Identification of naturally infected Lutzomyia intermedia and Lutzomyia migonei with Leishmania (Viannia) braziliensis in Rio de Janeiro (Brazil) revealed by a PCR multiplex non-isotopic hybridisation assay

To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.     

Molecular Characterization of a Non�CBabesia divergens Organism Causing Zoonotic Babesiosis in Europe

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.     

Transmission of human and macaque Plasmodium spp. to ex-captive orangutans in Kalimantan, Indonesia

Data are lacking on the specific diseases to which great apes are susceptible and the transmission dynamics and overall impact of these diseases. We examined the prevalence of Plasmodium spp. infections in semicaptive orangutans housed at the Orangutan Care Center and Quarantine, Central Kalimantan, Indonesia, by using a combination of microscopic and DNA molecular techniques to identify the Plasmodium spp. in each animal. Previous studies indicated 2 orangutan-specific Plasmodium spp., but our data show 4 Plasmodium spp. These findings provide evidence for P. vivax transmission between humans and orangutans and for P. cynomolgi transmission between macaques and orangutans. These data have potential implications for the conservation of orangutans and also for the bidirectional transmission of parasites between orangutans and humans visiting or living in the region.     

Bartonella spp. in pets and effect on human health

Among the many mammals infected with Bartonella spp., pets represent a large reservoir for human infection because most Bartonella spp. infecting them are zoonotic. Cats are the main reservoir for Bartonella henselae, B. clarridgeiae, and B. koehlerae. Dogs can be infected with B. vinsonii subsp. berkhoffii, B. henselae, B. clarridgeiae, B. washoensis, B. elizabethae, and B. quintana. The role of dogs as an important reservoir of Bartonella spp. is less clear than for cats because domestic dogs are more likely to be accidental hosts, at least in nontropical regions. Nevertheless, dogs are excellent sentinels for human infections because a similar disease spectrum develops in dogs. Transmission of B. henselae by cat fleas is better understood, although new potential vectors (ticks and biting flies) have been identified. We review current knowledge on the etiologic agents, clinical features, and epidemiologic characteristics of these emerging zoonoses.     

Hospital infestation by the cluster fly, Pollenia rudis sensu stricto Fabricius 1794 (Diptera: Calliphoridae), and its possible role in transmission of bacterial pathogens in Germany

The potential of the cluster fly, Pollenia rudis sensu stricto, to transmit bacterial pathogens was investigated during a mass infestation that took place in a German hospital. Cluster flies were individually examined for mesophilic bacteria carried on the exoskeleton. Bacterial growth could only be detected by using the enrichment culture technique to increase sensitivity, but not by direct intoculation of fly samples to agar plates. All 50 cluster fly samples that were tested carried opportunistic aerobic mesophilic Bacillus spp., whereas 41 fly samples were positive for Erwinia spp., 16 samples for Erwinia amylovara, 24 samples for Stenotrophomonas maltophilia, and 4 samples for Flavobacterium odoratum. Staphylococcus lugdunensis and Pseudomonas aeruginosa were found in 5 samples. No bacteriologically sterile cluster fly samples were obtained. The whole bacterial pattern found on P. rudis s. s. is known for its potential to cause opportunistic and/or nosocomial infections in humans. The results obtained led to the assumption that mass infestations of cluster flies occurring in sensitive areas, especially in hospitals, may cause a low, but not neglectable health threat due to mechanical transmission of bacterial pathogens.     

Evaluation of a recombinant salivary gland protein (thrombostasin) as a vaccine candidate to disrupt blood-feeding by horn flies

The potential for controlling blood-feeding by the cattle pest, Haematobia irritans irritans (horn fly), was tested by vaccination against thrombostasin (TS), an inhibitor of mammalian thrombin that is released into skin during horn fly blood-feeding. The increase in blood meal size that occurred for flies feeding on sensitized non-vaccinated hosts was blocked and egg development in female flies was delayed when horn flies fed on rabbits and cattle immunized with recombinant TS. This demonstration of the impact of disrupting TS action by vaccination provides a novel approach toward control of this veterinary pest and offers a paradigm for limiting blood-feeding in other medically-important insect species.     

Entomologic and serologic evidence of zoonotic transmission of Babesia microti,eastern Switzerland

We evaluated human risk for infection with Babesia microti at a site in eastern Switzerland where several B. microti-infected nymphal Ixodes ricinus ticks had been found. DNA from pooled nymphal ticks amplified by polymerase chain reaction was highly homologous to published B. microti sequences. More ticks carried babesial infection in the lower portion of the rectangular 0.7-ha grid than in the upper (11% vs. 0.8%). In addition, we measured seroprevalence of immunoglobulin (Ig) G antibodies against B. microti antigen in nearby residents. Serum from 1.5% of the 396 human residents of the region reacted to B. microti antigen (>1:64), as determined by indirect immunofluorescence assay (IgG). These observations constitute the first report demonstrating B. microti in a human-biting vector, associated with evidence of human exposure to this agent in a European site.     

Isolation of Onchocerca lupi in Dogs and Black Flies,California, USA

In southern California, ocular infections caused by Onchocerca lupi were diagnosed in 3 dogs (1 in 2006, 2 in 2012). The infectious agent was confirmed through morphologic analysis of fixed parasites in tissues and by PCR and sequencing of amplicons derived from 2 mitochondrially encoded genes and 1 nuclear-encoded gene. A nested PCR based on the sequence of the cytochrome oxidase subunit 1 gene of the parasite was developed and used to screen Simulium black flies collected from southern California for O. lupi DNA. Six (2.8%; 95% CI 0.6%–5.0%) of 213 black flies contained O. lupi DNA. Partial mitochondrial16S rRNA gene sequences from the infected flies matched sequences derived from black fly larvae cytotaxonomically identified as Simulium tribulatum. These data implicate S. tribulatum flies as a putative vector for O. lupi in southern California.     

Sand flies naturally infected by Leishmania (L.) mexicana in the peri-urban area of Chetumal city, Quintana Roo, México

The surveillance of prevalent Leishmania sand fly vectors is an important issue for epidemiological studies in populated areas where leishmaniasis is endemic. In this study, we collected sand flies from a peri-urban area in the southeast of Mexico. Natural infection with Leishmania (L.) mexicana was studied by PCR using a Leishmania internal transcribed spacer of the ribosomal RNA gene for amplification. Infected Lutzomyia olmeca olmeca, Lu. shannoni and Lu. cruciata sand flies were collected mainly during the high transmission season (November to March), coinciding with the highest sand fly densities. Additionally, positive specimens of Lu. olmeca olmeca were also captured during July and August. The infected sand flies were from primary forest (subperennial forest) and secondary forest (18-25 years old and 10-15 years old respectively). Sand flies collected with Disney and Shannon traps were the ones found to be infected with L. (L.) mexicana. We conclude that the high-risk period in which L. (L.) mexicana is transmitted in the peri-urban area of Chetumal City is from July to March and that transmission is associated with both the subperennial forest and the secondary forest.     

Disseminated Leishmaniasis Caused by Leishmania tropica in a Puppy from Karaj,Central Iran

A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was examined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large number of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT). DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.     

Epidemiological monitoring of American tegumentary leishmaniasis: molecular characterization of a peridomestic transmission cycle in the Amazonian lowlands of Bolivia

Human-made and environmental changes constitute a major risk factor for the (re-)emergence and spread of leishmaniasis; surveillance of the transmission cycle is essential in this context. This study integrated entomological and molecular parasitological techniques to document the transmission pattern of a peridomestic focus of Leishmania in the Isiboro Secure area of Bolivia. First the spatial distribution and relative density of phlebotomine sand flies, genus Lutzomyia, were established. Lutzomyia shawi was the predominant species in domestic and peridomestic environments (90% from all collections). Second, direct application of the hsp70 PCR to sand fly extracts detected Leishmania infections in Lu. shawi only, and gave an estimated infection rate of 0.21 to 0.38%. The cleavage of the hsp70 amplicon with restriction enzymes (hsp70 PCR-RFLP) allowed identification of Le. (V.) braziliensis and Le. (V.) guyanensis in Lu. shawi captured in the same village. These two parasite species were also found in humans from the study region, supporting the co-existence of two transmission cycles involving the same sand fly species. This study demonstrated the use of PCR-RFLP in the identification of Leishmania in sand fly pools which could lead to the development of methods for screening large sand fly populations in Latin America.     

Trachoma,flies and environmental factors in Burkina Faso

The presence of flies is one of the earliest risk factors for trachoma and it has been suggested that flies could act as vectors for transmission of chlamydiae. A national trachoma survey was conducted in 1997 in Burkina Faso to (i) study the relationship between trachoma occurrence, flies, dirty faces and some environmental factors, and (ii) investigate the role of flies in the presence of trachoma. The country was stratified into eight groups of provinces and a random sample of 30 clusters was selected in each group. All children aged < 10 years were examined for the diagnosis of active trachoma (trachomatous inflammation which was follicular and/or intense) and the dirtiness of the face and the presence of flies on the face were recorded. The children's carers were questioned about the number of baths given and daily face-washing. Household heads were asked about ownership of cattle and small ruminants. The presence of latrines, a stable, and garbage collection inside the yard was noted. Among 16,514 children examined, 27.0% had active trachoma and 3.3% intense inflammatory trachoma. Flies were present on 11.2% of children's faces and 82.4% and 19.7% of these children had active and intense inflammatory trachoma, respectively. Among the 30.2% of children with dirty faces, 70.2% had active and 10.2% intense inflammatory trachoma. In multivariate analysis, at least one daily bath showed a protective effect on both active and intense inflammatory trachoma. Face-washing twice daily was found to be significantly protective for active trachoma in some regions. A strong association was demonstrated between the presence of flies and dirty faces (odds ratio = 334, 95% confidence interval 202-546). The presence of flies on children's faces, dirty faces and trachoma appeared to be strongly associated. Although the presence of flies may be a marker of socio-economic status and is probably linked with other trachoma risk factors, our data indicated that interventions targeting fly control should be an important issue in controlling trachoma.     

Photorhabdus Species: Bioluminescent Bacteria as Human Pathogens?

We report two Australian patients with soft tissue infections due to Photorhabdus species. Recognized as important insect pathogens, Photorhabdus spp. are bioluminescent gram-negative bacilli. Bacteria belonging to the genus are emerging as a cause of both localized soft tissue and disseminated infections in humans in the United States and Australia. The source of infection in humans remains unknown.     

Bartonella spp. and Coxiella burnetii Associated with Community-Acquired,Culture-Negative Endocarditis,Brazil

We evaluated culture-negative, community-acquired endocarditis by using indirect immunofluorescent assays and molecular analyses for Bartonella spp. and Coxiella burnetii and found a prevalence of 19.6% and 7.8%, respectively. Our findings reinforce the need to study these organisms in patients with culture-negative, community-acquired endocarditis, especially B. henselae in cat owners.     

Occurrence of Potentially Pathogenic Bacterial-Endosymbionts in Acanthamoeba Spp.

Background:

Acanthamoeba- bacteria interactions enable pathogenic bacteria to tolerate harsh conditions and lead to transmission to the susceptible host. The present study was aimed to address the presence of bacterial endosymbionts of Acanthamoeba isolated from recreational water sources of Tehran, Iran. To the best of our knowledge this is the first study regarding occurrence of bacteria in environmental Acanthamoeba spp. in Iran.

Methods:

A total of 75 samples of recreational water sources were collected. Samples were cultured on non- nutrient agar 1.5% plates. Positive Acanthamoeba spp. were axenically grown. DNA extraction and PCR reaction was performed using JDP1-2 primers. All positive samples of Acanthamoeba were examined for the presence of endosymbionts using staining and molecular methods. The PCR products were then sequenced in order to determine the genotypes of Acanthamoeba and bacteria genera.

Results:

Out of 75 samples, 16 (21.3%) plates were positive for Acanthamoeba according to the morphological criteria. Molecular analysis revealed that Acanthamoeba belonged to T4 and T5 genotypes. Five isolates (35.7%) were positive for bacterial endosymbionts using staining method and PCR test. Sequencing of PCR products confirmed the presence of Pseudomonas aeruginosa and Agrobacterium tumefasiens.

Conclusion:

The presence of Acanthamoeba bearing pathogenic endosymbionts in water sources leads us to public health issues including improved sanitation and decontamination measures in recreational water sources in order to prevent amoebae-related infection. To the best of our knowledge this is the first report regarding the isolation of A. tumefasiens from Acanthamoeba in Iran and worldwide.     

Development of Sensitive Detection of Cryptosporidium and Giardia from Surface Water in Iran

Background

The protozoan parasites Cryptosporidium spp. and Giardia are known to occur widely in both raw and drinking waters. They are two of the causative agents of waterborne outbreaks of gastroenteritis throughout the world. In the present study, a PCR assay and FA were developed for detection of Cryptosporidium oocysts and Giardia cyst in environmental samples.

Methods

We have detected Cryptosporidium spp. oocysts and Giardia cysts in seeded and unseeded environmental water samples by PCR method. Water samples were spiked with oocysts (50, 100,300,500) and filtrated with a 1.2-µm pore size cellulose nitrate and follow by DNA extraction and purification by QIAamp DNA mini kit. Nested-PCR assay amplified an 850 bp fragment of 18s rRNA gene specific for Cryptosporidium and 435 bp fragment of glutamate dehydrogenase (GDH) target gene for Giardia. Also many river water from north of Iran, be checked by these methods.

Results

Cryptosporidium and Giardia DNAs were detected in seeded water sample and Giardia was detected in all 5 water samples from river in north of Iran by nested- PCR and FA. Also in one river water sample, Cryptosporidium was detected.

Conclusion

This protocol is effective for detection of these waterborne parasites in treated and untreated water samples. This study can also serve as a platform for further investigations and research water source in Iran.     

Molecular Epidemiology of Cryptosporidiosis in Iranian Children,Tehran, Iran

Background

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein (GP60) gene.

Methods

Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly.

Results

Out of 794 collected samples, 19 (2.40%) were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 (89.47%) of the positive isolates were Cryptosporidium parvum and 2 (10.52%) were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa (6/17) and IId (11/17). The most common allele in all 17 isolates belonged to IId A20G1a (41.18%). A22G1 (IF) subtype was detected in two C. hominis isolates of the children.

Conclusion

The predominancy of C. parvum species (specially, IId A20G1a subtype) in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran.     

Experimental control of Phlebotomus papatasi by spraying attractive toxic sugar bait (ATSB) on vegetation

The effect of attractive toxic sugar bait (ATSB) solution including fruit juice, sucrose and oral insecticides on populations of Phlebotomus papatasi (Scopoli), was studied in the central Jordan Valley, in a typical area with sparse desert vegetation. Three similar plots of land, each 35 hectares, were chosen for experiments: two for applications of ATSB and one as a control. Sand fly populations in all plots were monitored weekly from May to December. Experimental area I was sprayed three times between June and October, in patches covering about 10% of the vegetation. Experimental area II was sprayed twice with toxic baits, in August and again in October. The control area was also sprayed every second month with solution containing food dye marker instead of insecticide. After early toxin treatment, the population in area I dropped from ∼80 sand flies to ∼3 sand flies per trap in one month. In area II, the population declined about a month after treatment from ∼110 to ∼5 sand flies per trap. The control population was bimodal with peaks in July (∼135 flies per trap) and October (∼130 flies per trap). The food dye of the control bait marked an average of 65% to 79% of the sampled flies.     

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis among Humans and Domestic Dogs in Mazandaran Province,North of Iran

Background

New cases of visceral leishmaniasis (VL) have been reported recently in some parts of Mazandaran Province, north of Iran where the first human case of VL was reported in 1949. This study aimed to determine the present status of Leishmania infantum infection among humans and domestic dogs using serological and molecular methods in central parts of Mazandaran Province.

Methods

In this cross-sectional study, blood samples were randomly collected from 402 humans and forty-nine domestic dogs throughout 2009 and 2010 in the central part of Mazandaran Province including Semeskadeh and Kiakola districts where recent cases of human visceral leishmaniasis had been reported there. All the collected samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania infantum antibodies as well as convenience PCR assay on whole blood samples for detection of leishmanial infection and identification of Leishmania species.

Results

None of 402 collected human (402) and dog (49) blood samples showed anti Leishmania infantum antibodies at titers 1:3200 and 1:320 as cut-off values of DAT, respectively but only 2 of domestic dogs (4.1%) were found PCR-positive corresponding to L.infantum.

Conclusion

This study confirms the circulation of L. infantum at least among domestic dogs and highlights the sporadic pattern of VL in the studied areas. Further investigations regarding to sand flies fauna and wild canines as reservoir hosts of the disease, are recommended.