Emergence of Echinococcus multilocularis in Central Continental Croatia: A Human Case Series and Update on Prevalence in Foxes.

Human alveolar echinococcosis (HAE), caused by the metacestode stage of Echinococcus multilocularis, has emerged in many European countries over the last two decades. Here, we report the first data on the new HAE focus with increasing incidence in central Croatia, describe its clinical presentation and outcomes in diagnosed patients, and provide an update on the prevalence and geographic distribution of Echinococcus multilocuaris in red foxes. After the initial case in 2017 from the eastern state border, from 2019 to 2022, five new autochthonous HAE cases were diagnosed, all concentrated in the Bjelovar-Bilogora County (the county incidence in 2019 and 2021: 0.98/105, in 2022: 2.94/105/year; prevalence for 2019-2022: 4.91/105). The age range among four female and two male patients was 37-67 years. The patients' liver lesions varied in size from 3.1 to 15.5 cm (classification range: P2N0M0-P4N1M0), and one patient had dissemination to the lungs. While there were no fatalities, postoperative complications in one patient resulted in liver transplantation. In 2018, the overall prevalence of red foxes was 11.24% (28/249). A new focus on HAE has emerged in central continental Croatia, with the highest regional incidence in Europe. Screening projects among residents and the implementation of veterinary preventive measures following the One Health approach are warranted.

Diversity of Hepatozoon species in wild mammals and ticks in Europe.

BACKGROUND: Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe. METHODS: Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene. RESULTS: Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis (n = 22). CONCLUSIONS: Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.

Dirofilaria repens microfilaremia in humans: Case description and literature review.

INTRODUCTION: Dirofilaria repens is a vector-borne filaroid helminth of carnivorous animals, primarily domesticated dogs. Humans are considered to be accidental hosts in which D. repens rarely reach sexual maturity but induce local inflammation, mainly in subcutaneous and ocular tissues. METHODS: In the current study, we present the detection of multiple adults of D. repens, endosymbiont Wolbachia sp. and microfilariae by molecular analysis in peripheral tissues and bloodstream of a human host. A subsequent meta-analysis of published literature identified 21 cases of human infection with adult D. repens producing microfilariae. RESULTS: Within the study population, there were 13 (59.09%) males, eight (36.36%) females and, in one (4.55%) case, sex was not reported. A total of 11 (50.00%) cases had subcutaneous dirofilariasis, six (27.27%) had ocular dirofiliariasis, with single cases (4.55% each) of genital, mammary, lymphatic and a combination of subcutaneous and pulmonary dirofilariasis described. In one (4.55%) case, the primary anatomical site of adult D. repens could not be found. D. repens microfilariae were detected in the local tissue (local microfilariasis) in 11 (50.00%) cases and the peripheral blood (microfilaremia) in 11 (50.50%) cases. Final identification of D. repens microfilariae was based on morphological detection in 14 (63.64%) cases, and molecular detection in eight (36.36%) cases. CONCLUSION: The results of this study suggest that humans may act as a final host for D. repens, however its role as a source of D. repens infection is less clear.

The trends of human dirofilariasis in Croatia: Yesterday - Today - Tomorrow.

INTRODUCTION: Human dirofilariasis is a disease historically linked to the Mediterranean area. For the last few decades, however, Dirofilaria nematodes have been spreading, both in terms of prevalence and the geographical expansion in non-endemic areas. Currently, cases of human dirofilariasis are recorded in more than 40 countries worldwide. Croatia is considered an endemic area of the Adriatic basin. METHODS: In a nationwide investigation, new and previously published cases of human dirofilariasis in Croatia were analyzed. RESULTS: Since 1996, 30 cases of human dirofilariosis were reported in Croatia. A total of 14 (46,67%) cases were from the coastal and 16 (53,33%) from continental regions of the country. Based on anatomical location, 13 (43,33%) cases were subcutaneous, 12 (40%) were ocular and five (16,67%) occurred in the reproductive organs. In all 30 cases, Dirofilaria repens was identified as the causative agent. CONCLUSIONS: An increase in air temperature as climate change, changes in mosquito fauna, high prevalence of D. repens in dogs and limited use of chemoprophylaxis are possible risk factors for Dirofilaria infection in the Croatian population. Since reporting to epidemiological services is not mandatory in this country, the real number of human dirofilariasis cases is probably significantly higher than published. This emphasizes the need for mandatory reporting of human cases and surveillance of Dirofilaria infection in dogs and mosquitoes in Croatia, following the "One Health" concept.

First reports of indigenous lethal infection with Anaplasma marginale, Anaplasma bovis and Theileria orientalis in Croatian cattle.

Anaplasmosis and theileriosis are important diseases with great economic impact that affect the cattle industry worldwide. In this study, we describe the first molecularly confirmed clinical cases of anaplasmosis caused by Anaplasma marginale and of a concurrent infection with A. bovis and Theileria orientalis in Croatian cattle. Clinical signs of A. marginale-infected cows included fever, lethargy, dark urine, as well as icteric and reddish mucous membranes. Postmortem examination revealed icterus, urinary bladder filled with dark urine and splenomegaly. A marginale was observed within erythrocytes on Giemsa-stained spleen imprints. Three affected cows were successfully treated with oxytetracycline, after which no new deaths occurred in the herd. Three cows in a different herd died suddenly and were found to be concurrently infected with A. bovis and T. orientalis. Postmortem examination revealed generalized icterus and urinary bladder filled with dark urine. These cases of A. marginale, A. bovis and T. orientalis infection show that bovine anaplasmosis and theileriosis are present within Croatian cattle and should be included in differential diagnostic protocols.

Molecular detection of Babesia divergens and Mycoplasma wenyonii infection in cattle from Bosnia And Herzegovina.

We report two cases of bovine babesiosis caused by Babesia divergens in a region of central Bosnia and Herzegovina. The cases were detected in June 2017 and July 2018 from two small backyard farms. Routine clinical assessments, including physical examination and haematology, revealed lethargy, fever, anaemia, leukopenia and haemoglobinuria in the affected animals. Serum alterations included an elevation of aspartate aminotransferase and a decrease of serum phosphate or hypophosphatemia. Thrombocytopenia was detected in the first clinical case. Microscopic examination of blood smears revealed intracytoplasmic protozoan parasites from the genus Babesia. Molecular screening of both animals confirmed the presence of Babesia divergens, the causative agent of bovine babesiosis. B. divergens DNA was also detected in two engorged female Ixodes ricinus ticks removed from these animals. In addition, Mycoplasma wenyonii DNA was identified by molecular screening in the animal examined in June 2017, and in I. ricinus ticks feeding on this animal. This study provides molecular confirmation of B. divergens as a cause of piroplasmosis in cattle in South-East Europe. The detection of M. wenyonii DNA ain I. ricinus also provides the first evidence of this bacterium in ticks in Europe.

Seroprevalence of vector-borne pathogens in dogs from Croatia.

Canine vector-borne pathogens (VBPs) are a group of globally distributed and rapidly spreading microorganisms transmitted by arthropods. In the present survey, we investigated Anaplasma spp., Ehrlichia canis, Dirofilaria immitis and Borrelia burgdorferi sensu lato seroprevalence between three groups of dogs (asymptomatic, suspected and deceased) from continental and coastal regions of Croatia with the commercial point-of-care SNAP®4Dx®Plus for VBPs. The overall prevalence for tested pathogens in dogs was 6.1% with the highest prevalence detected for Anaplasma spp. (4.5%), while the remaining pathogens were found at a prevalence of less than 1% (E. canis 0.6%, B. burgdorferi s.l. 0.4%, D. immitis 0.6%). No statistically significant differences in VBP detection between dog cohorts could be found with the exception of D. immitis in the deceased group. Interestingly, no evidence of D. immitis could be found in the hearts of dogs in this group at necropsy, however. This study provides the first data on the seroprevalence of selected VBPs between dogs of different health statuses in Croatia. The results demonstrate that serological evidence of VBPs alone or in combination with co-infections were found just as frequently in asymptomatic dogs as those with suspected or confirmed evidence of VBP disease, raising questions about the pathogenic potential of these organisms in domesticated dogs.

Microscopic and molecular analysis of Babesia canis in archived and diagnostic specimens reveal the impact of anti-parasitic treatment and postmortem changes on pathogen detection.

BACKGROUND: Classification of Babesia parasites has traditionally relied on morphological differentiation based on piroplasm size and shape. Molecular typing has subsequently revealed a more complex taxonomy for these piroplasms than previously thought. To evaluate the factors that influence the morphology of Babesia species upon microscopic examination and hence, their taxonomic classification, we performed detailed characterizations of piroplasms from archival and prospective collections of cytological samples of dogs with piroplasmosis before and after death. Merozoite morphology and time of parasite disappearance following imidocarb dipropionate was also investigated. METHODS: The study was divided into a (i) review of archived cytological slides from confirmed cases of canine piroplasmosis, and (ii) a prospective study of smears and tissue imprints from 15 recently necropsied dogs. The latter group could be further sub-divided into a non-treated group and an imidocarb dipropionate-treated group. Exact times of treatment before death were reviewed. Additional blood smears prepared from the live dogs and taken before therapy were also evaluated in the latter group. Parasite burden per each slide was determined in both studies. The shape and size of merozoites were described from blood smears taken while the dogs were alive and from different organs during necropsy. The results of all measurements were statistically analyzed. RESULTS: The morphology and size of merozoites from live dogs corresponded to that of previously described 'large' Babesia. The morphology and size of merozoites were significantly different (P < 0.001) in postmortem samples, however, and more consistent in shape and size with piroplasm cells previously referred to as 'small' Babesia. PCR and sequencing confirmed B. canis as the causative agent of disease in all investigated dogs, including in postmortem negative tissue imprints from dogs treated at least 24 h before death. CONCLUSIONS: Changes in the morphology of 'large' B. canis to 'small'-like Babesia observed by light microscopy appear to represent a common postmortem change. Classification of Babesia parasites into 'large' and 'small' Babesia using only microscopy of postmortem slides should be treated with caution. PCR-based methodologies for detection and molecular typing of Babesia spp. may prove valuable for investigating suspected cases of babesiosis following necropsy.

Molecular detection of Anaplasma platys, Anaplasma phagocytophilum and Wolbachia sp. but not Ehrlichia canis in Croatian dogs.

The bacteria Anaplasma platys, Anaplasma phagocytophilum and Ehrlichia canis are tick-borne agents that cause canine vector-borne disease. The prevalence of these pathogens in South Eastern Europe is unknown with the exception of an isolated case of A. platys detected in a dog imported into Germany from Croatia. To gain a better insight into their presence and prevalence, PCR-based screening for these bacterial pathogens was performed on domesticated dogs from different regions of Croatia. Blood samples from 1080 apparently healthy dogs from coastal and continental parts of Croatia as well as tissue samples collected from 63 deceased dogs with a history of anaemia and thrombocytopenia were collected for molecular screening by an Anaplasmataceae-specific 16S rRNA conventional PCR. Positive samples were confirmed using a second Anaplasmataceae-specific PCR assay with the PCR product sequenced for the purpose of bacterial species identification. All sequenced isolates were georeferenced and a kernel intensity estimator was used to identify clusters of greater case intensity. 42/1080 (3.8%; CI 2.7-5.0) of the healthy dogs were PCR positive for bacteria in the Anaplasmataceae. Sequencing of the 16S rRNA gene amplified from these positive samples revealed the presence of A. platys in 2.5% (CI 1.6-3.4%, 27 dogs), A. phagocytophilum in 0.3% (CI 0-0.6%, 3 dogs) and a Wolbachia endosymbiont in 1.1% (CI 0.4-1.6%, 12 dogs) of dogs screened in this study. Necropsied dogs were free from infection. Notably, no evidence of E. canis infection was found in any animal. This survey represents a rare molecular study of Anaplasmataceae in dogs in South Eastern Europe, confirming the presence of A. platys and A. phagocytophilum but not E. canis. The absence of E. canis was surprising given it has been described in all other Mediterranean countries surveyed and raises questions over the regional vector capacity of the Rhipicephalus sanguineus tick.