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Abstract Background Cats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries. Methods From 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities. Results Overall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis. Conclusions This study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin. Graphical Abstract

Abstract Background Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. Methods Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer’s instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. Results The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test’s sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. Conclusions The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection. Graphical Abstract

Abstract Insect-based diets are gaining interest as potential ingredients in improving poultry gut health. This study assessed the dietary treatment with whole dried Tenebrio molitor larvae (TM) on broiler chickens’ gut microbiota and morphology. 120 Ross-308 broilers received treated diets with 5% (TM5) and 10% (TM10) replacement ratio in a 35-day trial. Intestinal histomorphometry was assessed, as well as claudin-3 expression pattern and ileal and caecal digesta for microbial community diversity. Null hypothesis was tested with two-way ANOVA considering the intestinal segment and diet as main factors. The TM5 group presented higher villi in the duodenum and ileum compared to the other two (P

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a zoonotic disease that affects various animal species, including dogs, that can serve as sentinels for indirectly estimating the environmental contamination. The current study aimed to determine the seroprevalence of T. gondii IgG and IgM antibodies in dogs across different regions of Greece and assess their living area as a potential risk factor. In total, 1282 blood samples were collected from dogs in urban and rural areas of Greece, including Attica and Thessaloniki. Serum samples were tested for T. gondii-specific IgG and IgM antibodies using an indirect immunofluorescent antibody test. A chi-square test was performed to assess the association between seropositivity for T. gondii and geographical location (urban/rural). The overall T. gondii seroprevalence was 47.6%, while the seroprevalence of IgG and IgM antibodies was 34.3% and 22.2%, respectively. Dogs from rural areas exhibited a significantly higher seroprevalence (53.8%) than those from urban areas (43.9%) (p < 0.001), with the estimated odds ratio being equal to 1.49 (95% CI, 1.18 to 1.65) and the relative risk increased by 22.4%. Dogs in Greece are highly exposed to T. gondii, particularly in rural areas. Measures to prevent canine infections are necessary, and basic hygiene practices, such as hand washing after petting dogs, are required to reduce human infection risk and safeguard public health.

While research on the parasitic fauna of wildlife in Greece is currently limited, conducted studies have provided valuable insights into the prevalence of parasitic infections in wild carnivores, omnivores, and herbivores. This review consolidates the existing data on the endoparasites detected in wild animals in Greece, specifically focusing on those that pose established or potential zoonotic risks. Over the last 60 years, various parasite species such as Leishmania infantum, Cryptosporidium spp., Toxoplasma gondii, Sarcocystis spp., Toxocara canis, Ancylostoma caninum, Capillaria spp., Baylisascaris spp., Trichinella spp., Thelazia callipaeda, Dirofilaria immitis, Echinococcus granulosus, Mesocestoides sp., Taenia spp., Alaria alata, and Dicrocoelium dendriticum have been identified in wildlife in Greece. These findings have become increasingly relevant due to the growing interaction between humans and wild animals, which now extends to urban environments, as well as the increased contact between wild and domestic animals. This is further complicated by the geographical expansion of vector-borne diseases due to global warming and the increased movements of humans and animals. Surveillance and monitoring of parasitic infections in Greek wildlife is warranted, and it should be based on interdisciplinary investigations considering the interconnectedness of human, wild, and domestic animals, as well as environmental health, in line with the One Health approach.

Digital PCR (dPCR) is an emerging technology that enables the absolute quantification of the targeted nucleic acids. The body of research on the potential applications of this novel tool is growing in human and veterinary medicine. Most of the research on dPCR applications in veterinary parasitology is concentrated on developing and validating new assays to detect and quantify parasites of great financial impact in the food-producing animal industry. Several studies describe the utility of dPCR for individualized medicine in companion animals. Most frequently, dPCR performance is assessed compared to quantitative PCR or Next Generation Sequencing platforms, while others also compare the accuracy of dPCR with traditional parasitological techniques considered gold standard methods. Other researchers describe dPCR assays for surveillance purposes, species identification, and quantification in mixed parasitic infections, the detection of mutations indicative of anthelmintic resistance, and the identification of new targets for drug development. This review provides an overview of the studies that employed dPCR in investigating animal parasites and parasitic diseases from a veterinary perspective and discusses how this novel technology could advance and facilitate diagnosis, surveillance, and the monitoring of response to treatment, or shed light on current gaps in our knowledge of the epidemiology of significant veterinary parasitic diseases.

Abstract 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. Graphical Abstract

Although Dirofilaria immitis in dogs is considered enzootic in northern Greece, the available data on the occurrence of infection in southern parts of the country demonstrate its scarcity. The aim of this study was to update the current knowledge on D. immitis infection in dogs in areas of Greece previously considered non-enzootic (Central Greece, Attica, Peloponnese, North Aegean, South Aegean, Crete and the Ionian islands). In total, 1528 dog blood samples were collected from the aforementioned areas and examined by Dirochek® ELISA (Synbiotics). Additionally, data published until 2022 on the prevalence of infection in these areas were compared to the data of this study. The ‘Wilson’ Score interval method (Epitools) was employed. Overall, 10.8% of dogs were positive for D. immitis. In detail, the prevalence was 21.7, 13.7, 10.7, 5.4, 4.7, 6.2 and 17.0% for D. immitis, in Central Greece, Attica, Peloponnese, North Aegean, South Aegean, Crete and the Ionian islands, respectively. Infection with D. immitis is recorded for the first time in Crete. The probability of a dog becoming infected has increased 4.1 times since 2022 in previously non-enzootic areas. This study denotes the spread of D. immitis and highlights the necessity for preventive measures.

Dermanyssus gallinae, the poultry red mite (PRM), is the most prevalent and harmful ectoparasite of laying hens globally. Although prevalence and risk factor studies can help veterinarians make decisions regarding farm treatments, relevant data are scarce. The present study investigated the prevalence and infestation severity of PRM in poultry farms across Greece and examined potential risk factors. AviVet traps were used to sample 84 farms (51 backyard, 33 industrial) over three years. Farm altitude, temperature, humidity, region, and production systems were assessed as potential risk factors with chi-square tests, initially for all the studied farms and then exclusively for backyard farms. The overall prevalence was 75.0% and was higher in backyard farms (80.4%) compared with industrial ones (66.7%), varying regionally from 66.7 to 90.9%. Altitude and temperature were not significant risk factors, but farms with humidity

The role of insects in animal nutrition has been reconsidered during recent years, paving the way for an increasing market for edible insects. Their protein and amino acid balance make them a promising source of protein for replacing high value proteins. Yellow mealworm, Tenebrio molitor L. (TM; Coleoptera: Tenebrionidae) larvae, have shown positive effects on broiler performance in several research studies and have a strong potential as a sustainable alternative protein source for monogastric animals. This study aimed to assess the effect of replacing various ratios of basal diets with T. molitor larvae on broiler performance as well as on several meat and welfare characteristics. For the study, 120 one-day-old male chicks (Ross 308) were randomly allocated in 3 treatments and 4 replications (10 birds per pen). Birds of the control group (basal diet) were fed with typical commercial maize and soymeal-based rations in mash form. The other 2 groups were treated with the same diet, after replacing 5% and 10% with dried TM larvae, respectively. On d 35 (end of trial), meat samples were collected and analysed. Body weight, feed intake, body weight gain and feed conversion ratio during the periods of 1 to 10 d (starter period), 11 to 24 d (grower period), 25 to 35 d (finisher period) and 1 to 35 d (total period) were assessed. Pododermatitis, diarrhoea, feather score and litter conditions were also assessed during the trial. The results indicated that TM larvae inclusion in the broilers' diet positively affected body weight gain values, as well as the carcass yield, the meat composition and various welfare traits. Additionally, the dietary treatments with TM larvae favourably affected meat composition and colour parameters, whereas there were also some positive effects on lipid and protein oxidation. Saturated fatty acids were decreased by the dietary supplementation whereas the polyunsaturated fatty acids to SFA ratio increased. In general, the study showed that whole TM larvae addition can provide a promising alternative to soybean meal in the diet of broilers, demonstrating a positive impact on growth, welfare and meat characteristics.

Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), affecting human health in Europe and Asia. Reports on canine clinical cases of TBE are rare, although dogs are used as sentinels for assessing human health risks. The first canine clinical TBE case in Greece is reported in this case report. The dog had a history of tick infestation and displayed neurological symptoms, particularly tetraparesis, neck hyperalgesia, and a sudden behavior change. Serum samples were obtained and examined in a commercial ELISA to detect anti-TBEV specific IgG and IgM antibodies. The dog tested seropositive for both IgG and IgM, and based on its history and compatible clinical signs, the diagnosis of TBE infection was reached. The prognosis was initially poor, and treatment included the administration of fluids, corticosteroids, and antibiotics, followed by physical therapy. After a 10-day hospitalization, the dog had a much better prognosis. This case highlights that TBEV does emerge in new locations, increasing human and animal infection risk. Veterinarians should include TBE in their differential diagnosis of canine patients with a history of tick infestations, progressive neurological symptoms, and abnormal behavior.

Abstract The poultry red mite (PRM), Dermanyssus gallinae, is arguably the most harmful, ubiquitous haematophagous ectoparasite infesting egg-laying hens. PRM is a vector of various microorganisms, with some being important for food microbiology and public health. The present study aimed to investigate the presence of specific pathogens, including Escherichia coli, Salmonella spp. and Listeria spp., carried by PRM infesting a chicken farm in Greece. Mites were caught using cardboard traps (Avivet), and 100 unwashed PRM were homogenized and used for microbiological cultures. Microbiological cultures were carried out on general and selective substrates to detect the above-mentioned bacteria. Specifically for Listeria spp., DNA was extracted from bacteria grown in Tryptone Soya Yeast Extract Agar using a commercial kit. The hly gene encoding the Listeriolysin O protein was amplified by PCR. Mites were identified as D. gallinae using morphological keys as well as by COI DNA barcoding. Microbiological cultures and PCR assays were positive for Listeria monocytogenes. No other bacteria were detected. The current study constitutes the first molecular isolation of L. monocytogenes from D. gallinae, confirming that PRM can carry this food-borne pathogen. PRM control measures and hygiene practices should be applied to minimize any possible contamination risk of poultry products with L. monocytogenes and safeguard public health.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

Tick-borne diseases (TBDs) are caused by pathogens of human and veterinary concern representing a major public health issue worldwide. Although feline medicine has progressed much in the recent decades, data on feline TBDs (FeTBDs) remain scant. Therefore, this study aimed to assess the prevalence of apicomplexan parasite infections, associated risk factors and clinical-hematological abnormalities in domestic feline populations from countries of the Mediterranean basin. Blood and serum samples from cats (n = 600) living in France, Greece, Israel, Italy, Portugal and Spain were collected along with animal data (i.e., age, sex, breed, housing conditions and geographical origin), clinical signs and laboratory blood test parameters. Cats were grouped according to their age as kitten (up to one year), young (between one and six years), mature (between seven and ten years) and senior (older than ten years). Blood samples were tested for Hepatozoon spp. and piroplasmids by conventional PCR targeting 18S rRNA gene. The overall prevalence of Hepatozoon spp. infection was 14.5%, being significantly higher in cats from Greece (30%) and Portugal (23%), followed by Spain (15%), Israel (15%) and France (4%). Cats from Italy scored negative. Hepatozoon felis was identified in 86 animals, with three different sequence types and H. silvestris was detected in one shelter cat from Portugal. No piroplasmid DNA was amplified. The risk of Hepatozoon spp. infection was related to feline geographical provenience, housing condition and age. No statistical correlation was reported with any clinical signs, while increased alanine aminotransferase (ALT) activity was the only laboratory abnormality significantly associated (p = 0.03) with the infection. Data suggest a high circulation of H. felis, and only occasionally of H. silvestris, within domestic feline populations in the Mediterranean basin, mainly in shelter or free roaming and young cats with asymptomatic or subclinical infection.

The Mediterranean monk seal (Monachus monachus) is classified as an endangered species by the IUCN, with a global population that does not exceed 800 individuals. There is limited understanding around the biology and health status of the species, rendering possible parasitic infections grave for its conservation efforts. The aim of the current study was the molecular identification of a parasitic nematode found in the digestive system of a sub-adult Mediterranean monk seal individual, that was found stranded in the area of Pagasitikos Gulf, Greece in 2019. Analysis of the stomach contents revealed the presence of two intact female nematode individuals. Standard protocols were followed as DNA extraction of the parasites was conducted and PCR amplification of the cytochrome oxidase subunit I (COI) mitochondrial gene was implemented. Sequencing analysis of a 585 bp-amplified product displayed a 96% similarity of the screened nematodes to the Pseudoterranova bulbosa species. Bayesian inference was implemented for the subsequent tree reconstruction. The phylogenetic tree revealed a clear genetic similarity between our parasitic nematode individuals named as Pseudoterranova spp. and Pseudoterranova bulbosa (bootstrap value: 82%), which is indicated for the first and only time as such, to be found in the waters of the Mediterranean Sea and also in the stomach of a Mediterranean monk seal.

Toxoplasma gondii is a zoonotic protozoon with a complex life cycle and the second most important foodborne pathogen in Europe. Surveillance of toxoplasmosis is based on national considerations since there are no mandatory controls along the food chain in the European Union, and underreporting of meat is still a problem in many countries like Greece. The current review provides an overview of T. gondii prevalence, associated risk factors, and surveillance in animals in Greece, focusing on the transmission role of meat and highlighting the control measures that should be adopted by consumers. Sows, wild boars, hares, equines, and cats had lower, while sheep and goats generally had higher seroprevalence than their respective pooled European and global values. Seroprevalence in chickens was similar between Greece and Europe, while there was high variation in cattle studies, with no data regarding dairy products. Though a comprehensive meat safety assurance system is the most effective approach to control the principal biological hazards associated with meat, such as T. gondii, the prerequisite risk categorisation of farms and abattoirs based on EFSA’s proposed harmonised epidemiological indicators has not materialised as yet in Greece. Therefore, comprehensive control strategies are still required to ensure food safety and safeguard public health.

Leishmaniosis caused by the protozoon Leishmania infantum that is transmitted through the bites of infected phlebotomine sandflies is of major veterinary concern in Greece. The country is endemic with particularly favourable environmental conditions for the spread of this infection. Moreover, Greece remains a popular touristic destination, and the continuous travel of pets raises concern regarding the possible dissemination of infection from endemic to non-endemic areas. Dogs are the main reservoir host, although other animal species, including humans, may also be infected. Canine leishmaniosis manifests as a visceral disease that can result in death if left untreated. Serological and molecular epizootiological studies have confirmed circulation of the parasite in Greek canine and feline populations as well as in other mammals. As a result, constant surveillance and identification of high-risk localities are necessary to establish chemoprophylactic protocols for travelling animals to safeguard animal and public health.

The aims of the present study were: (a) to describe the prevalence of helminth infections from pooled faecal samples from sheep flocks across Greece and (b) to evaluate flock-related factors potentially associated with the presence of these infections in the flocks. An extensive countrywide study was performed on 325 sheep farms throughout Greece; faecal samples were collected from ewes and processed for the identification of helminth parasites. Helminths were detected in samples from 92.9% of flocks; these included Dicrocoelium dendriticum (16.7% of flocks), Fasciola hepatica (0.6%), Paramphistomum cervi (2.2%), Moniezia spp. (18.8%), Trichostrongylidae (85.5%), Nematodirus spp. (18.8%), Strongyloides papillosus (7.1%), Trichuris spp. (20.0%) and lungworms (17.8%). Mean Trichostrongylidae counts across all flocks in the study were 215 epg. Specifically, for Trichostrongylidae, there were differences between flocks that had or had not received anthelmintics during the two months prior to sampling, as well as between flocks located in various areas of the country. In multivariable analyses, for the outcome ‘high (>300) epg counts in faecal samples’, the month into the lactation period at sampling and the application of reproductive control practices on the farm emerged as significant factors; for the outcome ‘high proportion (>63%) of Teladorsagia spp. in faecal samples’, the availability of straw bedding emerged as a significant factor; and for the outcome ‘high proportion (>63%) of Haemonchus contortus in faecal samples’, the age of the farmer emerged as a significant factor. For the outcome ‘presence of Trichuris spp. in faecal samples’, the provision of finished feed (concentrate) to animals emerged as a significant factor, whilst, finally, for the outcomes ‘presence of D. dendriticum in faecal samples’ and ‘presence of lungworms in faecal samples,’ no significant factors emerged.

Dermanyssus gallinae (PRM) is the most common blood-sucking ectoparasite in laying hens and is resistant against numerous acaricides. Silver nanoparticles (AgNPs) represent an innovative solution against PRM. The current study aimed to assess the in vitro acaricidal activity of AgNPs against PRM and describe their potential mechanism of action. Nanoparticles were produced using a wet chemistry approach. Mites were collected using AviVet traps from 18 poultry farms in Greece. Contact toxicity bioassays were carried out for 24 h with negative controls, 20, 40, 60, or 80 ppm AgNPs. Analysis of variance was used to compare the mortality rates of PRM between the control and treatment groups, while LC50, LC90, and LC99 values were estimated using probit regression analysis for the total farms jointly and separately. Nanoparticles displayed strong acaricidal activity, and mortality rates were significantly different between groups and increased by AgNPs concentration. Overall mean LC50, LC90, and LC99 values were 26.5, 58.8, and 112.3 ppm, respectively. Scanning electron microscopy on mites treated with 80 ppm AgNPs revealed cracks in their exoskeleton and limb detachments, presumably resulting from the interaction between AgNPs and the mites’ chitin. Future studies should focus on assessing AgNPs residues in chicken tissues before moving into field trials.

Abstract Background Infections by protozoans of the genus Giardia are a common cause of diarrhea in dogs. Canine giardiosis constitutes a disease with a zoonotic potential; however, it is often underestimated due to its challenging diagnosis. The objective of the study was to assess the diagnostic performance of an immunochromatographic strip test (SpeedTM Giardia, Virbac, France) comparing it with microscopy (zinc sulfate flotation) by utilizing the combination of an enzyme immunoassay (ProSpecTTM Giardia EZ Microplate Assay, Oxoid Ltd., UK) and the PCR as the gold standard. A positive result in both ELISA and PCR was set as the gold standard. Methods Initially, fecal samples from dogs with clinical signs compatible with giardiosis were tested with the SpeedTM Giardia test and separated into two groups of 50 samples each: group A (positive) and group B (negative). Thereafter, all samples were examined by zinc sulfate centrifugal flotation technique and assayed by the ProSpecTTM Giardia Microplate Assay and PCR. The performance of the SpeedTM Giardia and zinc sulfate centrifugal flotation tests were calculated estimating sensitivity, specificity, and positive and negative likelihood ratio; the chi-square and McNemar tests were used for the comparison of the two methods. Results Giardia cysts were not detected by microscopy in 16 out of the 50 samples (32%) of group A and in none of group B samples. Eight out of 50 samples in group B (16%) were tested positive both with the ProSpecTTM Giardia Microplate Assay and PCR. Fecal examination with the SpeedTM Giardia test was more sensitive (86.2%) than the parasitological method (58.6%, P < 0.001) while the specificity of both methods was 100%. Conclusions The SpeedTM Giardia test is an easy-to-perform diagnostic method for the detection of Giardia spp., which can increase laboratory efficiency by reducing time and cost and decrease underdiagnosis of Giardia spp. infections. This immunochromatographic strip test may be routinely exploited when a rapid and reliable diagnosis is required, other diagnostic techniques are unavailable and microscopy expertise is inefficient. In negative dogs with compatible clinical signs of giardiosis, it is recommended either to repeat the exam or proceed with further ELISA and PCR testing.

A female, 5 yr old Bactrian camel was presented to the Exotic and Wildlife Medicine Unit, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, with severe dehydration, depression, anorexia, mild dyspnea and diarrhea. Supportive treatment immediately initiated with fluids, electrolytes and broad-spectrum antibiotics. The general condition of the animal was stable for the next 3 days, but at 4th day became worse, since the camel remained in sternal recumbency, denied to drink water and abortion of a mummified fetus was noticed. The aborted fetus and fetal membranes were submitted for laboratory examinations (bacterial cultures, MZN, cytology, PCR) that revealed Toxoplasma gondii infection. Treatment with sulfadimidine improved the situation of the animal that returned to its farm 1 week later. This seems to be the first reported case in the literature of confirmed toxoplasmic abortion in camels.

Toxoplasma gondii is a ubiquitous zoonotic parasite, with felines being the only definitive hosts. Cats shed oocysts with their faeces, and seroprevalence studies can be used to indirectly assess the environmental contamination. The current study aimed to evaluate T. gondii seroprevalence in Greek cats and identify possible risk factors. In total, 1554 blood samples were analyzed from different cats across all nine geographic regions of Greece, and a short questionnaire was completed for each cat. A rapid immunochromatographic test was used to detect anti-T. gondii antibodies, IgG type, and 21.8% of cats were seropositive. Regarding risk factors, when chi-square tests were applied, seropositivity was significantly higher (p < 0.05) in rural cats, cats with outdoor access, and hunting cats. Gender, age, ownership, and raw feeding were not significant risk factors, although female, adult, stray, and raw-feeding cats had a higher seroprevalence than their counterparts. Binary logistic regression models were developed to adjust for the confounding effects of the initially recognized risk factors, and only hunting in urban areas remained a significant risk factor. Greek cats had lower seropositivity than the average European value, and the present research highlights the importance of updated seroprevalence and risk factor studies within the context of One-Health.

There is a scope to study protozoan infections in adult ewes and does, as these animals can act as reservoirs of infection for lambs and kids, for which these pathogens are harmful. The objectives of this study were to describe the prevalence of protozoan infections in faecal samples from adult sheep and goats on dairy farms across Greece and to evaluate farm-related factors potentially associated with the presence of protozoan infections in these animals. A cross-sectional study was performed on 325 sheep and 119 goat farms throughout Greece; faecal samples were collected from ewes and does and processed for the identification of protozoan parasites. Eimeria oocysts were found in faecal samples from 69% of farms (72% of sheep farms and 61% of goat farms), Giardia cysts in samples from 33% of farms (33% of sheep farms and 34% of goat farms) and Cryptosporidium oocysts in samples from 8% of farms (7% of sheep farms and 11% of goat farms). In a multivariable analysis, for the presence of Eimeria in samples from sheep farms, the lack of a designated building for lambs emerged as a significant factor; for the presence of Giardia in samples from goat farms, the availability of a main building for animals emerged as a significant factor; for the presence of Cryptosporidium, the lack of grazing and the management system emerged as the main significant factors in sheep and goat farms, respectively. Protozoa were found significantly more frequently in samples collected from farms on which farmers considered diarrhoea as an important health problem in their lambs/kids.

The objectives of the study were: (a) to study the effect of Haemonchus spp. on the growth performance characteristics of fattening calves, (b) to assess any potential effects on carcass characteristics and (c) to investigate the potential role of climatic conditions in the process of the infection. The study was conducted for 201 days in an intensively managed cross-Limousin herd. The animals were divided into two equal groups: those receiving anthelmintic treatment (AT) and the untreated (C) controls. The same nutritional regime was applied to both groups and the feed consumption was calculated daily. Standard parasitological examinations were performed at weekly intervals. At slaughter, carcasses were weighed and assessed for conformation and fat cover classes. Climatic variables were obtained for the location of the farm and a temperature-humidity index was calculated. Before anthelmintic treatment with ivermectin, there was no difference in parasitic burdens between the two groups: 544 (AT) vs. 554 (C) epg, whilst after it, counts were 0 and 450–700 epg, respectively, with over 96% of larvae identified as Haemonchus spp. and, later, confirmed as Haemonchus contortus. It was concluded that treated animals had a higher average total bodyweight gain, higher feed conversion ratio and carcass yield of superior quality than controls. There was a difference between the two groups in the pattern of reduction of dry matter intake as the temperature-humidity index increased.

The effects of nematodes and bacteria on intestinal health are of primary importance in modern swine production. The aim of the present study was to assess the seropositivity status of Ascaris suum infection in fatteners in intensive swine farms in Greece and address possible risk factors, including Lawsonia intracellularis as a predisposing factor to swine ascariosis. In total, 360 blood serum samples from pigs in the late fattening period, from 24 Greek swine farrow-to-finish farms (15 samples/farm) were collected and tested with Svanovir® A. suum antibody ELISA and Ileitis antibody ELISA. The results demonstrated 34.4% seropositive samples for A. suum and 42.2% for L. intracellularis. The analysis of predisposing risk factors suggested that the frequency of application of anthelminthic treatment to sows more than two times per year was significantly associated with the lower likelihood of A. suum infection, whereas a greater likelihood of A. suum infection was observed in pigs with concurrent L. intracellularis exposure. The results highlight the importance of proper anthelminthic metaphylaxis of the breeding stock, as well as the likely outcome of concurrent exposure to two intestinal pathogens in pigs, implying a possible association between intestinal nematodes and bacteria in swine.

Abstract Background Canine vector-borne diseases (CVBDs) represent a wide group of diseases of major significance for canine health. In addition to their veterinary importance, many of these diseases are of great zoonotic concern, posing a risk of potential transmission to humans. To date, there has been scant knowledge regarding the prevalence, distribution and risk factors of CVBDs in Greece. Therefore, the objectives of the present study were to update the current knowledge on the seroprevalence of Dirofilaria immitis, Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi (sensu lato) in dogs in Greece and, furthermore, to assess possible environmental and any other risk factors associated with these infections. A total of 1000 apparently healthy and randomly selected dogs, presented in veterinary clinics, were involved at the national level (n = 66 municipalities). Serum samples were obtained from each individual dog and were tested using the SNAP® 4Dx® Plus kit from IDEXX Laboratories. Possible risk factors were assessed using binary regression models, including dogs’ lifestyle, climatological parameters and the altitude of the region. Results Overall, 21.8% (95% CI: 19.4–24.5%) of the sampled dogs were found to be seropositive to at least one of the four pathogens examined. The most prevalent pathogen was Ehrlichia spp. (12.5%, 95% CI: 10.6–14.7) followed by D. immitis (9.0%, 95% CI: 7.8–11.5) and Anaplasma spp. (6.2%, 95% CI: 4.9–7.9). The lowest prevalence (0.1%) was recorded for B. burgdorferi (s.l.) where only one dog was found to be positive. Among the examined risk factors, low mean temperature was found to increase the prevalence of Ehrlichia spp. (P ≤ 0.001) and Anaplasma spp. (P ≤ 0.001), while low minimum temperature increased the prevalence of D. immitis (P ≤ 0.001). In addition, low total annual rainfall had an effect of the prevalence of Ehrlichia spp. (P ≤ 0.01). Altitude also had a significant effect on the prevalence of D. immitis (P ≤ 0.05) and Anaplasma spp. (P ≤ 0.01). Conclusions To our knowledge, this is the first large-scale seroepidemiological study of CVBDs in Greece. It has been evidenced that environmental factors such as temperature, rainfall and altitude can influence the prevalence and distribution of CVBDs.

Abstract Dirofilaria repens is a nematode affecting domestic and wild canids, transmitted by several species of mosquitoes. It usually causes a non-pathogenic subcutaneous infection in dogs and is the principal agent of human dirofilariosis in the Old World. In the last decades, D. repens has increased in prevalence in areas where it has already been reported and its distribution range has expanded into new areas of Europe, representing a paradigmatic example of an emergent pathogen. Despite its emergence and zoonotic impact, D. repens has received less attention by scientists compared to Dirofilaria immitis. In this review we report the recent advances of D. repens infection in dogs and humans, and transmission by vectors, and discuss possible factors that influence the spread and increase of this zoonotic parasite in Europe. There is evidence that D. repens has spread faster than D. immitis from the endemic areas of southern Europe to northern Europe. Climate change affecting mosquito vectors and the facilitation of pet travel seem to have contributed to this expansion; however, in the authors’ opinion, the major factor is likely the rate of undiagnosed dogs continuing to perpetuate the life-cycle of D. repens. Many infected dogs remain undetected due to the subclinical nature of the disease, the lack of rapid and reliable diagnostic tools and the poor knowledge and still low awareness of D. repens in non-endemic areas. Improved diagnostic tools are warranted to bring D. repens diagnosis to the state of D. immitis diagnosis, as well as improved screening of imported dogs and promotion of preventative measures among veterinarians and dog owners. For vector-borne diseases involving pets, veterinarians play a significant role in prevention and should be more aware of their responsibility in reducing the impact of the zoonotic agents. In addition, they should enhance multisectorial collaboration with medical entomologists and the public health experts, under the concept and the actions of One Health-One Medicine.

Clinical manifestations from the lower respiratory tract are rare in canine leishmaniosis (CanL), making bronchoscopy and lung fine-needle aspiration (FNA) seldomly justified. The aim of this prospective study was to investigate the involvement of Leishmania infantum in the lungs of dogs with naturally occurring CanL by bronchoscopy and examination of bronchoalveolar lavage fluid (BALF), bronchial mucosa biopsies, and FNA, using immunodiagnostics. Dogs with relevant concurrent diseases and azotemia were excluded. Cough was detected in 5/31 (16.1%) dogs. Lesions (hyperemia, edema, mucosal granularity, secretions) were identified upon bronchoscopy in 19/31 (61.3%) dogs. The cytology of BALF revealed histiocytic inflammation in 14/31 (45.2%) dogs; the parasite was identified in one dog (3.2%). The immunofluorescence antibody test in BALF was positive in 15/31 (48.4%) dogs. Histopathology of bronchial mucosa and/or adjacent alveoli revealed lesions (mononuclear cell infiltration, fibrosis, edema, thickening of the inter-alveolar septa) in 24/31 (77.4%) dogs, with no Leishmania amastigotes. Positive antigen staining was observed within the cytoplasm of mononuclear cells in immunocytochemistry and immunohistochemistry. Μononuclear cells showed antigenic positivity in bronchial mucosa (27/31; 87.1%), BALF (30/31; 96.8%), and lung FNA (27/31; 87.1%). In conclusion, lungs seem to be affected from CanL more commonly than previously believed, and bronchoscopy allows obtaining valuable samples for antemortem diagnosis.

Extensively reared ruminants are seasonally exposed to ticks. Tick-related production losses and health issues have been well documented in cows and sheep but not in goats where relevant literature is scarce. The objective here was to investigate the association of hard tick infestation with milk production and udder health of dairy goats reared extensively. A cross-sectional study was carried out during May and June, in two dairy goat farms. The farms were located in Central and Northern Greece and were representative of typical extensive production systems. A total of 304 goats (n = 152 from each farm) were randomly selected. Each goat was examined for presence of hard ticks. Daily milk yield and quality characteristics were recorded. Udder health status was determined by milk somatic cell count (SCC) and total viable count (TVC). Tick infestation prevalence was 28.6%; it was associated with a significant (p < 0.001) increase in SCC and TVC (84.0% and 78.6%, respectively). The latter meant that infested goats were 3.7 times more prone to udder health problems (p < 0.001). There were not any significant effects (p > 0.05) on milk production. Overall, results suggest that control of tick infestation in extensively reared dairy goat herds is important for enhancing health and welfare status.

This paper presents the results of an extensive countrywide investigation performed in 325 dairy sheep flocks and 119 goat herds throughout Greece. The objectives of the study were (a) to investigate fat and protein content in the bulk-tank raw milk of small ruminant farms in Greece and (b) to identify factors potentially influencing that content and factors that can contribute to increased content. The mean fat/protein contents in bulk-tank raw milk of sheep and goats were 6.16 ± 0.05%/4.43 ± 0.01% and 4.77 ± 0.44%/3.23 ± 0.30%, respectively. Significant differences were seen in protein content between farms in the various parts of the country. For sheep, multivariable analyses revealed breed and age of lamb removal from dams as significant for fat content, and somatic cell counts, management system in the farm, administration of anthelmintic treatment during the last stage of pregnancy, and farmer education as significant for protein content. For goats, significant factors were month into lactation period, age of kid removal from dams, and breed for fat content, and somatic cell counts, month into lactation, grazing duration, and % Teladorsagia larvae in faecal samples for protein content. For concurrently high fat and protein content, in multivariable analyses, the following emerged as significant factors: somatic cell counts in milk, numbers of parasite eggs in faeces, and veterinary collaboration (sheep), and month into lactation and somatic cell counts in milk (goats). The results indicate that high somatic cell counts in milk (reflecting the presence of mastitis) and gastrointestinal parasitic infections (mainly Teladorsagia infection) appear to exert a more significant influence on fat and protein content of milk, in comparison to non-infection-related factors.

Fly infestation remains a universal problem for dairy cattle herds, affecting the animals’ health and welfare status. Pre-weaned dairy calves are significantly challenged by the direct and indirect consequences of severe fly infestation, heat-stress and their interaction, which contribute to a stressful and fatiguing environment. Among several physiological, behavioral, clinical and biochemical traits, serum cortisol (SC) and creatine kinase (CK) levels, as well as feed consumption can be used as valid indicators of potential stressful and fatiguing conditions and, therefore, can be efficiently used for stress analysis studies. Hence, the objective of the study was to assess the fly-repellency effect of deltamethrin on pre-weaned dairy calves exposed to heat stress conditions, as well as its association with SC, CK concentrations and feed consumption. Two commercial dairy cattle herds of the Holstein breed in Central Macedonia (Greece) were involved in the study during summer months and under heat stress conditions. Deltamethrin administration resulted in (i) a decreased fly population (100% Musca domestica) landing on pre-weaned dairy calves, (ii) a reduced SC (stress indicator) and CK (fatigue indicator) concentration, and (iii) an increased consumption of feedstuff in deltamethrin treated animals compared to the untreated ones.

The poultry red mite (Dermanyssus gallinae), an obligatory blood feeding ectoparasite, is primarily associated with laying hens where it is estimated to cause losses of ~€231 million per annum to European farmers. Moderate to high infestation levels result in negative impacts on hen welfare, including increased cannibalism, irritation, feather pecking, restlessness, anemia, and mortality. Acaricides are currently the prevailing method of population control for D. gallinae, although resistance against some classes of acaricide has been widely reported. The development of resistance highlights a growing need for research into alternative control methods, including the development of a suitable and effective vaccine. Understanding the genetic structure of D. gallinae populations can support improved management of acaricide resistance and sustainability of future vaccines, but limited data are currently available. The aim of this study was to characterize D. gallinae isolates from Europe, targeting the cytochrome c oxidase subunit 1 (COI) gene to gain an insight into population structure and genetic diversity of currently circulating mites. Dermanyssus gallinae isolates were collected from Albania, Belgium, Croatia, Czech Republic, Denmark, France, Greece, Italy, the Netherlands, Portugal, Romania, Slovenia, Turkey and the United Kingdom. Genomic DNA was extracted from individual adult D. gallinae mites and a 681bp fragment of the COI gene was amplified and sequenced. Phylogenetic analyses of 195 COI sequences confirmed the presence of multiple lineages across Europe with 76 distinct haplotypes split across three main haplogroups and six sub-haplogroups. Importantly there is considerable inter- and intra-country variation across Europe, which could result from the movement of poultry or transfer of contaminated equipment and/or materials and husbandry practices.

Bacteriological, cytological and proteomics data have been obtained from ewes in two experiments, after intramammary challenge with Mannheimia haemolytica or Staphylococcus chromogenes. Animals were sampled before and sequentially after challenge. Conventional techniques were employed for bacterial isolation and somatic cell counting in milk samples; milk whey samples were subjected to proteomics evaluation by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. There was a correlation between leucocyte content and cathelicidin-1 spot densities in milk samples, although the protein was detected in milk earlier than the increase in leucocyte content. There was also a significant association between presence of mastitis in a mammary gland and detection of cathelicidin-1 in the respective milk sample; the degree of association was greater during the first 24 h post-inoculation. The data are further discussed in the research article “Detection of cathelicidin-1 in the milk as an early indicator of mastitis in ewes” [1]. Keywords: Biomarker, Diagnosis, Mastitis, Sheep, Somatic cell counts

Abstract Background Feline gastrointestinal parasitism constitutes an issue of concern for veterinarians since parasites are widespread and affect animals’ health and welfare. Furthermore, some of these pathogens have zoonotic potential. To provide detailed data on the current epizootiology of feline endoparasitism, a multicentric survey was conducted during 2016. Methods Faeces from 1150 cats were collected from all regions of Greece and examined by sedimentation and flotation techniques. Possible risk factors including gender, age, ownership status, living conditions and co-infections with other parasites were assessed using binary regression models for each one of the most prevalent parasites. Results The overall gastrointestinal parasite prevalence in cats was 50.7%. The study population consisted of cats of both sexes, different age groups, ownership status and living conditions. A total of 10 gastrointestinal parasitic species were detected and up to 5 different parasites were isolated in the same faecal sample. The most frequently identified parasites were Toxocara cati (n = 278; 24.2%), followed by Cystoisospora spp. (n = 189; 16.4%), Ancylostomatidae (n = 186; 16.2%), Aelurostrongylus abstrusus (n = 40; 3.5%), Giardia spp. (n = 26; 2.3%), Joyeuxiella pasqualei (n = 14; 1.2%), Capillaria aerophila (n = 8; 0.7%), Dipylidium caninum (n = 3; 0.2%), Toxascaris leonina (n = 2; 0.1%) and Troglostrongylus brevior (n = 2; 0.1%). The occurrence of co-infections was 11.6%. Concerning risk factors, the likelihood of T. cati infection was higher for female cats living outdoors and for cats being infected with Cystoisospora spp. In the same frame, young, stray, male and free of A. abstrusus cats were more likely to be infected with Cystoisospora spp. Correspondingly, stray, infected with Giardia spp. but free of Cystoisospora spp. cats were more likely to be infected with Ancylostomatidae. Regarding A. abstrusus infection, a higher probability was reported for cats living outdoors and for cats free of Cystoisospora spp., while Giardia spp. infections were more common in young and co-infected with Ancylostomatidae animals. Conclusions The prevalence of parasitized cats in Greece was high and thus consideration should be paid to control the risk factors, to implement targeted preventive antiparasitic treatments and educate cat owners on the value of prevention for the health and welfare of their cats.

The Mediterranean monk seal (Monachus monachus) is classified by the IUCN as “endangered,” with a global population estimated to number fewer than 800 individuals. Our understanding of the biology and health status of the species is still limited, rendering every medical case a challenge for conservationists and veterinary clinicians. Although studying and managing disease in wild marine hosts is complex and challenging, studying and mitigating the effects of any disease to the Mediterranean monk seal is of utmost importance for conservation. The aim of this study was to document for the first time the presence of the hookworm Uncinaria hamiltoni in rehabilitated Mediterranean monk seal pups in Greece. A detailed examination protocol was followed for all pups that live-stranded over 30 years in 22 different locations, including physical, parasitological, and other examinations. Hookworms (adults and/or eggs) were detected in all the fecal samples, from all animals. Molecular identification using MtDNA (COI) and ribosomal DNA (D2/D3 28S and internal transcribed spacer [ITS] regions) identified the nematode species as Uncinaria hamiltoni. The clinical impacts and the benefits of anthelmintic treatment as a tool for the conservation management of the species are discussed.

Canine leishmaniosis (Leishmania infantum) is a zoonotic disease that affects dogs worldwide. Greece is enzootic for this disease, and updated data for its current distribution are of major importance. The aim of this cross-sectional serological study was primarily to update the current knowledge of Leishmania infantum seropositivity status within the asymptomatic Greek canine population and, furthermore, to assess the possible climatological and other risk factors. In total, sera of 1265 asymptomatic dogs were collected from all prefectures of the country. A questionnaire that included all individual dog information was completed for all animals. The Speed Leish K® canine Leishmania antibody test kit (BVT Groupe Virbac, France) was employed. Potential risk factors were evaluated utilizing logistic regression models. Overall, 13.8% (n = 175) of the sampled dogs were seropositive to Leishmania infantum originating from all geographical departments of the country, whereas most prefectures had at least one seropositive animal. Outdoor living, high mean humidity, low mean wind speed and high total annual rainfall were found to increase the seropositivity status against the parasite. Conclusively, Leishmania infantum remains a common parasite challenge in the asymptomatic canine population of Greece, and therefore, its early diagnosis and effective prevention are significant in the country.

Domestic rabbits are commercially farmed for their meat whilst gastrointestinal diseases can hinder their production. Passalurusambiguus and Eimeria spp. are two common rabbit intestinal parasites that can cause diarrhoea, among other symptoms, and in severe cases, death. C. guttulatus is a commensal yeast of the rabbits’ stomach that is considered apathogenic but can worsen symptoms in rabbits suffering from coccidiosis. In the present case report, we describe an outbreak of deaths in three different age groups (A: lactating does, B: 58 days old and C: 80 days old) in an industrial rabbit farm in Greece. Symptoms included depression, diarrhoea, inappetence, weight loss, dehydration and ruffled furs. Using a faecal flotation technique, sick rabbits were found to be moderately to heavily infected with P. ambiguus, Eimeria spp. and C. guttulatus. Treatment with fenbendazole and oregano oil combined with hygiene control measures successfully controlled the infections and resolved clinical symptoms. A faecal flotation method or other reliable diagnostic technique should be used regularly in industrial rabbit farms to screen for gastrointestinal parasitic infections. Early diagnosis and control will help to maintain production levels and, therefore, limit financial losses for the farmer while ensuring animal welfare.

One-hundred and fifty, one-day-old Ross-308 female chicks were randomly allocated to five equal treatments: NCONTR negative control—not challenged; PCONTR positive control—challenged; PHERB1 and PHERB2 diets were supplemented with phytogenic formula (1 and 2 g/kg feed, respectively)—challenged; PSALIN diet was supplemented with salinomycin (60 mg/kg feed)—challenged. Challenge was made by oral inoculation with 3.5 × 104 E. acervulina, 7.0 × 103 E. maxima and 5.0 × 103 E. tenella oocysts, at 14 days of age. One week post inoculation, bloody diarrhea, oocysts numbers, and intestinal lesions were evaluated, along with intestinal microbiota, viscosity, and pH of digesta, and histopathology. PHERB2 had a comparable (p ≤ 0.001) growth performance and feed conversion ratio to PSALIN. PHERB1 and PHERB2 had similar (p ≤ 0.001) oocyst counts to PSALIN and lower than PCONTROL. PHERB2 and PSALIN had lower (p ≤ 0.001) jejunal, ileal, and cecal lesion scores compared to PCONTR. PHERB1 and PHERB2 had higher (p ≤ 0.001) jejunal and cecal lactobacilli and lower (p ≤ 0.001) coliform counts compared to other treatments. PCONTR had lower (p ≤ 0.001) jejunum villus height, height to crypt ratio, and villus goblet cells. Breast and thigh meat resistance to oxidation was improved (p ≤ 0.001) in PHERB1 and PHERB2 compared to the PCONTR. The polyherbal formula exerted a substantial improvement on growth performance and intestinal health of the Eimeria-challenged birds.

Abstract Background With the aim to assess the occurrence of hard ticks and the pathogens they may carry in dogs from Greece, ixodid specimens (n = 757) were collected from 310 animals living in six provinces across the Greek peninsula. All ticks were morphologically identified, and genomic DNA was extracted from 344 (45.5%) representative specimens, according to their species, engorgement status and sampling area. The occurrence of Anaplasma spp., Ehrlichia spp., Hepatozoon spp., Rickettsia spp., Babesia spp., Theileria spp. and Cercopithifilaria spp. was assessed by conventional and quantitative real-time PCR. Results Overall, 150 dogs (48.4%) were infested by ticks, with Rhipicephalus sanguineus (sensu lato) being the most prevalent (70.1%), followed by Haemaphysalis parva (14.7%), Rhipicephalus turanicus (11.4%), and Haemaphysalis concinna (2.4%). Out of 344 specimens molecularly examined, 41 (11.1%) were positive for at least one microorganism (i.e. 5.5% for Cercopithifilaria bainae, 2.9% for Hepatozoon canis, 1.7% for Rickettsia hoogstraalii, 1.2% for Hepatozoon felis, 0.6% for Rickettsia massiliae, 0.6% for Theileria ovis, 0.3% for Anaplasma platys and 0.3% for Coxiella like-endosymbiont). Conclusions The results of this study show that different tick species parasitize dogs in Greece, carrying a range of microorganisms potentially pathogenic for dogs and humans. Consequently, control strategies against ticks are of great importance to prevent the risk of tick-borne diseases. The relationship between ticks infesting dogs and associated microorganisms is described according to collection site and dog lifestyle.

Abstract Background A large number of fleas parasitize dogs living with sheep in Greece. The primary aim of this randomized, blinded, placebo-controlled trial was to examine the efficacy of a permethrin-fipronil combination (Effitix®) for the treatment and prevention of flea infestation in dogs living with sheep and the secondary aim was to examine the efficacy of this intervention on flea infestation, pruritus and skin lesions of the people in contact with these dogs. Methods Thirty dogs living with sheep and infested by at least 10 fleas and all 80 sheep living on the same premises were randomly allocated into equal groups. Group A dogs were treated three times, every 4 weeks, with a spot-on containing 54.5% permethrin and 6.1% fipronil, group A sheep were treated, on the same days, with a pour-on containing 1% deltamethrin, whereas group B dogs were sham-treated and group B sheep were placebo-treated. Flea counting was performed at the beginning of the trial (day 0) and after 14, 28, 56 and 84 days and the first five fleas from each animal were used for species identification. At the same time points, flea infestation, pruritus and skin lesions of the people in contact with the dogs were assessed. Results The percentage of dogs with zero flea counts was significantly higher in group A than in group B on days 14, 28, 56 and 84 and flea counts were significantly lower in group A dogs than in group B dogs at the same time points. The percent efficacy of the permethrin-fipronil combination was higher than 78% (arithmetic means) or than 96% (geometric means) throughout the study. No adverse reactions were recorded. Between the two flea species found on dogs, Ctenocephalides canis was predominant over C. felis. Flea-infected sheep were not found at the beginning or during the study and no significant changes in flea infestation, pruritus and skin lesions of the people in contact with the dogs were witnessed throughout the study. Conclusions A spot-on solution containing 54.5% permethrin and 6.1% fipronil is safe and effective for the treatment and prevention of C. canis and C. felis infestations in dogs living with sheep.

Dermanyssus gallinae (the poultry red mite, PRM) is an important ectoparasite in the laying hen industry. PRM can also infest humans, causing gamasoidosis, which is manifested as skin lesions characterized by rash and itching. Recently, there has been an increase in the reported number of human infestation cases with D. gallinae, mostly associated with the proliferation of pigeons in cities where they build their nests. The human form of the disease has not been linked to swallows (Hirundinidae) before. In this report, we describe an incident of human gamasoidosis linked to a nest of swallows built on the window ledge of an apartment in the island of Kefalonia, Greece. Mites were identified as D. gallinae using morphological keys and amplifying the Cytochrome C oxidase subunit I (COI) gene by PCR. Bayesian phylogenetic analysis and median-joining network supported the identification of three PRM haplogroups and the haplotype isolated from swallows was identical to three PRM sequences isolated from hens in Portugal. The patient was treated with topical corticosteroids, while the house was sprayed with deltamethrin. After one week, the mites disappeared and clinical symptoms subsided. The current study is the first report of human gamasoidosis from PRM found in swallows’ nest.

The paper reviews the challenges about haemonchosis—a significant and common parasitic infection of small ruminants. Haemonchus contortus is a highly pathogenic parasite that localises in the abomasum of affected animals and exerts its pathogenicity by blood-sucking activity, adversely affecting the health and productivity of animals. The first challenge is the uneven distribution of the infection globally, this being more prevalent in tropical and subtropical and warm temperate and summer rainfall regions than in cool and cold temperate and arid regions; hence, this leads in differences in the approaches required for its control. Another challenge is the widespread presence of Haemonchus strains resistant to the various anthelmintics available: Benzimidazoles, imidazothiazoles, macrocyclic lactones, closantel and monepantel, which makes the control of the infection difficult. The third challenge refers to the difficulty of diagnosing the disease, given that field evidence can provide suspicion about the infection, which needs to be subsequently confirmed by laboratory tests through parasitological or molecular techniques. The final challenge relates to the difficulties in the control of the infection and the necessity to use pharmaceutical products cautiously and with a planned approach, to avoid further development of anthelmintic resistance, also given that use of a recently licenced vaccine is not widespread. In conclusion, at the moment, we should be concerned, but not worried, about this infection, and apply correctly the appropriate health management plans.

Staphylococcus epidermidis is an important causal agent of ovine mastitis. A literature search indicated a lack of systematic studies of causal agents of the infection by using multi-locus sequence typing (MLST). The objectives were to analyse MLST-based data and evaluate the antimicrobial resistance of S. epidermidis isolates from ovine mastitis in Greece. The database included 1593 isolates from 46 countries: 1215 of human, 195 of environmental and 134 of animal origin, distributed into 949 sequence types (STs) and cumulatively with 450 alleles therein. Among mastitis isolates, bovine isolates were distributed into 36 different STs and ovine ones into 15 STs. The 33 isolates from ovine mastitis in Greece were in 15 different STs, 6 of these (ST677, ST678, ST700, ST 709, ST710, ST711) assigned for the first time; in addition, 5 alleles (65 for arcC, 59 for aroE, 56 and 57 for gtr and 48 for tpiA) were identified for the first time. The spanning tree of these isolates included 15 nodes and 14 edges (i.e., branches). Among these isolates, 19 showed resistance to antimicrobial agents (tetracycline, penicillin, fucidic adic, erythromycin, clindamycin, cefoxitin). Resistance-related genes (tetK, tetT, msrA, tetM, tetS, ermC, mecA) were detected. There was no association between STs and resistance to antimicrobial agents. Isolates with antimicrobial resistance were recovered more often from flocks where hand-milking was practised.

Intramammary infections (IMIs) caused by various pathogens may lead to clinical or subclinical mastitis, challenging the health and welfare status of infected animals and decreasing the quantity and quality of the produced milk. Additionally, the zoonotic potential of some of the pathogens isolated from IMI cases, the emergence of antibiotic resistance due to the extensive antibiotic use for IMI treatment, and the accumulation of antibiotic residues in milk and meat represent significant concerns for public health. Therefore, the investigation of IMI risk factors and the proposal of efficient measures to mitigate their effects on animal health and welfare is crucial. Although fly infestation is considered to play a significant role in the transmission of IMI pathogens, its adverse effects on udder health and the overall comfort status of dairy ewes have not been quantified and assessed on an evidential basis. Hence, the objectives of this study were to assess, for the first time, the fly repellent effect of deltamethrin and link it to: (i) the occurrence of common bacterial IMI; (ii) the somatic cell counts in milk; and (iii) the serum cortisol and creatine kinase levels (stress and fatigue indicators). The study was carried out in an intensive dairy sheep farm in northern Greece, during peak fly season. Deltamethrin treatment was associated with a reduced (i) number of flies (mostly Musca domestica) landing on treated ewes, compared to untreated ones (p < 0.05); (ii) colony-forming units in the case of Non-aureus Staphylococci IMIs (p < 0.05); and (iii) number of somatic cells in the milk (p < 0.001). Finally, serum cortisol and creatine kinase levels were significantly lower in deltamethrin-treated ewes (p < 0.001), indicating a less stressful environment for them.

The aim of this study was to investigate the effect of experimental Toxoplasma gondii infection on ram sperm quality. Five months old, pre-pubertal, rams were divided into four groups (n = 8 per group). Group A was the control group; the remaining animals received per os (p.o.) 5000 oocysts per ram. Group B did not receive treatment post-infection (p.i.). Group C received sulphadimidine (intermuscular injection (i.m.) 33 mg/kg for eight days; every 48 h) two months p.i. and Group D received the same drug twice (24 h p.i. and two months later). Blood samples were collected every 15 days to detect serum immunoglobulin G (IgG). Epididymal sperm samples were analyzed for concentration, kinetics, morphology/viability, functional membrane integrity, DNA integrity, and the presence of parasite DNA. Histopathological examination was performed on the testes. The IgG titres in infected groups raised two weeks p.i. and remained high for four months. Higher values were noticed in viability and functional membrane integrity in positive spermatozoa in the control group compared to other groups, level of significance p < 0.05. Abnormal sperm was higher in groups C and D vs. A and C vs. B (p < 0.05). T. gondii DNA was detected in three sperm samples of the infected rams (12.5%). Histopathology revealed similar findings with little variation among all infected groups, characterized mostly by increased interstitial connective tissue, non-purulent inflammation, and presence of seminiferous tubules with spermatogenic cell depletion, which increased gradually from D to C and B groups. In conclusion Toxoplasmosis in pre-pubertal age negatively affected mature ram sperm quality, while sulphadimidine administration failed to alter this.

The objective of this work was to develop, use and present a detailed questionnaire for the evaluation of health management in dairy small ruminants; it includes 442 questions organised in seven sections: general, infrastructure, animals, production characteristics, health management, nutrition, human resources. Consistency of replies was evaluated in 27 farmers, interviewed twice. Inconsistent replies were given by all farmers to 30 different questions (Cronbach’s coefficient alpha: 0.987). Then, interviews were performed in 444 farms around Greece. Mean duration of an interview was 63.6 min. Clarifications were requested by 273 farmers to 22 different questions (maximum per farmer: 8). The experience of the investigator, the primary language of farmers and asking clarifications by the farmers affected the duration of the interview. The questionnaire can be used for research work in the field, to record details in the farms under study. In accord with the needs of a particular study, it can be modified, by adding more specific questions or omitting others deemed of less importance. Moreover, it can also be used for routine monitoring purposes, as a useful means to record and maintain details of farms during clinical work. To the best of our knowledge, the questionnaire is the most extensive and detailed one available internationally for dairy small ruminants.

Intestinal parasites, helminths, and protozoa challenge health and welfare of pigs and deteriorate the sustainability of swine farms leading to monetary losses. A multicentric survey was conducted for approximately one year. Overall, 1150 fecal samples were collected from eight intensive farms in Greece at regular intervals and examined by flotation and Ziehl-Neelsen techniques. Age, season, and time of last recorded antiparasitic treatment were assessed as possible risk factors using binary regression models. The overall prevalence of intestinal parasitism in pigs was 44.7%. The most frequently detected parasites in the studied population were the protozoa Balantidium coli (37.8%), followed by Entamoeba spp. (8.3%), Cystoisospora suis (6.0%), and the nematodes Ascaris suum (3.7%), Trichuris suis (2.5%), and Oesophagostomum spp. (1.4%). Distribution of intestinal parasites in different age groups was as expected. In autumn, the prevalence of Balantidium coli infection enhanced whereas the prevalence of Entamoeba spp. and Cystoisospora suis infections increased in spring. Time of last recorded antiparasitic treatment influenced Balantidium coli and Trichuris suis infection levels. Our results demonstrated that swine intestinal parasitism in intensive farms of Greece seems to be relatively common and highlighted the importance of proper laboratory examinations, as well as the need for tailored made control programs.

The study investigated the presence of resistance to benzimidazoles in Haemonchus contortus helminths from ruminant species in Greece through the detection of the Phe/Tyr polymorphism in the amino acid at position 200 of the β-tubulin protein. In total, 288 adult female H. contortus helminths collected from the abomasum of various ruminant animals in Greece were tested. Of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and 48 from buffaloes. The frequencies of the homozygous and heterozygous resistant genotypes at the position 200 of the β-tubulin gene of helminths recovered from sheep were 96.9% and 3.1%, respectively. The frequencies of the homozygous and heterozygous resistant genotypes, respectively, were 100.0% and 0.0% in helminths from goats, 25.0% and 75.0% in helminths from cattle and 8.3% and 91.7% in helminths from buffaloes. In all parasitic populations, no homozygous susceptible genotypes were detected. The present study highlighted, for the first time, the emergence of benzimidazole-resistant H. contortus in goats, cattle, and buffaloes in Greece, using an allele-specific PCR. It is postulated that benzimidazole-resistant alleles were transferred from sheep or goats to cattle and buffaloes at the commonly grazing pastures in Greece.

Abstract Background Three flea species, Pulex irritans, Ctenocephalides canis and C. felis parasitize shepherd dogs living on sheep farms in Greece. The aim of this randomized, blinded, placebo-controlled trial was to examine the efficacy of spinosad, when administered three times every 4 weeks, as the only intervention to treat and prevent flea infestations in shepherd dogs living on sheep farms. Methods Thirty dogs living on sheep farms and infested by at least 24 fleas were randomly allocated into equal groups. Group A dogs received spinosad (45–70 mg/kg body weight), every 4 weeks for three administrations, whereas Group B dogs were placebo-treated. Flea counting was performed at the beginning of the trial (day 0) and after 14, 28, 56 and 84 days. The first five fleas from each dog and 2–6 fleas collected from 5–11 sheep were used for species identification. Results The percentage of dogs with zero flea counts was significantly higher in group A than in group B at days 14, 28, 56 and 84 and flea counts were significantly lower in group A than in group B at days 14, 28, 56 and 84. In group A, flea counts were significantly lower at days 14, 28, 56 and 84 compared to day 0 whereas there were no changes in flea counts of group B dogs. The percent efficacy of spinosad for the treatment and prevention of flea infestation was higher than 98 % (arithmetic means) or higher than 99 % (geometric means) throughout the study. No adverse reactions were recorded. C. canis was the predominant flea species of dogs at day 0. In group A the relative abundance of C. felis increased at day14 whereas in group B the relative abundance of P. irritans increased at days 14, 28, 56 and 84. Conclusions Spinosad is safe and effective for the treatment of C. canis and C. felis infestations and for the prevention of P. irritans, C. canis and C. felis infestations in shepherd dogs living in close proximity to sheep.

Eighteen mature ewes of the Mytilene breed and 18 mature Local- Damascus crossbred goats, seronegative for Toxoplasma gondii (T. gondii) by ELISA were used. All animals were mated after synchronization of estrus. On day 90 of pregnancy, animals were randomly assigned to 3 experimental groups; 6 ewes (S1) and 6 goats (G1) were orally inoculated by stomach tube with 1000 oocysts; 6 ewes (S2) and 6 goats (G2) were orally inoculated with a non-infected control inoculum. On day 140+2 of pregnancy, the remaining 6 ewes (S3) and 6 goats (G3) were inoculated by stomach tube with 3000 oocysts. Positive T. godii DNA was detected in 94% of fetal and maternal blood, 95% fetal tissue, 89% pre-colostral udder secretions and 12.5% milk samples using Polymerase Chain Reaction (PCR). Infected animals and their live newborns was seropositive (ELISA) until the end of the study. PCR was able to detect T. gondii DNA in maternal blood of infected animals 3-5 days before abortion occurred. This time period may be used to implement preventive and therapeutic measure to reduce abortion rate and associated economic losses. Since milk and milk products are important food sources in rural areas and in many cases it is used unpasteurized before consumption. The T. gondii DNA, detected by PCR in milk samples of infected animals, increases the possibility that the parasite is transmitted through consumption of unpasteurized milk which is a highly relevant result for public health considerations and providing valuable information for future research.