Rickettsia africae and Candidatus Rickettsia barbariae in ticks in Israel.

DNA of several spotted fever group rickettsiae was found in ticks in Israel. The findings include evidence for the existence of Rickettsia africae and Candidatus Rickettsia barbariae in ticks in Israel. The DNA of R. africae was detected in a Hyalomma detritum tick from a wild boar and DNA of C. Rickettsia barbariae was detected in Rhipicephalus turanicus and Rhipicephalus sanguineus collected from vegetation. The DNA of Rickettsia massiliae was found in Rh. sanguineus and Haemaphysalis erinacei, whereas DNA of Rickettsia sibirica mongolitimonae was detected in a Rhipicephalus (Boophilus) annulatus. Clinicians should be aware that diseases caused by a variety of rickettsiae previously thought to be present only in other countries outside of the Middle East may infect residents of Israel who have not necessarily traveled overseas. Furthermore, this study reveals again that the epidemiology of the spotted fever group rickettsiae may not only involve Rickettsia conorii but may include other rickettsiae.

Spotted fever group rickettsiae in ticks collected from wild animals in Israel.

We report molecular evidence for the presence of spotted fever group rickettsiae (SFGR) in ticks collected from roe deer, addax, red foxes, and wild boars in Israel. Rickettsia aeschlimannii was detected in Hyalomma marginatum and Hyalomma detritum while Rickettsia massiliae was present in Rhipicephalus turanicus ticks. Furthermore, a novel uncultured SFGR was detected in Haemaphysalis adleri and Haemaphysalis parva ticks from golden jackals. The pathogenicity of the novel SFGR for humans is unknown; however, the presence of multiple SFGR agents should be considered when serological surveillance data from Israel are interpreted because of significant antigenic cross-reactivity among Rickettsia. The epidemiology and ecology of SFGR in Israel appear to be more complicated than was previously believed.

A large Q fever outbreak in an urban school in central Israel.

BACKGROUND. On 28 June 2005, numerous cases of febrile illness were reported among 322 students and employees of a boarding high school located in an urban area in central Israel. Subsequent investigation identified a large outbreak of Q fever which started 2 weeks earlier. We describe the investigation of this outbreak and its possible implications. METHODS. We conducted a case-control study to identify risk factors for Q fever disease. Environmental sampling was conducted to identify the source and the mode of transmission of Coxiella burnetii, the infectious agent. RESULTS. Of 303 individuals, 187 (62%) reported being ill between 15 June and 13 July 2005. Serological evidence for C. burnetii infection was evident in 144 (88%) of the 164 tested individuals. Being a student, dining regularly at the school dining room, and boarding at school during a June religious holiday and the preceding weekend were all significant risk factors for contracting Q fever. C. burnetii DNA was detected using polymerase chain reaction on samples from the school dining room's air conditioning system, supporting contribution of the air conditioning system to the aerosol transmission of the infectious agent. CONCLUSIONS. We report a large outbreak of Q fever in an urban school, possibly transmitted through an air conditioning system. A high level of suspicion for C. burnetii infection should be maintained when investigating point source outbreaks of influenza-like disease, especially outside the influenza season.

Establishment of cell-based reporter system for diagnosis of poxvirus infection.

Poxvirus detection assays are based on morphology, viral antigens and specific nucleic acids, none of which indicates virus viability or infectious capacity. Determination of virus viability is achieved by propagation in cell cultures and subsequent analysis by the mentioned methods, a process that takes days. Thus, presented here the development of a new assay, named PILA (Poxvirus Infection Luciferase Assay), for rapid detection of infectious poxviruses which is a cell-based reporter assay. The assay is composed of two steps: (i) Transfection of cells with a poxvirus specific reporter vector which consists of the early 7.5-kDa-STR promoter, regulating the expression of luciferase gene; (ii) Infection with a poxvirus containing sample. Luciferase activity measured post infection, indicates the presence of infectious poxvirus in the sample. The assay can detect quantities as low as 100 PFU of VACV, six hours post infection. Orthopox virus universality was confirmed by detection of various Orthopoxviruses, and specificity was verified by using pox-specific neutralizing antibodies. The PILA is specific, rapid, simple, and suitable for detecting viable virus. The assay can be utilized for applications such as poxvirus titration, neutralizing assay and drug discovery. The assay was adjusted for live detection assay by using GFP as reporting gene.

Early diagnosis of severe Mediterranean spotted fever cases by nested-PCR detecting spotted fever Rickettsiae 17-kD common antigen gene.

We describe 3 cases of Mediterranean spotted fever (MSF) who presented with severe sepsis, in 2 of which the clinical diagnosis was unclear at presentation. In each case the diagnosis of MSF was made using a nested-PCR assay for Rickettsia conorii 17-kD protein gene. The nested-PCR based diagnosis preceded the serological results of MSF that were all negative at admission. The early diagnosis of MSF by specific PCR will facilitate an early institution of appropriate therapy, saving unnecessary tests and medications.

Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains.

The extent of knowledge regarding the diversity of globally distributed Ehrlichia canis strains has been limited to information gained from a few evolutionarily conserved genes. In this study, E. canis strains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S-->P and P-->T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain. E. canis whole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinct E. canis strains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in all E. canis isolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.

Fatal Rickettsia conorii subsp. israelensis infection, Israel.

Underdiagnosis of fatal spotted fever may be attributed to nonspecific clinical features and insensitive acute-phase serologic studies. We describe the importance of molecular and immunohistochemical methods in establishing the postmortem diagnosis of locally acquired Israeli spotted fever due to Rickettsia conorii subsp. israelensis in a traveler returning to Israel from India.

Rickettsia conorii in humans and dogs: a seroepidemiologic survey of two rural villages in Israel.

The prevalence of IgG-antibodies reactive with an Israeli strain of Rickettsia conorii (Israeli strain 487), the agent of Israeli spotted fever, was examined in humans and dogs from two rural villages in Israel where the disease has been reported in humans. Sixty-nine of 85 (81%) canine sera and 14 of 136 (10%) of human sera had anti-R. conorii antibodies. No direct association could be made between seropositivity of people and ownership of a seropositive dog. This study indicates that exposure to spotted fever group rickettsiae was highly prevalent among dogs compared with humans in the two villages examined, probably reflecting a greater exposure rate of canines to the tick vector. These results support a previous suggestion that canine serology could be a sensitive indicator for the presence and magnitude of human exposure to R. conorii.

Clusters of Mediterranean spotted fever in Israel.

Mediterranean spotted fever (MSF) usually occurs as sporadic cases. We report five clusters of MSF in Israel. Each cluster consisted of two to three patients. In two clusters, one patient died while the other recovered. In the other three clusters the patients presented with a benign course of the disease. The diagnosis of MSF in the fatal cases was confirmed by nested-polymerase chain reaction (PCR) tests performed on samples obtained from internal organs. Rickettsial DNA was also found in a tick obtained from a dog owned by one of the patients. MSF was diagnosed in the recovered patients by serology. The diagnosis of MSF fever in one family member should raise the awareness to the possibility of other cases in the vicinity.

Molecular evidence for Anaplasma phagocytophilum in Israel.

Sequences from the Anaplasma phagocytophilum 16S rRNA gene were detected in 5 ticks representing 3 species (Hyalomma marginatum, Rhipicephalus turanicus, and Boophilus kohlsi) collected from roe deer (Capreolus capreolus) in Mount Carmel, Israel. The sequences were all identical to those of Ap-variant 1 strain.

Concentration of Bacillus spores by using silica magnetic particles.

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.

Double labeling and simultaneous detection of B- and T cells using fluorescent nano-crystal (q-dots) in paraffin-embedded tissues.

A double immunohistochemical technique for the simultaneous detection of T- and B cells in paraffin-embedded mice tissues have been developed. This procedure is based on using fluorescent nano-crystals (q-dots). The benefit of using q-dots evolves from their unique fluorescence characteristics advantages: such as broad excitation spectrum, narrow emission band and high photo-bleaching threshold compare to organic fluorophores. T cells antigens (CD3) were stained using antibody-coated q-dots with max emission at 655 nm (GalphaRb-QD655). B cells antigens (CD45R/B220) were stained using streptavidin-coated q-dots with max emission at 585 nm (SA-QD585). The simultaneous detection of T- and B cells was demonstrated in paraffin-embedded lymph node using standard fluorescence microscope.

Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples.

A nested polymerase chain reaction (PCR) assay has been developed and used in the diagnosis of fatal and benign cases of Mediterranean spotted fever (MSF). The test was based on specific primers derived from a Rickettsia conorii 17-kD protein gene. A positive signal was obtained from spotted fever group (SFG) and typhus group (TG) rickettsiae. Discrimination between SFG and TG rickettsiae was based on a restriction fragment length polymorphism test. Other gram-negative bacterial species tested did not generate a signal, attesting for the specificity of the assay. The SFG-specific DNA fragment was detected in four of 29 acute-phase sera from serologically confirmed patients with MSF, while acute-phase sera from 25 patients without MSF were PCR negative. Acute-phase sera samples (five of five) and tissue autopsies (six of seven) from fatal suspected cases of MSF were PCR positive. The results demonstrate that sera and tissue samples are suitable specimens for the nested PCR tests, especially in fatal cases.