Rami M. Mukbel

Prevalence of hydatidosis among donkeys in northern Jordan

One hundred and thirty donkeys (Equus asinus), aged between 5 months and 14 years of age, from the Irbid Governorate in northern Jordan were necropsied between November 1997-May 1999. Of these animals, 16.9% had hydatid cysts in either their lungs and/or livers. No donkeys of 3 years of age or less were infected, where as 33.3% (22 of 66) aged 4 years or greater were infected. Intensity of infection increased with age in a linear fashion. The prevalence also increased with age approaching an asymptotic prevalence of 1 in the oldest animals. This implied there was minimal regulation of the parasite population by intermediate host immunity. The numbers of cysts in the donkeys were increasing at a rate of 0.48 cysts per year from 0.054 infections. The frequency distribution was highly aggregated, consistent with a negative binomial distribution indicating infection of donkeys was not random.

Complement Receptor 3 Deficiency Influences Lesion Progression during Leishmania major Infection in BALB/c Mice

ABSTRACT Leishmania major is an obligately intracellular protozoan parasite that causes cutaneous leishmaniasis. Like numerous intracellular pathogens, Leishmania exploits cell surface receptors as a means of entry into host cells. Complement receptor 3 (CR3; also called CD11b/CD18), a β2 integrin on phagocytic cells, is one such receptor. Ligation of CR3 has been shown to inhibit the production of interleukin-12, the cytokine that is pivotal in establishing the cell-mediated response necessary to combat intracellular infection. Here we investigate the role that CR3 plays in the establishment and progression of cutaneous leishmaniaisis in vivo. Dermal lesions of wild-type BALB/c mice are characteristically progressive and lead to extensive tissue necrosis coupled with elevated parasite burdens; CD11b-deficient BALB/c mice, however, demonstrate an intermediate phenotype characterized by chronic lesions and a reduced incidence of tissue damage. Infection followed by a reinfection challenge indicates that both susceptible (BALB/c) and resistant (C57BL/6) mice, regardless of CD11b status, develop resistance to L. major. In addition, CD11b does not bias the T helper cytokine response to L. major infection. Our results further indicate that CD11b is not necessary for disease resolution in resistant mice; rather, this protein appears to play a minor role in susceptibility.

A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.

Seasonal variations in the abundance of Gasterophilus spp. larvae in donkeys in northern Jordan.

One hundred and five donkeys were examined over a 12-month period for the presence of Gasterophilus spp. larvae in their stomachs. G. intestinalis and G. nasalis were found, but there was a distinct seasonal variation in their abundance. The numbers of second-stage larvae (L2) peaked during the early winter and early summer, but they were absent or at low levels during the early spring and early autumn. This suggests that there are two periods of adult fly activity (late autumn and late spring) for both G. nasalis and G. intestinalis. Third-stage larvae (L3) were found throughout the year, but the lowest levels were seen in the late summer and autumn. The parasites also had an aggregated distribution, which may be due to host selection by the fly. The numbers of larvae did not vary significantly with age, which may be due to a lack of acquired protective immunity or to tolerance after repeated exposure.

Genetic characterization of Cryptosporidium in animal and human isolates from Jordan

Little is known about the epidemiology of Cryptosporidium in Jordan and to date, only one genotyping study has been conducted on Cryptosporidium isolates from Jordanian children. In the present study, a total of 284 faecal samples from Jordanian cattle, sheep, goats and chicken and 48 human faecal samples were screened for the presence of Cryptosporidium using an 18S quantitative PCR (qPCR) and a C. parvum/C. hominis specific qPCR at a lectin locus. Of these, 37 of 284 animal faecal samples were positive by qPCR at the 18S locus giving an overall prevalence of 11.6%. The point prevalence of Cryptosporidium in chickens, sheep, horses, cattle and goats ranged from 4.8% (chickens) to 18.7% (cattle). A total of six species were detected; C. xiaoi (n=9),C. andersoni (n=7),C. ryanae (n=5),C. parvum (n=4),C. baileyi (n=1) and a genetically distinct and potentially novel species in two isolates from horses. Sub-genotype analysis of the 4 C. parvum isolates at the 60-kDa glycoprotein (gp60) locus identified subtype IIaA19G2R1 (n=2) and IIaA16GR1 (n=2). For the human samples, 4 positives (8.3% prevalence) were detected. Of these, two were C. parvum (subtypes IIdA20G1 and IIaA15G2R1) and two were C. hominis (subtypes 1bA9G3 and 1bA10G2R2). Further studies are required to better understand the epidemiology and transmission of Cryptosporidium in Jordan.

Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents.

Phlebotomus papatasi SP15: mRNA expression variability and amino acid sequence polymorphisms of field populations

BackgroundThe Phlebotomus papatasi salivary protein PpSP15 was shown to protect mice against Leishmania major, suggesting that incorporation of salivary molecules in multi-component vaccines may be a viable strategy for anti-Leishmania vaccines.MethodsHere, we investigated PpSP15 predicted amino acid sequence variability and mRNA profile of P. papatasi field populations from the Middle East. In addition, predicted MHC class II T-cell epitopes were obtained and compared to areas of amino acid sequence variability within the secreted protein.ResultsThe analysis of PpSP15 expression from field populations revealed significant intra- and interpopulation variation.. In spite of the variability detected for P. papatasi populations, common epitopes for MHC class II binding are still present and may potentially be used to boost the response against Le. major infections.ConclusionsConserved epitopes of PpSP15 could potentially be used in the development of a salivary gland antigen-based vaccine.

Human immune response to salivary proteins of wild-caught Phlebotomus papatasi

Phlebotomine sand flies are the known vectors of Leishmania parasites. New approaches in vaccination against Leishmania have investigated the possibility of integrating Phlebotomus papatasi salivary proteins to enhance the immune response and protect against the transmission of the infection. The aim of the present study was to screen human immune responses to wild sand fly saliva and evaluate immunogenic salivary proteins. Blood samples were collected from donors in control and sand fly infested areas. Antibodies specific for sand fly antigens in donor plasma were probed using immunoblotting. In addition, recall proliferation capability of peripheral blood mononuclear cells (PBMC) was tested after sand fly salivary homogenates stimulation. The significant immunogenic salivary proteins (SPs) identified by immunoblotting were SP28, SP32, and SP36. A specific proliferative response of PBMC after stimulation with sand fly salivary homogenates was evident in donors that have antibody responses against sand fly salivary proteins. Individuals with antibody recognition to a higher number of salivary proteins (i.e., 3 or more SP bands) showed lower PBMC proliferative responses after in vitro stimulation with salivary gland homogenates (SGH) only in the sand fly infested, leishmaniasis free area. Interestingly, the presence of a humoral immune response to many SP antigens inversely correlates with a strong cell-mediated immune response (CMI). It was also noticed that some other heavily expressed antigens, in sand fly salivary homogenate, lack or have weak humoral immune reactivity in exposed individuals. Therefore, considering these antigens alone as CMI activators, without including the immunodominant humoral immune response proteins, needs future investigation.

Population genetics analysis of Phlebotomus papatasi sand flies from Egypt and Jordan based on mitochondrial cytochrome b haplotypes

BackgroundPhlebotomus papatasi sand flies are major vectors of Leishmania major and phlebovirus infection in North Africa and across the Middle East to the Indian subcontinent. Population genetics is a valuable tool in understanding the level of genetic variability present in vector populations, vector competence, and the development of novel control strategies. This study investigated the genetic differentiation between P. papatasi populations in Egypt and Jordan that inhabit distinct ecotopes and compared this structure to P. papatasi populations from a broader geographical range.MethodsA 461 base pair (bp) fragment from the mtDNA cytochrome b (cyt b) gene was PCR amplified and sequenced from 116 individual female sand flies from Aswan and North Sinai, Egypt, as well as Swaimeh and Malka, Jordan. Haplotypes were identified and used to generate a median-joining network, FST values and isolation-by-distance were also evaluated. Additional sand fly individuals from Afghanistan, Iran, Israel, Jordan, Libya, Tunisia and Turkey were included as well as previously published haplotypes to provide a geographically broad genetic variation analysis.ResultsThirteen haplotypes displaying nine variant sites were identified from P. papatasi collected in Egypt and Jordan. No private haplotypes were identified from samples in North Sinai, Egypt, two were observed in Aswan, Egypt, four from Swaimeh, Jordan and two in Malka, Jordan. The Jordan populations clustered separately from the Egypt populations and produced more private haplotypes than those from Egypt. Pairwise FST values fall in the range 0.024–0.648.ConclusionThe clustering patterns and pairwise FST values indicate a strong differentiation between Egyptian and Jordanian populations, although this population structure is not due to isolation-by-distance. Other factors, such as environmental influences and the genetic variability in the circulating Le. major parasites, could possibly contribute to this heterogeneity. The present study aligns with previous reports in that pockets of genetic differentiation exists between populations of this widely dispersed species but, overall, the species remains relatively homogeneous.

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data.