations of each confirmed case, and the prevalence of tick infection with borreliae and the monthly incidence of TBRF were evaluated. Between 2000 and 2007, 148 cases of TBRF (each with fever, chills and headache) were passively detected in the town. Most (115) of these were confirmed by microscopy, with the other subjects categorized as probable (21) or suspected cases (12) of TBRF. Most (91%) of the 148 subjects were young people, and most came from rural areas and lived in large households in the old mud-and-thatch houses of Bijar. Most (82%) of the cases occurred during the summer or early autumn. Overall, 8543 soft ticks (Ornithodoros tholozani, O. lahorensis, Argas persicus and A. reflexus) were collected by clustered random sampling. When a random sample of the O. tholozani ticks (96 of the 577 collected) was checked for B. persica infection, by being crushed and then inoculated intraperitoneally into a mouse or suckling Syrian hamster, 19 were found infected. Peaks in the monthly incidence of TBRF occurred as the numbers of O. tholozani in the tick collections peaked, and it seems likely that most of the cases were caused by B. persica transmitted by O. tholozani. Further studies in Iran, to map the geographical variation in the prevalence of soft-tick infection with Borrelia and identify any Borrelia reservoirs, are recommended.

The PCR-based detection of Leishmania major infections in Meriones libycus (Rodentia: Muridae) from southern Iran.

Cutaneous leishmaniasis (CL) is an increasing public-health problem in the countries of the Mediterranean region. In southern Iran, for example, the incidence of CL has more than doubled over the last decade. As part of an investigation of this worrying trend, the leishmanial infections of the wild rodents that may act as reservoir hosts were investigated in the Iranian province of Fars. Overall, 82 rodents, 56 of them Libyan jirds (Meriones libycus), were collected, in live traps, from a new, hypo-endemic focus of zoonotic CL. When Giemsa-stained smears of ear tissue or any lesions on the rodents were prepared and checked under a light microscope, amastigotes were found in the smears from six of the rodents, all M. libycus. None of the other rodents investigated was found to be infected. The infected rodents were encountered in two of the three areas studied. In PCR-based assays, the amastigotes seen in the ear smears and those from two recent human cases of CL from the focus were all found to be identical to a reference strain of L. major. It therefore seems that M. libycus is the main active reservoir host for ZCL in the focus.