Mustafa Alsarraf

Rodents serve as hosts for many species of parasites, including cestodes, but the taxonomy of rodent-infecting tapeworms, especially in Central Europe, is still understudied. Therefore, the current study was conducted to identify larval and adult cestodes in free-living rodents from Northeastern Poland. The fieldwork was conducted between 2014 and 2023 in three forest sites. Cestodes were collected from five rodent species (Clethrionomys glareolus, Alexandromys oeconomus, Microtus agrestis, Apodemus agrarius and A. flavicollis, and were identified through morphology and by several genetic markers (12S rDNA, cox1, nad 1, 28S rDNA, 12S-16S rDNA) using phylogenetic analyses. A total of 58 cestode specimens were analyzed using molecular and phylogenetic analyses. Species identified included Catenotaenia henttoneni, Spasskijela kratochvili, Mesocestoides litteratus, Mesocestoides melesi, and others. Three Paranoplocephala specimens were identified as P. kalelai based on nad1 sequences. Morphological and genetic analyses indicated that the dominant adult cestode in bank vole was C. henttoneni and Mesocestoides spp. were the dominant larval cestodes. This study combined genetic and morphological methods to identify the cestodes infecting rodents in Poland. It confirmed the existence of P. kalelai in Poland, suggesting that previously reported Paranoplocephala spp., identified by morphological characters, may have corresponded to this molecularly identified species. Our study also highlighted gaps in genetic databases that currently still do not allow precise species identification.

Canine (K9) units are essential in law enforcement and military operations; they are often at higher risk of vector-borne infections due to their working and kennel environments. This study aimed to investigate the prevalence of vector-borne pathogens, including Babesia sp. and Mycoplasma spp., in military/police K9 dogs which visited the Adan veterinary hospital in Baghdad/Iraq in 2016-2017. 61 blood samples were collected on Whatman FTA cards and sent to the University of Veterinary Medicine in Vienna, Austria where they were tested with specific PCRs to get a better understanding about epidemiological situation of these vector-borne pathogens in K9s in Iraq. Two dogs tested positive, one with Babesia sp. and one with Mycoplasma haemocanis, making the prevalence 1.6% (1/61; 95% CI: 0.04-8.8%) for each parasite, the isolates were sequenced, and phylogenetic analysis was done to confirm their lineage with other sequences from the GenBank. This is the first study on vector-borne pathogens in K9s in Iraq and the first molecular record of infection with M. haemocanis in K9s.

ABSTRACT With the rising occurrence of skin diseases such as atopic dermatitis (AD), which are often associated with inflammation, skin barrier damage, and related complications, there is an urgent need for simple and effective early‐stage treatment strategies. Although current therapies, including moisturizers, injections, and corticosteroids, offer some relief, they present limitations such as side effects, high cost, or limited efficacy. Therefore, developing a straightforward therapeutic patch that promotes skin regeneration and enables efficient drug delivery is essential. In this study, electrospun nanofibrous patches composed of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and pectin (PEC) were fabricated and loaded with Hawthorn Extract (HE) and Dragon's Blood (DB) to enhance wound healing capacity. The patches were comprehensively characterized by morphological, chemical, and functional analyses. Surface wettability, swelling, and in vitro release studies confirmed improved hydrophilicity, rapid fluid uptake, and sustained delivery of the plant extracts. Antifungal activity against Candida albicans showed that dual‐extract patches completely inhibited fungal growth, while cytotoxicity assays with L929 fibroblasts confirmed biocompatibility. In vivo skin hydration testing on human volunteers demonstrated significant improvements, with dual‐extract patches enhancing skin moisture by up to 44% over a 6‐h period compared to the control group. These findings indicate that electrospun PVA/PVP/PEC patches, particularly those incorporating both HE and DB, combine hydration, antifungal properties, and biocompatibility. Such features highlight their strong potential for translation into clinical studies for treating AD and related chronic skin conditions.

INTRODUCTION: Since 24 February 2022, the day the Russian aggression against Ukraine began, millions of refugees and thousands of pets crossed the Polish-Ukrainian border. Additionally, an unknown number of shelter and stray dogs and cats were rescued and translocated to Poland by private persons and non-profit organizations. The aim of the present study was to examine rescued dogs and cats for presence of canine vector-borne parasites to determine the role of armed conflict in spreading these parasites. METHODS: In July 2022 blood samples were collected from two animal shelters in central Poland hosting dogs and cats rescued from Ukraine. Animals were imported from various regions of Ukraine, including eastern and southeastern Ukraine (military conflict area). Fifty-three dogs (51 from two shelters and two owned ones) and one shelter cat were examined by molecular methods (PCR and sequencing) for the presence of Babesia/Theileria spp., Dirofilaria spp. and Hepatozoon spp. DNA. RESULTS: We detected Dirofilaria immitis, a parasite species non-endemic in Poland, in two dogs translocated from Ukraine (2/53 = 3.8% [95% CI 0.5-13.0%]). One dog had a history of previous heartworm infection. High prevalence of Hepatozoon canis (51% [95% CI 36.8-64.9%]) was noted among translocated dogs. Prevalence of Dirofilaria repens in Ukrainian dogs (18.9% [95% CI 9.4-32.0%) was similar to prevalence in dogs from central Poland (12%). Co-infection of D. repens and D. immitis was found in two dogs and six dogs were co-infected by D. repens and H. canis. Hepatozoon canis infection was also identified in an imported cat. CONCLUSION: We confirmed that this military conflict has facilitated the spread of canine vector-borne parasites, including zoonotic species.

BACKGROUND: Canine hepatozoonosis caused by Hepatozoon canis is a common infection in dogs, with frequent case reports from the Mediterranean region and more recently from several Central European countries, such as Hungary and Germany. Despite the high prevalence of H. canis in red foxes, no infections have been reported to date in dogs in Poland. We describe here the first autochthonous cases of H. canis infection in dogs, including their clinical features, and report the prevalence of H. canis in grey wolves from different regions of Poland. METHODS: Thin smears prepared from blood samples collected from dogs were evaluated by microscopic examination. A total of 60 wolves and 47 dogs were tested. Infections were confirmed by PCR and sequencing. RESULTS: Gamonts of H. canis were found in > 50% of the neutrophils of two dogs and in < 10% of the neutrophils in another five dogs. Molecular typing by PCR sequencing of the 18S ribosomal RNA gene fragment confirmed infections in 11 dogs from different regions of Poland, in 2.7% of dogs attending veterinary practices in central Poland and in 35% of wolves from various geographical regions of Poland. Clinical features manifested mostly in older dogs, and the most common signs were anaemia and apathy. Young dogs usually remained asymptomatic. CONCLUSIONS: This is the first report of H. canis infection in dogs and wolves in Poland. Although the exact vector of the parasite is not known, veterinary practitioners should be aware of this new parasitosis and should consider appropriate diagnostics to confirm/exclude this infection. Further studies are needed to understand the transmission routes of H. canis in domestic and wild canids in Poland.

BACKGROUND: Dirofilaria immitis, also known as heartworm, is one of the most important parasitic nematodes of domestic dogs, causing a potentially serious disease, cardiopulmonary dirofilariosis, which can be lethal. This species seems to be less 'expansive' than its sister species Dirofilaria repens, and it is believed that climate change facilitates the spread of this parasite to new non-endemic regions. METHODS: In total, 122 heartworm isolates were analysed from nine endemic countries in Europe (Portugal, Spain, Italy, Greece, Hungary, Romania, Slovakia, and Ukraine) and a single isolate from Bangladesh by amplification and sequencing of two mitochondrial (mt) DNA markers: cytochrome c oxidase subunit 1 (COI) and dehydrogenase subunit 1 (NADH). The main aim of the current study was to determine the genetic diversity of D. immitis and compare it with D. repens haplotype diversity and distribution. DNA was extracted from adult heartworms or microfilariae in blood. Most isolates originated from dogs (Canis lupus familiaris) while 10 isolates originated from wildlife species from Romania, including eight isolates from golden jackals (Canis aureus), one isolate from a Eurasian otter (Lutra lutra) and one isolate from a red fox (Vulpes vulpes). RESULTS: Median spanning network analysis was based on the combined sequence (1721 bp) obtained from two mt markers and successfully delineated nine haplotypes (Di1-Di9). Haplotype Di1 was the dominant haplotype encompassing 91 out of the 122 sequences (75%) from all nine countries and four host species. Haplotype Di2 was the second most common haplotype, formed solely by 13 isolates from Italy. The remaining sequences were assigned to Di3-Di9 haplotypes, differing by 1-4 SNPs from the dominant Di1 haplotype. There was evidence for geographical segregation of haplotypes, with three unique haplotypes associated with Italy and four others associated with certain countries (Di4 and Di7 with Slovakia; Di8 with Greece; Di6 with Hungary). CONCLUSION: Diversity in D. immitis mt haplotypes was lower by half than in D. repens (9 vs. 18 haplotypes in D. immitis and D. repens, respectively), which may be associated with the slower expansion of heartworm in Central and NE Europe. NADH gene appears to be conserved in Dirofilaria sp. by showing lower genetic diversity than the analysed COI gene.

Dirofilaria repens is an expanding vector-borne zoonotic parasite of canines and other carnivores. Sub-clinically infected dogs constitute the most important reservoir of the parasite and the source of infection for its mosquito vectors. However, occurrence of D. repens infection in wild animals may contribute to the transmission of the parasite to humans and may explain the endemicity of filariae in newly invaded regions. The aim of the current study was to determine the occurrence of D. repens in 511 blood and spleen samples from seven species of wild carnivores (wolves, red foxes, Eurasian badgers, raccoons, raccoon dogs, stone martens, and pine martens) from different regions of Poland by means of a PCR protocol targeting the 12S rDNA gene. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodeships in four of the seven regions of Poland: Masovia, Lesser Poland, Pomerania and Warmia-Masuria. The highest prevalence was found in Masovia region (8%), coinciding with the highest previously recorded prevalence in dogs in Central Poland. The DNA of Dirofilaria was detected in 16 samples of three species (total prevalence 3.13%). A low and similar percentage of positive samples (1.9%, 4.2% and 4.8%) was recorded among badgers, red foxes, and wolves, respectively. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodships. Based on detection in different voivodeships, D. repens-positive animals were recorded in four out of the seven regions of Poland: in Masovia, Lesser Poland, Pomerania, and Warmia-Masuria. The highest prevalence of filariae was found in Masovia region (8%), reflecting the highest previously recorded prevalence in dogs (12-50%) in Central Poland. In summary, we conducted the first comprehensive study on the epidemiology of D. repens in seven species of wild hosts in all seven regions of Poland and identified the first case of D. repens infection in Eurasian badgers in Poland and the second in Europe.

The family Trombiculidae encompasses numerous and widespread mites, the larvae of which are obligatory parasites of vertebrates. Our research objective was to assess how trombiculid burdens on rodents inhabiting three forested and one open area in NE Poland vary in relation to host identity, sex and body size, and how they are influenced by qualities of the habitat. Trapped rodents (n = 240) were measured, weighted and screened for ectoparasites. Apodemus flavicollis (n = 42) and Myodes glareolus (n = 173) harbored a total of 4652 Hirsutiella zachchvatkini larvae. Statistical tests revealed that both prevalence and abundance were significantly higher in voles (93%, 27 larvae/ind.) than in mice (14%, 0.24 larvae/ind.) but there was no effect of host sex. Among the localities, H. zachvatkini was most prevalent (98%) and abundant (41 larvae/ind.) in rodents from the deciduous mixed forest in Tałty compared to fallow land (2.6%, 0.13 larvae/ind.). The highest prevalence and abundance were found in bank voles; in this host species in Tałty, prevalence reached 100% and the mean abundance was >42 larvae/ind. Significant positive correlations were found between the body mass and body length of voles and the number of attached larvae. Our results confirm the sylvan nature of H. zachvatkini and its preference for bank voles inhabiting deciduous forests with rich and humid undergrowth. The lack of a sex bias and the moderate correlation between mite burden and host size are in line with observations already made on other rodent-associated Trombiculidae.

Ticks are important vectors of numerous pathogens of medical and veterinary significance. The aim of the current study was to determine the prevalence of Babesia spp. and Borrelia burgdorferi s.l. in sled and pet dogs from Central and North-Eastern Europe. Neither Babesia spp. nor Borrelia burgdorferi s.l. infections were detected in sled dogs from seven countries (Poland, Lithuania, Latvia, Estonia, Belarus, Russia and Finland). The DNA of Babesia spp. was detected in 100% of symptomatic and 5.4% of asymptomatic pet dogs from Poland. Similarly, the DNA of Babesia spp. was identified in 82% of symptomatic and 3.8% of asymptomatic pet dogs from Ukraine. The DNA of Borrelia burgdorferi s.l. was detected in 4.4% of pet dogs. Molecular typing confirmed the presence of Babesia canis and Borrelia burgdorferi sensu stricto (s.s.) in selected samples. Four dogs were co-infected by B. canis and Borrelia burgdorferi s.l. Tick-borne pathogens constitute a serious health threat to pet dogs in Central and South-Eastern Europe, but were not observed among sled dogs from the same region of Europe nor in the Baltic countries.

Introduction and objective. Reports on tick-borne infections in free-living juvenile animals and their impact on survival of cervids in nature are lacking. The aim of the study was to detect and identify the Babesia and Anaplasma phagocytophilum species/ecotypes that may have contributed to the death of juvenile animals from a wildlife rescue centre in spring 2020. Materials and method. PCR amplification and sequencing of two genetic markers (18S rDNA and cox1 for Babesia, 16S rDNA and groEL for A. phagocytophilum) were used for screening eleven samples derived from juvenile animals which died in a rescue centre (seven roe deer Capreolus capreolus, one elk Alces alces, one red squirrel Sciurus vulgaris, one European beaver Castor fiber, one red fox Vulpes vulpes). Phylogenetic analysis of full-length 18S rDNA sequence was performed to enable differentiation between two closely-related species infecting wild ungulates, Babesia capreoli and Babesia divergens (zoonotic).

In recent years, a new focus of the relict tick Haemaphysalis concinna was discovered in Western Poland, near Wolsztyn, Greater Poland voivodeship. This species may play an important role in the circulation of pathogens of medical and veterinary importance. In the present study we tested 880 juvenile ticks collected from rodents, including 427 H. concinna, 443 Ixodes ricinus and 10 Dermacentor reticulatus for three of the most common pathogens vectored by ticks in Poland: Rickettsia and Babesia spp. and Borrelia burgdorferi s.l. Additionally, molecular techniques were applied for accurate identification of tick host species (the voles Microtus and Alexandromys). Our study found differences in the range and prevalence of vectored pathogens between the three tick species. DNA of all three pathogens was found in I. ricinus. In juvenile H. concinna, DNA of Babesia microti, Borrelia afzelii and Rickettsia sp. was identified. Moreover, DNA of a new unnamed Babesia species related to B. crassa, was found in two H. concinna nymphs. This genotype of Babesia was previously identified in H. concinna in the Far East and then in Central Europe. DNA of Rickettsia raoulti and B. afzelii was detected in D. reticulatus nymphs. Among rodent hosts, Alexandromys oeconomus seems to be host of the highest significance for juvenile tick stages and was the only host species with B. afzelii detected in blood samples. Using phylogenetic methods, we confirmed a clear division between rodents from the genera Microtus and Alexandromys. Moreover, we found that A. oeconomus trapped in Western Poland clustered with a Central European A. oeconomus allopatric phylogroup.

Dirofilariasis is a fast-spreading disease of dogs and humans in Europe. We investigated whether Dirofilaria spp. have spread northwards in Europe, invading the Baltic countries. Altogether, 424 blood samples were collected from eight countries in the period 2017-2019, including 227 samples from sled dogs and 197 samples from other dogs. PCR amplification and sequencing were conducted employing three genetic markers (mitochondrial [mt] 12S rDNA, mt cytochrome c oxidase subunit I [COI] gene and mt dehydrogenase subunit I [NAD1] gene). The SNAP test (IDEXX) for detection of D. immitis infections was also implemented. The DNA of D. repens was detected in 59 of 424 dogs (prevalence 13.9%). D. repens was found in sled dogs from Lithuania, Latvia, Poland and Belarus. Only one dog from Estonia was infected, apparently an imported case. The highest prevalence was recorded in Lithuania (38%). Among pet dogs from the Ukraine, six dogs tested positive (3.8%). Our study has revealed a high prevalence of D. repens infections in Lithuania and Latvia, but no evidence for spread of the heartworm D. immitis. We conclude that sled dog kennels constitute hot spots for D. repens transmission.

BACKGROUND: Rodents constitute an important part of the diet of many carnivore species. This predator-prey food chain is exploited by helminth parasites, such as cestodes, whose larval stages develop in rodents and then mature to the adult stage in predators. The main aim of our study was to use molecular techniques for identification of cestode species recovered from both intermediate and definitive hosts, with a particular focus on the genus Mesocestoides. METHODS: Larval cestodes were obtained during our long-term studies on rodent helminth communities in the Mazury Lake District in the north-east Poland in 2000-2018. Cestode larvae/cysts were collected from body cavities or internal organs (e.g. liver) during autopsies. Adult tapeworms were derived from nine red foxes, three Eurasian badgers and one Eurasian lynx. PCR amplification, sequencing and phylogenetic analyses were conducted employing three genetic markers: 18S rDNA, mitochondrial (mt) 12S rDNA and the mt cytochrome c oxydase subunit 1 (cox1) gene fragment. RESULTS: Altogether 19 Mesocestoides samples were analyzed, including 13 adult tapeworms from definitive hosts and six larval samples from 4 bank voles and 2 yellow-necked mice. Phylogenetic analyses revealed three well-supported trees of similar topology. In each case the Mesocestoides samples formed two separate clades. All isolates from foxes, the lynx isolate and two isolates from rodents grouped with Mesocestoides litteratus. Four isolates from rodents and all three isolates from Eurasian badgers were resolved in a separate clade, most similar to North American M. vogae (syn. M. corti). Examination of fixed, stained adult specimens from Eurasian badgers revealed consistency with the morphology of Mesocestoides melesi. Therefore, this clade is likely to represent M. melesi, a species first described in 1985 from the Eurasian badger Meles meles. Molecular analysis allowed also the identification of Taenia crassiceps, Hydatigera kamiyai and Cladotaenia globifera among larvae derived from rodents. CONCLUSIONS: Molecular and phylogenetic analyses support the recognition of M. melesi as a valid species. Our data represent the first record of the larvae of this species in rodents. This is the first report on the occurrence of H. kamiyai in rodents from Poland.