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.

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.

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.

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.

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.

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.

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.

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.

Angiotensin II Blood Serum Levels in Piglets, after Intra-Dermal or Intra-Muscular Vaccination against PRRSV.

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces apoptosis in different organs. Angiotensin II (Ang II) is the main effector of the renin-angiotensin system and participates in apoptosis. Thus, this study aimed to investigate changes in piglet serum Ang II levels following intradermal (ID) and intramuscular (IM) vaccination with a commercial PRRS modified live virus (MLV) vaccine. The trial was conducted in a commercial pig farm, including 104 piglets which were randomly allocated to four groups: Group A-Porcilis PRRS ID, Group B-Porcilis PRRS IM, Group C-Diluvac ID and Group D-Diluvac IM. The study piglets were either vaccinated or injected at 2 weeks of age and they were tested by qRT-PCR for PRRSV and by ELISA for Ang II. The results indicated differences in viremia of tested piglets at 7 weeks of age, while piglets at 10 weeks of age were all found qRT-PCR positive for PRRSV. In addition, significant differences were noticed in Ang II in 7-week-old piglets. In conclusion, the present study provides evidence that ID vaccination induces less tissue damage, based on the lower measurements of Ang II in the serum of ID vaccinated piglets.

Redox Status of Pregnant Ewes after Vaccination against Clostridial Diseases.

The redox status shortly after the vaccination of pregnant ewes is rather unexploited. Thus, the present study was designed to evaluate the fluctuation of redox status after the administration of the annual booster dose of a polyvalent clostridial vaccine in pregnant ewes, 3 to 4 weeks before lambing, with or without a simultaneous injection of Vit E/Se. In total, 24 pregnant Lacaune ewes 3-4 weeks before lambing were randomly allocated into four equal groups: the V (vaccinated with a polyvalent clostridial vaccine), VE (vaccinated and injected IM with Vit E/Se), E (injected IM with Vit E and Se), and C (neither vaccinated nor injected with Vit E/Se). The study period lasted for 21 days, starting on the day of administration. Four redox biomarkers, the antioxidant capacity (TAC), the thiobarbituric acid reactive substances (TBARS), the reduced glutathione (GSH), and the catalase (CAT) were evaluated in blood samples collected from all ewes before the injections (0 h) and then at 12 (12 h), 24 (D1), and 48 h (D2), and thereafter on days 4 (D4), 6 (D6), 10 (D10), 14 (D14), and 21 (D21). The results reveal that the TAC was the only biomarker evaluated that was significantly affected by group and significantly lower in vaccinated animals (V and VE groups) compared to non-vaccinated (E and C groups). The absence of an increase in the TBARS values after vaccination in groups V and VE indicates the absence of significant oxidative stress. Overall, it can be assumed that annual booster immunizations against clostridial diseases do not impose acute oxidative stress on pregnant ewes in the last month of pregnancy.

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.

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.

Investigation of Serotype Prevalence of Escherichia coli Strains Isolated from Layer Poultry in Greece and Interactions with Other Infectious Agents.

Colibacillosis is the most common bacterial disease in poultry and it is caused by avian pathogenic Escherichia coli (APEC), which is assigned to various O-serogroups. Previous studies have shown that APEC strains are more often related to certain O-serogroups such asO78, O2 and O1. E. coli has been reported to act either as a primary or secondary agent in complicating other infections. The aim of this study was to investigate the occurrence of and characterize the O-serogroups of E. coli strains isolated from commercial layer and layer breeder flocks showing macroscopic lesions of colibacillosis and increased or normal mortality in Greece. Furthermore, we attempted to assess the interaction between infectious agents such as Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), infectious bronchitis (IBV) and infectious laryngotracheitis (ILT) with E. coli infections in layer flocks with increased mortality. Our study revealed that in addition to the common serogroups (O78, O2), many other, and less common serogroups were identified, including O111. The O78, O111 and O2 serogroups were frequently detected in flocks with lesions of colibacillosis and increased mortality whereas O2, O88 and O8 were reported more commonly in birds with colibacillosis lesions but normal mortality rates. These data provide important information for colibacillosis monitoring and define preventative measures, especially by using effective vaccination programs because E. coli vaccines are reported to mainly offer homologous protection. Finally, concerning the association of the four tested infectious agents with E. coli mortality, our study did not reveal a statistically significant effect of the above infectious agents tested with E. coli infection mortality.

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.

Effects of Phenolic Phytogenic Feed Additives on Certain Oxidative Damage Biomarkers and the Performance of Primiparous Sows Exposed to Heat Stress under Field Conditions.

The aim of this study was to investigate the effects of two commercial phenolic phytogenic feed additives (PFAs) on sows under heat stress conditions of high summer temperatures for seven days before and seven days after the farrowing. The PFA-1 product was a mixture based on the plants Emblica officinalis, Foeniculum vulgare, Citrus sinensis and nut fiber, while the PFA-2 product was a mixture based on plants Andrographis paniculata, Glycyrrhizia glabra, Tinospora cordifolia and nut fiber. A total of 48 primiparous sows were divided into three groups: T1-control group: regular gestation (GF) and lactation feed (LF); T2 group: regular GF and LF supplemented with PFA-1; T3 group: regular GF and LF supplemented with PFA-2. Each sow in the T2 and T3 groups received 5 g daily of the PFA-1 and PFA-2 product, respectively, for seven days before and seven days after the farrowing. Blood samples were collected from all groups 24 h after farrowing. Thiobarbituric acid--reactive substances (TBARS) and protein carbonyl (CARB) concentrations were determined in the sow plasma. The body condition scoring (BCS) and the backfat of sows on the farrowing and weaning days along with reproductive parameters and litter characteristics were recorded. The highest number of stillborn piglets and the largest interval from weaning to estrus were observed in the T1 group. The lowest number of alive 24 h after birth and weaning piglets and the lowest BCS and backfat at weaning were also recorded in the T1 group. TBARS and CARB concentrations were significant higher in the T1 group compared to all other groups. In conclusion, the use of phenolic PFAs seems to reduce oxidative damage caused by heat stress and ameliorate performance in primiparous sows.

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.

Investigation of Fas (APO-1)-Related Apoptosis in Piglets Intradermally or Intramuscularly Vaccinated with a Commercial PRRSV MLV.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces apoptosis through the activation of death receptors, including cell-surface Fas receptor. The aim of this study was to investigate the impact of intradermal (ID) and intramuscular (IM) vaccination with a commercial PRRSV-modified live vaccine in piglets on Fas-related apoptosis. The study included 104 suckling piglets from a commercial farrow-to-finish pig farm, suffering from positive unstable PRRSV status. Animals were assigned in four groups: group A-Porcilis PRRS ID-vaccinated pigs, group B-Porcilis PRRS IM-vaccinated pigs, group C-Diluvac ID adjuvant-administered pigs, and group D-Diluvac IM adjuvant-administered pigs. Vaccines were administered at 2 weeks of age. Blood samples were collected from the same pigs at 4, 7, and 10 weeks of age. Sera were examined by quantitative real-time reverse transcription-PCR (qRT-PCR) for PRRSV and by ELISA for soluble Fas (sFas). At 4 weeks of age, all groups were negative qRT-PCR for PRRSV; at 7 weeks only group A was negative; and at 10 weeks all groups were positive. sFas was significantly increased in groups C (4 vs. 7, 4 vs. 10, and 7 vs. 10 weeks) and D (7 vs. 10 weeks). Significant differences among groups were noticed only at 10 weeks (A vs. C, A vs. D, B vs. C, B vs. D). A significant positive and moderate correlation between PRRSV viral load and Fas level was observed. In unvaccinated piglets, increased serum sFas levels reveal apoptotic suppression compared with vaccinated piglets. In the latter, vaccine-derived antibodies limit the infection and may attribute to the reduced Fas expression, suggesting a weak induction of lymphocyte-mediated cytotoxicity.

Evaluation of Intradermal PRRSV MLV Vaccination of Suckling Piglets on Health and Performance Parameters under Field Conditions.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in weaning and growing pigs. A vaccination against PRRSV is one of the most important control measures. This trial aimed to evaluate the effect of the intradermal (ID) administration of a PRRSV-1 modified live virus (MLV) vaccine in comparison to the intramuscular (IM) administration on the piglets' health and performance. A total of 187 suckling piglets of a PRRSV-positive commercial farrow-to-finish farm were assigned to four groups: group A­PRRSV ID, group B­PRRSV IM, group C­control ID, and group D­control IM. At 2 weeks of age, all the study piglets were either vaccinated with a PRRSV-1 MLV vaccine or injected with the vaccine adjuvant (controls). The collected blood serum samples were tested by ELISA and qRT-PCR. The side effects, body weight (BW), average daily gain (ADG), mortality rate, and lung and pleurisy lesions scores (LLS, PLS) were also recorded. The ELISA results indicated that the vaccination induced an important seroconversion at 4 and 7 weeks. Significant differences in the qRT-PCR results were noticed only at 10 weeks in group A vs. group C (p < 0.01) and group B vs. group C (p < 0.05). High viral loads, as evidenced by the qRT-PCR Ct values, were noticed in animals of both non-vaccinated groups at 7, 10, and 13 weeks. An ID vaccination has a positive impact on the BW at the piglets' slaughter, while both an ID and IM vaccination had a positive impact on the ADG. The mortality rate was lower in vaccinated groups at the finishing stage. The LLS and PLS were significantly lower in the vaccinated groups. In conclusion, our study demonstrated that the ID vaccination of suckling piglets with a PRRSV-1 MLV vaccine has a positive effect on the piglets' health and performance, including an improved BW and a lower LLS and PLS index at their slaughter, as well as a decreased mortality rate at the growing/finishing stage.