Alternative protein sources in aquafeed: Current scenario and future perspectives.

Fish meal represents the main protein source for most commercially farmed aquatic species, as it is characterized by high nutritional value and lack of anti-nutritional factors. However, its availability and the market price have been recognized as serious problems at least for over a decade, making it necessary to search for non-conventional protein sources, as an alternative to fish meals. This review aims to comprehensively examine and critically revise the use of fish meal and all alternative protein sources explored to date on the health, welfare, and growth performance of the major aquatic species commercially interesting from a global scenario. The investigation revealed that the inclusion levels of the different protein sources, plant- and animal-derived, ranged from 10 to 80 % and from 2 to 100 % respectively, in partial or complete replacement of fish meal, and generated positive effects on health, welfare, growth performance, and fillet quality. However, the results showed that above a certain level of inclusion, each protein source can negatively affect fish growth performance, metabolic activities, and other biological parameters. Moreover, it is likely that by mixing different protein sources, the combination of each ingredient causes a synergistic effect on the nutritional properties. Therefore, the future of aquatic feed formulation is expected to be based on the blend of different protein sources. Overall, the analysis highlighted the need for additional research in the field of replacing fish meals with new protein sources, given that many knowledge gaps are still to be filled on aquatic species, which deserve to be investigated.

Content of Toxic Elements (Arsenic, Cadmium, Mercury, Lead) in Eggs from an Ethically Managed Laying Hen Farm.

Domestic chicken farming has been promoted and spread in several Italian municipalities and worldwide as an aid to the self-consumption of domestically produced food. This study investigated the levels of four toxic elements (As, Cd, Hg, and Pb) in eggs from an ethical laying hen farm, comparing the element concentrations with those possibly present in supermarket eggs. A total of 201 eggs, 141 from the farm and produced by different hen genotypes, and 60 from the supermarket, were collected. The levels of the toxic elements were evaluated in the yolk, albumen, and eggshells of all eggs. The results show that the supermarket eggs' yolk and albumen were more contaminated with lead, compared to the rural eggs. Contrarily, the mean content of arsenic was higher in the albumen and eggshells of the rural eggs, compared to the supermarket eggs. The cadmium content was below the LOQ (0.005 mg/kg) in all samples. The mercury content was below or around the LOQ in all rural eggs. Overall, the supermarket egg albumens were significantly more contaminated than the rural ones. No significant differences were found in quality parameters for both types of eggs. The toxic element values that were detected were in line with other studies in the literature. However, despite the concentrations found not representing a risk to the consumers' health, the results of this study raise a potential food safety issue, and it would be desirable to set specific MRLs for eggs for consumers' protection.

Systemic Oxidative Stress in Subacute Stroke Patients Undergoing Rehabilitation Treatment.

The imbalance in oxidative stress in acute stroke has been extensively studied; on the contrary, its investigation in the subacute phase is limited. The aim of this study was to analyse the variation in the systemic oxidative status in subacute post-stroke patients before (T0) and after a six-week rehabilitation treatment (T1) and to investigate the relationship between systemic oxidative status and rehabilitation outcomes. We enrolled 109 subjects in two different centres, and we analysed their serum hydroperoxide levels (d-ROMs), biological antioxidant power (BAP), thiol antioxidant components (-SHp), and relative antioxidant capacity (OSI and SH-OSI indices). Activity of Daily Living (ADL), hand grip strength, and walking endurance were evaluated using the modified Barthel Index, the Hand Grip test, and the 6-min walk test, respectively. At T0, most of the patients showed very high levels of d-ROMs and suboptimal levels of the BAP, OSI, and SH-OSI indices. Comparing the T1 and T0 data, we observed an improvement in the rehabilitation outcomes and a significant decrease in d-ROMs (549 ± 126 vs. 523 ± 148, p = 0.023), as well as an improvement in the OSI and SH-OSI indices (4.3 ± 1.3 vs. 4.7 ± 1.5, p = 0.001; 11.0 ± 0.4 vs. 1.2 ± 0.4, p < 0.001). In addition, significant correlations were seen between the oxidative stress parameters and the rehabilitation outcomes. These results suggest monitoring the systemic oxidative stress status in post-stroke patients in order to plan a tailored intervention, considering its relationship with functional recovery.

Muscle quality improvement in subacute post-stroke patients after rehabilitation: Usefulness of segmental phase angle from bioelectrical impedance analysis.

BACKGROUND & AIMS: During stroke rehabilitation, the whole-body Phase Angle (PhA) from Bioelectrical Impedance Analysis (BIA) is measured to assess whole-body muscle quality, which reflects cellular integrity and function. The segmental BIA is a valuable method for assessing the body composition of specific body segments, such as the arms, legs, and hemisoma. After a stroke insult, patients frequently experience hemiparesis, and segmental PhA from segmental BIA appears to be an appropriate parameter for examining the muscle quality of affected and unaffected limbs separately. This study aims to investigate whether segmental PhA is more informative than whole-body PhA in (a) assessing the deterioration of muscle quality in post-stroke patients and (b) monitoring its recovery following rehabilitative treatment. METHODS: This longitudinal study recruited subacute post-stroke patients who were admitted to our rehabilitation center. At admission, demographic, anamnestic, and clinical information, such as the presence of comorbidities, were recorded. BIA was used to evaluate the whole-body PhA and segmental PhA of the affected and unaffected hemisoma, arms, and legs at admission (T0) and after a six-week rehabilitation program (T1). The modified Barthel Index (mBI), Fugl-Meyer Assessment for the Upper Extremity (FMA-UE), and Motricity Index of the Upper (MI-UE) and Lower (MI-LE) Extremities were evaluated at T0 and T1 to determine the patient's Activity of Daily Living (ADL) performance, upper limb motor performance, and upper and lower limb muscle strength, respectively. RESULTS: We evaluated segmental and whole-body BIA in 70 subacute post-stroke patients (women n = 34, ischemic n = 56, mean age 70 ± 11) at T0 and T1. Whole-body PhA values of the patients were below the normal range. Considering segmental data, the affected hemisoma, arm, and leg had considerably lower PhA values as compared to the unaffected body segments. Furthermore, at T1, the PhA values of all affected body segments improved, while those of the unaffected ones and whole-body PhA did not. At both T0 and T1, the segmental PhA values of the affected body segments showed to be related with all clinical outcome measures, while whole-body PhA correlated only with mBI. CONCLUSIONS: This study emphasizes the significance of measuring segmental PhA in hemiparetic subacute stroke patients undergoing rehabilitation treatment. Segmental PhA is a more accurate parameter to evaluate rehabilitation treatment in patients with hemiparesis because it can distinguish affected from unaffected body segments, hence facilitating accurate monitoring of muscle quality improvements resulting from a rehabilitation program.

Restoring Activity of Milk Thistle (Silybum marianum L.) on Serum Biochemical Parameters, Oxidative Status, Immunity, and Performance in Poultry and Other Animal Species, Poisoned by Mycotoxins: A Review.

Grains are major farm animals' diet ingredients, and one of the main concerns is when are mycotoxin (MyT) contaminated, compromising animals' health, performance, and product safety. Among the natural phytocompounds that are being used to prevent MyT damage, silymarin (SIL), an extract from the seed of the milk thistle (MT) is a suitable candidate. This review aims to examine the scientific evidence concerning the anti-MyT toxicity effects of MT/SIL in poultry and livestock. In vitro and in vivo studies (n = 27) showed that MT whole plant, seed, or SIL-standardized extract had positive effects on animal health, performance, and restoring the hepatic activity, with a reduction of organ lesions caused by MyT. Furthermore, showed utility for combating MyT-immunodepression, improving intestinal health, and limiting the excretion of toxins residues in food of animal origin, although in some cases, MT/SIL supplementation does not produce appreciable effects. The use of MT in animal nutrition can be useful since the bioactive compounds, also if present in variable amounts, can help the animals to counteract the effects of MyT. The use of the phytoextract of SIL, due to its cost, can be useful if it reported the specific bioactive compounds, recognize for their pharmacological activities.

Use of Milk Thistle in Farm and Companion Animals: A Review.

Milk thistle, Silybum marianum, is a medicinal plant grown for its bioactive compounds with well-documented antioxidant and hepatoprotective properties. Milk thistle has a well-established pharmacological reputation for treatments of human liver disease, but it is also used in animals. This review summarizes the experimental evidence of milk thistle's effects on animals when administered as silymarin extract (feed additive) or a feed ingredient, if administered as seed or expeller/cake with the seed residue still containing the bioactive components. The use as a feed additive or feed ingredient is motivated by the complexity of silymarin registration as a veterinary drug. In farm animals, the drug improves the animals' performance and product quality and oxidative stability, supports liver function during the productive life-cycle, improves gut-health and morphology, and can reduce intestinal pathogens. In dogs and cats, the treatment is focused on acute and chronic liver diseases including the detoxification processes and support of drug treatments including chemotherapy. In equine athletes, milk seed cake showed positive effects and a faster return of cortisol to the resting values before exercise occurred. In aquaculture, it confirms its usefulness in supporting animal health and performance. In certain studies it is not clear what has been administered, and the composition and doses are not always clearly reported. A few studies reported no effects, but none reported problems connected to milk thistle administration. However, the overall picture shows that the use of milk thistle results in improved or restored health parameters or better animal performance.

Development of Histamine in Fresh and Canned Tuna Steaks Stored under Different Experimental Temperature Conditions.

Among biogenic amines, histamine is most frequently involved in foodborne intoxication. To evaluate histamine formation in tuna, several storage conditions were reproduced. An LC-MS/MS method was used for analytical determinations. Fresh tuna samples (not contaminated and grafted with tuna muscle naturally incurred with histamine at 6000 mg/kg) were stored at 4, 12, and 20 °C, and daily samples were collected for 6 days. The development of histamine was observed only in grafted tuna samples. At 4 °C, histamine formation progressed from 12.8 mg/kg (day 1) up to 68.2 mg/kg (day 6). At 12 °C, higher concentrations developed (23.9 mg/kg on day 1 up to 2721.3 mg/kg on day 6) relative to 20 °C (from 12.0 to 1681.0 mg/kg). It was found that at 4 °C, if grafted tuna was submerged in oil, histamine formation progressed more slowly. In a naturally contaminated sample, it was observed that the histamine distribution was uniform, while the normal cooking process did not affect the histamine level. Furthermore, it was found that the use of histamine-contaminated equipment for food handling may result in histamine formation in food. These results confirm the importance of implementing good hygiene practices and respecting the cold chain.

Relationship between Nutritional Status, Food Consumption and Sarcopenia in Post-Stroke Rehabilitation: Preliminary Data.

After a stroke, patients can suffer from sarcopenia, which can affect recovery. This could be closely related to an impairment in nutritional status. In this preliminary analysis of a longitudinal prospective study, we screened 110 subjects admitted to our rehabilitation center after a stroke. We then enrolled 61 patients, who underwent a 6-week course of rehabilitation treatment. We identified a group of 18 sarcopenic patients (SG), according to the European Working Group on Sarcopenia in Older People 2 (EWGSOP2), by evaluating muscle strength with the handgrip test, and muscle mass with bioelectrical impedance analysis (BIA). With respect to the non-sarcopenic group (NSG), the SG at admission (T0) had worse muscle quality, according to the BIA-derived phase angle, and a lower score of MNA®-SF. In contrast to the NSG, the SG also exhibited lower values for both BMI and the Geriatric Nutritional Risk Index (GNRI) at T0 and T1. Moreover, 33% of the SG had a major risk of nutrition-related complications (GNRI at T0 < 92) and discarded on average more food during the six weeks of rehabilitation (about one-third of the average daily plate waste). Of note is the fact that the Barthel Index's change from baseline indicated that the SG had a worse functional recovery than the NGS. These results suggest that an accurate diagnosis of sarcopenia, along with a proper evaluation of the nutritional status on admission to rehabilitation centers, appears strictly necessary to design individual, targeted physical and nutritional intervention for post-stroke patients, to improve their ability outcomes.

Evaluation of the Acaricidal Effectiveness of Fipronil and Phoxim in Field Populations of Dermanyssus gallinae (De Geer, 1778) from Ornamental Poultry Farms in Italy.

The poultry red mite Dermanyssus gallinae is the most important blood-sucking ectoparasite in egg-laying hen facilities. The aim of this study was to evaluate the in vitro acaricidal activity of different concentration of authorized (phoxim, ByeMite®, 500 mg/mL) and unauthorized (fipronil, Frontline® 250 mg/100 mL spray) molecules on 14 field isolates of D. gallinae collected from different ornamental poultry farms from different Italian regions. The sensitivity test was performed by contact exposure to four concentrations of each insecticide diluted at 1:5 (10,000-2000-400-80 ppm for phoxim, 500-100-20-4 ppm for fipronil) on a filter paper. The effectiveness of the treatment was significantly (p < 0.0001) associated with the dose of the pesticide used. Considering the mean lethality, phoxim has greater efficacy compared to fipronil (p < 0.001). A great variability of lethality rate was observed with the increase in fipronil dilution; conversely, for phoxim, some outliers were observed, particularly in one farm, suggesting the hypothesis that a certain degree of resistance in the mite population could occur possibly as a consequence of the continual contact with the molecule. This underlines the importance of the use of licensed products administered at correct dosages and the need for alternative molecules to avoid the onset of drug resistance phenomena.

Supplementation of Boswellia serrata and Salix alba Extracts during the Early Laying Phase: Effects on Serum and Albumen Proteins, Trace Elements, and Yolk Cholesterol.

Extracts from Boswellia serrata (Bs) and Salix alba (Sa) are used as supplements in poultry feed. The aims of this research were to study the possible effects of dietary supplementation with Bs and Sa extracts on serum and albumen proteins, zinc and iron, and yolk cholesterol content in Leghorn hens during the critical phase of the onset of laying. A total of 120 pullets, 17 weeks of age, were assigned to two groups (control (C) and treated (T), n = 60 each). The T group received a supplement containing Bs (5%) and Sa (5%) for 12 weeks. The study lasted 19 weeks. Serum proteins were fractionated using agarose gel electrophoresis (AGE) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Trace elements were determined in serum using atomic absorption spectrometry, and yolk cholesterol was determined using a colorimetric test. No significant differences were observed between control and supplemented hens for the analyzed biochemical indices. Moreover, the supplementation with phytoextracts did not negatively affect the physiological variations in serum proteins; therefore, it can be safely used as a treatment to prevent inflammatory states at onset and during the early laying phase.

Occurrence of Histamine in Commercial Cat Foods under Different Storage Conditions.

In fish-based foods, one of the effects of inappropriate storage can be the formation of biogenic amines. Among these, histamine is considered one of the most toxic. The purpose of the present study is to assess the occurrence of histamine in fish-based pet foods, and to evaluate the changes in histamine content during storage at different temperatures. For the analysis, an LC-MS/MS method was used. Fifty-eight pet foods were purchased, and an aliquot of them was analyzed just after opening the packages. Histamine was detected in 12 samples and concentrations ranged from 1.5 to 30.1 mg/kg. The remaining of each positive sample was divided into seven subsamples. One of them was used as a control sample and kept at -20 °C, while the other six were exposed to different environmental conditions. Samples exposed to room temperature showed no significant changes in histamine levels, while samples exposed to high temperatures showed significant increases in histamine content. Finally, samples exposed to refrigerator temperature showed a slight decrease in histamine levels. Under the experimental conditions, the EU limit of 100 mg/kg established for fishery products was never exceeded. These results seem to indicate a low risk of histamine intoxication in cats fed fish-based pet food.

Influence of Dietary Supplementation with Boswellia serrata and Salix alba on Performance and Blood Biochemistry in Free-Range Leghorn Laying Hens.

This study was conducted to evaluate the safety and the beneficial effects of dietary supplementation with Boswellia serrata (Bs) and Salix alba (Sa) in Leghorn hens during the critical pre-laying and laying phases. A total of 120 pullets, 17 weeks of age, were assigned to two groups (Control­C; Treated­T, n = 60 each). For 12 weeks, the T group received a diet supplemented with 0.3% of dry extracts of Bs (5%) and Sa (5%). The study lasted 19 weeks. Productive performance, serum analytes, H/L ratio, IgA and anti-IBV antibodies were investigated. Water intake was significantly higher, while body and egg weight was significantly lower for the T group (p < 0.05). No other differences were detected in performance parameters, serum analytes, IgA and H/L ratio excluding t0, with a significantly (p < 0.05) higher H/R ratio and higher titers of anti-IBV antibody for the T group. Overall, the data obtained in this study show that the supplementation with Bs and Sa was safe and resulted in an increase in water consumption, a decrease in egg weight, and a sedative effect in the hens. In the future, it would be interesting to test this supplement in hens reared on intensive farms.

Occurrence of Ochratoxin A in Different Types of Cheese Offered for Sale in Italy.

The detection of Ochratoxin A (OTA) in the milk of ruminants occurs infrequently and at low levels, but its occurrence may be higher in dairy products such as cheese. The aim of this study was to investigate the presence of OTA in cheeses purchased in the metropolitan city of Bologna (Italy) and the surrounding area. For the analysis, a LC-MS/MS method with a limit of quantification (LOQ) of 1 µg/kg was used. OTA was detected in seven out of 51 samples of grated hard cheese (concentration range 1.3-22.4 µg/kg), while it was not found in the 33 cheeses of other types which were also analysed. These data show a low risk of OTA contamination for almost all types of cheese analysed. To improve the safety of cheese marketed in grated form, more regulations on cheese rind, which is the part most susceptible to OTA-producing moulds, should be implemented or, alternatively, producers should consider not using the rind as row material for grated cheese. It would be interesting to continue these investigations particularly on grated hard cheeses to have more data to update the risk assessment of OTA in cheese, as also suggested by EFSA in its 2020 scientific opinion on OTA.

Use of Flubendazole and Fenbendazole for Treatment of Lung Severe Infection by the Gapeworm Cyathostoma bronchialis (Nematoda: Syngamidae) in Branta hutchinsii, Anser indicus and B. leucopsis Exotic Geese: An Interesting Case.

A 6-year-old female goose (Branta hutchinsii) from a group of ornamental exotic geese was found dead due to severe respiratory failure, followed by emission of haemorrhagic sputum and blood clots from the beak and nostrils, and then collapse. At necropsy, the cause of death was attributed to a total of 76 helminth parasites found in the trachea and lungs, then identified as Cyathostoma bronchialis. The flock was initially treated by feed with flubendazole (1200 g/1000 kg/feed) for 7 consecutive days but, at the reappearance of the respiratory symptoms 10 days after, the animals were given fenbendazole by drinking water (300 mg/L) for 7 consecutive days, but at the reappearance of the respiratory symptoms 10 days after, the animals were given fenbendazole via drinking water (300 mg/L) for 7 consecutive days. Despite these treatments, the respiratory symptoms continued to relapse 10-15 days after the end of drug administration. In the literature, there are no data regarding drugs for the treatment of C. bronchialis infestations in geese, and the use of these drugs in exotic birds occurs as "off-label" use. This case study provides information on C. bronchialis life cycle, which is still poorly studied and poorly documented today. In particular, the case provides useful suggestions for evaluating an appropriate protocol for the treatment of C. bronchialis in geese.

Effect of Beta 3 Adrenoreceptor Modulation on Patency of the Ductus Arteriosus.

ß3-adrenoreceptor (ß3-AR), a G-protein coupled receptor, has peculiar regulatory properties in response to oxygen and widespread localization. ß3-AR is expressed in the most frequent neoplasms, also occurring in pregnant women, and its blockade reduces tumor growth, indicating ß3-AR-blockers as a promising alternative to antineoplastic drugs during pregnancy. However, ß3-AR involvement in prenatal morphogenesis and the consequences of its blockade for the fetus remain unknown. In this study, after the demonstrated expression of ß3-AR in endothelial and smooth muscle cells of ductus arteriosus (DA), C57BL/6 pregnant mice were acutely treated at 18.5 of gestational day (GD) with indomethacin or with the selective ß3-AR antagonist SR59230A, or chronically exposed to SR59230A from 15.5 to 18.5 GD. Six hours after the last treatment, fetuses were collected. Furthermore, newborn mice were treated straight after birth with BRL37344, a ß3-AR agonist, and sacrificed after 7 h. SR59230A, at the doses demonstrated effective in reducing cancer progression (10 and 20 mg/kg) in acute and chronic mode, did not induce fetal DA constriction and did not impair the DA ability to close after birth, whereas at the highest dose (40 mg/kg), it was shown to cause DA constriction and preterm-delivery. BRL37344 administered immediately after birth did not alter the physiological DA closure.

Occurrence of Aflatoxin M1 (AFM1) in Donkey Milk Collected in Northern Italy.

Aflatoxin M1 (AFM1) is a well-known mycotoxin that can be found in the milk of animals that have ingested feed contaminated with aflatoxin B1 (AFB1). In Italy, the development of donkey farms is mainly due to growing request of donkey milk, which is considered an incomparable substitute for human mother's milk for its chemical composition and organoleptic characteristics. The aim of this study was to assess the occurrence of AFM1 in donkey milk produced in a farm in Northern Italy, also in view of the few data available about the presence of this mycotoxin in this type of milk. Therefore, 63 milk samples were collected and analyzed using a fast and sensitive HPLC and fluorescence detection (FLD) method previously optimized and validated. None of the milk samples collected were found to be contaminated at a level above the limit of quantification (LOQ) (0.0125 ng/mL), while only one sample showed traces of the mycotoxin at a concentration between the limit of detection (LOD) and LOQ (0.0044 ng/mL), well below the legal limit established for infant milk and follow-on milk (0.025 ng/mL). These results are in line with those of the few similar surveys carried out on donkey milk and seem to indicate a low risk of AFM1 contamination for this food.

Molecular detection and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae strains in backyard poultry in Italy.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) represent the most important avian Mycoplasma species in the poultry industry, causing considerable economic losses. In Italy, the presence of MG or MS has been investigated especially in commercial poultry farms. To our knowledge, no systematic investigations on MG or MS presence using highly specific diagnostic assays have been performed in backyard poultry. The aim of this study was to detect and molecularly characterize MG and MS strains in 11 backyard poultry flocks located in different regions of Italy. Tracheal swabs were collected and DNA was extracted. For MS, a PCR targeting a vlhA gene fragment was performed, and typing and subtyping was attempted. The presence of MG was investigated by a screening PCR, then MG typing by gene-targeted sequencing (GTS). All the amplicons were sequenced, then MG and MS dendrograms were constructed. All the flocks examined resulted Mycoplasma positive: 5 out of 11 (45.45%) were MG and MS positive, 3 (27.27%) were MG positive, and the remaining 3 (27.27%) were MS positive. The MS detections were assigned to types C, D, and F. All strains of type D belonged to subtype D1 and 2 unknown subtypes were identified. A MS sequence showed peculiar characteristics, which did not allow assignment to a known MS type or subtype. MG GTS analysis identified 6 MG strains belonging to 5 subclusters circulating in Italian backyards chicken flocks. The results of this study provide evidence of a risk for commercial poultry farms, especially in areas where backyard and commercial farms are close, suggesting the implementation of biosecurity measures.

Assessment of Ochratoxin A Exposure in Ornamental and Self-Consumption Backyard Chickens.

Ochratoxin A (OTA) is a mycotoxin that may be present in various food and feed of plant and animal origin, including chicken meat. In Italy, backyard poultry farming is rather widespread. Animals are raised for meat, eggs and for ornamental purpose, and they are often fed with home-made diets not subject to official controls. The purpose of this study was to evaluate exposure of ornamental and backyard chickens to OTA using biliary ochratoxin A as a biomarker. Therefore, bile samples, in addition to kidney, liver and muscle, were collected from 102 chickens reared in 16 farms located in 6 Italian regions. High-performance liquid chromatography method and fluorimetric detection (HPLC-FLD) analysis were carried out firstly on bile from all animals, and OTA was detected in 12 chickens (concentration range 3.83-170.42 µg/L). Subsequently, the kidneys of these chickens were also analysed, and the mycotoxin was not detected. The analytical detection limits (LODs) of OTA in bile and kidney were 2.1 µg/L and 0.1 µg/kg, respectively. In conclusion, these animals were exposed to OTA but their meat can be considered safe, given that this mycotoxin, if present, concentrates highest in kidneys. Biliary ochratoxin A confirms its use as a valid biomarker to assess exposure of poultry to OTA.

Molecular characterization of the meq gene of Marek's disease viruses detected in unvaccinated backyard chickens reveals the circulation of low- and high-virulence strains.

Marek's disease (MD) is an important lymphoproliferative disease of chickens, caused by Gallid alphaherpesvirus 2 (GaHV-2). Outbreaks are commonly reported in commercial flocks, but also in backyard chickens. Whereas the molecular characteristics of GaHV-2 strains from the commercial poultry sector have been reported, no recent data are available for the rural sector. To fill this gap, 19 GaHV-2 strains detected in 19 Italian backyard chicken flocks during suspected MD outbreaks were molecularly characterized through an analysis of the meq gene, the major GaHV-2 oncogene. The number of four consecutive prolines (PPPP) within the proline-rich repeats of the Meq transactivation domain, the proline content, and the presence of amino acid (aa) substitutions were determined. Phylogenetic analysis was performed using the Maximum Likelihood method. Sequence analysis revealed a heterogeneous population of GaHV-2 strains circulating in Italian backyard flocks. Seven strains, detected from birds affected by classical MD, showed a unique meq isoform of 418 aa with a very high number of PPPP motifs. Molecular and clinical features are suggestive of a low oncogenic potential of these strains. The remaining 12 strains, detected from flocks experiencing acute MD, transient paralysis, or sudden death, had shorter Meq protein isoforms (298 or 339 aa) with a lower number of PPPP motifs and point mutations interrupting PPPP. These features allow us to assert the high virulence of these strains. These findings reveal the circulation of low- and high-virulence GaHV-2 strains in the Italian rural sector.

Chlamydiosis in Backyard Chickens (Gallus gallus) in Italy.

Until recently, Chlamydia psittaci was considered to be the only etiological agent of avian chlamydiosis, but two new avian species, Chlamydia gallinacea and Chlamydia avium, have recently been described in poultry and pigeons or psittacine birds, respectively. The aim of this study was to explore the occurrence of C. psittaci and C. gallinacea in backyard chickens in Italy. Cloacal swabs were taken from 160 asymptomatic chickens reared in 16 backyard farms. Samples were tested for C. psittaci and C. gallinacea by specific real-time polymerase chain reaction assays, with 24 (15%) of the 160 chickens resulting positive for C. gallinacea. To attempt chlamydial isolation, new samples were obtained from two farms harboring a high prevalence (60% and 70%, respectively) of C. gallinacea-positive chickens. In total, eight C. gallinacea and one C. psittaci isolates were successfully recovered from 13 chickens. C. gallinacea was confirmed to be the endemic chlamydial species in chickens, with a high ompA intraspecies diversity. The presence of viable C. psittaci and C. gallinacea demonstrated by isolation from chickens in backyard farms poses a potential public health problem.