Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

Association of lameness with milk yield and lactation curves in Chios dairy ewes.

The objective of the study was twofold: (i) to quantify the differences in daily milk yield (DMY) and total milk yield (TMY) between lame and non-lame dairy ewes and (ii) to determine the shape of lactation curves around the lameness incident. The overall study was a prospective study of lameness for the surveyed sheep population, with a nested study including the selection of matching controls for each lame ewe separately. Two intensively reared flocks of purebred Chios ewes and a total of 283 ewes were used. Data, including gait assessment and DMY records, were collected on a weekly basis during on-farm visits across the milking period. A general linear model was developed for the calculation of lactation curves of lame and non-lame ewes, whereas one-way ANOVA was used for the comparisons between lame ewes and their controls. Lameness incidence was 12.4 and 16.8% on Farms A and B, respectively. Average DMY in lame ewes was significantly lower (213.8 g, P < 0.001) compared with the rest of the flock, where DMY averaged 1.340 g. The highest DMY reduction in lame ewes was observed during the week 16 of the milking period (P < 0.001), whereas the reduction of DMY, for lame ewes, remained significant at P < 0.001 level from week 8 to week 28 of milking. Comparisons between lame and controls revealed that at the week of lameness diagnosis a significant DMY reduction (P ≤ 0.001) was observed in lame ewes (about 32.5%), which was maximised 1 week later (35.8%, P ≤ 0.001) and continued for several weeks after recovery, resulting in 19.3% lower TMY for lame ewes for the first 210 d of the milking period (P < 0.01). Moreover, at flock level, TMY for non-lame and lame ewes, as calculated by the general linear model, was 318.9 and 268.0 kg, respectively. The results of this study demonstrate evidence of significant financial losses in dairy sheep due to lameness which, however, need to be accurately estimated in further, more detailed, analyses.

Spatial Distribution of Dermanyssus gallinae Infestations in Greece and Their Association with Ambient Temperature, Humidity, and Altitude.

Dermanyssus gallinae, the poultry red mite (PRM), is the most prevalent and harmful ectoparasite of laying hens globally. Although prevalence and risk factor studies can help veterinarians make decisions regarding farm treatments, relevant data are scarce. The present study investigated the prevalence and infestation severity of PRM in poultry farms across Greece and examined potential risk factors. AviVet traps were used to sample 84 farms (51 backyard, 33 industrial) over three years. Farm altitude, temperature, humidity, region, and production systems were assessed as potential risk factors with chi-square tests, initially for all the studied farms and then exclusively for backyard farms. The overall prevalence was 75.0% and was higher in backyard farms (80.4%) compared with industrial ones (66.7%), varying regionally from 66.7 to 90.9%. Altitude and temperature were not significant risk factors, but farms with humidity <60% had a lower infestation risk. Infestation severity did not significantly differ by risk factors. The poultry red mite is highly prevalent across Greek poultry production systems and regions. In the future, global warming, reduced acaricide options, and a ban on cage systems will all threaten a wider spatio-temporal distribution of the PRM, justifying the urgent need for effective monitoring and control methods to protect hen production and welfare and workers' health.

Prediction of carcass traits in fattening Chios and Serres lambs using real-time ultrasonography and live body weight measurements pre-slaughter.

The objective of this study was to assess the capability of predicting carcass traits and meat cuts weights, in fattening lambs of indigenous Greek dairy sheep breeds, using ultrasound measurements and live body weight measurements pre-slaughter. A total of 187 lambs of Chios and Serres breeds were involved in the study. Body condition score, live body weight (LBW), and ultrasound measurements of Longissimus lumborum muscle depth (LMD) and subcutaneous fat thickness (SFT) at the lumbar region were recorded pre-slaughter. After slaughter, the carcasses were classified using five-degree grading systems for muscle development and fat deposition, while hot (HCW) and cold carcass (CCW) and meat cuts weights were measured. The statistical analyses included descriptive statistics and linear regression models to estimate the fixed effects of sex and the covariances of LBW, BCS, and ultrasound measurements on the studied traits. High R2 values (0.60 ≤ R2 ≤ 0.92) were observed in the models predicting HCW, CCW, forequarter, leg chump on shank off, the short loin, the eye of the short loin, and foreshank weights. Among the models estimated LMD, SFT, and LBW as significant predictors, the ones predicting hot and cold carcass weights, the short loin, the eye of the short loin, and the eye of the rack weights were successfully validated. Other models including BCS, LBW, sex, and either one or none of the ultrasonography measurements as predictors were also validated and presented.

Beyond Borders: Dirofilaria immitis Infection in Dogs Spreads to Previously Non-Enzootic Areas in Greece-A Serological Survey.

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

Genetic Characterization of Small Ruminant Lentiviruses Isolated from Dairy Sheep in Greece.

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains.

Seroepidemiology of Maedi-Visna in Intensively Reared Dairy Sheep: A Two-Year Prospective Study.

The objective of this study is to prospectively evaluate the seroepidemiology of maedi-visna (MV) infections in intensively reared dairy sheep. A total of 407 purebred Chios and Lacaune ewes from four farms were surveyed for two consecutive years and were serologically tested semiannually with an indirect ELISA at pre-mating and pre-lambing. The farms' structure and management practices were similar and animal traits (age, breed, and production stage) were recorded. Based on the serological status, morbidity frequency measures were estimated, and ewes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or as animals with an intermittent presence of antibodies. During the study, period seroprevalence, incidence rate, and cumulative incidence were 84.8%, 33.6 new cases per 100 sheep-semesters, and 64.2%. Point-seroprevalence ranged from 48.5% to 96.0% among the studied farms and sampling occasions, and they increased by age. Increased morbidity frequency measures indicate the significance of horizontal transmission in intensive dairy sheep farms. A remarkable percentage of infected animals seroreverted (8.1%) or presented an intermittent presence of antibodies (10.3%) during the study, confirming the risk of misdiagnosis in cross-sectional studies and in the currently implemented testing and elimination programs. The serological patterns observed in our study need to be considered when studying MV epidemiology and for the designing of efficient MV elimination programs.

A Review of Toxoplasma gondii in Animals in Greece: A FoodBorne Pathogen of Public Health Importance.

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

Leishmaniosis 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.

A Longitudinal Cohort Study of Risk Factors Associated with Small Ruminant Lentivirus Seropositivity in Intensively Reared Dairy Ewes in Greece.

A two-year longitudinal cohort study was conducted on a total of 407 purebred Chios and Lacaune ewes from four intensive dairy sheep farms to assess potential risk factors for small ruminant lentiviruses (SRLVs) seropositivity. Ewes were serologically tested semiannually at pre-mating and pre-lambing, and their age, breed, and body condition score (BCS) were recorded. Εwes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or animals with an intermittent presence of antibodies. Mixed binary logistic regression models were used to estimate the adjusted relative risks of the studied risk factors for (i) the individual ewes' seropositivity, (ii) the manifestation of specific serological patterns, and (iii) the occurrence of seroconversion and seroreversion incidents. Increased age was associated with seropositivity and constantly seropositive status (p < 0.001 in both cases). On the other hand, age was negatively associated with constantly seronegative pattern, seroconversion incident, and the intermittent presence of antibodies (p < 0.05 in all cases). Moreover, breed was recognized as a risk factor: Lacaune ewes demonstrated increased seropositivity, whereas Chios ewes were more likely to demonstrate an intermittent presence of antibodies (p < 0.01 in both cases). Seropositive status (p < 0.001), seropositivity in animals with an intermittent presence of antibodies (p = 0.001), and seroconversion incidents (p < 0.001) were significantly increased at pre-lambing compared to pre-mating. The risk factors recognized in our study contribute to a better understanding of SRLVs epidemiology and the evidence-based designation of SRLVs' control programs in intensive dairy sheep farms in Greece.

In Vitro Acaricidal Activity of Silver Nanoparticles (AgNPs) against the Poultry Red Mite (Dermanyssus gallinae).

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

Circulation of Pestiviruses in Small Ruminants from Greece: First Molecular Identification of Border Disease Virus.

The incidence of small ruminant pestivirus infections in Greece remains unknown as they have not been diagnosed in the country since 1974 when the most recent Border Disease Virus (BDV) outbreak was reported. The objective of our study was to explore the possible occurrence of pestiviral infections among sheep and goat farms in Greece and to further determine the variants of major concern. Thus, serum samples were collected from 470 randomly selected animals belonging to 28 different flocks/herds. ELISA on p80 antibody revealed the existence of seropositive animals in four out of the 24 studied sheep flocks, whereas all the goats in the four studied herds were seronegative. Viral RNA and antigens were detected in two sheep out of the four seropositive flocks by RT-PCR and ELISA, respectively. Sequencing and phylogenetic analysis showed that the newly identified Greek variants were closely related to the strains of the BDV-4 genotype. One of the BDV-positive sheep demonstrated the diagnostic profile of a persistently infected (PI) animal, providing additional information regarding the source of the infection. This is the first molecular identification of BDV isolates in Greece. Our findings indicate that BDV infections are likely to remain undiagnosed, highlighting the need for further epidemiological studies and active surveillance programs to determine the prevalence and impact of BDV infections on a countrywide level.

Point-of-Care Diagnostics for Farm Animal Diseases: From Biosensors to Integrated Lab-on-Chip Devices.

Zoonoses and animal diseases threaten human health and livestock biosecurity and productivity. Currently, laboratory confirmation of animal disease outbreaks requires centralized laboratories and trained personnel; it is expensive and time-consuming, and it often does not coincide with the onset or progress of diseases. Point-of-care (POC) diagnostics are rapid, simple, and cost-effective devices and tests, that can be directly applied on field for the detection of animal pathogens. The development of POC diagnostics for use in human medicine has displayed remarkable progress. Nevertheless, animal POC testing has not yet unfolded its full potential. POC devices and tests for animal diseases face many challenges, such as insufficient validation, simplicity, and portability. Emerging technologies and advanced materials are expected to overcome some of these challenges and could popularize animal POC testing. This review aims to: (i) present the main concepts and formats of POC devices and tests, such as lateral flow assays and lab-on-chip devices; (ii) summarize the mode of operation and recent advances in biosensor and POC devices for the detection of farm animal diseases; (iii) present some of the regulatory aspects of POC commercialization in the EU, USA, and Japan; and (iv) summarize the challenges and future perspectives of animal POC testing.

Repeatability of Health and Welfare Traits and Correlation with Performance Traits in Dairy Goats Reared under Low-Input Farming Systems.

The objectives of the study were to estimate the repeatability of health and welfare traits and investigate their association with performance in three breeds of dairy goats reared under low-input farming systems in Greece. A total of 1210 goats of Eghoria (n = 418), Skopelos (n = 429), and Damascus (n = 363) breeds were assessed. Udder health, parasitic resistance, welfare, milk yield and quality, and body condition score were recorded monthly for two milking periods. Udder health records included somatic cell count (SCC) and total viable count (TVC). Based on combinations of SCC and TVC and thresholds set at >106 cells/mL and >2 × 104 cfu/mL, respectively, additional udder health phenotypes were defined. Parasitism included myiasis, tick infestation, gastrointestinal nematode (GIN) and cestode faecal egg count (FEC), and lungworm faecal larval count (FLC). Infection with each of the endoparasites was defined based on FEC/FLC. Welfare assessment parameters included the presence of ear and horn injuries, ocular and nasal discharge, body and udder abscesses, injury and lesions on the skin of different regions, diarrhoea, hernias, overgrown hooves, arthritis, lameness, and udder asymmetry. Trait repeatability and animal correlations were estimated. Significant (p < 0.05) repeatability was reported for all udder health and most welfare traits in all breeds, GIN and cestode FEC, and GIN and lungworm infection in Eghoria, and myiasis in Skopelos. Correlations of health and most of welfare traits with performance were non-significant or favourable. Overall, results demonstrate potential to improve health and welfare of the studied breeds without compromising performance.

Deltamethrin Application on Pre-Weaned Calves Improves Feed Consumption, Stress and Fatigue Status under Heat Stress Conditions.

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

Association of Hard Ticks (Ixodidae) Infestation with Milk Production and Udder Health of Extensively Reared Dairy Goats.

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

Feline Toxoplasmosis in Greece: A Countrywide Seroprevalence Study and Associated Risk Factors.

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

The Effects of Replacing Soybean Meal with Rapeseed Meal, Cottonseed Cake, and Fava Beans on the Milk Yield and Quality Traits in Milking Ewes.

The replacement of soybean meal (SBM) from intensively reared dairy sheep diets has emerged as a significant challenge for sustainable production. However, the effects of this replacement on milk production have not been sufficiently elucidated. The objective of this study was to prospectively assess the effects of replacing SBM with a mixture of alternative protein sources on the milk yield (MY) and the milk quality traits (MQT) in intensively reared dairy sheep. A total of 112 multiparous, purebred milking ewes of the Chios and Frizarta breeds, from two intensive dairy sheep farms, were involved in the study, postweaning, and were assigned to either the control (CR) or the experimental ration (ER) group. In the ER, 3/4 of the SBM was replaced by a mixture of rapeseed meal, cottonseed cake, and fava beans, producing a ration of a similar nutritional value. MY, MQT, and body condition scores were recorded for each individual ewe monthly for a period of 4 months during lactation. The experimental ration was associated with beneficial effects on daily and 100-day fat yields and on the electrical conductivity of milk as an improved udder health status indicator, with no adverse effects on any of the rest of the studied milk production traits.