Amer Alić

A molecular survey of vector-borne pathogens in red foxes (Vulpes vulpes) from Bosnia and Herzegovina.

BackgroundRed foxes (Vulpes vulpes) have recently been recognized as potential reservoirs of several vector-borne pathogens and a source of infection for domestic dogs and humans, mostly due to their close vicinity to urban areas and frequent exposure to different arthropod vectors. The aim of this study was to investigate the presence and distribution of Babesia spp., Hepatozoon canis, Anaplasma spp., Bartonella spp., ‘Candidatus Neoehrlichia mikurensis’, Ehrlichia canis, Rickettsia spp. and blood filaroid nematodes in free-ranging red foxes from Bosnia and Herzegovina.MethodsSpleen samples from a total of 119 red foxes, shot during the hunting season between October 2013 and April 2014 throughout Bosnia and Herzegovina, were examined for the presence of blood vector-borne pathogens by conventional PCRs and sequencing.ResultsIn the present study, three species of apicomplexan parasites were molecularly identified in 73 red foxes from the entire sample area, with an overall prevalence of 60.8%. The DNA of B. canis, B. cf. microti and H. canis was found in 1 (0.8%), 38 (31.9%) and 46 (38.6%) spleen samples, respectively. In 11 samples (9.2%) co-infections with B. cf. microti and H. canis were detected and one fox harboured all three parasites (0.8%). There were no statistically significant differences between geographical region, sex or age of the host in the infection prevalence of B. cf. microti, although females (52.9%; 18/34) were significantly more infected with H. canis than males (32.9%; 28/85). The presence of vector-borne bacteria and filaroid nematodes was not detected in our study.ConclusionThis is the first report of B. canis, B. cf. microti and H. canis parasites in foxes from Bosnia and Herzegovina and the data presented here provide a first insight into the distribution of these pathogens among the red fox population. Moreover, the relatively high prevalence of B. cf. microti and H. canis reinforces the assumption that this wild canid species might be a possible reservoir and source of infection for domestic dogs.

The spread of zoonotic Thelazia callipaeda in the Balkan area

BackgroundThelazia callipaeda (Spirurida, Thelaziidae), also known as “oriental eyeworm”, is a small nematode parasite that lives in the conjunctival sac of domestic and wild carnivores, rabbits and even humans, causing mild (e.g., conjunctivitis, epiphora, and ocular discharge) to severe (e.g., keratitis, and corneal ulcers) ocular disease. This study reports, for the first time, the occurrence of T. callipaeda infection in the Balkan regions (i.e., Bosnia and Herzegovina and Croatia), it provides genetic evidence on the origin of the infection in that area and discusses potential expansion pathways in the near future.MethodsThis survey was conducted in two Western Balkan countries, Bosnia and Herzegovina and Croatia. At necropsy, from January 2011 to April 2014, a total of 184 carcasses of red foxes were examined throughout the study area and worms were collected from the conjunctival sac. In the same period, worms were also collected during clinical examination from the conjunctival sac of four dogs and a cat from Bosnia and Herzegovina and two dogs from Croatia. All nematodes collected were morphologically identified and molecularly characterized by sequencing of partial cox 1 gene.ResultsT. callipaeda was observed in 51 (27.71%) foxes and the highest prevalence (50.0%) was in the region of East Bosnia. Beside the 4 cases of hyperemia (7.84%), most of the infected animals had no signs of ocular infection (n = 47, 92.15%). A total of 417 adult nematodes collected (364 from foxes, 51 from dogs, 2 from cat) were morphologically and molecularly identified as T. callipaeda haplotype 1.ConclusionThis is the first report of autochthonous cases of T. callipaeda infection in red foxes, dogs and cat in Bosnia and Herzegovina and Croatia and data presented here suggest that reports of thelaziosis in other Balkan areas are, as yet, not diagnosed most likely due to the lack of awareness of practitioners. In addition, data regarding the spread of the infection in Europe over the last ten years suggests that an increasing pattern in the distribution of this disease in domestic and wild animals should be expected in the future.

Hepatozoon silvestris sp. nov.: morphological and molecular characterization of a new species of Hepatozoon (Adeleorina: Hepatozoidae) from the European wild cat (Felis silvestris silvestris).

SUMMARY Based on morphological and genetic characteristics, we describe a new species of Hepatozoon in the European wild cat (Felis silvestris silvestris), herein named Hepatozoon silvestris sp. nov. The study also provides the first data on the occurrence of H. felis in this wild felid. Hepatozoon meronts were observed in multiple cross-sections of different organs of four (44%) cats. Additionally, extracellular forms, resembling mature gamonts of Hepatozoon, were found in the spleen and myocardium of two cats. Furthermore, tissues of six animals (67%) were positive by PCR. Hepatozoon felis was identified infecting one cat (11%), whereas the 18S rRNA sequences of the remaining five cats (56%) were identical, but distinct from the sequences of H. felis. Phylogenetic analyses revealed that those sequences form a highly supported clade distant from other Hepatozoon spp. Future studies should include domestic cats from the areas where the wild cats positive for H. silvestris sp. nov. were found, in order to investigate their potential role to serve as intermediate hosts of this newly described species. Identification of its definitive host(s) and experimental transmission studies are required for elucidating the full life cycle of this parasite and the possible alternative routes of its transmission.

Troglostrongylus brevior in an Eurasian lynx (Lynx lynx) from Bosnia and Herzegovina

BackgroundIn the past few years the interest of the scientific community on lungworms of the genus Troglostrongylus has grown due to the increased number of unexpected cases of infections with Troglostrongylus brevior in domestic cats from Mediterranean Europe, likely due to a spill-over from wild reservoirs. Thus, there is a merit to increase our knowledge on the occurrence of this parasite in felids from European regions. The present paper describes lung lesions associated with T. brevior infection in the endangered Eurasian lynx (Lynx lynx) from Bosnia and Herzegovina.FindingsThe carcass of an illegally killed 3-year-old male Eurasian lynx was presented for necropsy at the Faculty of Veterinary Medicine of Sarajevo (Bosnia and Herzegovina). Grossly, multiple, multinodular, consolidated and firm, tan to grey areas, occupying the caudal third of caudal lung lobes, were observed. At cut section, the catarrhal fluid was draining from the airways. Larvae of T. brevior were found in tracheal scraping. The histopathological examination revealed multifocal to coalescing areas, centered on bronchi and bronchioles, and expanded alveoli filled with necrotic debris, degenerated inflammatory cells, mostly neutrophils and macrophages, and multiple cross sections of parasite larvae and thin-walled morulated eggs of lungworms. The paraffin-embedded lung samples were molecularly positive for T. brevior.ConclusionThis paper describes the first record of T. brevior in the Eurasian lynx and the associated host lung pathology. Given its pathogenic potential and the lack of data on troglostrongylosis in lynx populations, the occurrence and impact of Troglostrongylus spp. on wildlife health as well as the role of L. lynx as reservoir of infection for other felids, should be further investigated.

Occurrence of Cryptosporidium spp. and Giardia duodenalis in red foxes (Vulpes vulpes) in Bosnia and Herzegovina.

Abstract The aim of this study was to determine the occurrence and distribution of Cryptosporidium spp. and Giardia duodenalis among free-living red foxes (Vulpes vulpes) in Bosnia and Herzegovina. For this purpose, a total of 123 fecal samples from red foxes, shot during hunting seasons between January 2011 and March 2012 were examined using immunofluorescent microscopy. Overall, observed prevalences of Cryptosporidium spp. and Giardia duodenalis were 3.2 % (4/123) and 7.3% (9/123), respectively. The results show that foxes might play the role of potential reservoirs of Cryptosporidium and Giardia parasites, but further molecular analysis are necessary to elucidate the source of infection, routes of transmission and zoonotic potential of these two pathogens.

Red fox (Vulpes vulpes) as a potential reservoir host of cardiorespiratory parasites in Bosnia and Herzegovina.

Red fox (Vulpes vulpes) is considered as reservoir of different cardiorespiratory parasites of veterinary and medical importance. Since data on cardiorespiratory parasites in foxes in Bosnia and Herzegovina are still lacking, the aims of the present study were to (i) investigate the prevalence and geographical distribution of these parasites, (ii) determine genetic diversity of detected parasite species, and (iii) to estimate the role of foxes in the transmission cycle to companion animals and humans. Four species, morphologically and molecularly identified as Eucoleus boehmi (64.6%; 51/79), Eucoleus aerophilus (69.7%; 154/221), Crenosoma vulpis (45.7%; 101/221) and Linguatula serrata (1.3%; 1/79) were retrieved from nasal cavity and lungs in 184 (83.3%) animals. The occurrence of heartworms, Angiostrongylus vasorum and Dirofilaria immitis was not detected by necropsy or PCR. Furthermore, three distinct haplotypes of E. aerophilus (I, III, XV) and two of C. vulpis (I, II) previously reported in pet animals and wild carnivores were confirmed in this study. A new haplotype of C. vulpis (designated as haplotype V) was also identified based on 12S rRNA gene for the first time. The present study indicates a high prevalence and wide distribution of nasal and lung nematodes in fox population in Bosnia and Herzegovina, and supports the existence of transmission patterns between wildlife and pet animals.

High diversity of blood-associated parasites and bacteria in European wild cats in Bosnia and Herzegovina: A molecular study

Wild felids may play a significant role in the transmission of various pathogens to domestic cats, in particular, but also to other animals and humans. However, since data on the occurrence of blood-borne organisms in European wild cats (Felis silvestris silvestris) are scarce, the present study aimed to provide an insight into the genetic diversity of the agents carried by this sylvatic species in Bosnia and Herzegovina and to elucidate their pathogenic impact. Tissue samples from 18 adult wild cats were collected and examined by PCR and histopathology. Five species of apicomplexan parasites belonging to three genera (Babesia sp., Cytauxzoon sp., Hepatozoon silvestris, H. felis, Hepatozoon sp.), as well as two different sequence types of undescribed hemotropic mycoplasmas (designated as type A and type B), were identified in 15 animals (83%). Histopathology revealed no relevant lesions associated with any of the agents detected. The results clearly showed that European wild cats can harbour a broad range of blood-associated parasites and bacteria. However, further studies are required to investigate the possible implication of hematophagous arthropod vectors in their transmission and to clarify the true pathogenic significance of these organisms. Direct transmission of the agents by bites should also be considered as an alternative, non-vectorial route of transmission in wild cats.

Fatal pulmonary cysticercosis caused by Cysticercus longicollis in a captive ring-tailed lemur (Lemur catta)

Here we describe fatal pulmonary cysticercosis caused by Cysticercus longicollis, the larval stage of Taenia crassiceps in a 15-year-old female ring-tailed lemur (Lemur catta) from Sarajevo Zoo. After sudden death, the lemur was subjected to necropsy and large multicystic structure, subdivided with fibrous septa and filled with numerous translucent, oval to ellipsoid bladder-like cysts (cysticerci), almost completely replacing right lung lobe was observed. In addition, numerous free and encysted cysticerci were found in the thoracic cavity. Histopathology revealed connective tissue outlined cavities that compress lung parenchyma. Each cavity contained several thin walled cysticerci with single inverted protoscolex, one or more suckers and rostelum with two rows of hooks. In many of the cysticerci one or several exogenous buds of daughter cysticerci were observed. Based on morphology and microscopic appearance the parasite was identified as C. longicollis. Subsequent molecular analysis and sequencing confirmed presumptive diagnosis. To our knowledge, this case represents the first report of T. crassiceps and cysticercosis caused by C. longicollis in Bosnia and Herzegovina.

Eulimdana clava (Nematoda: Filarioidea) infection in domestic pigeons (Columba livia domestica): Molecular characterization and pathological changes

Filarial nematodes of the Eulimdana genus inhabit subcutaneous tissue of various avian species, mostly Charadriiforme birds. In domestic pigeons, E. clava is the only species recorded in the subcutaneous tissue in a number of isolated cases. In the present study, we discuss the morphology and histopathology of filarial nematodes recovered from subcutaneous tissue of domestic pigeons in Bosnia and Herzegovina. In total 110 pigeons were submitted to necropsy at the Department of Pathology of the Faculty of Veterinary Medicine in Sarajevo. At necropsy, in four pigeons (3.6%) numerous thread-like 0.9-2.1 cm long nematode parasites were observed in the subcutaneous tissue, peritracheal and periesophageal connective tissue. In one pigeon, the parasites were also found free in the body cavity around the heart and lungs. In addition, several 80-90 μm long microfilariae were noted in the tissue cross-sections. No significant lesions were observed associated with adult parasites or microfilariae. Based on morphology, host species and localization detected parasites were identified as E. clava. Molecular analyses of the cox1 and 12S rRNA nucleotide sequences herein generated revealed the close genetic relationship to other filarioid nematodes. The importance of the nematodes in pigeons and the lack of sequences in genetic databases for comparison of avian filarial parasites are emphasized.

Echinococcus ortleppi, the cattle strain in a crested porcupine (Hystrix cristata): A new host record

Echinococcus granulosus sensu lato, a dog tapeworm is a species complex causing cystic echinococcosis or hydatid disease in a great variety of mammalian intermediate hosts, including humans. This complex comprises five species including Echinococcus ortleppi (G5 genotype or cattle strain). In the present paper, we report the first case of infection with the larval stage of latter cestode in a captive crested porcupine (Hystrix cristata), molecularly confirmed by PCR and sequencing of the cox1 and nad1 genes. The food contaminated with the parasites eggs is the most likely source of the infection. Our data broaden the knowledge on the host range and geographical distribution of this rarely reported species of Echinococcus in Europe.