Friday sermons, family planning and gender equity attitudes and actions: evidence from Jordan.

BACKGROUND: To assess the effects of a communication intervention designed to enhance Jordanian religious leaders' (RLs) communication about family health. METHODS: Programmatic effects on RLs were evaluated with a panel study design; 245 male and 145 female RLs participated in both baseline and end-line surveys in 2011. To assess effects on mosque attendees, a nonequivalent, post-intervention only with control group design was utilized; 431 intervention and 426 control respondents were interviewed in 2012. RESULTS: Although RLs in the intervention site reported higher levels of preaching and teaching about the family health topics at end line than at baseline, their congregants were no more likely than control congregants to report having heard such messages over the previous 6-month period, contrary to the anticipated outcome. Yet, intervention congregants compared with controls were more likely to take action related to the topics to which they were exposed. CONCLUSIONS: Despite the mixed findings, intervention mosque goers who recalled messages were more likely to report taking relevant actions. These findings suggest that trained RLs compared with their counterparts were more effective in message dissemination. Thus, the findings support broader implementation of this type of intervention once the programmatic changes recommended in this article are made.

The characterization of the Phlebotomus papatasi transcriptome.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome.

The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure.

Some pathogens (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, Leishmania spp) have been shown to persist in their host after clinical cure, establishing the risk of disease reactivation. We analyzed the conditions necessary for the long term maintenance of Leishmania major in genetically resistant C57BL/6 mice after spontaneous healing of their dermal lesions. Interleukin (IL)-10 was found to play an essential role in parasite persistence as sterile cure was achieved in IL-10-deficient and IL-4/IL-10 double-deficient mice. The requirement for IL-10 in establishing latency associated with natural infection was confirmed in IL-10-deficient mice challenged by bite of infected sand flies. The host-parasite equilibrium was maintained by CD4+ and CD8+ T cells which were each able to release IL-10 or interferon (IFN)-gamma, and were found to accumulate in chronic sites of infection, including the skin and draining lymph node. A high frequency of the dermal CD4+ T cells released both IL-10 and IFN-gamma. Wild-type mice treated transiently during the chronic phase with anti-IL-10 receptor antibodies achieved sterile cure, suggesting a novel therapeutic approach to eliminate latency, infection reservoirs, and the risk of reactivation disease.

Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis.

We have developed a model of cutaneous leishmaniasis due to Leishmania major that seeks to mimic the natural conditions of infection. 1,000 metacyclic promastigotes were coinoculated with a salivary gland sonicate (SGS) obtained from a natural vector, Phlebotomus papatasii, into the ear dermis of naive mice or of mice preexposed to SGS. The studies reveal a dramatic exacerbating effect of SGS on lesion development in the dermal site, and a complete abrogation of this effect in mice preexposed to salivary components. In both BALB/c and C57Bl/6 (B/6) mice, the dermal lesions appeared earlier, were more destructive, and contained greater numbers of parasites after infection in the presence of SGS. Furthermore, coinoculation of SGS converted B/6 mice into a nonhealing phenotype. No effect of SGS was seen in either IL-4- deficient or in SCID mice. Disease exacerbation in both BALB/c and B/6 mice was associated with an early (6 h) increase in the frequency of epidermal cells producing type 2 cytokines. SGS did not elicit type 2 cytokines in the epidermis of mice previously injected with SGS. These mice made antisaliva antibodies that were able to neutralize the ability of SGS to enhance infection and to elicit IL-4 and IL-5 responses in the epidermis. These results are the first to suggest that for individuals at risk of vector-borne infections, history of exposure to vector saliva might influence the outcome of exposure to transmitted parasites.

Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein.

Leishmania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leishmania infection, while immunity to the saliva protects against infection as determined by coinoculation of parasites with vector salivary gland homogenates (SGHs) or by infected sand fly bites (Kamhawi, S., Y. Belkaid, G. Modi, E. Rowton, and D. Sacks. 2000. Science. 290:1351-1354). We have now characterized nine salivary proteins of Phlebotomus papatasi, the vector of Leishmania major. One of these salivary proteins, extracted from SDS gels and having an apparent mol wt of 15 kD, was able to protect vaccinated mice challenged with parasites plus SGH. A DNA vaccine containing the cDNA for the predominant 15-kD protein (named SP15) provided this same protection. Protection lasted at least 3 mo after immunization. The vaccine produced both intense humoral and delayed-type hypersensitivity (DTH) reactions. B cell-deficient mice immunized with the SP15 plasmid vaccine successfully controlled Leishmania infection when injected with Leishmania plus SGH. These results indicate that DTH response against saliva provides most or all of the protective effects of this vaccine and that salivary gland proteins or their cDNAs are viable vaccine targets against leishmaniasis.

Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies.

Despite the fact that Leishmania are transmitted exclusively by sand flies, none of the experimental models of leishmaniasis have established infection via sand fly bites. Here we describe a reproducible murine model of Leishmania major infection transmitted by Phlebotomus papatasi. Prior exposure of mice to bites of uninfected sand flies conferred powerful protection against Leishmania major that was associated with a strong delayed-type hypersensitivity response and with interferon-gamma production at the site of parasite delivery. These results have important implications for the epidemiology of cutaneous leishmaniasis and suggest a vaccination strategy against this and possibly other vector-borne diseases.

Leishmania vaccine development: exploiting the host-vector-parasite interface.

Visceral leishmaniasis (VL) is a disease transmitted by phlebotomine sand flies, fatal if untreated, and with no available human vaccine. In rodents, cellular immunity to Leishmania parasite proteins as well as salivary proteins of the sand fly is associated with protection, making them worthy targets for further exploration as vaccines. This review discusses the notion that a combination vaccine including Leishmania and vector salivary antigens may improve vaccine efficacy by targeting the parasite at its most vulnerable stage just after transmission. Furthermore, we put forward the notion that better modeling of natural transmission is needed to test efficacy of vaccines. For example, the fact that individuals living in endemic areas are exposed to sand fly bites and will mount an immune response to salivary proteins should be considered in pre-clinical and clinical evaluation of leishmaniasis vaccines. Nevertheless, despite remaining obstacles there is good reason to be optimistic that safe and effective vaccines against leishmaniasis can be developed.

Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model.

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.

The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections.

Sand fly saliva contains a rich array of pharmacologically active compounds whose primary function is to prevent the hemostatic mechanisms of the host. Several studies have ascribed immunosuppressive properties to sand fly saliva as well as an exacerbative effect on Leishmania infectivity for their mammalian hosts. This review provides a comprehensive account of sand fly salivary components, the immunomodulatory properties exhibited by some of its molecules, and describes the findings concerning the influence of saliva on Leishmania infections. The potential use of saliva as part of an anti-Leishmania vaccine for the mammalian host is also addressed.

Echinococcus granulosus: possible formation of a shelled egg in vitro.

What appeared to be the early stages in the formation of a single egg with a striated embryophore was observed in an in vitro culture of Echinococcus granulosus protoscoleces isolated from sheep hydatid cysts in North Jordan. The 'egg' measured 19 x 19 microns in diameter and was formed in an intermediate vesicular/monozoic form which was never previously reported from a culture. This is the first report of an apparently shelled egg forming in an in vitro culture, but although promising, cannot be regarded as being unequivocal and will require confirmation by further work.

Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi.

Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector-parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1-Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40-60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.

Short report: outbreak of cutaneous leishmaniasis in a nonimmune population of soldiers in Wadi Araba, Jordan.

An outbreak of zoonotic cutaneous leishmaniasis (ZCL) occurred in a battalion of 80 soldiers posted at Qurayqira camp in Wadi Araba in southern Jordan. The battalion spent an intermittent period of five and a half months in the area, during which 45.0% (36 of 80) of the soldiers showed clinical disease. Of the 44 clinically negative soldiers, 31 were tested with leishmanin and 11 (35.5%) were leishmanin positive. The number of lesions in infected soldiers ranged from one to 15 and were mostly on the face and extremities. This report shows the level of transmission of ZCL in Wadi Araba, which is presently undergoing economic expansion and development following the peace process of the Arab-Israeli conflict.

Novel antigens for neurocysticercosis: simple method for preparation and evaluation for serodiagnosis.

Neurocysticercosis (NCC), which is caused by infection with the larval stage of the pork tapeworm (Taenia solium), is now recognized as a major cause of neurologic diseases in countries where the infection is endemic. Migration of persons from these countries is resulting in diagnosis and local transmission in nonendemic countries at increasing rates. In the present study, immunoblotting and an ELISA were carried out using antigens of T. solium cysticerci fractionated by isoelectric focusing and serum samples from patients with NCC, alveolar (AE) or cystic echinococcosis (CE), and other diseases. Immunoblot analysis revealed antigens fractionated by isoelectric focusing (pH 9.2-9.6) either from cyst fluid of T. solium cysticerci or from intact cysts had unique components (glycoproteins) highly specific and sensitive for detection of NCC exclusively. All confirmed NCC serum samples (53 of 53) recognized at least three major bands of 10-26-kD of fractions with pH 9.2-9.6 from either intact cysts or cyst fluid. These bands were not recognized by sera from patients with other parasitic diseases including AE (0 of 34), CE (0 of 36), or other heterologous parasitoses (0 of 77), patients with hepatoma (0 of 19) or sarcoidosis (0 of 11), or sera from healthy controls (0 of 29). The ELISA using the antigens showed the same sensitivity and specificity for differentiation of NCC (53 of 53) from other diseases (0 of 107) or healthy individuals (0 of 29). Both immunoblotting and the ELISA using the fractionated antigens readily differentiated all NCC from AE or CE in a blind test of 29 serum samples of persons with NCC, CE, and AE. Antigens fractionated from cyst fluid of T. solium cysticerci by a simple, single-step isoelectric focusing (pH 9.2-9.6) are highly specific and sensitive for differential serodiagnosis of NCC in immunoblotting and/or an ELISA.

A new focus of cutaneous leishmaniasis caused by Leishmania tropica in northern Jordan.

An endemic focus of cutaneous leishmaniasis caused by Leishmania tropica was identified in the district of Bani Kinana, northern Jordan. Fifty-five autochthonous cases were detected from February to September 1994 in a population of about 50,000. Most cases (74.5%) were from the villages of Malka and Um-Quais. The lesions were typically small, dry and mostly located on the face and extremities. Multiple lesions were common. Incidence was higher in females than in males and familial clustering of cases was observed in houses on the outskirts of villages. A leishmanin survey showed a 23.3% positivity rate in the populations of Malka and Um-Quais. The rate of positivity was low in the younger age groups and increased with age. There was some evidence that the disease is a zoonosis in this focus.

Species composition of sand flies and population dynamics of Phlebotomus papatasi (Diptera: Psychodidae) in the southern Jordan Valley, an endemic focus of cutaneous leishmaniasis.

The species composition of sand flies and the seasonality of Phlebotomus papatasi Scopoli were studied in the southern Jordan Valley from May to November 1992 using CDC light traps. Eleven species of sand flies were recorded, including P. kazeruni Theodor & Mesghali and P. tobbi Adler & Theodor, which are new records for the study area, and Sergentomyia squamipleuris Newstead, which is reported for the first time from Jordan. P. papatasi was the most abundant Phlebotomus species collected from domestic habitats as well as Psammomys obesus Cretzschmar burrows, comprising 89.4 and 99.5% of the total Phlebotomus catches, respectively. The catch of P. papatasi in CDC light traps was compared among domestic habitats, P. obesus burrows in an agriculturally modified semiarid rural habitat, and P. obesus burrows in a natural semiarid rural habitat. Peak P. papatasi abundance occurred in September and October and then declined sharply by late November. The abundance and temporal association of P. papatasi activity with the prevalence of cutaneous leishmaniasis in the study area pointed to the significance of this sand fly in the transmission of the parasite.

Urbanization--how does it affect the behaviour of sandflies?

The effect of building activities on the abundance of sandflies was investigated in Jordan. A total of 3442 sandflies were collected. The largest collections were made from natural habitats, uninhabited and inhabited houses. Collections from the latter two often included a larger number of Phlebotomus species. It was evident from this study that sandfly populations are able to recover to, and often exceed their original natural densities in man-made artificial habitats.

The behaviour and dispersal of sandflies in Ras el Naqb, south Jordan with particular emphasis on Phlebotomus kazeruni.

The behaviour and dispersal of sandflies from an enclosed cave were investigated during the summer of 1988 in Ras el Naqb by the mark-release-recapture technique. Nine species of sandflies were reported during this study including 5 Phlebotomus and 4 Sergentomyia species. P. sergenti, P. alexandri, S. adleri/S. clydei and S. taizi were reported for the first time from Jordan. P. kazeruni was the most abundant sandfly species inside the cave. The maximum distance travelled by a single P. kazeruni was 340m. However, the majority were recaptured within 10m of the cave. It seems likely that an independent "population" of sandflies which breeds inside the cave is developing.

Diagnosis of canine echinococcosis: comparison of coproantigen detection with necropsy in stray dogs and red foxes from northern Jordan.

The sandwich enzyme linked immunosorbent assay (ELISA) was used as a diagnostic test for Echinococcus granulosus infection by detecting coproantigens in 94 stray dogs Canis familiaris and eight red foxes (Vulpes vulpes) from northern Jordan. The results were analyzed in relation to actual helminth infection as revealed by necropsy. The infection rate of dogs with E. granulosus was 13.8% with a worm load ranging between 3-> 10,000 per infected dog. In contrast, eight of 13 E. granulosus infected dogs were coproantigen positive (overall sensitivity 61.5%). The sensitivity increased to 87.5% and 100% in dogs harboring > 20 and > 100 worms/dog, respectively. The specificity of coproantigen-ELISA was 91%. The greatest cross-reactivity was found in dogs infected with Dipylidium caninum. The positive and negative predictive values for the coproantigen-ELISA test were 50% and 94.2%, respectively. Thus, a coproantigen negative dog is most probably truly negative for E. granulosus. In contrast, a coproantigen positive dog may not be truly positive for E. granulosus, except if it has a high worm burden of > 100 worms/animal.

Environmental manipulation in the control of a zoonotic cutaneous leishmaniasis focus.

An investigation is currently underway to assess the efficiency and practicality of combined burrow destruction and removal of chenopods in the control of a zoonotic cutaneous leishmaniasis (ZCL) focus. Karameh and Sweimeh, situated in the southern Jordan Valley, are stable endemic foci for ZCL. Control measures have been initiated in Karameh and will be maintained for two years. Sweimeh will be used as control. Karameh combines a desert habitat with a high water table created by extensive irrigation. This resulted in an unnatural abundance of chenopods and active rodent colonies throughout the year. Some 50 Psammomys obesus and 6 Meriones tristrami were captured from various colonies. At present, chenopods are being uprooted and burrows destroyed to a depth of 0.5 to 1 meter within a perimeter of 2 Km from Karameh. However, man-made changes of the topography has made the application of control measures more difficult. The effect of control will be assessed through comparison of leishmanin skin tests (LST) positivity in children below the age of six years, from both foci, prior to and post control. Precontrol LST have shown a positivity rate of 33.3% in Karameh and 80.2% in Sweimeh.

Characterization of a blood activated chitinolytic system in the midgut of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi.

We characterized a cDNA from Phlebotomus papatasi, PpChit1, which encodes a midgut specific chitinase and show the presence of a functional, blood-induced chitinolytic system in sand flies. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293-F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4-methyl-umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase displayed similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65-70%.