Optimization of Eva Green real-time mPCR for differentiating C. jejuni/coli directly from feces.

OBJECTIVE: To develop and optimize a rapid molecular method for diagnosing campylobacteriosis directly from a clinical fecal sample and at the same time for determining the most common causing agents - C. jejuni/coli. MATERIALS AND METHODS: 38 clinical fecal samples from hospitalized patients with diarrheal syndrome were tested using a rapid immunochromatographic test. All positive samples were tested for confirmation by culturing in a microaerophilic atmosphere. The Eva Green real-time mPCR reaction of a direct fecal sample was conducted using the "IQ5TM Real-Time PCR System" apparatus. RESULTS: Out of 38 clinical fecal samples which were ICT positive, 18 strains were isolated by culture, namely, 17 of C. jejuni and 1 of C. coli. The Eva Green real-time mPCR reaction also reported 18 positive samples for Campylobacter, out of which 17 were of C. jejuni and only one of C.coli. CONCLUSION: We developed and optimized the Eva Green real-time mPCR for the detection and species differentiation of C. jejuni/coli directly from a clinical fecal sample. The molecular analysis we described has a 100% sensitivity and specificity when comparing the results obtained by it to those of the culture method, which is currently the "gold standard" in the diagnosis of campylobacteriosis (Tab. 2, Fig. 1, Ref. 6).

Epidemiological characteristics of brucellosis in sheep and goats in Bulgaria: 2005-2008.

AIM: To analyse and present the epidemiological and epizootological situation of brucellosis in humans, sheep and goats caused by Brucella melitensis. METHODS: The veterinary health services analyzsd epidemiological reports, as well as other official documents, in relation to the epidemiology and the measures undertaken for control of brucellosis in Bulgaria, 2005-08. RESULTS: In Bulgaria animal infections caused by Brucella melitensis have been eradicated since 1941. In 2005 several Bulgarian citizens with Brucella infection were diagnosed in the National Reference Laboratory. All the cases were imported, mostly by employees on sheep and goat farms in Greece. For a period of four years, the number of infected humans was 120 and distributed in 12 districts. Epidemiological investigations suggested that 45 persons (37.5%) were infected in Greece and 4 in Cyprus, Turkey, Italy and Tanzania respectively. All other patients were considered to be infected in Bulgaria. They were owners of infected sheep and goats or consumers of contaminated dairy products with Brucella melitensis. In June 2006, the first outbreaks of brucellosis in sheep and goat farms were recorded in the Smolyan district. By the end of 2008, the infection was registered in 16 villages of 4 districts. The average prevalence rate in the infected herds was 12.2% in goats and 1.8% in sheep. In order to achieve eradication 496 goats and 117 sheep were slaughtered. CONCLUSION: A farm where goats from Greece were introduced illegally in 2005 was identified as the primary source of the infection. the negative epidemiological aspects of free movement of goods and persons in the European Union are considered.

Persistence and multiplication of obligate anaerobe bacteria in amebae under aerobic conditions.

After co-cultivation of Mobiluncus curtisii, an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4-6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4-7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7-14 days the amount of bacterial cells increased to 1x10(5)-1x10(6) CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.

On the vascular action of crataemon.

The vascular action of crataemon is studied. The effect of the drug on organ blood flow is examined using isotope methods. It is established that under conditions of blood loss, 10 mg/kg crataemon sharply inceases the blood supply to the myocardium and liver. Under the same conditions the blood flow in the kidneys and intestines decreases after administration of the drug. Its effect is weaker in case of normal blood pressure. In the case of perfusion of cat hind legs the flavonoid mixture studied has a biphasic effect manifested in the form of initial brief hypertension, followed by a more prolonged and stronger depressor reaction. In larger oral doses (100 and 200 mg/kg) crataemon increases the resistance of the capillaries and decreases capillary permeability in albino rats. The experiments carried out confirm the predominantly action of crataemon on some vascular regions (heart and liver). It has biphasic effect on peripheral vessels, which may be attributed to the presence of different components. The capillary-tonic action is most probably due to the presence of rutin and rutin-like flavonoids.