Evidence of Horse Exposure to Anaplasma phagocytophilum, Borrelia burgdorferi, and Leishmania infantum in Greece through the Detection of IgG Antibodies in Serum and in an Alternative Diagnostic Sample-The Saliva.

Among the various zoonotic pathogens that infect horses, Anaplasma phagocytophilum, Borrelia spp. and Leishmania spp. have gained scientific interest, and relevant molecular and serological studies in horses have been conducted worldwide. Moreover, human and veterinary medicine have extensively applied alternatives to serum diagnostic samples-such as saliva-for detecting pathogens or antibodies. In this study, we investigated the exposure of horses in Greece to A. phagocytophilum, B. burgdorferi, and L. infantum, and we assessed the diagnostic accuracy of saliva compared to serum in detecting IgG antibodies against the abovementioned pathogens. Paired saliva and serum samples were collected from 317 horses from different regions in Greece. The paired samples were examined using the indirect fluorescent antibody test (IFAT) for detecting IgG antibodies against A. phagocytophilum, B. burgdorferi, and L. infantum. Sensitivity, specificity, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were determined to assess the validity of saliva as an alternative to serum. The receiver operating characteristic (ROC) curve revealed that the optimal cut-off value for detecting antibodies against all the examined pathogens in saliva was 1/10. Higher seropositivity rates were found for B. burgdorferi (15.14%) and A. phagocytophilum (14.19%) compared to L. infantum (1.26%). The detection of IgG antibodies using IFAT in saliva samples had a good test performance compared to serum. The two sample types had a substantial to almost perfect agreement. Although the sensitivity was moderate (70.83-75.56%) in all cases, the specificity was almost perfect to perfect (99.63-100%). This study provides the first evidence that horses in Greece are exposed to A. phagocytophilum and B. burgdorferi and confirms that the seroprevalence of L. infantum in horses in Greece remains low. Our findings suggest that saliva sampling coupled with IFAT could be successfully applied for detecting IgG antibodies against these important zoonotic pathogens in large-scale epidemiological studies in horses, at the population level, as an alternative to serum.

Leishmaniosis in Greece: The Veterinary Perspective.

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.

Hepatic Coccidiosis in Wild Rabbits in Greece: Parasite Detection on Liver Imprints and the Associated Biochemical Profile.

(1) Background: Rabbit hepatic coccidiosis, caused by Eimeria stiedae, is a devastating disease with high morbidity and mortality rates. The disease is well described in rabbits, but little is known about E. stiedae infection in wild rabbits. In this study, we investigated the presence of E. stiedae infection in wild rabbits from the island of Lemnos, Greece, where this species is overpopulated, and the effects of infection on common hepatic biomarkers. (2) Methods: We used liver impression smears to detect the coccidian oocysts, and we defined the liver biochemical profile of the infected individuals. (3) Results: Overall, 13.3% of the liver imprints examined were positive for the presence of coccidial oocysts. The activities of liver enzymes, that is, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamyltransferase (GGT), as well as globulins (GLOB), were increased while the concentrations of albumins (ALB), total proteins (TP) and the albumin to globulin (A/G) ratio were decreased in the infected individuals compared to the non-infected ones. (4) Conclusions: This study adds to the current knowledge on the pathogens affecting wild rabbits and those circulating in this population on the island of Lemnos, Greece. Moreover, we showed that E. stiedae infection exerts pathological effects on the hepatocyte integrity and liver function of wild rabbits, as reflected by the abnormal values of liver injury and dysfunction biomarkers.

Natural Co-Exposure to Borrelia burgdorferi s.l. and Anaplasma phagocytophilum: Unraveling the Hematological Profile in Sheep.

The occurrence of co-infected hosts and questing ticks with more than one tick-borne pathogen-as in the case of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato-is expected in endemic regions. Their synergy-in terms of pathogenesis and disease severity-has been suggested previously in humans. Limited data exist on the clinicopathological alterations in co-infected sheep. In this study, we investigated the impact of A. phagocytophilum and B. burgdorferi s.l. seropositivity, alone and in combination, on the hematological parameters of naturally infected sheep. A complete blood count was performed, and indirect immunofluorescence assays were used to detect IgG antibodies against A. phagocytophilum and IgG and IgM antibodies against B. burgdorferi s.l. Single natural exposure to B. burgdorferi s.l. was characterized by low Packed Cell Volume (PCV) values and platelet (PLT) counts, while single exposure to A. phagocytophilum was characterized by low PCV values, low white blood cell (WBC) counts, and an increased risk for leukopenia and neutropenia. Co-exposure resulted in the most severe blood abnormalities; all the blood parameters decreased, and the sheep presented an increased risk for anemia. Our study showed that natural co-exposure to A. phagocytophilum and B. burgdorferi s.l. in sheep leads to more severe blood abnormalities and enhances the pathogenic processes. More studies are needed to clarify the possible background mechanisms.

Current Data on Rickettsia felis Occurrence in Vectors, Human and Animal Hosts in Europe: A Scoping Review.

Rickettsia felis is an emerging pathogen with increasing reports of human cases and detection in arthropod and animal host species worldwide. In this scoping review we record the newest data reported for R. felis in Europe: the vector and host species found to be infected, and the geographical distribution and prevalence of R. felis infection in vectors and hosts. A total of 15 European countries reported the occurrence of R. felis in hosts and vectors during 2017−2022. The vectors found to be infected by R. felis were flea, tick and mite species; Ctenocephalides felis and Ixodes ricinus were the dominant ones. The hosts found to be infected and/or exposed to R. felis were humans, cats and small mammals. Physicians should be aware of the epidemiology and include illness caused by R. felis in the differential diagnosis of febrile disease. Veterinarians should keep training pet owners on the need for effective year-round arthropod control on their pets, especially for fleas.

Evaluation of Heller's reaction and the urine dipstick method alone or in combination to detect proteinuria in sheep.

BACKGROUND: Urine dipstick and Heller's reaction are easy first-line screening tests for the detection of proteinuria; however, the performance of these methods in alkaline ovine urine is largely unknown. OBJECTIVE: The aim of this study was to evaluate the diagnostic performance of Heller's reaction alone or in combination with dipstick for the detection of proteinuria in sheep, using the urine protein to creatinine ratio (UP/C) with two cut-off values as the reference method. METHODS: Ninety-eight urine samples were collected from sheep using the transient apnea method. Heller's reaction, the dipstick method, and the UP/C ratio were used to assess proteinuria. The results were statistically analyzed twice, based on two different UP/C cut-off values of 0.2 and 0.5. Cohen's kappa value was used to determine the agreement between the UP/C ratios and Heller's reaction, the dipstick method, or the combination of methods. Sensitivity, specificity, and positive and negative likelihood ratios were calculated. ROC curves were also generated, and the areas under the curve (AUC) were evaluated to determine the optimal threshold for the numerical values of the two methods. RESULTS: Heller's reaction is more specific (96.67% and 96.00% when the cut-off value is 0.2 and 0.5, respectively) than the dipstick method, while the dipstick method was more sensitive (91.18% and 91.30%, when the cut-off value was 0.2 and 0.5, respectively) than Heller's reaction for the detection of proteinuria. Both tests were accurate when any grade >0 was considered positive. CONCLUSIONS: Proteinuria can almost be excluded in ovine urine samples with negative Heller's reaction and dipstick test.

Detection of Antibodies Against Toxoplasma gondii in Filter Paper-Dried Blood Dot Spots Compared with Serum in Pigs and Assessment of Variation Associated with Packed Cell Volume.

The aim of this study was to assess the agreement between anti-Toxoplasma gondii IgG antibody detection in serum and filter paper (FP) blood spots using the indirect immunofluorescence antibody assay (IFA) and to evaluate the potential impact of the packed cell volume (PCV) on antibody detection in FPs. A pair of a serum and an FP sample was collected from 96 sows at various farms in Greece, with previously identified high seropositivity and/or risk factors associated with high seropositivity against T. gondii. The PCV value was determined using the microhematocrit method. IFA was used for the detection of antibodies against T. gondii. T. gondii-specific antibodies were detected in 45.8% serum samples and 41.6% FP samples showing almost perfect agreement. Detection in FP samples presented high sensitivity (87.1-92.8%) and excellent specificity (100%) when compared with detection in serum, regardless of the PCV values. The findings of this study support the reliability of FPs for the evaluation of the serological status of swine against T. gondii. FPs could be a good alternative sample type compared with serum for large-scale epidemiological studies.

Evaluation of an Indirect Immunofluorescence Assay for the Detection of Anaplasma phagocytophilum Antigen in Ovine Buffy Coat Smears.

Diagnosis of anaplasmosis is challenging considering the great variation in clinical signs and the limitations of the available diagnostic assays, while the detection of carrier animals that play a significant role in disease epidemiology as reservoirs is of great significance. In this study, we evaluated the diagnostic accuracy of a newly developed indirect immunofluorescent assay (Ag-IFAT) for the detection of A. phagocytophilum antigens in buffy coat specimens, alone and in combination with cytology, using PCR as a reference. Blood samples were collected from 138 sheep of the Chios breed from six farms in Greece. A buffy coat was extruded from the centrifuged blood. Buffy coat smears were used for cytological examination and the Ag-IFAT assay. The Ag-IFAT assay presented excellent specificity (100%) and high sensitivity (85.4%) for the detection of A. phagocytophilum antigens in buffy coats, and it has an almost perfect agreement with PCR and cytology (κ value = 0.88 and 0.85, respectively). A. phagocytophilum antigens are likely to be detected using Ag-IFAT in a PCR-positive animal, as indicated by the good performance of the assay. Overall, this assay presents high diagnostic accuracy, and it could be used for the detection of animals during the early stage of infection.

Wild Rabbit Exposure to Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi Evidenced by Serum and Aqueous Humor Antibody Detection.

Wild rabbits (Oryctolagus cuniculus) can be important sentinel species for the presence of zoonotic pathogens. Therefore, we collected blood samples from wild rabbits harvested by hunters during the hunting season 2019-2020 on the island of Lemnos, to determine exposure of wild rabbits to the zoonotic pathogens Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi, as well as aqueous humor to assess its diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios. Antibodies against these pathogens were detected by Indirect Immunofluorescence Antibody (IFA) assay. Out of the 72 wild rabbits included in the study, 4.2%, 5.5%, 18% and 9.7% were seropositive to L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively. Although less frequently, antibodies were also detected in aqueous humor of wild rabbits. The antibody detection in aqueous humor presented 100% specificity but decreased sensitivity compared to serum suggesting that aqueous humor could be successfully used in epidemiological studies to confirm exposure at the population level but has little diagnostic value at the individual level. This is the first report on the seropositivity of wild rabbits to A. phagocytophilum and B. caballi and the detection of antibodies against A. phagocytopylum, L. infantum, T. gondii and B. caballi in the aqueous humor.

European Brown hare (Lepus europaeus) as a source of emerging and re-emerging pathogens of Public Health importance: A review.

European brown hare (Lepus europaeus, EBH) is probably the most important game animal in Europe throughout its historical distribution. The decline in its populations across its geographic range in Europe have been attributed to factors such as reproductive rate and the ability for adaptation, climate, feed availability, predators, anthropogenic factors and diseases. Apart from common diseases of hares with a high impact on their mortality such as European Brown hare Syndrome, EBH has been involved in the epidemiology of pathogens with zoonotic potential. In this work, the role of EBH as a source of Crimean-Congo haemorrhagic fever virus (CCHFV), Hepatitis E virus (HEV), Yersinia spp., Brucella spp., Francisella tularensis, Toxoplasma gondii and Leishmania infantum is discussed. Hares may significantly contribute to the epidemiology of important emerging zoonotic pathogens through maintenance of high endemicity levels as in the case of CCHFV, as a reservoir of important pathogens such as Yersinia spp., B. suis, F. tularensis and L. infantum and as a potential source of T. gondii for other animals, especially for carnivores but also for humans. However, EBH may also be a host of minor importance as in the case of HEV. The continuous surveillance of hare populations will enable the collection of information on the population health status and the pathogens currently circulating in the area posing risk for wildlife, domestic animals and humans. The possible live animal translocations of infected hares, the fact that this species acts as a host of vectors (fleas, ticks, mosquitoes and sandflies) and the prey of carnivores and omnivores that travel in great distances getting into contact with domestic animals and humans, further highlights the need to be included in surveillance studies. Besides, the hunter-harvested EBH population is an excellent indicator for recent pathogen transmission due to its short lifespan.

Antimicrobial practices among small animal veterinarians in Greece: a survey.

BACKGROUND: The inappropriate use of antibiotics is a major issue in clinical practice in Greece with serious implications for public health and animal health. The purpose of the present study was to provide a first insight into the use of antibiotics by small animal practitioners in Greece and assess their compliance with general rules for the rational use of antibiotics. This is the first survey of its kind in Greece. METHODS: A questionnaire was designed to collect basic information on the use of antibiotics by pet veterinarians. The questionnaire was sent to a total of 70 veterinarians mainly operating in the region of Attica, a region that comprises almost 50% of the Greek population and where veterinarians are engaged solely in small animal practice. The questionnaire consisted of 37 closed questions dealing with various aspects on the use of antibiotics. RESULTS: The majority of practitioners report cases where the pet owner initiated antibiotic treatment without veterinary prescription. Almost every clinician reported owner-compliance challenges. Regarding microbiological analysis, 73% of respondents initiate empirical treatment while waiting for laboratory results or use antibiogram only when the treatment is unsuccessful. Eighty-eight per cent declared to use antimicrobials postoperatively in clean surgical procedures. Different types of antimicrobials and treatment durations than the ones proposed by guidelines on rational use of antibiotics are preferred for various organ systems e.g. in urinary and gastrointestinal infections. CONCLUSIONS: Our findings suggest the need for guidelines on antibiotic use in small animal practice in Greece, and the deployment of systematic surveillance on antimicrobials use and resistance to inform the initial choice of antibiotics upon local antimicrobial resistance profiles. Targeting the other end of the problem, pet owners, our findings indicate the need to educate them on the rational use of antibiotics and, critically, stop antibiotic availability without prescription.

Investigation of the Role of the European Brown Hare in the Epidemiology of Bacterial Zoonotic Pathogens: A Serological and Molecular Survey in Greece.

The aim of this study was to investigate the occurrence of Bartonella spp, Brucella spp, Coxiella burnetii, and Francisella tularensis in European Brown hares (Lepus europaeus) hunter harvested during 2-year hunting periods in northern and central Greece. Serum samples were examined for the presence of IgG antibodies by using an immune fluorescence test and/or an enzyme-linked immunosorbent assay. PCR was used to detect Bartonella spp DNA in blood samples and Brucella spp, C. burnetii, and F. tularensis DNA in liver samples. Antibodies against Bartonella spp were detected in 12 hares (12/105); whereas none of the hares examined was seropositive for Brucella spp, C. burnetii, and F. tularensis. The presence of Bartonella spp, Brucella spp, C. burnetii, and F. tularensis DNA was not detected in the samples examined. This study did not provide any evidence that the European Brown hare is involved in the epidemiology of Brucella spp, C. burnetii, and F. tularensis in Greece. However, our results suggest that this species is exposed to Bartonella spp, which gives the impetus for further investigation of its role as another host of this bacterium.

Phylogenetic Analysis of Bird-Virulent West Nile Virus Strain, Greece.

We report the full polyprotein genomic sequence of a West Nile virus strain isolated from Eurasian magpies dying with neurologic signs in Greece. Our findings demonstrate the local genetic evolution of the West Nile virus strain responsible for a human disease outbreak in the country that began in 2010.

Serological and molecular investigation of selected parasitic pathogens in European brown hare (Lepus europaeus) in Greece: inferring the ecological niche of Toxoplasma gondii and Leishmania infantum in hares.

The occurrence of infection or exposure to Toxoplasma gondii, Neospora caninum, and Leishmania infantum was investigated in European brown hares (Lepus europaeus, EBH) hunter-harvested over two consecutive hunting seasons in northern and central Greece. Geographical information system was used along with the ecological niche model to define the geographical distribution of seropositive hares relative to environmental parameters and to identify high-risk areas for hare exposure. Molecular analysis showed that 3.8% and 9.6% of the examined hares were infected with N. caninum and L. infantum, respectively, while, 5.7%, 0.95%, and 12.4% of the hares tested positive for the presence of antibodies against T. gondii, N. caninum, and L. infantum respectively. None of the examined hares was polymerase chain reaction-positive for T. gondii. Mixed exposure against both T. gondii and L. infantum was found in 2.9% of the hares examined. Rainfall indices and land uses significantly influenced the exposure of hares to T. gondii and L. infantum. This is the first molecular and serological survey of protozoan pathogens in EBH in Greece. Furthermore, we report the environmental parameters related to hare seropositivity and present a risk map for hare exposure to T. gondii and L. infantum in northern and central Greece. The ecological niches of T. gondii and L. infantum in the hares presented herein could be applied to other regions with similar environmental and climatic conditions.

Histograms of Complete Blood Counts in Dogs: Maximizing Diagnostic Information.

Histograms, which are an integral part of the automated complete blood count, are now available through most of the automatic hematology analyzers used in veterinary clinical practice. Data concerning the size and number of blood cells are graphically presented in histograms, and their variations are also illustrated. Important information that is not apparent from numerical results are sometimes provided by histograms. Histograms are also referred to as frequency distribution curves and are essentially graphs resulting from the placement of the sizes of cells on the x-axis and the number of cells on the y-axis. Typically, automated analyzers provide histograms for each class of blood cells, that is, for erythrocytes, leukocytes, and platelets. Thus, when the erythrocyte histogram shows asymmetry with a right shift, it means the size of the erythrocytes is greater than normal (macrocytosis); when it presents a left shift, the size of the erythrocytes is less than normal (microcytosis). When two peaks are found in the curve, two populations of erythrocytes coexist, as in the case of a blood transfusion or therapeutic response. In the leukocyte histogram, three peaks are found: the closest to the y-axis (left) corresponds to the lymphocytes, the middle to the monocytes, and the right to the polymorphonuclear cells (neutrophils, eosinophils, and basophils). Finally, in platelet histogram, asymmetry with a right shift suggests the presence of giant platelets or schistocytes. Although the histogram is not recommended as a stand-alone test, it allows the practitioner to observe abnormalities in the distribution curve that correspond to abnormalities in the size or number of cells, and to quickly make diagnostic or therapeutic decisions that are particularly important in emergencies.

Environmental parameters as risk factors for human and canine Leishmania infection in Thessaly, Central Greece.

Thessaly, Central Greece, is an endemic area for leishmaniasis with higher incidence rate during the last years. We herein investigated the geographical distribution of human leishmaniasis cases and Leishmania infected dogs in relation to environmental parameters to identify high-risk areas. All the human leishmaniasis cases (n = 82) reported to Hellenic Centre for Disease Control and Prevention from 2007 to 2014 and 85 Leishmania polymerase chain reaction positive dogs were included in this study. To analyse the data geographical information system (GIS) together with the Ecological Niche Model (ENM) were used. The most important findings of the study were: (i) Central plain of Thessaly together with the coast line and the western and eastern lowlands were identified as high-risk geographical areas. (ii) The highest percentage of the high-risk areas was found in low altitude (<200 m above sea level) and in irrigated and cultivated agricultural areas. (iii) A total of 20% of the human settlements was found in high-risk areas. (iv) The maximum temperature of the warmest month contributes the highest per cent to define both environmental niche profiles for humans and dogs. (v) The ENM could be a useful tool for the epidemiological study of leishmaniasis. Spatial analysis may allow the design of entomological studies and identify target population in order to implement preventive measures.

Diagnostic performance of a rapid in-clinic test for the detection of Canine Parvovirus under different storage conditions and vaccination status.

Canine parvovirus (CPV) is one of the most common causes of acute haemorrhagic enteritis in young dogs, while clinical diagnosis is often indecisive. The aim of our study was to evaluate the diagnostic accuracy of an in-clinic rapid test in the detection of CPV infection in dogs. To this end, we compared the Rapid Diagnostic Kit of Canine Parvovirus, Coronavirus and Rotavirus antigen (Quicking(®)) to PCR, which is considered as the most reliable diagnostic method. A total of 78 duplicated faecal samples were collected from diarrhoeic dogs. Vaccination history within a month prior to the onset of diarrhoea was reported for 12 of the sampled dogs. The rapid diagnostic test was performed in 23 of the faecal samples directly, while the rest were placed into a sterile cotton tipped swab suitable for collection and transportation of viruses (Sigma Σ-VCM(®)) and stored at -20 °C. The sensitivity of the Quicking rapid diagnostic test compared to PCR in the total number of samples, in samples from non-vaccinated dogs and in samples tested directly after collection were 22.22% (95% CI: 13.27-33.57%), 26.67% (95% CI: 16.08-39.66%) and 76.47% (95% CI: 50.10-93.04%) respectively, while the specificity of the test was 100% in any case. In conclusion, negative results do not exclude parvoenteritis from the differential diagnosis, especially in dogs with early vaccination history, but a positive result almost certainly indicates CPV infection. An improved sensitivity may be expected when the test is performed immediately.