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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The diversity of patogenic bacteria within the Anaplasmataceae family in dogs from Croatia
No full textPorodica Anaplasmataceae dio je obitelji malih unutarstaničnih proteobakterija koja obuhvaća rodove Anaplasma, Ehrlichia, Neorickettsia, Wolbachia, Aegiptianella i Candidatus Neoehrlihia. Ove bakterije preživljavaju kao unutarstanični endosimbionti, no istovremeno mogu uzrokovati bolesti ljudi i životinja. Bakterije roda Anaplasma prenose se vektorima (krpelji šikare), a na području Europe najznačajniji je krpelj vrste Ixodes ricinus. Vrste iz porodice Anaplasmataceae je složeno uzgojiti na staničnim kulturama kao i dokazati u kliničkim uzorcima različitim bojanjima, stoga se u istraživanjima koriste molekularne metode. U ovom diplomskom radu analizirani su uzorci DNA izolirani iz krvi pasa arhive Laboratorija za parazitologiju Hratskog veterinarskog instituta. DNA je naprije umnožena lančanom reakcijom polimeraze „nested“ protokolom, a dijelovi 16S rRNA gena potom su sekvencionirani te analizirani odgovarajućim programima. Budući da ne postoje podaci o vrstama prisutnim u RH, cilj je bio istražiti koje su vrste iz roda Anaplasmataceae prisutne u pasa u Hrvatskoj te utvrditi učestalost pojedinih vrsta. Od ukupno 68 uzoraka pasa u njih 22 pronađena je DNA bakterija iz porodice Anaplasmataceae. Najzastupljenija je bila vrsta A. platys (64 %) a pronađene su i vrste A. phagocytophilum (18 %) te Wolbachia, endosimbiont Dirofilaria repens (18 %).Anaplasmataceae is part of a family of small intracellular proteobacteria which includes the genera Anaplasma, Ehrlichia, Neorickettsia, Wolbachia, Aegyptianella and Candidatus Neoehrlihia. These bacteria survive as endosymbionts in other cells, but at the same time can cause diseases in humans and animals. Bacteria of the genus Anaplasma are transmitted by vectors (ticks), and the most important in Europe is the tick species Ixodes ricinus. Species of family Anaplasmataceae are complicated to grow in cell cultures as well as to prove them in clinical samples by different coloring, therefore the research is based on molecular methods. This thesis analyzed the samples of DNA isolated from the blood of dogs belonging to the archives of the Laboratory of parasitology of the Croatian veterinary institute. DNA was amplified by polymerase chain reaction "nested" protocol, 16S rRNA gene parts sequenced and analyzed in the appropriate programs. As there are no data on the species present in Croatia the goal was to investigate which species of the genus Anaplasmataceae are present in dogs from Croatia and to determine the frequency of certain species. From a total of 68 samples of dogs in 22 of them the DNA of bacteria from the family Anaplasmataceae was found. The most abundant species was A. Platys (64 %) and species A. phagocytophilum (18 %) and Wolbachia, endosimbiont of Dirofilaria repens (18 %) also were found
Molecular Typing for Piroplasms Detected in Giemssa Stained Postmortem Obtained Tissue Inprints and Peripheral Blood Smears Originateing from Dogs
No full textBabezioza je najčešći uzročnik hemolitičkih anemija u Hrvatskoj. Temeljem morfologije merozoita babezije/teilerije dijele se na velike i male piroplazme. Za razliku od razmazaka periferne krvi u otiscima organa nije moguće razlikovati velike od malih babezija, jer mijenjaju svoja morfološka obilježja. Morfološki nije moguće razlikovati vrste odnosno podvrste , stoga je nužno koristiti molekularne metode za gensku tipizaciju uzročnika hemolitičke anemije u pasa.Ovo se najčešće provodi iz svježih uzoraka krvi no kojiput je potrebno prevesti gensku tipizaciju iz razmazaka krvi odnosno otisaka organa i tkiva uginulih pasa. U ovom istraživanju provedena je genska tipizacija iz 22 strugotine arhivskih citoloških uzoraka s tri različita protokola. U svim uzorcima prethodno je mikroskopski analizirana staničnost uzorka, prisutnost merozoita te njihova morfologija. Od 13 citološki pozitivnih uzoraka DNK je različitim protokolima umnožena u 12 uzoraka. Genska tipizacija je provedena na 9 uzoraka uspješno umnoženih trećim protokolom. DNK babezija je umnožena pomoću tri različita protokola: (I) jednostruka PCR reakcija (II) dvostruka PCR reakcija „nested“ (III) jednostruka PCR reakcija iz pročišćenih i koncentriranih DNK uzoraka. Uspješnost umnažanja je bila različita i iznosila je 31%, 70% i 70%. Također su dokazane razlike u uspješnosti umnažanja između otisaka organa i krvnih razmazaka istim protokolom. Tako je drugim protokolom iz krvnih razmazaka uspješnost umnažanja iznosila 60%, a iz otisaka organa 75%. Trećim protokolom iz krvnih razmazaka je uspješnost bila 40%, a iz otisaka 88%. Različita uspješnost umnažanja ukazuje na nužnost primjene različitih molekularnih protokola za dokazivanje piroplazmi u otiscima organa i razmascima krvi. Uspješnost umnažanja je potvrđena analizom nukleotidnih sljedova i dokazivanjem protozona B. canis canis. Ovim istraživanjem dokazana je prikladnost genske tipizacije i analize nukletidnih sljedova patogena. Analiza nukleotidnih sljedova prikladna je za dokazivanje vrste/ podvrste patogena iz arhivskih citoloških uzorcaka obojenih po Giemsi ili Diff Quick®.Babesiosis is common infectious agents of haemolytic anaemia in dogs in Croatia. Based on morphology of merozoites, Babesia/Theilera species are divided on big and small piroplasms. Differentiation between big of small piroplasms is not possible in the tissue imprints, unlike in peripheral blood smear, because they change their morphologic characteristic. The use of molecular methods for genetic typing of causative agent of haemolytic anaemia in dogs is necessary because morphologically is not possible to distinguish species, or subspecies of piroplasms. This is generally done from the fresh blood samples, but sometimes is necessary to perform genetic typing from the blood smear, or post-mortem tissue imprints. In this study genetic typing is performed from 22 grinds of cytological samples using three different protocols. Previously, cellularity, merozoites finding and their morphology have been analysed in all samples by microscopic method. Using different PCR protocols DNA is duplicated in 12 samples from 13 cytological positive samples. Genetic typing is performed on 9 samples successfully duplicated in third protocol. DNA of Babesiosis is duplicated using three different protocols: (I) simple PCR reaction (II) double PCR reaction „nested“ (III) simple PCR reaction from purified and concentrated DNA samples. The successfulness of DNA multiplication was different and amounted 31%, 70% and 70%. Significant differences are established in successfulness of multiplication between tissue imprints and blood smear using the same protocol. Using the second protocol successfulness of multiplication in blood smear samples was 60%, and from tissue imprints 75%. In third protocol successfulness of multiplication in blood smears samples was 40% and in tissue imprints samples 88%. The difference in the successfulness of duplication points on necessity of using different molecular protocols for detection of piroplasms in the tissue imprints and blood smear. The successfulness is confirmed with analysis of nucleotide sequences and detection of protozoon B. canis canis. In this study suitability of genetic typing and analysis of nucleotide sequences of pathogens is detected. The analysis of nucleotide sequences is suitable for detection of species/ subspecies of piroplasms from archival cytological samples stained by Giemsa or Diff Quick®
The ecological and genetic characteristics of ectoparasites of wild ungulates from different habitats in Croatia
No full textRadi utvrđivanja raznolikosti faune ektoparazita, njihove rasprostranjenosti, učestalosti pojavljivanja, spolne strukture, razvojnih stadija, područja prihvaćanja te vektorskog potencijala, pregledane su 1723 jedinke 6 vrsta divljih papkara (jelen obični, jelen lopatar, srna obična, svinja divlja, muflon, divokoza), na 45 lokacija u 3 biogeografske regije. Ektoparaziti su determinirani do razine vrste, a 170 ih je podvrgnuto genetskoj analizi sekvenciranjem odsječka 16S rRNA i odsječka COI gena. Sa 664 invadirane jedinke divljih papkara (38,53%) prikupljeno je 5477 ektoparazita, od čega je 11 vrsta pronađeno u kontinentalnoj regiji, u alpinskoj 7 i u mediteranskoj 8 vrsta. Među 10 vrsta krpelja dominantni je Ixodes ricinus (77,49%), slijede Dermacentor reticulatus (10,31%), Haemaphysalis concinna (8,77%), Ixodes gibbosus (0,91%), Haemaphysalis inermis (0,62%), Haemaphysalis punctata (0,51%), Dermacentor marginatus (0,45%), Rhipicephalus bursa (0,37%), Ixodes hexagonus (0,31%) i Hyalomma marginatum (0,22%). Od ostalih vrsta najzastupljeniji su kukci Lipoptena cervi (33,63%), Haematophinus suis (0,96%), Hippobosca equina (0,93%), Damalinia spp. (0,32%) te pijavica Haemopis sanguisuga (0,05%). Sekvencioniranjem su dokazana 3 haplotipa za I. ricinus, 1 za H. inermis te 2 za R.bursa. je Za dvije vrste divljih papkara je po prvi puta potvrđeno da su nespecifični nositelji ektoparazita: jelen obični za H. suis i divlja svinja za H. sanguisuga, što je prvo bilježenje ove pijavice na sisavcima. Pronalazak vrsta I. gibbosus u kontinentalnoj, D. reticulatus u mediteranskoj i Hy. marginatum u alpinskoj regiji značajno odstupa od dosad poznate rasprostranjenosti tih vrsta u Hrvatskoj, što ukazuje na učinke klimatskih promjena na biologiju i ekologiju ektoparazita i divljih papkara.In order to determine diversity of ectoparasite fauna, its distribution, frequency of occurrence, sexual structure, developmental stages, attachment sites and vector potential, 1723 individuals of 6 species of wild ungulates (red deer, fallow deer, roe deer, wild boar, mouflon, chamois) were examined at 45 locations in 3 biogeographical regions. Ectoparasites were determined to the species level and 170 were subjected to genetic analysis by sequencing the 16S rRNA segment and the COI gene segment. From 664 infested individuals (38.53%) of wild ungulates, 5477 ectoparasites were collected, of which 11 species were found in the Continental region, 7 in the Alpine and 8 in the Mediterranean region. Among 10 tick species, Ixodes ricinus was dominant (77.49%), followed by Dermacentor reticulatus (10.31%), Haemaphysalis concinna (8.77%), Ixodes gibbosus (0.91%), Haemaphysalis inermis (0.62%), Haemaphysalis punctata (0.51%), Dermacentor marginatus (0.45%), Rhipicephalus bursa (0.37%), Ixodes hexagonus (0.31%) and Hyalomma marginatum (0.22%). Among other species, the most common were insects Lipoptena cervi (33.63%), Haematophinus suis (0.96%), Hippobosca equina (0.93%), Damalinia spp. (0.32%), and leech Haemopis sanguisuga (0,05%). Sequencing revealed 3 haplotypes of I. ricinus, 1 of H. inermis and 2 of R. bursa. For the first time it was confirmed that two species of wild ungulates are nonspecific carriers of ectoparasites: red deer for H. suis and wild boar for H. sanguisuga, which is the first record of this leech on mammals. Finding of I. gibbosus in Continental region, D. reticulatus in Mediterranean and Hy. marginatum in Alpine region significantly deviates from their hitherto known distribution in Croatia, indicating the effects of climate change on biology and ecology of ectoparasites and wild ungulates
Pathological, parasitological and molecular research of feline verminose pneumonias
No full textVerminozne pneumonije mačaka su kronične upale pluća uzrokovane oblićima iz rodova Aelurostrongylus i Troglostrongylus. Često su sporedan nalaz obdukcije, a podaci o stvarnoj učestalosti ove bolesti u populaciji mačaka u svijetu i u Hrvatskoj su dvojbeni. Morfološke sličnosti ličinki (L1) oblića Troglostrongylus spp. i A. abstrusus otežavaju razlikovanje ovih vrsta parazita. Obzirom da je u RH provedeno samo jedno istraživanje verminozne pneumonije mačaka, uzročnici verminozne pneumonije u šest mačaka istraženi su patološkim, parazitološkim i molekularnim metodama kako bi ih se identificiralo. Ovo istraživanje predstavlja prvu gensku potvrdu (18SrRNA, ITS2) A. abstrusus, dok isključuje prisutnost T. brevioru RH. Također predstavlja prvu primjenu umjetne probave u svrhu izolacije i determinacije plućnih nematoda.Verminose pneumonias are chronic pneumonias caused by nematodes of the Aelurostrongylus and Troglostrongylus genus. They are often an incidental postmortal finding, and the data on the actual incidence of this disease in cats in the world and in Croatia are dubious. The morphological similarities of the larvae (L1) of the Troglostronylus spp. and A. abstrusus nematodes render differentiating these species difficult. As only one research of feline verminose pneumonia has been conducted in Croatia, the causal agents of verminose pneumonia in six cats have been subjected to pathological, parasitological and molecular investigation in order to identify them. This research presents the first genetic proof of (18SrRNA, ITS2) A. abstratus, while excluding the presence of T. brevior in Croatia. It also presents the first application of artificial digestion with the goal of isolating and determining these lung nematode
THE INFLUENCE OF PROTOZOA GIARDIA DUODENALIS ON APPEARANCE AND INTENSITY OF GASTROINTESTINAL SYMPTOMS IN DOGS
No full textKako paraziti (naročito iz roda Giardia) pripadaju u izrazito česte uzročnike
probavnih simptoma u pasa vrlo je važno odrediti utjecaj G. duodenalis na pojavnost i
intenzitet probavnih simptoma u pasa. U ovo istraživanje su bila uključena 82 psa. Svi
uzorci stolice pretraženi su na postojanje parazita metodom flotacije te neposrednom
imunoflourescencijom, a tipizacija svih giardija provedena je izdvajanjem ukupne
DNA, umnažanjem specifičnog odsječka DNA i određivanjem nukleotidnog sljeda
pročišćenog proizvoda za svaki pojedini izolat. Psi su podijeljeni na dvije osnovne
skupine: psi s probavnim simptomima (simptomatski, n=42) i psi bez prisutnih
probavnih simptoma (asimptomatski, n=40). Formirane su skupine pasa ovisno o
pojedinom genskom tipu G. duodenalis. U svrhu kliničke procjene intenziteta bolesti
korišten je bodovni sustav koji obuhvaća 12 kliničkih simptoma.
U pretraživanih pasa dokazane su giardije vrsno specifičnih genskih skupina C
i D. Nije utvrđena statistički značajna razlika u pojavnosti G. duodenalis između pasa
sa simptomima i pasa bez kliničkih simptoma, kao niti razlike u učestalosti pojedinih
kliničkih simptoma između skupine simptomatskih pasa u kojih je izolirana G.
duodenalis genske skupine C ili D, osim učestalosti pojave sluzi u stolici koja je
učestalija u pasa invadiranih genskom skupinom C. Intenzitet kliničkih simptoma
statistički se značajno ne razlikuje između pasa s i bez utvrđene giardije. U skupini pasa
invadiranih giardijom veći je udio slučajeva kroničnog tijeka bolesti. Statistički je bila
značajna povezanost istovremenih invazija G. duodenalis s Criptosporidium spp (<0.0001). Vrijednosti intenziteta kliničkih simptoma vrlo slabo su povezane s pojavom
različitih parazita u stolici pasa, a psi s giardijom istovremeno su invadirani većim
brojem drugih parazita, nego psi bez giardije. Nađena je statistički značajno viša
aktivnost lipaze i CPK te statistički značajno niža koncentracija ureje i aktivnosti ALT u
serumu pasa invadiranih s G. duodenalis. Opažene razlike u vrijednostima hematoloških
i biokemijskih pokazatelja krvi između pasa invadiranih genskim C i D skupinama nisu
statistički značajne.Introduction
Giardia has a wide range of host species and is a common cause of diarrheal
disease in humans and animals. Molecular data have defined seven genetic assemblages
of Giardia duodenalis, named A-G. Humans are infected with assemblages A and B,
dogs primarily with C and D, but dogs can also be infected with assemblages A and B,
and are able to transmit these zoonotic assemblages to their owners. The epidemiology
and clinical impact of infections with this parasite in dogs is still not completely
understood due to variable results across different studies. The aim of this study was to
analyze giardia assemblage prevalence in dogs and influence of giardia assemblage on
clinical symptoms and its intensity. The aim of this study was also to analyze whether
co-invasion of different giardia assemblages with other parasites/pathogens has
influence on clinical symptoms and its intensity.
Materials and methods
This research included 82 dogs (older than one year) that were admitted to the
Clinic for Internal Medicine, University of Zagreb. The dogs were symptomatic (n=42)
and asymptomatic (n=40). All fecal samples were examined for intestinal parasites by
direct microscopic examination after the flotation technique and by
immunofluorescence, and giardia positive samples were subjected to further genetic
characterization by using PCR of small subunit ribosomal DNA (Ssu rRNA gene) genes.
Stool samples were examined for the presence of C. perfringens by fecal culture in 5% sheep blood agar. Data on clinical symptoms, blood hematology and biochemistry were
collected from the database of the Clinic for Internal Medicine, and a scoring system for
the intensity of 12 gastrointestinal symptoms was constructed.
Statistical analysis was done by Stata 13.1 (Stat Corp. USA).
Results
Prevalence rates for giardia were 30.4% (25/82) in all dogs: 30% in asymptomatic dogs, and 31% in symptomatic dogs. Giardia was more prevalent in younger animals, and there was no sex predisposition for giardia infection. Ten dogs were molecularly positive for G. duodenalis assemblage C, and 15 dogs were positive for G. duodenalis assemblage D. Zoonotic assemblages A or B were not found. The following parasites/pathogens were found: Giardia duodenalis (30.4%), Trichuris vulpis (9.76%), Cryptosporidium spp (12.19%), Isospora canis (4.88%), Ancylostoma spp/Uncinaria spp (2.44%); Toxocara canis (7.32%), Toxascaris leonina (1.21%); Clostridium perfringens (70.73%). Co-invasion of two or more parasites/pathogens was found in 35.9% of dogs with diarrhea, and giardia positive dogs were more often infected with Cryptosporidium spp than giardia negative dogs. There was no correlation between the presence of giardia or its different assemblage and the intensity and appearance of gastrointestinal symptoms in dogs, with the exception of symptoms duration: giardia positive dogs had chronic symptoms more often than giardia negative dogs. Statistically relevant co-invasion of giardia and cryptosporidium was found. Statistically important differences in concentration of BUN and activity of lipase, CPK and ALT were found in giardia positive dogs.
Discussion/conclusions
Almost the same prevalence of giardia in symptomatic and asymptomatic dogs shows the need to test all dogs for giardia, and also to perform treatment on all positive dogs. In our research we found only host adapted assemblages C and D with the explanation being that host adapted assemblages replicate faster and “push out” other assemblages. The offered hypothesis is that transmission of host adapted species may be favored by intensive contact between animals and that these prevail over other non-host adapted species.
VII
Although some research of human giardiasis showed correlation between giardia assemblage and intensity or appearance of gastrointestinal symptoms in humans, according to our research these models can not be translated to dogs. However, in this research we found only assemblages C and D, so this conclusion can not be accepted for assemblages A and B without further investigation. This study has identified an increased risk in dogs of Giardia spp. and co-infections with Cryptosporidium spp, probably due to their life cycle and contamination of same invasion reservoirs (water). From that comes the need to test all giardia positive dogs for Cryptosporidium spp
GENOTYPING OF TICKS AND TICK BORNE PATHOGENS ON THE TERRITORY OF THE REPUBLIC OF CROATA
No full textKrpelji se nakon komaraca smatraju najvažnijim biološkim vektorima uzročnika
bolesti u svijetu. Dosadašnja podjela se temeljila na identifikaciji vrsta temeljem
specifičnih morfoloških obilježja. Učestalija primjena molekularnih metoda dovela je
do promjena u sistematizacija, ali i do dokaza novih patogena u krpeljima, životinjama i
ljudima.
Kako bi se utvrdio značaj, vektorski potencijal i raznolikost krpelja na području
RH istraživanje je provedeno na 509 arhivskih prethodno morfološki determiniranih
krpelja. Krpelji su pojedinačno analizirani sekvenciranjem odsječka 16S rRNA gena. U
svakog pojedinačnog krpelja istražena je prisutnost patogena iz rodova Babesia,
Theileria, Anaplasma, Ehrlichia, Hepatozoon, Mycoplasma, Borrelia, Rickettsia,
Francisella i Coxiella, te je provedeno mapiranje krpelja i dokazanih patogena.
Sekvenciranjem je dokazano šest vrsta krpelja iz roda Ixodes: I. gibbosus, I.
canisuga, I. kaiseri, I. ventalloi, I. hexagonus i I. ricinus. Unutar vrsta I. hexagonus i I.
ricinus su dokazana tri i 24 različita genotipa. Rod Dermacentor je bio zastupljen sa
dvije vrste, D. marginatus i D. reticulatus. Unutar roda Rhipicephalus dokazana je vrsta
R. bursa i skupina R. sanguineus sensu lato (s.l.), sa četiri različita genotipa. Tri skupine
sekvenci tri genotipa su bile slične sekvencama R. turanicus, a četvrta R. sanguineus
sensu stricto (s.s.). Hyalomma margintum je bila jedina vrsta roda Hyalomma, dok su
unutar roda Haemaphysalis dokazane: H. parva, H. inermis i H. concinna.
Vrste iz rodova Babesia i Theileria su dokazane u 6,3% i 6,1% krpelja. Rod Babesia je
bio zastupljen s osam skupina sekvenci koje su odgovarale B. canis, B. vulpes, B.
microti, B. venatorum, Babesia sp. „Badger type A” i „Badger type B”, B. ovis, Babesia
cf. crassa i Babesia sp „tavsan“. Unutar roda Theileria dokazane su četiri vrste: T. ovis,
T. orientalis (buffeli/sergenti), T. equi i T. capreoli. U 18% krpelja su dokazane
proteobakterije iz porodice Anaplasmataceae: Anaplasma capra, A. phagocytophilum,
Ehrlichia canis, Ehrlichia sp., „Candidatus Neoehrlichia lotoris“ i endosimbionti
Wolbachia sp. i Midichloria mitochondrii. Anaplasma capra je dokazana u R. bursa, te
R.turanicus 2 i R. turanicus 3, a E. canis u R. bursa. Tri vrste iz roda Hepatozoon su
sekvenciranjem dokazane u 6,1% krpelja: H. canis, H. felis i Hepatozoon „Badger
type“. Pet vrsta iz roda Rickettsia, R. slovaca, R. raoultii, R. aeschlimannii, R. massiliae
i R. monacensis su dokazane u 11,7% krpelja. U R. turanicus 3 s Pelješca dokazana je
Francisella tularensis subsp. holartica, dok je endosimbiont „Francisella-sličan“
dokazan u 1,5% D.reticulatus i D. marginatus. Na području kontinentalne Hrvatske u
genotipovima I. ricinus su dokazane Borrelia afzelii i B. valaisiana u 1,9% i 0,8%
krpelja. Istraživanjem nisu dokazane bakterije iz roda Mycoplasma i vrsta Coxiella
burnetii. Ovo istraživanje predstavlja prvo opsežno istraživanje krpelja i patogena u
krpeljima u Hrvatskoj.Ticks are obligate hematophagous ectoparasites of mammals, birds, amphibians and
reptiles, and currently are after mosquitoes considered as most important biological vectors of
pathogens in the world.Overall 920 species of ticks have been described so far, classified
within three families (Ixodidae, Argasidae and Nuttalliellidae), among which Ixodidae (hard
ticks) represents the most important family. About 10% of known tick species can transmit
various pathogens such as viruses, rickettsiae, bacteria and parasites. With development of
molecular methods, a new species, strains or genetic variants of pathogens/microorganisms,
are being detected in ticks worldwide, and the spectrum of potential tick transmitted
pathogens affecting domestic animals and humans that can cause disease important not only
in veterinary medicine but also within public health aspects, continues to increase.
Current classification of ticks was based on species identification according to morphological
features, but frequent use of molecular methods has led to changes in systematization. For
example, sequence analysis of 12S rRNA gene, classified the genus Boophilus in the genus
Rhipicephalus, while analysis of 12S rRNA, 16S rRNA and COX1showed that Rhipicephalus
sanguineus is considered a complex group (sensu lato) and includes at least 17 related species.
So far in Croatia, studies based on morphological determination of ticks have described 21
tick species grouped within five genera, while molecular studies are scarce. The first genetic
study on ticks within R. sanguineus complex showed presence of two genetic lines
Rhipicephalus sp. phylogenetically classified in group II with subgroups Rhipicephalus sp. IIa
and Rhipicephalus sp. IIb. Within the genus Ixodes, two species of ticks, I. canisugaand I.
hexagonus have been confirmed. Numerous studies in Croatia focused on detection of of tick
transmitted pathogens in animals, while studies on pathogens in tick, and even tick species
present in Croatia are lacking.
The aim of the study was to investigate tick species present in Croatia based on
sequencing of 16S rRNA gene fragment and to compare results with results of morphological
determination. Furthermore, to investigate pathogens in individual ticks in order to obtain
more precise data on ticks and pathogens in ticks they may carry for the first time in Croatia
and to compare detected pathogens with tick “genetic” lines. One of the aims was to visualize
ticks and detected pathogens by mapping, as well.
The study was carried out on 509 archived morphologically identified ticks. Ticks
from different locations were collected from domestic and wild animals, dogs, cats, humans
and environment during period of 2014 to 2017. DNA was extracted manually from each tick
and analyzed individually based on PCR amplification of 16S rRNA gene fragment. All
extracted DNA from each tick was screened for presence of Babesia, Theileria, Anaplasma,
Ehrlichia, Hepatozoon, Mycoplasma, Borrelia, Rickettsia, Francisella and Coxiella DNA
using conventional PCR and subsequent sequencing. Amplified products were analysed using
capillary electrophoresis, purified with ExoSAP-IT®
PCR Clean-Up Reagent kitand sequenced
in both directions in Macrogen Europe. Sequences were assembled using.Lasergene®
software, edited with SeqmanTM
, and compared with available squences in GenBank®
using
the BLAST®
. Obtained results were visualised by mapping with QGIS® software.
In the current study we have detected 15 tick species belonging to five genera.
Genus Ixodes (31,2%; 159/509) was the most representative genus with six species: I.ricinus,
I. canisuga, I. hexagonus, I. gibbosus, I. kaiseri and I. ventalloi. Ixodes gibbosus, I. kaiseri
and I. ventalloi were confirmed molecularly for the first time in Croatia. The number of
species is not surprising, because previous studies morphologically described eight species in
Croatia.
Out of the five known species in Europe, three species from subgenus Pholeoixodes, I.
canisuga, I. hexagonus, and I. kaiseri were detected in this study. Presence of I. kaiseri from
red fox in the continental region represents the first confirmation of species in Croatia, but
also one of the rarest findings in Europe. So far has been detected from foxes and dogs from
Germany, Romania, Hungary and Serbia.
Ixodes canisuga (4,5%; 23/509) ticks were geographically limited to continental
Croatia, and as other species from subgenus Pholeoixodes, werecollected from red foxes,
except one tick from environment.Sequences were identical to sequences from other
European countries, including neighboring countries such as Serbia, Bosnia and Herzegovina
and Hungary.
The last species, I. hexagonus (3,7%, 19/509) was detected in all regions, except Istria.
Unlike I. canisuga, sequence analysis showed presence of three different groups of sequences
grouped within three isolates I. hexagonus 1, I. hexagonus 2 and I. hexagonus 3. Sequences
showed up to two nucleotide differences from each other and shared 99% similarity. Ixodes
hexagonus 1 dominated in all areas and was present in southern Adriatic (Mljet). In central
Adriatic, it appeared together with I. hexagonus 2, while in the continental region with I.
hexagonus 2 and I. hexagonus 3. Detection of I. hexagonus in central and southern Adriatic
represents the first finding of tick species in the coastal region. Sequences of I. hexagonus 1
and I. hexagonus 3 were identical to sequences from European countries, while I. hexagonus 2
with the same sequences shared a slightly lower similarity of 99,7%.
One of the interesting findings represent genetic confirmation of I. ventalloi (1,2%;
6/509) from european hares from Vir. Besides its first molecular confirmation in Croatia,
represents the first finding in Southeastern Europe. Current study confirmed presence of I.
gibbosus (0,8%, 4/509) in Croatia. So far has been described only on Brač, therefore its
finding on sheeps from Cres, Rab and Pag indicates its possible presence in other coastal
areas.Sequences shared 99% similarity with sequences from Turkey and Greece.
Sequencing confirmed 24 different groups of sequences of I. ricinus (20,8%; 106/509),
similar to other European studies, for the first time in Croatia. Sequences shared 98%
similarity and differed up to two nucleotides. Genotypes were geographically located in
continental region, besides two ticks from Gorski kotar and central Adriatic. Ticks were
mostly collected from the environment, but also from canids and wild ruminants, except for
one tick collected from humans. The most frequent isolate was I. ricinus 1 (71,7%; 76/106),
followed by I. ricinus 7 (4,7%; 5/106), I. ricinus 6 (2,8%; 3/106) and I ricinus 3 (1,9%;
2/106). The remaining 20 genotypes were detected each in one tick. Sequences of I. ricinus 1,
I. ricinus 7, I. ricinus 15, I. ricinus 16 and I. ricinus 20 were identical to I. ricinus sequences
in Europe, while sequences of 19 isolates showed similarity from 98% to 99%.
Dermacentor reticulatus is considered as tick species which has the fastest expansion
throughout Europe, and whose southern limits have not been determined precisely. This study
confirmed its presence in all investigated areas, besides Istria and southern Adriatic.
Confirmation of tick in northern (Senj, mouflon) and central Adriatic (Vir, european hare)
represents the southernmost finding of the species in Croatia. All 16S rRNA sequences were
identical and shared 99% to 100% similarity with D. reticulatus sequences from Europe.
Another member of genus, D. marginatus has been detected southwards, on Hvar from wild
boarand throughout continental Croatia.
Rhipicephalus bursa and R. sanguineus sensu lato (s.l.) were confirmed within genus
Rhipicephalus. Rhipicephalus bursa (11,2%; 57/509) was present in almost entire coastal
area. In continental region was collected from foxes in Zagreb and Daruvar and from sheep
from Slunj, which represents its first confirmation outside coastal area and evidence of its
spreading from south to north. Sequences shared 99% similarity with sequences of R.
bursafrom Europe, such as Turkey, France and Spain.
One third of the ticks belonged to the Rhipicephalus sanguineus s.l. (32,2%; 164/509).
Group of 149 sequences belonged to the R. turanicus. Differences within sequences varied up
to three nucleotides and were classified into three groups: R. turanicus 1, R. turanicus 2 and
R. turanicus 3. The most frequent isolate R. turanicus 3 was found in central and southern
Adriatic, but also in Zagreb area. Confirmation of R. turanicus in Zagreb from fox is the first
description of species in continental Croatia. Remaining two genotypes have been confirmed
throughout central and southern Adriatic, as well. Sequences were identical to the sequences
of R. turanicus from Turkey and R.turanicus found on Murter.
Fourth group of sequences (9,1%; 15/164) shared similarity of 94% with the other sequences
and corresponded to the sequences of R.sanguineus s.s. Rhipicephalus sanguineus s.s. was
collected only from dogs from the northern (Rovinj, Premantura) and central Adriatic (Zadar,
Obrovac Sinjski, Hvar), and in continental region (Zagreb, Vinkovci). Sequences were
identical to sequences of Rhipicephalus sanguineus from dogs from Serbia and Croatia.
Hyalomma marginatum (6,9%; 35/509) was the only species from the genus
Hyalomma. Ticks were present in coastal region and collected mainly from cows, then horses,
goats, donkeys and dogs. Sequences were identical to each other but also to other sequences
from Europe but at the same time shared 99,7% similarity with the 16S rDNA sequences of
Hy. rufipes from Afrika and Hy. turanicum from Irak.
For the first time in Croatia three species from Haemaphysalis genus were confirmed:
H. parva (1,5%; 8/509), H. inermis (1,2%; 6/509) and H. concinna (0,4%; 2/509). Ticks from
eastern Croatia were collected from the environment, except H. concinna from foxes (Požega)
and H. inermis from foxes and horses (Jastrebarsko, Zadar). Sequences of H. concinna were
identical with sequences from Hungary, while sequences of H. inermis and H. concinna
shared 99,5% similarity with sequences from Australia and Turkey.
Members from family Anaplasmataceae were the most commonly detected
microorganisms occuring in 18% (92/509) of ticks. Sequencing revealed several species of
anaplasma Anaplasma capra, A. phagocytophilum, Ehrlichia canis, Ehrlichia sp.,
“Candidatus Neoehrlichia lotoris” and endosymbionts Wolbachia sp. and Midichloria
mitochondrii. The most significant finding is the detection of DNA of A. capra and E. canis
for the first time in Croatia. Zoonotic A. capra was detected in 5,1% of ticks belonging to R.
bursa, R. turanicus, D. marginatus and H. inermis. Ticks from Rhipicephalus genus were
present in central and southern Adriatic (sheeps, goats), while D. marginatus and H. inermis
in continental Croatia (sheep, fox). DNA of Ehrlichia canis was detected in 0,4% of R. bursa
from sheep and goats from central Adriatic.
Piroplasms (12,4%; 63/509) were the second most common group of pathogens
detected in 12,4% of ticks in this study. Sequencing showed almost equal prevalence of
Babesia spp. (6,3%) and Theileria spp. (6,1%). Babesia genus included eight group of
sequences corresponding to B. canis, B. vulpes, B. microti, B. venatorum, Babesia sp. “Badger
type A” and “Badger type B”, B. ovis, Babesia cf. crassa and Babesia sp. "tavsan". Babesia
canis represents well known piroplasm in dogs from Croatia. Sequencing revealed two
genotypes of B. canis, B. canis 1 (1,9%) and B. canis 2 (0,4%). Babesia canis DNA was
detected in D. reticulatus (fox, brown bear, dog) as expected, but also in D. marginatus (dog,
human), R. bursa (goat, cattle), R. turanicus (goat), I. hexagonus (fox) and I. ricinus (fox).
Zoonotic babesia B. venatorum and B. microti were detected in I. ricinus from foxes and
environmentin continental Croatia.
Within the genus Theileria, four species have been detected: T. ovis, T. orientalis
(buffeli/ sergenti), T. equi and T. capreoli. The most common species T. ovis was detected in
3,5% of ticks: R. turanicus (sheep), D. reticulatus (foxes), R. bursa (cow, sheep), Hy.
marginatum (dog) and I. ricinus (red deer). Pathogen was more abundant in central and
southern Adriatic. Theileria orientalis (buffeli/sergenti) was detected in 1,9% of ticks: D.
reticulatus, D. marginatus and I. gibbosus. Pathogen was geographically limited to
continental Croatia, with the southernmost finding in D. reticulatus from Otočac and I.
gibbosus from Cres. Theileria capreoli was so far confirmed in grey wolves but and dogs in
Croatia, but never in ticks. Detection in one D. marginatus from environment represents its
first detection in ticks in Europe.
Three species of genus Hepatozoon were confirmed in 6,1% of ticks: H. canis, H. felis
and Hepatozoon „Badger type". Sequence analysis showed two different H. canis genotypes,
H. canis 1 and H. canis 2. Both genotypes were geographically limited to continental region,
except one H. canis 1 detected in R. sanguineus s.s. from central Adriatic (Medviđa).
Pathogen was detected in ticks collected from foxes (I. hexagonus, I. ricinus, I. canisuga, D.
reticulatus, horse (I. ricinus), environment (I. ricinus) and in two ticks from dogs, I. ricinus
and R. sanguineus s.s. Rhipicephalus turanicus 3 from cats from Dugi otok was found to
harbour DNA of H. felis (0,4%).
Five species from Rickettsia genus were detected in 11,7% of ticks namely: R.
slovaca, R. raoultii, R. aeschlimannii, R. massiliae and R. monacensis. The causative agents
of TIBOLA syndrome R. slovaca and R. raoultii have been detected in 0,9% and 4,7% of
ticks. Rickettsia slovaca DNA was found in D. reticulatus (fox, mouflon) from northern
Adriatic and D. marginatus (human, cattle, sheep) from continental Croatia. Rickettsia
raoultii was expectedly frequentin D. reticulatus collected from various animal species and
environment. Besides D. reticulatus, rickettsia DNA was present in D. marginatus and H.
parva from environment in continental region.So far, DNA of zoonotic rickettsia from spotted
fever group R.aeschlimannii was detected in Split and Sinj area. This study confirmed its
DNA in Hy. marginatum throughout coastal area. Rickettsia DNA was also present in R.
turanicus from southern Adriatic. Rickettsia massiliae was detected in 2,4% R. turanicus ticks
from domestic ruminants from southern Adriatic. Rickettsia monacensis (0,9%) was detected
in I. ricinus from continental region and in Hy. marginatum from Sinj.
Francisella tularensis (0,2%) was detected in one R. turanicus 3 from Pelješac.
Sequence was identical to F. tularensis subsp. holartica from D. marginatus (BIH, Portugal),
H. concinna (Hungary), I. ricinus (Germany, Serbia) and european hare (Spain). In 1,5% of
D. reticulatus (golden jackal, wild boar, horse, fox, environment) and D. marginatus (human)
from continental Croatia sequences were identical to „Francisella-like“ endosymbiont from
D. reticulatus collected from environment in Poland.
Borrelia burgdorferi s.l. DNA was detected in 2,7% ticks. Sequencing revealed higher
prevalence of B. afzelii (1,9%) than B. valaisiana (0,8%). Similar to previous studies, both
species have been detected in various genotypes of I. ricinus in continental Croatia. Most
ticks were collected from environment, except two ticks from dog and fox. Bacteria from
Mycoplasma genus, as well as Coxiella burnetti were not detected.
This study is one of the most comprehensive studies on genetic diversity of ticks
collected from different species of domestic and wild animals, dogs, cats, humans, and the
environment, not only in Croatia but also in Europe, which includes comparison of
morphological determination and genotyping of 16S rDNA gene fragment. Sequencing of 16S
rRNA and comparison with the BLAST®
has shown to be the most suitable method for
detecting tick species and at the same time the analyzed gene is sufficiently heterogeneous to
enable the differentiation of potential genotypes within the species. This study confirmed
presence of D. reticulatus, D. marginatus, I. ricinus, I. hexagonus, I. canisuga, R. bursa, R.
turanicus sensu lato, R. sanguineus sensu strico, Hy. marginatum, but for the first time
species such as I. gibbosus, I. kaiseri, I. ventalloi, H. concinna, H. inermis and H. parva, have
been confirmed molecularly. The genetic approach showed genetic diversity within the
population of I. ricinus and R. turanicus in Croatia, and also the presence of at least two
species within the R. sanguineus sensu lato complex. The systematic approach in detection of
tick transmitted pathogens in individual ticks showed presence on numerous pathogens,
important not only in veterinary medicine but also within public health aspects. This research
contributed to the knowledge of tick species, their distribution and tick transmitted pathogens
Bacterial culturing and genotyping of bacteria from the genus Bartonella isolated from cats in Republic Croatia : BACTERIAL CULTURING AND GENOTYPING OF BACTERIA FROM THE GENUS BARTONELLA ISOLATED FROM CATS IN REPUBLIC CROATIA
No full textPripadnici roda Bartonella su Gram - negativne bacilarne bakterije, a danas je
poznato više od 40 vrsta, podvrsta i kandidata. Najznačajnija vrsta, Bartonella henselae,
glavni je uzročnik bolesti mačjeg ogreba (BMO) u ljudi. Primarni rezervoari bakterije
su mačke, asimptomatski nositelji uzročnika u svojim eritrocitima. Iako su protokoli za
dokazivanje bartonela različiti, uzgoj još uvijek predstavlja „zlatni standard”
dijagnostike. Ujedno predstavlja metodu dobivanja dostatne količine DNA za daljnje
genske analize, kao što je analiza sekvenci više gena (MLST). Zbog dugotrajnog rasta i
zahtjevnog umnažanja bartonela, uzgoj na hranjivim podlogama predstavlja
dijagnostički izazov.
Bartonele su proširene u cijelom svijetu i predstavljaju opasnost za zdravlje
ljudi. No kulturelna ili molekularna istraživanja vrsta u Hrvatskoj nisu do sada sustavno
provedena. Istovremeno serološka istraživanja i razne kliničke manifestacije ljudi
dokazuju prisutnost uzročnika u Hrvatskoj. Stoga je ovo prvo istraživanje takve vrste u
Hrvatskoj. B. henselae genski je heterogena i dijeli se na 37 sekvencijskih tipova (ST).
Osim istraživanja učestalosti i utvrđivanja optimalnog uzgojnog protokola, cilj ovog
rada je genski tipizirati izolate, steći uvid u raznolikost i rasprostranjenost genotipova te
doprinijeti poznavanju genetike samog uzročnika u jugoistočnoj Europi. Određivanje
sekvencijskih tipova izoliranih iz mačaka u Hrvatskoj značajno je, jer se oni razlikuju u
patogenosti i zoonotskom potencijalu.
Ukupno je pretraženo 279 mačaka. Metoda uzgoja uzoraka krvi 189 živih
mačaka s 13 lokacija u Republici Hrvatskoj izvedena je korištenjem pet čvrstih i dvije
bifazične hranjive podloge, a pretraženi su i lančanom reakcijom polimerazom.
Uzgojem na istim hranjivim podlogama analizirano je i 90 uzoraka krvi iz srca uginulih
mačaka. Za svaku životinju dostavljen je upitnik s anamnestičkim, kliničkim i
epizootiološkim podacima, s ciljem određivanja čimbenika rizika povezanih s
infekcijom mačaka. Za identifikaciju Bartonella sp. iz krvi i izolata konvencionalnim
PCR-om korištene su početnice za ciljne gene 16SrRNA i ITS (16S-23S rRNA).
Dokazivanje vrste B. henselae i njenih ST-ova provedeno je MLST metodom na osam
genskih lokusa iz izolata uzgojenih iz krvi 31 mačke. Aleli i ST-ovi određivani su korištenjem PubMLST baze podataka za bakteriju B. henselae i uspoređeni s
postojećima iz ostalih regija svijeta. Umnažanjem i sekvenciranjem 16SrRNA gena i
ITS regije dokazana je B. henselae i kod uginule mačke.
Bartonele su izdvojene iz 31 žive (16,4%) i jedne uginule (1,1%) mačke što daje
ukupnu učestalost infekcije od 11,5%. Istovremeno lančanom reakcijom polimerazom u
krvi 189 mačaka nije dokazan odsječak DNA Bartonella sp. Na svim korištenim
čvrstim hranjivim podlogama u primoizolaciji zabilježen je porast kolonija nakon četiri
do 56 dana inkubiranja. Najveća učestalost izdvajanja bakterije B. henselae utvrđena je
na BH (87,5%) i COL (82,4%) agaru. MLST analizom identificirano je 30 potpunih
alelnih profila iz kojih je proizašlo pet različitih sekvencijskih tipova. Najučestaliji ST5
izdvojen iz 17 (56,7%) mačaka, a dokazani su i ST6 (23,3%), ST1 (13,3%) i ST24
(3,3%). Usporedba alelnih profila rezultirala je jednim potpuno novim genotipom
(ST33), pripisanom jednom od tri klonalna kompleksa vrste B. henselae (CC2).
Područje, dob i po prvi puta prisutnosti crijevnih parazita utvrđeni su kao
čimbenici rizika za infekciju mačaka bakterijom B. henselae (p<0,05). Mačke starosti
≤12 mjeseci najučestalije su bile inficirane, a uočen je trend smanjivanja prevalencije s
porastom dobi mačaka. Statistički značajna povezanost s infekcijom utvrđena je u
mačaka s primorskih područja u odnosu na mačke s kontinenta, kao i u mačaka
uzorkovanih u razdoblju od siječnja do travnja u odnosu na ostatak godine.
Multivarijabilnom analizom prisutnost crijevnih parazita utvrđena je kao čimbenik
najsnažnije povezan s bakterijemijom mačaka. Metoda uzgoja korištenjem različitih
hranjivih podloga pokazala se uspješnom u izdvajanju većeg broja izolata. Dokaz
zoonotskog genotipa ST5 kod najvećeg broja mačaka predstvalja javnozdravstveni
značaj, osobito jer se u nekoliko mačaka dovodi u vezu s oboljenjima ljudi od BMO-a.Introduction. Bacteria of the genus Bartonella sp. are small, short,
pleomorphic, Gram-negative coccobacilli (0.5 by 1 to 2 µm), and today almost 40
species are known, along with numerous unnamed or Candidatus species. Bartonella
henselae is the most important species of zoonotic significance, the principal etiologic
agent of cat scratch disease (CSD) in humans, most commonly manifested by localized
lymphadenopathy with or without bacteremia.
The main reservoirs of bacteria B. henselae are cats, and the causative agent is
most often transmitted to humans by contamination of the scratch site with feces of the
cat flea (Ctenocephalides felis), which contains viable multiplied pathogens. Cats are
chronic asymptomatic carriers of bacteria B. henselae, which infects their erythrocytes,
making isolation from the cat’s blood on blood agar plates more successful than from
other animals and humans.
Epizootiological studies have shown a prevalence of Bartonella infection in
cats of 10-30% by culture from cat’s blood. Although protocols for proving Bartonella
are different, isolation by cultue on agar plates is still considered the "gold standard" of
diagnosis. It is also a method by which sufficient amounts of DNA can be obtained for
further genetic analysis of bacteria B. henselae, such as analysis of multiple gene loci.
Due to the slow growth and fastidious cultivation, isolation on culture media is a
diagnostic challenge.
In spite of Bartonella are widespread throughout the world and pose a threat to
human health, research on species / isolates in Croatia has not been carried out
systematically so far. Serological studies and various clinical manifestations of people
has proven the presence of pathogens in Croatia. To date, no studies by culture and / or
molecular investigation of bacteria B. henselae in cats, or genotyping methods of
species / isolates have been performed, so this is the first study of its kind in Croatia.
B. henselae isolates show a considerable genetic heterogeneity and bacteria is
divided into sequence types (ST), which today are most often determined by multilocus sequence typing (MLST) analysis. Prior to the start of this study, 32 different STs were
known, and today there are a total of 37 enrolled in the PubMLST database
(https://pubmlst.org/organisms/bartonella-henselae). Beside to study the prevalence of
infection and optimal culture procedures, the aim of this paper is to perform B.
henselae-genotyping, gain insight into the diversity and distribution of sequence types
and contribute to knowledge of the genetics of the bacteria B. henselae in Southeast
Europe. Determining the sequence types isolated from cats in Croatia is important,
because they differ in virulence and zoonotic potential.
Material and methods. A total of 279 cats were searched. 189 blood samples
of live cats collected in 20 veterinary clinics from 13 different locations in the Republic
of Croatia were analyzed. Samples were inoculated on five types of solid and two types
of biphasic culture media, previously tested using ATCC strains of bacteria B. henselae
(49882) and B. clarridgeiae (700095), and analysed in parallel by polymerase chain
reaction (PCR). Any colony growth indicating species of the genus Bartonella was
screened by PCR, and isolates were stored in pure culture by freezing at –80 °C, in
broths with added glycerol, or in a commercial bacterial storage system. In addition to
samples of live cats, 90 samples of blood from the hearts of dead cats were analyzed,
tested only by culture. Each sample was accompanied by a questionnaire which contains
anamnestic, clinical and epizootiological data on animals, in order to determine the risk
factors associated with infection of cats with bacteria of the genus Bartonella sp.
Five solid culture media were used, Columbia agar with 5% sheep blood
(COL), Brain heart agar with 5% rabbit blood (BH), Chocolate agar with 10% sheep
blood (ČOK), Tryptic soy agar with 5% sheep blood (TSA) and Esculin blood agar with
5% sheep blood (EKA). In addition, two biphasic media were used, made by a
combination of liquid and solid culture media: Tryptic soy broth with Tryptic soy agar
(TSA+TSB) and Brucella broth with Brain heart agar (BH+BB). To identify Bartonella
sp. from blood and isolates by conventional PCR, primers for the target genes 16SrRNA
and ITS (16S-23S rRNA) were used.
Detection of B. henselae species and sequence types determination was
performed by multi locus sequence typing (MLST) at eight gene loci (16SrRNA, batR,
ftsZ, gltA, groEL, nlpD, ribC and rpoB) from isolates obtained by culture of blood samples of 31 cats. Nucleotide sequence determination was performed at the
commercial company Macrogen Inc. (Amsterdam, The Netherlands). The obtained
sequences were processed using the computer program BioNumercis, which compared
the nucleotide sequences of both directions, in order to obtain a unique nucleotide
sequence.
Alleles and STs were determined using the MLST online database
(https://pubmlst.org/organisms/bartonella-henselae) for bacteria B. henselae and were
assigned an eight-digit numerical code, consisting of a combination of alleles at each
locus. Alleles were compared with each other on the basis of categorical coefficient and
UPGMA, and presented in the form of dendrograms and MST (minimum spanning
tree). The obtained STs with this research were compared with the existing STs from
different countries and regions of the world.
In the statistical processing of epizootiological data, we used the computer
program STATA 13.1. Univariate and multivariante logistic regression was used to
analyze the statistical correlation of epizootiological data from the questionnaires and to
determine risk factors associated with B. henselae infection. Independent variables
statistically significant in univariate analysis (p-value <0,05) were included in
multivariable model and further tested to evaluate if remained associatied with feline
Bartonella bacteremia.
Results. Out of a total of 279 cats searched, Bartonella spp. were isolated
from 31 live and one dead cat. Determined prevalece was 16,4% (CI 11,074 – 21,730)
(total frequency of isolation was 11,5%, in live cats 16,4% and in dead 1,1%). On all
types of solid culture media used in the primoisolation a growth of colonies was
recorded, and the typical colonies for species B. henselae (hard, dry, difficult to break,
and firmly embedded to the surface) were observed after four to 56 days of incubation.
B. henselae was identified by amplification and sequencing of the 16SrRNA gene and
the ITS region. By analyzing the nucleotide sequences of eight gene loci of all isolates
uing the MLST method, five different sequence types (ST) of bacteria B. henselae were
identified. Out of 31 infected live cats, a total of 30 complete MLST allelic profiles
were obtained, and one was incomplete and was not assigned a sequence type with
certainty.
The most common genotype was ST5, isolated from 17 (56,7%) cats, and of
the other sequence types ST6 was detected in seven (23,3%) cats, ST1 in four (13,3%)
and ST24 in one (13,3%). The genotype of one cat was completely new due to a new
combination of alleles, and was deposited into an online database
(https://pubmlst.org/organisms/bartonella-henselae) as a sequence type ST33. Based on
the determined combination of alleles, MLST analysis assigned a numerical code 2-3-3-
1-2-1-1-2, and phylogenetic analysis determined affiliation to one of the three
previously described CCs of B. henselae species, clonal complex 2 (CC2).
The best efficiency of isolation of B. henselae – bacteria on seven types of
culture media, comparing the growth rate (days of incubation) and the isolation rate
(share of isolates obtained from infected blood samples on specific media), in the
primary isolation was on BH agar (87.5%, 21/24 isolates) and COL agar (82.4%, 14/17
isolates). Cat’s isolates with varying level of bacteremia were grown on both culture
media, on BH agar with a range of 16 to 1960 CFU / mL, and on COL agar from 40 to
3050 CFU / mL. Slightly lower isolation rate was observed on EKA (80,0%) and ČOK
(77,3%) agar and on biphasic medium TSA+TSB (80,0%). The advantage of a certain
culture medium in the isolation of a particular sequence type of B. henselae has not been
observed. B. henselae species was identified from an isolate grown on ČOK agar plate
from a blood sample of a dead cat by PCR and sequencing. By examining the blood of
live cats by PCR, we were unable to amplify the DNA fragment of Bartonella.
By processing epizootiological data from the live cat by survey questionnaires,
a statistically significant association (p <0,05) was found between the infection of cats
with B. henselae and the area, age of cats and and first-time findings of intestinal
parasites. The highest prevalence of infected animals was found in the cities of Zabok
(66,7%, 2/3), Rijeka (50,0%, 5/10), Pula (40,0%, 8/20) and Jastrebarsko (30,4 %, 7/23),
while in four out of the 13 locations (Osijek, Split, Vukovar and Velika Gorica) there
were no cats infected with Bartonella. Cats ≤12 months of age were the most frequently
infected (25,0%), and a trend of declining prevalence with increasing age of cats was
observed. In the coastal area, cats were significantly more frequently infected than on
the mainland, as were cats sampled between January and April compared to the rest of
the year. Multivariate analysis revealed the strongest association between feline
bacteremia and the presence of intestinal parasites.
Conclusion. The prevalence of cat infection with bacteria B. henselae
obtained on culture media (16,4%) is within the range found in similar studies in cats in
Europe and the rest of the world, and it is the third most common infection in the
domestic cat group. A slightly higher rate of isolation was observed in cats with respect
to lifestyle (going out, lack of owners). It was found that coastal cats were more
frequently infected with B. henselae than continental. Such a finding could be
associated with an average warmer climate of coastal locations, which is more
conducive to the life of cat fleas, the main vectors in Bartonella transmission. Infection
with intestinal parasites and age up to one year are risk factors that are significantly
associated with infection of cats with B. henselae. On the other hand, factors such as
sampling in the summer months, antibiotic administration, flea control treatments, and
age above one year are possible causes of differences in prevalence between sites.
The identification of five different sequence types from isolates of 30 cats
confirmed the genetic diversity of the bacterium B. henselae in the territory of the
Republic of Croatia. For the first time in the Republic of Croatia and for the first time
globally from the cat’s blood has been proven the ST33 genotype. It was not observed
association of other genotypes with particular location because they were equally
distributed. The culture method has proven successful for detecting B. henselae bacteria
from the blood of cats. Combined use of several different culture media, with extended
incubation time up to 60 days, are recommended for the isolation of bacteria B. henselae
by culture from the blood of the cats. The zoonotic genotype ST5 was isolated from
three cats in households with CSD patients suggesting that it is a common and virulent
zoonotic genotype
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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