Josipa Habuš

Multiple Co-infections of Rodents with Hantaviruses, Leptospira, and Babesia in Croatia

Hantaviruses, Leptospira spp., and Babesia spp. are rodent-borne pathogens present worldwide. We studied multiple co-infections of small rodents in Croatia with all three pathogens. Twenty-eight Apodemus flavicollis and 16 Myodes glareolus were tested for the presence of hantavirus RNA by real-time RT-PCR, Leptospira strains by renoculture method and Babesia DNA by PCR. Anti-hantavirus antibodies and anti-Leptospira antibodies were detected by serological methods. Very high infection rates with each pathogen were found in A. flavicollis: 20 of 28 rodents (71%) were infected with Dobrava virus, 13 rodents (46%) were infected with Leptospira, and 5 rodents (18%) were infected with Babesia. Multiple co-infections with all three pathogens were found in 3 of 28 (11%) A. flavicollis animals, suggesting that the same rodent host can be infected with several pathogens at the same time. Dual infections with both hantaviruses and Leptospira were found in 7 of 44 rodents (16%), with hantaviruses and Babesia in 2 rodents (5%), and double infection with both Leptospira and Babesia were found in 1 rodent (2%). Since hantaviruses, Leptospira, and Babesia have similar geographical distributions, it is to be expected that in other parts of the world multiple co-infections, representing a serious threat to public health, can be found.

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia

Croatia is a focus for many rodent-borne zoonosis. Here, we report a survey of 242 rodents and small mammals, including 43 Myodes glareolus, 131 Apodemus flavicollis, 53 Apodemus agrarius, three Apodemus sylvaticus, six Sorex araneus, four Microtus arvalis, one Microtus agrestis, and one Muscardinus avellanarius, collected at eight sites in Croatia over an 8-year period. Multiplex MassTag polymerase chain reaction (PCR) was used for detection of Borrelia, Rickettsia, Bartonella, Babesia, Ehrlichia, Anaplasma, Francisella tularensis, and Coxiella burnetii. Individual PCR assays were used for detection of Leptospira, lymphocytic choriomeningitis virus, orthopoxviruses, flaviviruses, hantaviruses, and Toxoplasma gondii. Of the rodents, 52 (21.5%) were infected with Leptospira, 9 (3.7%) with Borrelia miyamotoi, 5 (2%) with Borrelia afzelii, 29 (12.0%) with Bartonella, 8 (3.3%) with Babesia microti, 2 (0.8%) with Ehrlichia, 4 (1.7%) with Anaplasma, 2 (0.8%) with F. tularensis, 43 (17.8%) with hantaviruses, and 1 (0.4%) with an orthopoxvirus. Other agents were not detected. Multiple infections were found in 32 rodents (13.2%): dual infections in 26 rodents (10.7%), triple infections in four rodents (2.9%), and quadruple infections in two rodents (0.8%). Our findings indicate that rodents in Croatia harbor a wide range of bacteria and viruses that are pathogenic to humans.

Epizootiological survey of small mammals as Leptospira spp. reservoirs in Eastern Croatia.

In this survey we investigated a population of small mammals in Eastern Croatia in order to determine Leptospira carriage rates and identify circulating serovars. Out of 67 trapped animals, 20 (29.9%) isolates were obtained. Identification of isolates using microscopic agglutination test, pulsed field gel electrophoresis and multi locus sequence typing revealed that 10 (50.0%) isolates belong to serogroup Pomona, serovar Mozdok, 6 (30.0%) isolates to serogroup Australis, serovar Jalna, 2 (10.0%) isolates to serogroup Sejroe, serovar Saxkoebing, and 1 (5.0%) isolate to serogroup Grippotyphosa, serovar Grippotyphosa. One isolate from serogroup Bataviae was unable to be identified to the serovar level. Amplification of a 331-bp region of the locus LA0322 using real-time polymerase chain reaction determined that 12 (60.0%) isolates belong to L. kirschneri, 6 (30.0%) isolates to L. interrogans, and 2 (10.0%) isolates to L. borgpetersenii. Leptospira carriage rate was high (29.9%), which corresponds to a high incidence of human and domestic animal leptospirosis in Eastern Croatia. Furthermore, 90.0% of the isolates belong to serogroups Pomona, Australis and Sejroe which are also the most prevalent serogroups in humans in this area. These findings suggest that small mammals might be an important source of Leptospira spp. infection in Eastern Croatia.

Electrocardiographic changes in hospitalized patients with leptospirosis over a 10-year period.

Summary Background The aim of this study was to investigate the incidence and type of ECG changes in patients with leptospirosis regardless of clinical evidence of cardiac involvement. Material/Methods A total of 97 patients with serologically confirmed leptospirosis treated at the University Hospital for Infectious Diseases „Dr. Fran Mihaljević‟ in Zagreb, Croatia, were included in this retrospective study. A 12-lead resting ECG was routinely performed in the first 2 days after hospital admission. Thorough past and current medical history was obtained, and careful physical examination and laboratory tests were performed. Results Abnormal ECG findings were found in 56 of 97 (58%) patients. Patients with abnormal ECG had significantly elevated values of bilirubin and alanine aminotransferase, lower values of potassium and lower number of platelets, as well as more frequently recorded abnormal chest x-ray. Non-specific ventricular repolarization disturbances were the most common abnormal ECG finding. Other recorded ECG abnormalities were sinus tachycardia, right branch conduction disturbances, low voltage of the QRS complex in standard limb leads, supraventricular and ventricular extrasystoles, intraventricular conduction disturbances, atrioventricular block first-degree and atrial fibrillation. Myopericarditis was identified in 4 patients. Regardless of ECG changes, the most commonly detected infection was with Leptospira interrogans serovar Australis, Leptospira interrogans serovar Saxkoebing and Leptospira kirschneri serovar Grippotyphosa. Conclusions The ECG abnormalities are common at the beginning of disease and are possibly caused by the direct effect of leptospires or are the non-specific result of a febrile infection and metabolic and electrolyte abnormalities. New studies are required for better understanding of the mechanism of ECG alterations in leptospirosis.

New trends in human and animal leptospirosis in Croatia, 2009–2014

Leptospirosis is a geographically widespread and globally underestimated zoonosis that affects humans and variety of animals. To identify trends and possible risk factors, joined medical and veterinary teams investigated epidemiology and epizootiology of leptospirosis in Croatia. Retrospective analysis of data obtained from referent diagnostic laboratories included a total of 1917 human and 123964 animal sera tested in the period from 2009 to 2014. We found high human leptospirosis average incidence rate of 1.53/100000 with clear predominance of male patients older than 40 years (sex ratio M/F:3.2; median age 51±15.1years). Statistical analysis revealed seasonal and annual variations of incidence in humans that were primarily associated with favourable weather conditions (temperature 10-19, 9°C and precipitation above 100mm/m2). Majority of infections in humans were caused by serogroups Sejroe, Australis and Icterohaemorrhagiae. Notable variations in seroprevalence and changing trends in prevailing serogroups were recorded in most of the domestic animals and during the entire period of investigation. All of the observed findings underline leptospirosis as a significant human and veterinary public health threat and emphasize the importance of continuous multidisciplinary surveillance. We also argue that only input from both professions improves our overall knowledge on leptospirosis and leads to better and more efficient prevention and control strategies.

Survey for hantaviruses, tick-borne encephalitis virus, and Rickettsia spp. in small rodents in Croatia.

In Croatia, several rodent- and vector-borne agents are endemic and of medical importance. In this study, we investigated hantaviruses and, for the first time, tick-borne encephalitis virus (TBEV) and Rickettsia spp. in small wild rodents from two different sites (mountainous and lowland region) in Croatia. In total, 194 transudate and tissue samples from 170 rodents (A. flavicollis, n=115; A. agrarius, n=2; Myodes glareolus, n=53) were tested for antibodies by indirect immunofluorescence assays (IIFT) and for nucleic acids by conventional (hantaviruses) and real-time RT-/PCRs (TBEV and Rickettsia spp.). A total of 25.5% (24/94) of the rodents from the mountainous area revealed specific antibodies against hantaviruses. In all, 21.3% (20/94) of the samples from the mountainous area and 29.0% (9/31) from the lowland area yielded positive results for either Puumala virus (PUUV) or Dobrava-Belgrade virus (DOBV) using a conventional RT-PCR. All processed samples (n=194) were negative for TBEV by IIFT or real-time RT-PCR. Serological evidence of rickettsial infection was detected in 4.3% (4/94) rodents from the mountainous region. Another 3.2% (3/94) rodents were positive for Rickettsia spp. by real-time PCR. None of the rodents (n=76) from the lowland area were positive for Rickettsia spp. by real-time PCR. Dual infection of PUUV and Rickettsia spp. was found in one M. glareolus from the mountainous area by RT-PCR and real-time PCR, respectively. To our knowledge, this is the first detection of Rickettsia spp. in small rodents from Croatia. Phylogenetic analyses of S- and M-segment sequences obtained from the two study sites revealed well-supported subgroups in Croatian PUUV and DOBV. Although somewhat limited, our data showed occurrence and prevalence of PUUV, DOBV, and rickettsiae in Croatia. Further studies are warranted to confirm these data and to determine the Rickettsia species present in rodents in these areas.

Phylogenetic characterisation of feline immunodeficiency virus in naturally infected cats in Croatia indicates additional heterogeneity of subtype B in Europe

This study was performed on 29 domestic cats with a variety of clinical signs, possibly related to FIV infection. Blood samples were tested by a rapid immunochromatographic (ICA) procedure for detection of FIV antibodies. Subsequently, polymerase chain reaction (PCR) was performed to amplify a portion of the proviral gag gene. All 11 positive PCR products were sequenced and compared with previously reported FIV sequences. Croatian proviral isolates that could be amplified were clustered within subtype B, and additional heterogeneity was confirmed by the formation of three separate clusters. Phylogenetic analysis of circulating strains in Croatia and in southeast Europe is necessary to improve diagnostic methods and selection of the appropriate vaccinal strains.

Epidemiological and clinical features of leptospirosis in a highly endemic area over three time periods

To present the features of human leptospirosis over three time periods (1970–1975; 2000–2005; 2010–2015), to compare the collected data and to determine whether the incidence, seasonal and spatial distribution, prevalence of presumptive infective serogroups and clinical features have changed over the last 50 years.