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Krajem svibnja 2015. godine u Laboratorij za parazitologiju, Hrvatskog veterinarskog instituta, Zagreb, Hrvatska dostavljena je krv ovna iz okolice Benkovca sa sumnjom na piroplazmozu. Ovan je uz potpornu terapiju vitaminima AD3E i B-kompleksa liječen imidokarbom (Imizol® 0,5 mL s/c) i oksitetraciklinom (Geomicin retard® 5 mL i/m) te je za pet dana prestao pokazivati kliničke znakove bolesti. U razmazu krvi pronađena su periferno smještena intraeritrocitarna tjelešca, a sekvenciranjem odsječka 16S rRNK i MSP4 gena dokazali smo vrstu Anaplasma ovis, dok DNK drugih uzročnika nije bila prisutna. Ovim istraživanjem smo po prvi puta dokazali vrstu A. ovis u Hrvatskoj, ali i u Jugoistočnoj Europi. Od danas priznatih osam vrsta anaplazmi, anaplazmozu malih preživača uzrokuju tri vrste A. phagocytophilum, A. capra i A. ovis od kojih je A. phagocytophilum dokazana u Hrvatskoj. Prema navodima veterinara Veterinarske ambulante Benkovac opisani klinički znaci bolesti su česti u ovaca i ovnova dopremljenih s drugih područja Hrvatska i nikada nisu uočeni u životinja mlađih od pet mjeseci. Treba naglasiti da osim Imizolom veterinari na području Benkovca oboljele ovce uvijek liječe i tetraciklinima što predstavlja i lijek izbora, a oboljele životinje uspješno izliječe ukoliko lijekove apliciraju unutar pet dana od pojave kliničkih znakova bolesti. Vektor je krpelj Rhipicephalus bursa prisutan diljem Republike Hrvatske ujedno pronađen i na oboljelom ovnu. Stoga treba očekivati da je uzročnik prisutan i u drugim dijelovima Hrvatske., In late May 2015, a sample of ram blood with suspected piroplasmosis from the vicinity of Benkovac was delivered to the Laboratory for Parasitology of the Croatian Veterinary Institute in Zagreb, Croatia. Therapy with Imidocarb (Imizol 0.5 mL s/c) and oxytetracycline (Geomycin retard 5 mL i/m) was started immediately, with supportive therapy with vitamins AD3E and B-complex. The health status improved and clinical signs disappeared after five days, and the ram fully recovered. Peripherally located intraerythrocyte bodies were found in the blood smear, and Anaplasma ovis was confirmed by sequencing the 16S rRNA and MSP4 gene, while the DNA of other pathogens was not present. This research proved the presence of A. ovis in Croatia and in Southeast Europe for the first time. Of the eight species of Anaplasma recognized today, small ruminant anaplasmosis is caused by three species, A. phagocytophilum, A. capra and A. ovis, and to date only A. phagocytophilum has been proven in Croatia. According to veterinarians from the Benkovac Veterinary practice, the described clinical signs of the disease are common in sheep and rams introduced from non-endemic areas and have never been observed in animals younger than five months. It should be emphasized that in addition to Imisol, veterinarians in the Benkovac area always treat sick sheep with tetracyclines, the drug of choice for Anaplasma treatment, and sick animals are successfully cured if treatment is administered within five days of the onset of clinical signs. The vector is the tick Rhipicephalus bursa, which is present throughout the Republic of Croatia and was also found on the infected ram, so it is to be expected that the causative agent is also present elsewhere in Croatia.

Krpelji 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 dokazana su 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, 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 s Pelješca dokazana je Francisella tularensis subsp. holartica, dok je endosimbiont bakterije „Francisella-sličan“ dokazan u 1, 5% D. reticulatus i D. marginatus. Na području kontinentalne Hrvatske u genotipovima I. ricinus dokazane su Borrelia afzelii i B. valaisiana u 1, 9% i 0, 8% krpelja. Istraživanjem nisu dokazane bakterije iz roda Mycoplasma i vrsta Coxiella burnetii. Istraživanje predstavlja prvo opsežno istraživanje krpelja i patogena prenosivih krpeljima u Hrvatskoj. Sustavnim pristupom u pojedinačno analiziranih krpelja su po prvi puta molekularno dokazane DNK patogena koji su od značaja ne samo u veterinarskoj medicini, već i za područje javnog zdravstva.

Piroplasmosis is one of most important tick-borne disease in dogs. Except well known Babesia canis small piroplasms B. gibsoni, B. vulpes have been detected in asymptomatic dogs in Croatia. Here we report first B. gibsoni and B. vulpesclinical cases in Croatia, molecular characterisation and treatement efficiency. Blood samples of four dogs with confirmed small merozoites showing anemia and thrombocytopenia were analyzed using SNAP®4DX® Plus, SNAP® Leishmania and conventional PCR to Anaplasma/Ehrlichia, Babesia/Theileria and Mycoplasma sp.. Positive samples were sequenced. Small merozites were initially assumed to be B. gibsoni. Sequencing revealed B. gibsoni in three (Dog 1, 2, 3) and B. vulpes in one dog (Dog 4). Lethargy, weakness, pale mucous membranes, splenomegaly, anemia and severe thrombocytopenia were observed in two B. gibsoni (Dog 1, 2) infected dogs. First one had a history of long-term immunosupressive therapy due to cataract. Dog 3 co-infected with M. haemtoparvum and B. gibsoni had chronical skin problems with scars all over the body. B. vulpes infected dog showed only anorexia, lethargy and anemia. Several drug combinations (imidocarb diproprionate, imidocarb diproprionate and doxycycline in Dog 1 and 2 ; doxycycline, metronidazole and enrofloxacine in Dog 1) in B. gibsoni cases resulted in improvment of health condition but not in elimination of pathogens. Final combination of atovaquone and azythromicine resulted in full clinical recovery, parasite clearance and permanent health improvement during follow up period (Dog 1 and 3). In case of B. vulpes infection doxycycline administration suggested an apparent clinical recovery. Described cases clearly showed that small piroplasms should always be included in differential diagnosis in cases of anorexia, lethargy, weakness, anemia and thrombocytopenia. Due to almost identical morphology of merozoites species have to be confirmed with molecular methods because of different drug efficiency.

Introduction: Apicomplexan hemogregarines of the genus Hepatozoon, described in more than 300 animal species, are transmitted by various hematophagous arthropods. In the current report we provide the first morphological description, pathological changes and molecular characterization of a Hepatozoon sp. from a stone marten. Materials and Methods: A paralyzed young adult wild female stone marten from Western Croatia was euthanized for determination of cause of illness. During necropsy representative tissue samples were collected for histology and molecular analyses. Abscess content was sampled for bacterial culture and ectoparasites for morphological determination. Results: Necropsy revealed cachexia, head and neck abscesses at the site of bite wounds and severe ectoparasitic infestation. Severe emaciation was caused by massive concurrent bacterial and parasitic infections. Cultivation and histology results confirmed β-hemolytic Streptococcus pyogenes and E. coli within multifocal to coalescing subcutaneous purulent lesions. Although no gross lesions were visible in the heart and cervical muscles, microscopic examination revealed numerous Hepatozoon meronts and released merozoites from ruptured meronts associated with granulomatous inflammation. Macrophages often contained 1.17- 3.12 µm round to oval intracytoplasmatic merozoites. Meronts were sporadically found in the liver, spleen and kidneys without an apparent local inflammation. Sequences of 666 bp fragment of 18S rRNA from the spleen, liver, heart and cervical muscle were identical to Hepatozoon “Badger type” (KU198330). Identical Hepatozoon DNA was confirmed with sequencing in Ixodes ricinus ticks from the stone marten, while fleas from the animal were negative. Conclusions: Infection with Hepatozoon sp. from a stone marten showed similar lesions and organ distribution to those described from different Hepatozoon sp. in other marten species.

Danas se B. henselae dijeli na sekvencijske tipove (eng. sequence type, ST) koji se međusobno razlikuju prema virulentnosti, odnosno zoonotskom potencijalu. U Hrvatskoj do sada nisu provedena molekularna istrižavanja izolata B. henselae ni iz mačaka, niti iz ljudi, stoga je cilj ovog rada prikazati gensku tipizaciju B. henselae iz dvije „zdrave“ mačke u vlasništvu pacijenata oboljelih od bolesti mačjeg ogreba u Hrvatskoj.Rad prikazuje dva slučaja oboljele djece s dvije različito teške kliničke slike. Sumnja se da su izvor zaraze mačke u vlasništvu obitelji pacijenata, prethodno invadirane buhama. Iz obiju mačaka kulturelno je izdvojena i analizom sekvenci više gena (MLST) dokazana Bartonella henselae ST5.Ovo je prvi prikaz klinički različitih slučajeva BMO u Hrvatskoj, najvjerojatnije uzrokovanih istim genotipom B. henselae, i ujedno prvi dokaz ST5 u jugoistočnoj Europi. Prikazana je brza i osjetljiva dijagnostika B. henselae iz uzgojenih izolata, a kronično inficirane mačke invadirane buhama dokazane su kao izvor zaraze za ljude.

Fleas and ticks are important vectors of vector-borne pathogens that affect cats and dogs. In the present study ticks and fleas collected from dogs and cats were investigated for presence of vector-borne pathogens and endosymbionts they harbor. The ectoparasites were collected from 14 deceased dogs and 22 deceased cats with gross findings of hemolytic anemia. After the morphological determination of ectoparasites, DNA was extracted for molecular detection of Babesia/Theileria sp., Anaplasma/Ehrlichia sp., Hepatozoon sp., Mycoplasma sp. and Bartonella sp. Ticks Dermacentor reticulatus (4/7, 57%), Ixodes ricinus (4/7, 57%) and Rhipicephalus sanquineus s.l. (1/7, 14%) were collected from 7 out of 14 dogs. Fleas were present in 64% of dogs: Ctenocephalides felis in 67% and C. canis in 33%. In three dogs co-infestation with more than one ectoparasite were detected: (i) C. felis and I. ricinus, (ii) C. canis and D. reticulatus, (iii) D. reticulatus, I. ricinus and R. sanquineus. Babesia canis was detected in four ectoparasites collected from four dogs (4/14, 29%): D. reticulatus (2/4, 50%), I. ricinus (1/4, 25%) and C. canis (1/4, 25%). Rickettsia raoultii was found in a single D. reticulatus (1/14, 7%). Candidatus Midichloria mitochondrii was detected in all collected I. ricinus (4/14, 29%). Two groups of endosymbionts were detected in canine fleas: (i) identical to Wolbachia pipientis, endosymbiont found in C. felis (4/14, 29%) and C. canis (2/14, 14%), (ii) identical to Wolbachia endosymbiont of Xenopsylla sp. found in C. felis (2/14, 14%) and C. canis (1/14, 7%). In two cases co-infections were detected (i) B. canis and W. pipientis in C. canis, (ii) B. canis and Can. Midichloria mitohondrii in I. ricinus. Flea C. felis was present on all examined cats as well as a single I. ricinus tick. Sequences revealed the presence of two endosymbiont groups in C. felis: one identical to W. pipientis (9/22, 41%) and second to W. endosymbiont of Xenopsylla sp. (13/22, 59 %). Anaplasma phagocytophilum was detected in I. ricinus (5%, 1/22), Bartonella clarridgeiae in four (4/6, 67%) and B. hensellae in two fleas (33%). In six cases co-infections were detected (i) W. endosymbiont of Xenopsylla sp. and B. clarridgeiae (4/6, 67%), (ii) W. pipientis with B. hensellae (33%, 2/6).

The lancet liver fluke Dicrocoelium dendriticum occurs in various domestic (sheep, goats, cattle, horses, rabbits, dogs) and wild (deer, wild boar, buffalo, camels) mammals and humans around the globe. Since reports of D. dendriticum in large carnivores are limited, like in the brown bear (Ursus arctos), we performed the study to investigate the presence and the prevalence of D. dendriticum in free ranging brown bears from different Croatian regions. Data on age, gender, location at the time of death, season, intensity of infection and pathohistological changes have been collected from infested animals. Parasites from bears, goat and sheep were further genotyped to investigate possible genetic difference among D. dendriticum isolates. Between February 2014 and December 2016, we sampled the complete gall bladders and liver samples of 136, hunted and dead due to other causes, brown bears across the bear range in Croatia. Each gallbladder and content was washed through the sieve to detect and count each parasite. We found that 60% of the examined brown bears were infected, ranging from 1- 103 parasites (mean=16.31, median=1) without differences in prevalence related to location, gender or age class. Seasonal distribution of Dicrocoelium infestation indicated a significantly higher percentage of infestation in autumn (mid August-December, 72%) compared to spring (February-May, 45% ; p=0.00192). Sequencing of ITS-1 of individual D. dendriticum from 16 bears, 5 sheep and one goat didn’t show any heterogeneity. COX1 sequences were identical from all bears from various regions showing differences in 7 nucleotide position with similarity of 98% with small ruminant isolates. To evaluate possible genetic variability of D. dendriticum within single bear COX1 was sequenced from 3-5 adult flukes from 10 bears without any SNP. Histopathology showed granulation of the surface of the liver and hepatomegaly in infested individuals, as well as enlarged gall bladders with firmer consistency of bile ducts surrounding the parenchyma. In the current survey we have presented evidence that bears could serve as final host of potential new “bear” genotype of D. dendriticum. This finding also suggests possible adaptation of D. dendriticum and unique life cycle of “bear” isolate.

Vector-borne pathogens are widely distributed and represent emerging group of diseases in dogs. For veterinary practitioners they represent a substantial diagnostic challenge because clinical signs might be often concealed and overlapping. Although prevalence of antibodies against vector-borne pathogens have already been reported in apparently healthy dogs from Croatia, here we report results of survey on blood samples with SNAP®4Dx®Plus collected from three groups of dogs: (i) apparently healthy (52%, 753/1, 448), (ii) suspected dogs with clinical signs of the disease (44%, 632/1, 448), (iii) deceased dogs with the history of anemia and thrombocytopenia (4%, 63/1448). In total 1, 448 dogs were tested for presence of Dirofilaria immitis antigen, antibodies to Anaplasma phagocytophilum/Anaplasma platys, Borrelia burgdoferi and Ehrlichia canis. Highest prevalence was detected in group of apparently healthy dogs with 6% (47/753) serologically-positive. Seroprevalence was: A. phagocytophilum/A. platys 5% (41/753), B. burgdoferi 0.2% (2/753), E. canis 0.6% (5/753) and D. immitis 0.3% (3/753). Within the group of „suspected dogs“ slightly lower prevalence of 5% (30/632) was noted with similar distribution on pathogens: A. phagocytophilum/A. platys in 3% (21/632), B. burgdoferi 0.4% (3/632), E. canis 0.6% (4/632) and D. immitis in 0.6% (6/632). Deceased dogs showed identical seropositivity (6%, 4/63) with apparently healthy group: A. phagocytophilum/A. platys (3%, 2/63), B. burgdoferi (2%, 1/63) and D. immitis (3%, 2/63). Co-infections were found in 0.5% (4/753) of apparently healthy dogs, 0.3% (2/632) within group of „suspected dogs“ while in deceased dogs only one case was registered (2%, 1/63). In all tested groups A. phagocytophilum/A. platys was the most prevalent pathogen, followed by E.canis in apparently healthy and „suspected dogs“, while the lowest prevalence was for D. immitis and B. burgdoferi. These findings raise question on role of selected vector borne diseases in development of anemia and thrombocytopenia since prevalence among groups was almost identical.

Fleas and ticks represent common ectoparasites of cats. However, pathogens which they transmit have not been investigated in Croatian cats. The aim of this study was to investigate vector-borne pathogens from Croatian feline patients and to determine their pathogenicity based on pathologic and molecular findings. The study included 37 cats with gross findings of anemia and/or jaundice submitted for necropsy upon owners request in a period of two years. During necropsy representative tissue samples were collected for histopathology and molecular analyses. Blood, bone marrow, liver, lungs, myocardium and spleen were screened by conventional PCR for presence of Anaplasma/Ehrlichia, Babesia/Theileria, Bartonella, Hepatozoon, Mycoplasma and Rickettsia DNA. All amplified samples were sequenced in both directions and assembled. Cause of death was determined following results of pathologic and molecular investigation. In 25 cats out of 37 investigated (68%) at least one pathogen was detected: Candidatus Mycoplasma haematoparvum (64%), Can. M. haemominutum (32%), Babesia canis (16%), Can. M. turicensis (12%), M. haemofelis (8%) and Bartonella clarridgeiae (4%). Hemoplasmas were detected in different organs, while B. canis was detected only in blood and B. clarridgeiae in one liver. Co-infections were found in nine cats (9/25=36%). In three cats (3/25=12% of positive cats and 3/24=13% of cats with hemoplasmas) causes of death were hypoxia and circulatory collapse linked to hemoplasma infection: M. haemofelis infection, Can. M. haemominutum and M. haemofelis co-infection, and Can. M. haemominutum and Can. M. haematoparvum co-infection. In the remaining 23 cats cause of death wasn’t associated with detected pathogens, which raises question of their pathogenicity in these animals. Although vector-borne pathogens are common in Croatian cats, it seems they mostly present incidental findings, but not primary causes of death. However, in minority of animals infected with one or more hemoplasmas, hemolytic anemia can cause death as a consequence of massive erythrocyte destruction.

Dogs can harbor a variety of parasites in digestive tract, which cause gastro-intestinal disturbances. At the same time several species have a zoonotic potential. In the current study fecal samples from 2, 335 dogs were analyzed in the period from 2012 to 2015. The fecal samples were collected by the dog owners and delivered to Croatian Veterinary Institute Laboratory for parasitology, through veterinary clinics, due to gastrointestinal disorders, mostly from the Zagreb area. Coprological examinations were conducted with the flotation technique, using magnesium sulfate with direct immunofluorescence for detection of Giardia cysts and Cryptosporidium oocysts. The results are presented by year for better visibility. The overall prevalence in 2012 was 48.1%: Giardia duodenalis (23.8%), Cryptosporidium spp. (8.0%), Toxocara canis (4.5%), Isospora spp. (4.7%), Toxascaris leonina (0.7%), Ancylostoma/Uncinaria (0.5%), Trichuris vulpis (5.2%), Sarcocystis sp. (0.5%), Capillaria spp. (0.2%). A single-pathogen was detected 55.2%, and co-infection with two and more than two pathogens in 44.8%. The overall prevalence in 2013 was 57.7%: Giardia duodenalis (26.5%), Cryptosporidium spp. (10.8%), Toxocara canis (5.0%), Isospora spp. (5.3%), Toxascaris leonina (0.9%), Ancylostoma/Uncinaria (12.0%), Trichuris vulpis (5.3%), Sarcocystis sp. (0.5%), Capillaria spp. (1.1%), Strongyloides stercoralis (0.5%). A single-pathogen was detected in 60.4%, and co-infection with two and more than two pathogens in 38.6%. The overall prevalence in 2014 was 64.9%: Giardia duodenalis (26.7%), Cryptosporidium spp. (18.7%), Toxocara canis (4.7%), Isospora spp. (5.0%), Toxascaris leonina (1.5%), Ancylostoma/Uncinaria (3.2%), Trichuris vulpis (3.9%), Sarcocystis sp. (0.2%), Capillaria spp. (0.7%), Strongyloides stercoralis (0.1%). A single-pathogen was detected in 51.8%, and co-infection with two and more than two pathogens in 48.2%. The overall prevalence in 2015 was 56.6%: Giardia duodenalis (26.3%), Cryptosporidium spp. (16.4%), Toxocara canis (3.6%), Isospora spp. (4.8%), Toxascaris leonina (0.6%), Ancylostoma/Uncinaria (1.6%), Trichuris vulpis (1.4%), Sarcocystis sp. (1.8%), Capillaria spp. (0.2%). A single-pathogen was detected in 45.1%, and co-infection with two and more than two pathogens in 54.9%. The most common co-infection in all years was with Giardia duodenalis and Cryptosporidium sp. The results of current study clearly show the need for regular fecal analysis to exclude parasites as common agents causing GI problems. Also, it is quite important to reconsider routine dechelminisation programs since protozooan parasites were most frequently detected.

Dirofilaria repens is highly prevalent in Croatian dogs. Infection is asymptomatic but dermatitis could appear. Eye lesions so far have not been described. Ophthalmological examination of the left eye in four years old, mixed breed, male dog from Vukovar revealed single nonpruritic nodule. Located episclerally, close to limbus, pink tumor of elastic consistency and botryoidal surface, measured 8 mm in diameter. Complete excisional biopsy and tissue formalin fixation were performed. On the tumor cut surface irregular pseudo cyst wall covering coiled white nematode was found. Tissue was processed for routine histopathology and few nematode segments 2 mm in diameter were microscopically identified as immature female Dirofilaria sp. according to outer cuticular longitudinal ridges and lack of microfilaria. Dirofilaria repens was confirmed based on morphology of anterior part and longitudinal stripes. Histology showed concentric 3 mm thick outer layer of collagenous wall. Inner layer consisted of loosely arranged collagenous fibers, network of capillaries and fibroblasts surrounded by macrophages, eosinophils, neutrophils and plasma cells. Eosinophilic proteinaceous fluid filled the pseudo cyst space and surrounded fragments of coiled nematode. On transversal sections of fragments evenly spaced cuticular ridges were present over pronounced muscular layer, lateral chords and segments of digestive and immature reproductive system. This findings confirmed dirofilariasis as a cause of granulomatous episcleritis. Clinical, hematological, biochemical, radiological as well as cardiac sonographic and modified Knott’s tests excluded presence of other adult nematodes, microfilaria or lesions consistent with canine dirofilariasis, respectively. In cases of nodular ocular lesions except neoplasms, Dirofilaria repens has to be considered as possible etiology in Croatian dogs. In this unusual case immature adult caused chronic inflammation, despite the lack of microfilaria previously described as cause of strong inflammatory response in ocular lesions.

Retrovirus infections caused by feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are common in domestic cats in Croatia. The IDEXX SNAP Combo FeLV Antigen/FIV Antibody Test® is used in routine diagnostics from whole blood, serum or plasma (clinical samples), but not from cats presented for necropsy. We therefore performed a study to investigate usefulness the SNAP test on postmortally obtained blood and body fluids. Samples were collected from two group of cats: (A) cats with known status of FeLV/FIV-N=12 ; (B) cats without known history of FeLV/FIV testing-N=19. In group A results from live cats were compared to test results from postmortally collected blood. In group B FeLV/FIV status was determined on postmortal results of blood samples and compared to results obtained from body fluids including lung juice, ascites, thoracic or pericardial effusion. In group A, tests performed during life and after death gave the same result in 67% (8/12) of animals. In four cats from same group results didn’t match: i) two cats showed FIV infection on clinical samples and FeLV/FIV coinfection on postmortal samples ; ii) single cat was positive for FeLV and FIV during life, while it was only positive for FeLV after death ; iii) clinical sample from a cat was negative, while postmortal blood was positive for FeLV. In group B, results from postmortal blood matched with body fluids in 94% (16/17) of tested cats. Lung juice from one cat was negative, while postmortal blood showed infection with FeLV. This study presents preliminary results which indicate that IDEXX SNAP Combo test could be useful in postmortal diagnostics of FeLV/FIV status. However, a systematic research which includes live and dead positive and negative cats (with determined viral status by serologic and molecular methods) should be performed for definite conclusion on the accuracy of this SNAP test on postmortal samples.

Insects of the genus Culicoides belong to the most numerous haematophagous insects in the world. Nearly 1400 different species of Culicoides have been described worldwide. Insects of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are biological vectors of bluetongue virus, transmitting it by sucking blood from the infected ruminant host. An entomological survey of insects of the genus Culicoides as bluetongue virus biological vectors was conducted in 2016 in order to determine the Culicoides fauna in Croatia. The Culicoides identified morphologically were compared with the selected species COX1 gene sequences obtained. A total of 76977 Culicoides specimens were collected and morphologically identified ; 200 specimens were identified by molecular methods. The presence was demonstrated of 21 Culicoides known species and 11 new species showing 84% to 94% similarity to the sequences available in the NCBI GeneBase. The C. obsoletus and C. scoticus, and 1 Culicoides showing 94% similarity to C. obsoletus were identified by the C. obsoletus group vector sequencing. The C. pulicaris group vector sequencing identified the C. pulicaris, C. punctatus, C. lupicaris and C. newsteadi, and 1 specimen showing 94% similarity to C. newsteadi. Sequencing of other Culicoides species identified the following species: C. nubeculosus, C. riethi, C. puncticollis, C. fagineus, C. flavipulicaris, C. circumscriptus, C. fascipennis, C. achrayi, C. parroti, C. salinarius, C. griseidorsum, C. simulator, C. picturatus, C. festivipennis, C. alazanicus, 1 Culicoides with 94% similarity to C. paradoxalis, 1 Culicoides with 85% similarity to C. oxystoma, 1 Culicoides with 85% similarity to C. circumscriptus, C. salinarius and C. manchuriensis species, 2 Culicoides with 86% similarity to Culicoides sp., and 4 new Culicoides spp. Data on the Culicoides species present in Croatia, obtained by morphological and molecular identification, are now available in Croatia for the first time. This entomological survey revealed predominance of the vectors of C. obsoletus and C. pulicaris groups as a major risk factor for the spread of bluetongue virus, which is important for the epidemiology of bluetongue as an infectious disease. Finding of various Culicoides species is highly relevant for the knowledge of fauna and biodiversity of Culicoides insects in Croatia.

Pseudogravidnost Hydrometra)je patološko stanje koje je karakterizirano nakupljanjem različite količine sterilne tekućine u negravidnoj maternici, uz prisustvo žutog tijela i stalno povišenim koncentracijama progesterona u krvnoj plazmi (više od 3 ng/mL). Predstavlja jedan od najčešćih uzroka smanjene plodnosti u koza. Pojavnost pseudogravidnosti je učestalija u stadima mliječnih pasmina koza i kreće se od 3% do 5% (7, 69%). Etiologija stanja još nije sasvim razjašnjena, ali se kao mogući uzroci navode prisutnost perzistentnog žutog tijela, embrionalna smrtnost s posljedičnom resorpcijom embrija i učestala hormonalna manipulacija spolnim ciklusom. Tipični klinički simptom pseudogravidnosti je znatno povećanje maternice s tankom stijenkom u kojoj fluktuira rijetko sluzavi, vodenasti, aseptični sadržaj. Transabdominalna ultrazvučna pretraga je najbolja dijagnostička metoda pseudogravidnosti. Izvodi se na lijevom ventrolateralnom dijelu abdomena, između 40. i 70. dana nakon parenja. Karakteristika ultrazvučne slike kod pseudogravidnosti prikazuje ne-ehogena tekućinom ispunjena područja koja su međusobno odvojena dvostrukim tkivom tankih stijenki, a predstavljaju dilatirane maternične rogove. Ne uočavaju se placentomi, fetalne membrane niti fetus. Pseudogravidnost u koza se uspješno liječi aplikacijom prostaglandina (PGF2α), koji dovode do luteolize žutog tijela i utječu na evakuaciju aseptične tekućine iz uterusa. Druga injekcija PGF2α se daje 12 dana nakon prve, njome se postiže dobar postotak koncepcije (do 85%) i smanjuje se mogućnost ponovne pojave pseudogravidnosti. Prognoza pseudogravidnosti je povoljna ako se na vrijeme dijagnosticira, većina životinja nakon učinkovite terapije prostaglandinima očituje znakove estrusnog vladanja i nakon parenja koncipira.

Pseudogravidnost (Hydrometra) je patološko stanje koje je karakterizirano nakupljanjem različite količine sterilne tekućine u negravidnoj maternici, uz prisustvo žutog tijela. Predstavlja jedan od najvažnijih uzroka smanjene plodnosti u koza. Cilj ovoga istraživanja bio je utvrditi postotak pojave pseudogravidnosti koza na hrvatskim farmama Sanskih koza kao predstavnika visoko mliječne pasmine. Istraživanje je provedeno kroz period od sredine kolovoza 2010. godine do kraja studenog 2011. godine. Tijekom istraživanja analiziran je progesteronski profil koza kroz period od mjesec i pol dana tijekom početka rasplodne sezone (prijelazni period) i van sezone nakon provedenog svjetlosnog tretmana te je napravljena ultrazvučna dijagnostika gravidnosti 35. do 50. dana nakon parenja. Istraživanje na farmi OPG Moravec je uključivalo 79 koza (39 koje su se ojarile tijekom prve polovice ožujka 2010. godine, te 40 koza koje su se ojarile tijekom prve polovice ožujka 2011. godine). Na farmama OPG-u Zadravec i OPG-u Malinarić istraživanje je uključivalo 42 koze (22 +20). Na farmi OPG Moravec pred početak sezone 2010. godine, niti jedna koza nije imala konstantno povišenu razinu progesterona. Kasnije, nakon provedene ultrazvučne dijagnostike gravidnosti utvrđeno je da tri od 39 koza imaju pseudogravidnost, sa incidencijom od 7.69%. Tijekom 2011. godine na svakoj farmi je nakon analize progesteronskog profila zabilježena jedna koza sa konstantno povišenom razinom progesterona, a kasnije je ultrazvučno potvrđena pseudogravidnost, sa ukupnom incidencijom od 3.65% (3/82). Svim kozama kojima je dijagnosticirana pseudogravidnost su aplicirani prostaglandini (PGF2α). Životinje su izbacile tekućinu i pokazivale znakove estrusnog vladanja unutar 1.5 do 2.5 dana od terapije, bile su parene u drugom ciklusu i sve su ostale su gravidne. Prognoza pseudogravidnosti je povoljna, jer ako se na vrijeme dijagnosticira, pogotovo u prijelaznom preiodu, a i kasnije tijekom sezone, životinje budu normalno parene i mogu koncipirati.

Poaching is one of the main threats to the strictly protected grey wolf (Canis lupus) population in Croatia. It is generally difficult to obtain hard evidence to convict individuals suspected of illegal hunting or trading threatened species. Veterinary forensics often includes species determination from trace evidence like blood, saliva, hair or bone fragments. We report a case of species determination from two canid skulls (marked as H1 and H2). Both animals were hunted near Zagreb, Croatia in 2010. Hunter reported a legal harvest of two golden jackals (Canis aureus), but hunting inspection doubted species identification. Our task was to determine which of the three canid species present in Croatia these skulls belong to – strictly protected grey wolf, golden jackal which is a game species or a dog. Because of similar morphology and evolutionary relatedness distinguishing the three canid species is particularly difficult, even on a DNA level. For species determination we used craniometrical analysis, combined with analysis of several DNA markers - mitochondrial DNA control region, Y chromosome and MHC genes. As skulls were exposed to high temperatures during trophy preparation, DNA (extracted from tooth rooth) was damaged and aggravated DNA analysis. Both craniometrical measurements and DNA analysis suggested that the skull marked as H1 belongs to the wolf. Based on craniometrical analysis we concluded that skull marked as H2 is not a purebred jackal or a purebred wolf, while DNA analysis of maternally inherited mitochondrial DNA control region confirmed dog maternal ancestry. Due to the damaged DNA amplification of other markers was not successful and we were not able to confirm paternal ancestry of this individual.

Predstavnici roda Canis u Hrvatskoj su vuk (Canis lupus), pas (Canis lupus familiaris) i čagalj (Canis aureus). Zbog slične veličine i građe tijela, razlikovanje ova tri kanida na temelju morfologije je ponekad otežano. Istraživanjem na dvije lubanje kanida nepoznate vrsne pripadnosti testirali smo hipotezu da se kombinacijom kraniometrijske analize i analize kontrolne regije mitohondrijske DNK pouzdano može utvrditi vrsna pripadnost kanida, u slučaju kada nam je dostupna samo lubanja istraživanje životinje. Na temelju rezultata kraniometrijskih mjerenja zaključili smo da lubanja pod oznakom H1 pripada vuku, dok smo za lubanju pod oznakom H2 utvrdili da nije čistokrvni čagalj, niti čistokrvni vuk. Analizom DNK utvrdili smo da se slijed kontrolne regije mitohondrijske DNK izolirane iz lubanje oznake H1 podudara s vučjim haplotipom WCRO1, dok se slijed lubanje H2 poklapa s psećim haplotipom DCRO8. Ovim istraživanjem potvrdili smo našu hipotezu da se kombinacijom kraniometrije i analize mitohondrijske DNK može utvrditi vrsna pripadnost kanida u slučaju kada nam je dostupna samo lubanja istraživane životinje, s naglaskom da je za utvrđivanje očinske linije kod hibrida potrebna analiza dodatnih genskih markera.