Apicomplexan haemoparasites in domestic cats in Romania.

BACKGROUND: Apicomplexan haemoparasites are protozoans that infect a variety of domestic and wild animal species, as well as humans. Data regarding haemoprotozoans in domestic cats are limited; therefore, the aim of this study was to assess the occurrence of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. in domestic cats in Romania using molecular tools. METHODS: Blood samples from 371 domestic cats were screened for the presence of piroplasmids. All samples that yielded a visible band in agarose gels were subsequently tested by specific assays targeting the 18S rDNA of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. Moreover, nested PCR assays targeting mitochondrial genes of Babesia spp. were used for screening of all Babesia spp. 18S rDNA-positive samples. RESULTS: From the total number of sampled cats, 19.4% were positive in the PCR assay targeting piroplasmids. Babesia spp. were identified in 15.1% of cats, while 0.5% were positive for Hepatozoon spp. Molecular analyses confirmed the presence of Babesia canis. No samples were positive for Cytauxzoon spp. CONCLUSIONS: The high infection rates of domestic cats with Babesia spp. and the need for species differentiation highlight the importance of mitochondrial genes as targets for molecular protocols.

New geographical records for tick-borne pathogens in ticks collected from cattle in Benin and Togo.

BACKGROUND: Ticks are obligate hematophagous arthropods capable of transmitting a great variety of endemic and emerging pathogens causing diseases in animals and humans. OBJECTIVES: The aim of this study was to investigate the presence of Bartonella spp., Rickettsia spp., Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in ticks collected from cattle in Benin and Togo. METHODS: Overall, 396 (148 males, 205 females and 43 nymphs) ticks were collected from cattle in 17 districts (Benin and Togo) between 2019 and 2020. Ticks were pooled into groups of 2-6 ticks per pool according to individual host, location, species and developmental stage. The DNA of each pool was extracted for molecular screening. RESULTS: PCR results revealed that 20 tick pools were positive for Bartonella spp. (Benin and Togo) and 23 tick pools positive for Rickettsia spp. (Benin), while all pools were negative for A. phagocytophilum and B. burgdorferi s.l. Sequence analysis of positive Rickettsia samples revealed the presence of Rickettsia aeschlimannii. CONCLUSIONS: The present study highlights the presence of zoonotic agents in ticks collected from cattle in Benin and Togo. This information will raise awareness of tick-borne diseases among physicians and veterinarians, stimulate further studies to monitor these pathogens, and advise on necessary measures to control the spread of these zoonoses.

Efficacy of a topical formulation containing esafoxolaner, eprinomectin and praziquantel (NexGard Combo®) against natural infestations with the cat louse, Felicola subrostratus under field conditions.

Felicola subrostratus is the only species of louse affecting domestic cats. Although it is considered a rare ectoparasite of pet cats, it occurs mainly in stray or shelter animals and is sometimes associated with intense pruritus and secondary bacterial infections. The aim of the present study was to evaluate the efficacy of a single dose of the topical formulation of esafoxolaner, eprinomectin and praziquantel (NexGard Combo®, Boehringer Ingelheim) in cats for the treatment of naturally acquired chewing lice infestation in a multi-site, positive-control, blinded clinical field study. Thirty-one domestic cats presenting natural F. subrostratus infestation were included in the study. The animals had not been treated with any ectoparasiticide within the previous 60 days. After inclusion, each cat was randomly assigned to one of the two groups: group 1, 14 cats treated with NexGard Combo® or group 2, 17 cats treated with Frontline Combo®. A clinical evaluation was performed at days 0 (inclusion), 14 and 30 and consisted in scoring the skin lesions and symptoms and scoring the presence of lice. On day 30, all cats from both groups scored 0 for the presence of live lice and no dead lice were found, demonstrating 100% efficacy. The clinical scores significantly improved from day 0 to day 30 in both groups. During the 30 days of surveillance, no reinfestations due to the hatching of eggs were observed and none of the cats had any adverse reactions. Esafoxolaner demonstrated high efficacy for the treatment of F. subrostratus infestation.

Title: Efficacité d'une formulation topique contenant de l'esafoxolaner, de l'éprinomectine et du praziquantel (NexGard Combo®) contre les infestations naturelles par le pou du chat, Felicola subrostratus, dans des conditions de terrain. Abstract: Felicola subrostratus est la seule espèce de pou affectant les chats domestiques. Bien que ce pou soit rare comme ectoparasite chez les chats de compagnie, il infeste principalement les animaux errants ou en refuge et est parfois associé à un prurit intense et à des infections bactériennes secondaires. Le but de la présente étude était d'évaluer l'efficacité d'une dose unique de la formulation topique d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard Combo®, Boehringer Ingelheim) chez le chat pour le traitement de l'infestation naturelle par les poux broyeurs dans une étude de terrain multi-sites, randomisée, avec un contrôle positif. Trente et un chats domestiques présentant une infestation naturelle par F. subrostratus ont été inclus dans l'étude. Les animaux n'avaient été traités avec aucun ectoparasiticide au cours des 60 jours précédents. Après inclusion, chaque chat a été assigné au hasard à l'un des deux groupes : groupe 1, 14 chats traités avec NexGard Combo®, ou groupe 2, 17 chats traités avec Frontline Combo®. Une évaluation clinique a été réalisée aux jours 0 (inclusion), 14 et 30 et a consisté à un score clinique des lésions et symptômes cutanés et à noter la présence de poux. Au jour 30, tous les chats des deux groupes ont obtenu un score de 0 pour la présence de poux vivants et aucun pou mort n'a été trouvé, démontrant une efficacité de 100 %. Les scores cliniques se sont significativement améliorés du jour 0 au jour 30 dans les deux groupes. Au cours des 30 jours de surveillance, aucune réinfestation due à l'éclosion des œufs n'a été observée et aucun des chats n'a présenté d'effet indésirable. L'esafoxolaner a démontré une efficacité élevée pour le traitement de l'infestation par F. subrostratus.

Efficacy of afoxolaner (NexGard®) against natural infestations with Trichodectes canis in dogs under field conditions.

BACKGROUND: Trichodectes canis is a small chewing louse found globally that primarily infests dogs. Limited information is available on the efficacy of isoxazolines against infestation with the chewing louse. In the present study, we evaluated the efficacy of afoxolaner, an isoxazoline class compound, in naturally infested domestic dogs. METHODS: The field study was carried out in Romania. Between September 2021 and December 2021, 43 dogs with confirmed T. canis infestation were included in the study. On the day of the inclusion (day 0), each animal was clinically examined and randomly treated with a control product labeled for use against lice [fipronil-(S)-methoprene combination (Frontline Combo®; Boehringer Ingelheim)] or with the investigational product [chewable tablets containing afoxolaner (NexGard®; isoxazoline)]. Each animal was evaluated for the presence of lice at 15 and 30 days post-inclusion. RESULTS: Of the 48 dogs initially included in the study, 43 completed the treatment period [18 in the control group (CG) and 25 in the investigational group (IG)]. At day 14, no living T. canis lice were detected on the dogs in either group. At day 14, dead lice were detected in four dogs in the IG, while eggs were present in two dogs in the IG and in one dog in the CG. At day 30, no lice were detected in either group, while eggs were still present in one dog in the CG. CONCLUSION: These results suggest that afoxolaner is a feasible treatment option against chewing lice in dogs, providing 100% curative efficacy.

Spotted Fever Group Rickettsia spp. Diversity in Ticks and the First Report of Rickettsia hoogstraalii in Romania.

Tickborne bacterial pathogens have been described worldwide as risk factors for both animal and human health. Spotted fevers caused by Rickettsiae may cause non-specific symptoms, which make clinical diagnosis difficult. The aim of the current study was to evaluate and review the diversity of SFG Rickettsiae in ticks collected in 41 counties in Romania. A total of 2028 questing and engorged ticks collected in Romania belonging to five species were tested by PCR amplification of Rickettsia spp. gltA and 17-D gene fragments: Ixodes ricinus (n = 1128), Dermacentor marginatus (n = 507), D. reticulatus (n = 165), Rhipicephalus rossicus (n = 128) and Haemaphysalis punctata (n = 100). Five Rickettsia species were identified following DNA sequence analysis: R. helvetica, R. monacensis, R. slovaca, R. raoultii, and R. hoogstraalii. The most common species detected was R. monacensis. Moreover, R. hoogstraalii was detected for the first time in Romania and in R. rossicus ticks. The detection of R. raoultii and R. monacensis in questing larvae of Hae. punctata suggests the possible transovarial transmission of these Rickettsia species in ticks. The detection of R. hoogstraalii for the first time in Romania increases the reported SFG Rickettsia diversity in the country.

What Is the Real Influence of Climatic and Environmental Factors in the Outbreaks of African Swine Fever?

African swine fever has a significant economic, social and environmental impact due to official regulation of the disease, namely the mass depopulation of all pigs in confirmed outbreaks. The main objective of the present study was to statistically analyze the possible correlation between the number of outbreaks and infected pigs from backyard farms with the altitude, seasonal average annual humidity, and average temperature during spring, summer, autumn and winter, as well as the distance from forests, rivers, and lakes in Romania. The study included all infected backyard pigs with African swine fever virus (n = 7764) and outbreaks (n = 404) that occurred in Romania between 6 February 2020 and 2 March 2021. The number of infected pigs and ASF outbreaks were significantly higher in localities at altitudes between 100 and 500 m, average annual humidity between 60% and 80%, average spring temperature between 10 and 14 °C, average summer temperature between 16 and 22 °C, average autumn temperature between 10 and 13 °C, average winter temperatures between -1 and 2 °C, and at distances of less than 5 km from the forests, less than 15 km from lakes and less than 5 km from the rivers. The number of affected pigs decreased significantly at summer temperatures below 16 °C.

Genotyping of African Swine Fever Virus (ASFV) Isolates in Romania with the First Report of Genotype II in Symptomatic Pigs.

The World Organisation for Animal Health has listed African swine fever as the most important deadly disease in domestic swine around the world. The virus was recently brought from South-East Africa to Georgia in 2007, and it has since expanded to Russia, Eastern Europe, China, and Southeast Asia, having a devastating impact on the global swine industry and economy. In this study, we report for the first time the molecular characterization of nine African swine fever virus (ASFV) isolates obtained from domestic pigs in Mures County, Romania. All nine Romanian samples clustered within p72 genotype II and showed 100% identity with all compared isolates from Georgia, Armenia, Russia, Azerbaijan, Ukraine, Belarus, Lithuania, and Poland. This is the first report of ASFV genotype II in the country.

Seasonal dynamics of Phlebotomus neglectus (Diptera: Psychodidae) in cave microhabitats in Romania and the rediscovery of Sergentomyia minuta (Rondani, 1843) after 50 years.

BACKGROUND: In a countrywide study aiming to update the knowledge on diversity of sand fly species in Romania, a sand fly population was observed in an isolated system of cave microhabitats. The caves are located in the protected area of Canaraua Fetii, Dobrogea region, southeastern Romania. The highest sand fly diversity was recorded in this area between 1968 and 1970. This work presents a study conducted to estimate the seasonal variation of the sand fly species in correlation with the particular environmental factors of the isolated system of cave microhabitats. METHODS: Sand flies were collected between May and October 2020 from one trapping site of interest in Canaraua Fetii. The trapping site consisted of a cave entrance. CDC miniature light traps and sticky traps were used to collect insects from the exterior walls of the cave entrance. Species identification of collected sand flies was done using morphological keys. Statistical analysis of the trapping and climatic data was performed. RESULTS: From all collected sand flies, 99.7% (818/822) were Phlebotomus neglectus, 0.1% (1/822) Ph. balcanicus and 0.2% (2/822) Sergentomyia minuta. Sand fly activity was first observed on 2 July and last on 24 September. A monomodal abundance trend was present, with the peak activity between 16 and 17 July. The analysis of the climatic data showed correlations between the total number of captured sand flies and both average temperature and average relative humidity. The total number of collected specimens was statistically higher when CDC miniature light traps were used compared to sticky traps. The number of females on the sticky traps was significantly higher than the number of males on the same trap type. Compared with the sticky traps, significantly more males were collected by CDC miniature light traps. This is the first record of Se. minuta in Romania after 50 years of no records (despite the trapping effort of the last 5 years in the country). Also, Ph. sergenti, previously present in this location, was not found. CONCLUSIONS: In the investigated natural habitat, the diversity of the sand fly species appears to have changed, with the predominance of Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the two predominant species in 1968-1970). A monomodal abundance trend was observed as in other regions of the country. The sand fly activity in this particular cave microhabitat appears to be longer than in other regions in Romania. Longer sand fly activity increases the zoonotic risk of various pathogenic species' transmission, with an impact on public health, as sand flies are important insect vectors.

Babesia pisicii n. sp. and Babesia canis Infect European Wild Cats, Felis silvestris, in Romania.

Haemoparasites of the genus Babesia infect a wide range of domestic and wild animals. Feline babesiosis is considered endemic in South Africa, while data on Babesia spp. infection in felids in Europe is scarce. Using samples from 51 wild felids, 44 Felis silvestris and 7 Lynx lynx, the study aimed to determine the presence and genetic diversity of Babesia spp. in wild felids in Romania by analyzing the 18S rDNA and two mitochondrial markers, cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) genes. By 18S rDNA analyses, Babesia spp. DNA was detected in 20 European wild felids. All sequences showed 100% similarity to B. canis by BLAST analysis. Conversely, Cytb and COI analyses revealed the presence of two Babesia spp., B. pisicii n. sp., which we herein describe, and B. canis. The pairwise comparison of both mitochondrial genes of B. pisicii n. sp. showed a genetic distance of at least 10.3% from the most closely related species, B. rossi. Phylogenetic analyses of Cytb and COI genes revealed that B. pisicii n. sp. is related to the so-called "large" canid-associated Babesia species forming a separate subclade in a sister position to B. rossi.

Three new species of Cytauxzoon in European wild felids.

Protists of the genus Cytauxzoon infect a wide variety of wild and domestic felids worldwide. While the American Cytauxzoon felis has been well described, data on the European isolates of Cytauxzoon are still scant. The aim of the current study was to determine the genetic diversity of European Cytauxzoon spp. in wild felids across Europe by analyzing one nuclear and two mitochondrial genes, along with representative complete mitochondrial genomes. Overall, 106 biological samples from wild felids (92 from Felis silvestris and 14 from Lynx lynx) from Germany, Romania, Czech Republic, and Luxembourg were collected and screened for the presence of Cytauxzoon spp. using nested PCR protocols, targeting the highly conserved 18S rDNA, mitochondrial cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) genes. Furthermore, 18 previously confirmed wild felid biological samples from Europe, and comparative material from USA positive for C. felis, were included in the study. In 18S rDNA sequences analyses, Cytauxzoon spp. from felids formed two separate clades of New World and Old World isolates, with a low inner diversity of the European clade. In contrast to 18S rDNA, the phylogenetic analyses of CytB and COI genes affirmatively revealed three highly supported clades, resulting in three defined genotypes. Similar intra- and interspecific variability of CytB and COI genes was observed in the case of different Babesia spp. Considering geography, host species and analyses of three genes, we conclude that the three detected genotypes of Cytauxzoon in European wild felids represent three new species, which we herein describe.

First report of Cytauxzoon sp. infection in Germany: organism description and molecular confirmation in a domestic cat.

Cytauxzoonosis is described as an emerging tick-borne disease of domestic and wild felids caused by protozoans of the genus Cytauxzoon. While in the Americas the condition is described as a fatal disease, in Europe, reports on the clinical expression of the infection are scarce. This study describes the first case of Cytauxzoon sp. infection in Germany, in a domestic cat. A 6-year-old male domestic cat living in Saarlouis (Saarland) was presented with anorexia, lethargy and weight loss. The cat had an outdoor lifestyle and had not travelled abroad. Serum clinical chemistry analysis revealed azotaemia with markedly increased symmetric dimethylarginine, hypercreatinemia, hyperphosphatemia and hypoalbuminemia. Moreover, a mild non-regenerative anaemia was present. Approximately 1 year prior to these findings, the domestic cat was diagnosed with a feline immunodeficiency virus (FIV) infection. These results pointed toward a decreased glomerular filtration rate, presumably as a result of kidney dysfunction. Round to oval signet ring-shaped intraerythrocytic organisms, morphologically suggestive for a piroplasm, were revealed during blood smear evaluation with a degree of parasitaemia of 33.0%. PCR analyses and sequencing of a region of the 18S rRNA gene confirmed the presence of a Cytauxzoon sp. infection, with 99-100% nucleotide sequence identity with previously published Cytauxzoon sp. isolates. As this is the first molecularly confirmed Cytauxzoon sp. infection in a domestic cat in Germany, these findings suggest that cytauxzoonosis should be considered as a differential diagnosis in cases of anaemia in outdoor domestic cats, particularly in areas where wild felid populations are present.