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.

Repeatability and reproducibility of microscopic examination of adhesive tape strip cytology slides for the quantification of Malassezia spp. in canine skin.

BACKGROUND: The optimal microscopic magnification and number of optical fields of adhesive tape strip cytological slides that should be examined when searching for Malassezia yeasts on canine skin are unknown. OBJECTIVES: To determine the optimal magnification and the minimum number of optical fields that should be examined to maximise intraobserver repeatability and interobserver reproducibility. MATERIALS AND METHODS: Seven experienced examiners counted, twice, the number of yeasts in 10, 20, 30, 40 and 50 optical fields of 40 slides at ×400 and ×1000 magnification. RESULTS: The number of yeasts per unit surface area was significantly higher at ×1000 compared to ×400 magnification. Repeatability and reproducibility for counting the yeasts was very poor. CONCLUSIONS AND CLINICAL RELEVANCE: Adhesive tape strip cytological slides should be examined microscopically for Malassezia spp. at ×1000 magnification. The repeatability of this examination for counting the yeasts is poor.

Contexte - Le grossissement microscopique optimal et le nombre de champs optiques des lames cytologiques de bandes adhésives à examiner lors de la recherche de levures Malassezia sur la peau de chien sont inconnus. Objectifs - Déterminer le grossissement optimal et le nombre minimal de champs à examiner pour maximiser la répétabilité intra-observateur et la reproductibilité inter-observateur. Matériels et méthodes - Sept examinateurs expérimentés ont compté, deux fois, le nombre de levures dans 10, 20, 30, 40 et 50 champs de 40 lames aux grossissements ×400 et ×1 000. Résultats - Le nombre de levures par unité de surface était significativement plus élevé au grossissement ×1 000 par rapport au grossissement ×400. La répétabilité et la reproductibilité du comptage des levures étaient très médiocres. Conclusions et pertinence clinique - Les lames cytologiques de bandes adhésives doivent être examinées au microscope pour Malassezia spp. à un grossissement ×1 000. La répétabilité de cet examen de comptage des levures est faible.

Introducción- se desconoce el aumento microscópico óptimo y el número de campos ópticos de los portaobjetos citológicos en tiras de cinta adhesiva que deben examinarse al buscar levaduras Malassezia en la piel canina. Objetivos- determinar el aumento óptimo y el número mínimo de campos ópticos que deben examinarse para maximizar la repetibilidad intraobservador y la reproducibilidad interobservador. Materiales y métodos- siete examinadores experimentados contaron dos veces el número de levaduras en campos ópticos de 10, 20, 30, 40 y 50 de 40 portaobjetos con aumentos de x ×400 y ×1000. Resultados- el número de levaduras por unidad de superficie fue significativamente mayor con un aumento de ×1000 en comparación con un aumento de ×400. La repetibilidad y reproducibilidad para contar las levaduras fue muy pobre. Conclusiones y relevancia clínica - Los portaobjetos citológicos en tiras de cinta adhesiva deben examinarse microscópicamente para detectar Malassezia spp. con un aumento de ×1.000. La repetibilidad de este examen para contar las levaduras es pobre.

Contexto - A ampliação microscópica ideal e o número de campos ópticos das lâminas citológicas de fita adesiva que devem ser examinados nas pesquisas de leveduras do gênero Malassezia em cães são desconhecidos. Objetivos - Determinar a magnificação ideal e o número mínimo de campos ópticos que devem ser examinados para maximizar a repetibilidade intraobservador e a reproducibilidade interobservador. Materiais e métodos - Sete examinadores experientes contaram duas vezes o número de leveduras em 10, 20, 30, 40 e 50 campos ópticos de 40 lâminas nas magnificações de x400 e x1000. Resultados - O número de leveduras por unidade de área de superfície foi significativamente maior em x1000 em comparação com a ampliação de x400. A repetibilidade e a reprodutibilidade para a contagem de leveduras foi muito pobre. Conclusões e relevância clínica - Lâminas de citologia por fica adesiva devem ser examinadas microscopicamente para Malassezia spp a uma magnificação de x1.000. A repetibilidade deste exame para contagem de leveduras foi pobre.

Toxoplasma gondii Infection in Swine: Implications for Public Health.

Toxoplasmosis, due to Toxoplasma gondii, is a parasitic disease with global importance. Among livestock, chronic T. gondii infection has been reported in higher rates in pigs and small ruminants, but with subclinical infections in case commonly encountered in pigs. Seroprevalence in the global pig population ranges according to the age or species of pigs, geographical distribution, production programs, and systems. Generally, T. gondii infections are noticed in low prevalence rates in conventional pig farms with high hygiene standards. In contrast, higher prevalence is common on free-ranging farms, outdoor or backyard small pig fams, as well as in farmed or hunted wild boars. The T. gondii average worldwide seroprevalence in pigs is reported to be 13% in Europe, 21% in Africa, 25% in North America, 21% in Asia, and 23% in South America. Human toxoplasmosis outbreaks have been correlated with the consumption of raw or undercooked meat, especially from infected pigs or wild boars, as well as of contaminated drinking water. The risk of infection in processed pork products is lower compared with fresh pork, as meat processing can reduce or inactivate T. gondii tissue cysts. Hence, the prevalence of T. gondii in the pig population may be a useful indicator of the risk of human toxoplasmosis associated with the consumption of pork products. The lack of obligatory screening methods at farm level for the detection of antibodies in farmed animals or the viable T. gondii in carcasses at slaughterhouse level increases the risk of contaminated pork or meat products. For this reason, the application of biosecurity and surveillance programs at farm level is very important to prevent a T. gondii infection.

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.

Phlebotomine sandflies and factors associated with their abundance in the leishmaniasis endemic area of Attiki, Greece.

Leishmaniasis is a parasitic disease of animals and humans caused by several Leishmania species and transmitted by phlebotomine sandflies. The aim of the present study was to identify the species of field collected phlebotomine sandflies in the endemic area of the Attiki during 4 consecutive years, to isolate the Leishmania parasites from the infected sandflies, and identify possible factors associated with sandfly abundance in the area. A total of 542 trappings were made in 46 collection sites, in purely urban areas, periurban areas, and purely rural areas in Attiki. Out of the 3254 sandflies trapped, 1448 (44.43%) were female and 241 (16.64%) of the females were blood fed while Leishmania infantum DNA was detected in the 0.41% of them. Regarding sandfly species, the most prevalent was Phlebotomus tobbi (41.52%) followed by Sergentomyia minuta (27.44%), P. neglectus (14.83%), P. simici (11.08%), P. papatasi (3.68%), P. similis (0.89%), and P. alexandri (0.56%). Periurban areas were found to have the highest density of sandfly populations.

Urban stray cats infested by ectoparasites with zoonotic potential in Greece.

A large population of stray cats is encountered in many urban areas sharing the same environment with people, usually being in a close direct contact with them. A variety of ectoparasites can infest such cats, causing mild dermatological abnormalities to more severe systemic disorders. In order to determine the extent of which stray cats carry ectoparasites, particularly those of zoonotic potential, 341 stray cats originating from the urban area of Thessaloniki, Greece, were examined between 2012 and 2014. The signalment of each cat such as gender, hair length, and roughly estimated age were recorded. From a total of 341 examined stray cats, 127 (37.24%; 95% confidence interval (CI) 32.14-42.64) were infested with at least one of the following ectoparasites: mites-Otodectes cynotis (15.8%), Notoedres cati (2.35%), Cheyletiella blakei (2.05%); fleas-Ctenocephalides felis (24.3%); ticks-Rhipicephalus sanguineus (0.88%); and lice-Felicola subrostratus (0.59%). A significantly higher prevalence of ectoparasites was observed in long-haired individuals (p < 0.00001). The above ectoparasites may either cause or transmit diseases not only in cats but also in humans Therefore, antiparasitic control should be included in stray cat neutering campaigns while public health education for taking preventive measures will decrease the risk of transmission to humans.

Conservation considerations for a management measure: an integrated approach to hare rearing and release.

Wildlife managers are challenged with the task of deciding whether a management measure is appropriate or not, and furthermore they have to convince others about the merits of their decision. Population decline of some hare species (genus Lepus) has resulted in their Red Listing and conservation measures are being undertaken. Release or restocking is a frequent measure in some countries, and thousands of hares are released annually, mainly for hunting purposes. These hares can be obtained by either intensive or extensive rearing or translocation of the wild animals. Each method entails difficulties and different survival rates in the wild. Survival rates in the wild are low for hares intensively reared in cages but are higher for hares reared extensively in enclosures and wild translocated hares. The benefits of the hare release practice are significantly lower than the action's implementation cost. Hare releases have not increased significantly the wild hare population or the hunting harvest in areas where the practice has been applied. The risk of genetic and evolutionary degradation and pathogen transmission is possible in wild populations. The need for wise management of this practice is evident and the term 'Permitted Transferring Units' should be introduced to denote regions where hares should not be transferred for rearing and release.

Evaluation of indirect immunofluorescence antibody test and enzyme-linked immunosorbent assay for the diagnosis of infection by Leishmania infantum in clinically normal and sick cats.

Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P < 0.001). The diagnostic sensitivity of all serological tests, using PCR as the gold standard, was very low, but ELISA and IFAT for anti-Leishmania IgM had 100% specificity. The diagnostic sensitivity of all serological tests could not be improved by changing the cut-off values. Seropositivity for Leishmania spp was not associated with signalment, living conditions, season of sampling and health status of the cats or with seropositivity to feline leukemia virus, feline immunodeficiency virus, feline coronavirus, Toxoplasma gondii and Bartonella henselae. In conclusion, because of their low sensitivity and very high specificity two of the evaluated serological tests (ELISA for anti-Leishmania IgG and IFAT for anti-Leishmania IgM) may be useless as population screening tests but valuable for diagnosing feline infection by L. infantum.

Subacute Rumen Acidosis in Greek Dairy Sheep: Prevalence, Impact and Colorimetry Management.

Subacute rumen acidosis (SARA) is a current issue in intensive livestock farming, and it is particularly associated with feeding high-concentrate diets. This study investigated the prevalence and impact of SARA in forty-two Greek dairy sheep flocks by recording rumen pH, milk composition, and milk yield over a period of nine months. Moreover, it explored the use of computerized rumen colorimetry as a management and diagnostic tool for SARA in dairy sheep. In culled ewes, computerized rumen mucosal colorimetry was applied, and rumen wall samples taken for histological examination. SARA cases were identified in 19 farms (45%, n = 42). Farms with SARA cases had lower milk fat levels, while milk yield and milk protein levels did not differ based on the SARA status of the farms. In culled ewes, rumen color was significantly associated with the flock's SARA status, and affected ewes showed increased thickness in non-keratinized and total epithelial layers. It was concluded that computerized rumen mucosal colorimetry in aged, culled ewes shows promise as an indicator, post mortem, of SARA present in dairy sheep flocks whose impact can be minimized by making significant changes in dietary management.

Comparative Evaluation between Visual and Automated Dipstick Urinalyses in Dogs.

Urine test strips are commercially available and can be assessed with semi-automated analyzers or by visual assessment. This study aimed to compare the visual and automated evaluations of dipstick variables in canine urine samples. One hundred and nineteen urine samples were evaluated. Automated analysis was performed on a veterinary urine analyzer URIT-50Vet (URIT Medical Electronic) with UC VET13 Plus strips. Multistix 10 SG dipsticks (Siemens Healthcare GmbH, Erlangen, Germany) were used for visual evaluation, along with a refractometer (Clinical Refractometer Atago T2-Ne, Atago Co., Tokyo, Japan) for urine specific gravity measurements. A linear relationship was observed between the pH measurements (p = 0.2) of the two methods; the Passing-Bablok procedure was valid since neither proportional nor systematic significant errors were observed. Comparing the two methods, the correlation for urine specific gravity was poor (p = 0.01, CI 0.667-1.000). Moderate agreement was demonstrated for proteins (κ = 0.431), bilirubin (κ = 0.434) and glucose (κ = 0.450). Agreement was substantial for blood (κ = 0.620) and poor for leukocytes (κ = 0.100). Poor agreement was observed for ketones (κ = -0.006). Apart from the pH analysis, visual and automated dipstick urinalyses should not be used interchangeably. Multiple urine samples obtained from the same dog during the day should be evaluated using the same method to overcome erroneous results.

Association of Paraoxonase-1 and NT-proBNP with Clinical, Clinico-Pathologic and Echocardiographic Variables in Dogs with Mitral Valve Disease.

The objective of the present study was to measure the concentration of Paraoxonase-1 (PON-1) and N-terminal-prohormone-B-type natriuretic peptide (NT-proBNP), in the serum of dogs with degenerative Mitral Valve Disease (MVD), in order to identify their association with the clinical stage and specific clinico-pathologic and echocardiographic findings.Eighty dogs diagnosed with MVD and staged according to the ACVIM (American College of Veterinary Internal Medicine) consensus statement (B1, B2, C and D), based on their clinical, radiographic, and echocardiographic findings, were included in the study. NT-proBNP was measured only in stage B1 and B2 dogs. Clinical stage did not have a significant effect on PON-1 concentrations (p = 0.149), but NT-proBNP levels were lower in B1 dogs (p = 0.001). A significant correlation between PON-1 and total plasma proteins (p = 0.001), albumin (p = 0.003) and white blood cell count (p = 0.041) was detected, whereas there was no significant correlation (p = 0.847) between PON-1 and NT-proBNP concentrations. PON-1 showed a significant but weak negative correlation with normalized left ventricular internal diameter at diastole (LVIDdn) (p = 0.022) and systole (LVIDsn) (p = 0.012), as well as mitral valve E to A wave velocity ratio (MV E/A) (p = 0.015), but not with Left Atrial to Aortic root ratio (LA/Ao) (p = 0.892) or fractional shortening (FS%) (p = 0.944). PON-1 seems to be an insensitive marker of clinical stage and disease severity in MVD, but can be indicative of some clinico-pathological and echocardiographic changes. NT-proBNP changes are independent of oxidative stress.

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.

Effects of Injectable Administration of Dexamethasone Alone or in Combination with Vitamin E/Se in Newborn Low Birth Weight Piglets.

Increasing litter size may lead to low-birth-weight piglets (LBW) and further negative long-term effects. This study aimed to evaluate the effects of intramuscular administration (IM) of dexamethasone (Dexa) alone or in combination with vitamin E/Se on LBW piglets during the early postnatal period. The study included a total of 100 LBW piglets that were divided into 5 groups (20 LBW piglets per group) and treated with IM Dexa alone or in combination with vitamin E/Se (Vit E/Se) after birth as follows: (a) Group A-Cont: Control group, (b) Group B-Dexa1: Dexa on D1 (1st day of life), (c) Group C-Dexa3: Dexa on D1, D2, D3 (D2: 2nd day of life, D3: 3rd day of life), (d) Group D-Dexa + VitE/S1: Dexa + Vit E/Se on D1, and (e) Group E-Dexa + VitE/S3: Dexa + Vit E/Se (IM) on D1, D2, D3. Body weight (BW) and the Average Daily Weight Gain (ADWG) were recorded for all piglets on days 1, 7, 14, and 25, and vitality score (VS) was recorded on days 1, 2, 3, 4, and 14. A significant increase in BW and ADWG in Group E-Dexa + VitE/S3 and a significant reduction in Group C-Dexa3 were noticed in comparison to other groups. VS in groups Group B-Dexa1 and Group C-Dexa3 were significantly lower in comparison to other groups. Furthermore, piglets of Group C-Dexa3 had a significantly higher frequency of clinical findings compared to other groups. In conclusion, the administration of Dexa and vitamin E/Se combined after the birth of LBW piglets for 1-3 days has beneficial effects on their growth and survival scores.

Feeding Flaxseed and Lupins during the Transition Period in Dairy Cows: Effects on Production Performance, Fertility and Biochemical Blood Indices.

Flaxseed and lupin seed were offered as an alternative dietary approach in dairy cows, through the partial substitution of soybean meal. Milk production and fertility traits were investigated. A total of 330 animals were allocated into two groups, treated (n = 176) and control (n = 154). From each group, 30 animals were selected for hematological and cytological studies. The experimental feeding period lasted for 81 days (25 days prepartum and 56 days postpartum). The control ration (group C) contained corn, barley, soybean meal, rapeseed cake, corn silage and lucerne hay; whereas, in the treatment group (group T), 50% of the soybean meal was replaced by an equal mixture of flaxseed and lupins. The two rations were formulated to be isonitrogenous and isoenergetic. Milk samples were analyzed for chemical composition, somatic cell count (SCC) content and total colony forming units (CFU). Blood samples were collected, and serum was analyzed for non-esterified fatty acids (NEFA), acute phase proteins (haptoglobin and serum amyloid) and lipid oxidation indices, namely thiobarbituric-acid-reactive substances (TBARS) and catalase activity. To assess polymorphonuclear neutrophils (PMN) numbers, endometrial samples from each cow were collected on days 21 and 42. No difference was recorded between groups in milk yield (p > 0.05). In multiparous cows, NEFA (mMol/L) concentrations were significantly lower in group T than in group C on day 14 (p > 0.009) and on day 42 (p = 0.05), while no difference was detected in the group of primiparous cows. At all time points, serum TBARS and catalase values were similar in both groups (p > 0.05). Multiparous cows in group T expressed the first postpartum estrus and conceived earlier than cows in group C (p ≤ 0.05). Between days 21 to 42 postpartum, the PMN reduction rate was higher in group T animals (p ≤ 0.05). Acute phase protein levels were in general lower in group T animals, and at specific time points differed significantly from group C (p ≤ 0.05). It was concluded that the partial replacement of soybean meal by flaxseed and lupins had no negative effect on milk yield or milk composition, and improved cow fertility; which, along with the lower cost of flaxseed and lupins mixture, may increase milk production profitability.

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.

Investigation of comorbidities in dogs with leishmaniosis due to Leishmania infantum.

In endemic areas, dogs with leishmaniosis due to Leishmania infantum frequently have comorbidities, including mostly neoplastic, infectious, and parasitic diseases. The aim of this study was to compare the prevalence of comorbidities among dogs that are not infected by L. infantum, dogs that are infected but do not present leishmaniosis, and dogs with leishmaniosis, and to examine if certain comorbidities are independent risk factors for the infection by L. infantum and/or for the development of canine leishmaniosis (CanL). A total of 111 dogs, older than 1-year and non-vaccinated against CanL, were allocated into three groups: group A (n = 18) included dogs that were not infected by L. infantum, group B (n = 52) included dogs that were infected by L. infantum but did not present CanL, and group C (n = 41) included dogs with CanL. Signalment and historical data were obtained using a structured questionnaire. Laboratory examinations included complete blood count, serum biochemistry, urinalysis, fecal parasitology, modified Knott's test, microscopic examination of capillary blood, buffy coat, lymph node, bone marrow and conjunctival smears, qualitative serology for Dirofilaria immitis, Anaplasma phagocytophilum/A. platys, Borrelia burgdorferi and E. canis, IFAT for L. infantum, ELISA for Babesia spp. and Neospora caninum, and real-time PCR for L. infantum in bone marrow, skin biopsies and conjunctival swabs. A variety of comorbidities were found in all three groups. No independent risk factors for infection by L. infantum were found. On the contrary, among dogs infected by L. infantum, being a mongrel [odds ratio (OR): 11.2], not receiving prevention for dirofilariosis (OR: 26.5) and being seropositive to N. caninum (OR: 17.1) or to Babesia spp. (OR: 37.6), were independent risk factors for presenting CanL. Although no comorbidities influence the probability of canine infection by L. infantum, certain comorbidities may be precipitating factors for the transition from the subclinical infection by L. infantum to the overt CanL.

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.

Histopathological Pulmonary Lesions in 1st-Day Newborn Piglets Derived from PRRSV-1 MLV Vaccinated Sows at the Last Stage of Gestation.

Modified live virus (MLV) vaccines for the control of porcine respiratory and reproductive syndrome virus (PRRSV) have been associated with the vertical and horizontal transmission of vaccine viruses. The present study aimed to describe pathological lung lesions in piglets born by gilts vaccinated with PRRSV-1 MLV. In total, 25 gilts were vaccinated at late gestation (100th day) and were divided into five groups according to the different vaccines (Vac) used: no vaccine-control group, Vac-1-strain DV, Vac-2-strain VP-046 BIS, Vac-3-strain 94881, Vac-4-strain 96V198. Within the first 0-9 h of the farrowing, blood samples were collected from all newborn piglets and lung samples were exanimated grossly, histopathologically and with scanning electron microscopy. PRRSV (RT-PCR-positive) and antibodies were detected in the serum of piglets from gilts vaccinated with Vac-2. In these piglets, moderate to severe interstitial pneumonia with thickened alveolar septa was noticed. Type II pneumocyte hyperplasia was also observed. The rest of the trial piglets showed unremarkable lung lesions. Phylogenetic analysis revealed the 98.7% similarity of the PRRSV field strain (GR 2019-1) to the PRRS MLV vaccine strain VP-046 BIS. In conclusion, the Vac-2 PRRSV vaccine strain can act as an infectious strain when vaccination is administrated at late gestation, causing lung lesions.

The Use of Stromal Vascular Fraction in Long Bone Defect Healing in Sheep.

The objectives of the present study were to evaluate (a) the feasibility of using stromal vascular fraction (SVF) and nanocrystalline hydroxyapatite (nHA) paste in combination for the treatment of segmental bone defect, (b) the quality of the callus produced, (c) the potential improvement of the autograft technique, and (d) the direct comparison of the biomaterial to the use of autogenous cancellous bone. Unilateral, segmental mid-diaphyseal bone defect was created on the right metatarsus of skeletally mature sheep animals (n = 24) under anesthesia (D0). Residual segments were stabilized by stainless-steel plates and appropriate screws. Defects were managed as follows: group A: use of nHA paste to filling, group B: use of autogenous bone graft mixed with nHA bone paste, placed in defect, group C: use of SVF mixed with nHA bone paste injected into defect, group D: use of bone graft and SVF with nHA paste before apposition in bone defect. SVF had been previously isolated from adipose tissue of the animals intra-operatively after digestion with collagenase solution and neutralization. Animals were evaluated clinically and by X-raying and ultrasonographic examination of the defect, at regular intervals, until D90. Ultrasonographic assessment performed along the length of the defect included calculation of the length of the bone defect and assessment of vascularization. SVF was successfully isolated from group C and D animals, with the average yield being 1.77 × 106 cells. The comparison of clinical scores (based on the 'Kaler scale') on each post-operative day indicated significant differences between the four groups on D1 to D30 (p < 0.01); the median clinical score within group A was 2.5 for D1-D30 and 1 for the entire period; respective scores for other groups were 1.5 (p = 0.07) and 0 (p = 0.033). Differences in radiographic assessment scores were significant for scores obtained on D60 (p = 0.049) and D90 (p = 0.006). There was a significant difference between the four groups in the length of the bone defect, as assessed ultrasonographically, for the entire length of the study; median values were 8, 8.5, 6, and 8 mm for groups A, B, C, and D, respectively (p = 0.008). There was a significance in the differences between median scores obtained during the histopathological examination: 2, 11, 13.5, and 12 for group A, B, C, and D (p = 0.022). There was an inverse correlation between the overall scores of histopathological evaluations and the length of the bone defect (observed on D90) (p < 0.0001) and a correlation between the overall scores and the radiographic assessment scores (obtained on D90) (p < 0.0001). This is the first study in which the efficacy of fresh autologous Stromal Vascular Fraction (SVF) from adipose tissue in enhancing bone healing in a long, weight-bearing, diaphyseal bone was evaluated. It is concluded that the lumbosacral region was an attractive site for harvesting adipose tissue, the use of SVF contributed to faster rehabilitation post-operatively, and SVF significantly enhanced bone formation; in general, the results indicated an osteogenic potential of SVF comparable to the gold standard autologous bone graft.

Serological and molecular investigation into the role of wild birds in the epidemiology of West Nile virus in Greece.

BACKGROUND: A West Nile virus (WNV) disease outbreak occurred in 2010 in northern Greece with a total of 262 laboratory-confirmed human cases and 35 deaths. A serological and molecular surveillance was conducted on samples of hunter-harvested wild birds prior to and during the outbreak. FINDINGS: Serum and tissue samples from 295 resident and migratory wild birds, hunter-harvested during the 2009-2010 and 2010-2011 hunting seasons at the epicenter of the outbreak in northern Greece, were tested for the presence of WNV-specific antibodies by immunofluorescence assay and virus neutralization test. WNV neutralizing antibodies were detected in 53 avian samples. Fourteen positive sera were obtained from birds hunter-harvested up to 8 months prior to the human outbreak. Specific genetic determinants of virulence (His249Pro NS3 mutation, E-glycosylation motif) were recognized in a WNV lineage 2 strain isolated from a hunter-harvested Eurasian magpie and a nucleotide mismatch was revealed between this strain and a mosquito WNV strain isolated one month earlier in the same area. CONCLUSIONS: This is the first report regarding exposure of wild birds to WNV prior to the 2010 outbreak, in Greece. Results provide evidence of the implication of wild birds in a local enzootic cycle that could allow maintenance and amplification of the virus before and during the outbreak. Findings of past exposure of migratory birds to WNV upon their arrival in Greece during autumn migration, suggest avian species with similar migration traits as candidates for the introduction of WNV into Greece. The possibility that an endemic circulation of WNV could have caused the outbreak, after an amplification cycle due to favorable conditions cannot be excluded.