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The possibility that Dermanyssus gallinae , the poultry red mite, could act as a vector of infectious disease-causing pathogens has always intrigued researchers and worried commercial chicken farmers, as has its ubiquitous distribution. For decades, studies have been carried out which suggest that there is an association between a wide range of pathogens and D. gallinae , with the transmission of some of these pathogens mediated by D. gallinae as vector. The latter include the avian pathogenic Escherichia coli (APEC), Salmonella enterica serovars Enteritidis and Gallinarum and influenza virus. Several approaches have been adopted to investigate the relationship between D. gallinae and pathogens. In this comprehensive review, we critically describe available strategies and methods currently available for conducting trials, as well as outcomes, analyzing their possible strengths and weaknesses, with the aim to provide researchers with useful tools for correctly approach the study of the vectorial role of D. gallinae . Graphical Abstract

Background Babesia spp. are widespread tick-borne intraerythrocytic protozoa, infecting a broad range of vertebrate hosts. Red foxes are reservoirs of Babesia vulpes, belonging to the Babesia microti -like group (clade I), and play an important role in the epidemiology of canine and wildlife babesiosis. Besides B. vulpes , another species of this genus was molecularly reported in red foxes from Israel and Iraq and provisionally named “ Babesia sp. MML-2014”; however, no morphological description of this small Babesia species was provided, preventing a proper species naming. Methods Infection with piroplasmid species was detected and described by microscopy of stained blood smears in one red fox from Southern Italy. Molecular characterization of the Babesia sp. and differentiation from B. vulpes was performed through PCR amplification of nuclear ( 18S rRNA , ITS1-5.8S-ITS2) and mitochondrial (cytochrome c oxidase subunit 1, cox1 ) gene markers, followed by DNA sequencing and phylogenetic analysis. In addition, Ixodes kaiseri ticks collected from the infected fox were screened for piroplasmids by PCR. Results Sequence comparison of piroplasmids showed 98–99% identity with the undescribed Babesia sp. MML-2014 and phylogenetic analyses confirmed that this taxon belongs to the Western group (clade III) and is differentiated by B. vulpes . Morphological and morphometric analyses further demonstrated that Babesia sp. nov. is a distinct small piroplasm and is characterized by unique Maltese cross forms. Based on the above, we named Babesia banethi sp. nov. as a new taxon. In addition, Babesia sp. nov. DNA was detected in the intestine of one engorged I. kaiseri specimen. Conclusions This study provides genetic and morphological findings of B. banethi sp. nov. A morphological description with measurements of the parasite forms in red fox erythrocytes, differential diagnosis supplemented by genetic characterization, and the deposition of the holotype in suitable collections have been made in compliance with the ICZN guidelines. Graphical Abstract

Background The life cycle of Leishmania infantum is maintained mainly in dogs in anthropogenic environments and in many other wild animals in the sylvatic cycle. The ecological plasticity of some wild canids facilitates their role as hosts for Leishmania spp. in different endemic regions. Although red foxes ( Vulpes vulpes ) frequently test positive for L. infantum in Europe, little is known about their clinical presentation, immune response, or treatment outcomes. This study investigated the prevalence, clinical, and immunological features of L. infantum infection in foxes from southern Italy, complemented by an in vitro evaluation of cytokine responses in fox macrophages. Methods Wild foxes from a wildlife rehabilitation center in southern Italy were molecularly and serologically screened for L. infantum . One sick fox underwent a complete diagnostic confirmation, treatment, and follow-up through hematological, biochemical, cytological, and molecular evaluations. In addition, peripheral blood mononuclear cells (PBMCs) isolated from a healthy fox were differentiated into macrophages and experimentally infected with L. infantum to assess early host–parasite interactions and cytokine gene expression profiles at two time points (4 h and 48 h). Results Overall, 13 out of 54 foxes (24.1%) were molecularly positive for L. infantum , with a prevalence of 25% (5/20) in necropsied animals and 22% (9/41) in live animals. One individual tested seropositive for L. infantum and Ehrlichia sp. using the SNAP Leish 4Dx ® test (2.4%, 1/41). The sick fox treated with meglumine antimoniate and allopurinol showed marked clinical and laboratory improvement. In the in vitro evaluation, the percentage of infected macrophages decreased from 32.8% at 4 h (2.25 parasites/cell) to 21.5% at 48 h (2 parasites/cell). Cytokine gene expression at 4 h and 48 h showed an increase in interleukin-6 (IL-6) (0.164–0.552) and IL-10 (0.828–4.245), stable IL-4 (0.505–0.708), a decrease in IL-12 (1.793–1.223) and IFN-γ (1.507–0.613), and consistently low TNF-α (0.377–0.411). Conclusions The present study confirmed a high L. infantum prevalence of infection in red foxes from southern Italy. Serological findings herein and in the literature, together with in vitro cytokine gene expression, suggested that most foxes may remain subclinically infected. The high molecular positivity and the synanthropic nature of red foxes reinforce their role as reservoirs of L. infantum in endemic areas. Graphical Abstract

In the poultry industry, control of the red mite D. gallinae primarily relies worldwide on acaricides registered for use in agriculture or for livestock, and those most widely used are carbamates, followed by amidines, pyrethroids and organophosphates. Due to the repeated use of acaricides--sometimes in high concentrations--to control infestation, red mites may become resistant, and acaricides may accumulate in chicken organs and tissues, and also in eggs. To highlight some situations of misuse/abuse of chemicals and of risk to human health, we investigated laying hens, destined to the slaughterhouse, for the presence of acaricide residues in their organs and tissues. We used 45 hens from which we collected a total of 225 samples from the following tissues and organs: skin, fat, liver, muscle, hearth, and kidney. In these samples we analyzed the residual contents of carbaryl and permethrin by LC-MS/MS.Ninety-one (40.4%) samples were positive to carbaryl and four samples (1.7%) were positive to permethrin. Concentrations of carbaryl exceeding the detection limit (0.005 ppm) were registered in the skin and fat of birds from two farms (p<0.01), although these concentrations remained below the maximum residue limit (MRLs) (0.05 ppm) (p<0.01). All organs/tissues of hens from a third farm were significantly more contaminated, with skin and muscle samples exceeding the MRL (0.05 ppm) (p<0.01) of carbaryl in force before its use was banned. Out of 45 chickens tested, 37 (82.2%) were found to be contaminated by carbaryl, and 4 (8.8%) by permethrin. The present study is the first report on the presence of pesticides banned by the EU (carbaryl) or not licensed for use (permethrin) in the organs and tissues of laying hens, which have been treated against red mites, and then slaughtered for human consumption at the end of their life cycle.

Background The poultry red mite Dermanyssus gallinae (De Geer, 1778) is a major ectoparasite of poultry. Infestations are found in most laying hen farms in Europe, and breeder flocks have also been reported to be affected. Mite infestation has detrimental effects on animal welfare, it causes significant economic losses, and, additionally, D. gallinae is often considered as a vector for pathogens. Despite suspicion of a close relationship between the poultry red mite and Salmonella enterica enterica serovar Gallinarum biovar Gallinarum (serovar Gallinarum), the causative agent of fowl typhoid disease (FT), there has been no definitive proof of mite-mediated transmission. Therefore, an investigation was conducted to determine if D. gallinae -mediated transmission of serovar Gallinarum could be demonstrated among four different hen groups. Methods Two groups of 8 hens (A and B) were experimentally infected with serovar Gallinarum in two isolators. After 7 days, when birds showed signs of FT, about 25,000 mites were introduced. After 3 days, mites were harvested and used to infest two other hen groups of 8 (C and D), in two separate isolators. The health status of hens was constantly monitored; detection and quantification of serovar Gallinarum were performed by PCR and qPCR from mites and organs of dead hens. The maximum likelihood estimation of the infection rate and mite vectorial capacity were calculated. Results Clinical disease was observed in groups infected with serovar Gallinarum (A and B) and in hens of groups C and D infested with mites harvested from the isolators containing groups A and B. In all four groups, serovar Gallinarum was detected from liver, spleen, ovary, and cecum of hens, thus confirming the diagnosis of FT. Mite analysis demonstrated the presence of the pathogen, with an estimated infection rate ranging between 13.72 and 55.21 infected per thousand mites. Vectorial capacity was estimated to be 73.79. Conclusions Mites harvested from birds infected with serovar Gallinarum were shown to carry the mite, and then to transfer serovar Gallinarum to isolated groups of pathogen-free birds that subsequently showed signs of FT. Mite vectorial capacity was high, demonstrating that D. gallinae should be considered an effective vector of FT.

Background Dermanyssus gallinae poses a significant global threat to poultry production by affecting animal health and welfare. Conventional acaricidal treatments are limited by the emergence of resistant mite populations and by the risk of chemical residues in eggs and meat. In this study, we evaluated an innovative control strategy based on a hydroxyethyl cellulose (HEC)- hydrogel as a delivery matrix for Eucalyptus globulus essential oil (EO) and the entomopathogenic fungus Beauveria bassiana . Mites were collected from naturally infested hen farms and divided by life stage. Bioassays were conducted under controlled laboratory conditions to compare the effects of treatments applied both with and without pre-incubation of the HEC matrix, expressed as lethal times (LT₅₀ and LT₉₀). Results The EO treatment alone or in combination with B. bassiana and without pre-incubation achieved an LT₅₀ of 7.4 ± 0.2 days in adult mites, while the B. bassiana -only treatment exhibited a slightly prolonged LT₅₀ of 8.8 ± 0.2 days. Notably, pre-incubation markedly enhanced efficacy ( p  < 0.001); the combined treatment reduced the LT₅₀ to 4.9 ± 0.2 days and the LT₉₀ to 10.5 ± 0.3 days, underscoring the synergistic action of EO and B. bassiana . Similar patterns were observed in nymphs, which generally exhibited higher lethal times compared to adults, with strong statistical support. Conclusions The findings indicate that HEC-based hydrogel enriched with E. globulus EO and/or B. bassiana conidia is efficacious against D. gallinae . The significant reduction in LT₅₀ and LT₉₀ in adult mites highlights the potential of this approach for integrated pest management. This novel formulation, by harnessing the synergistic effects of E. globosus EO and B. bassiana within a stable hydrogel matrix, may provide a tool for controlling D. gallinae in the poultry industry, thus reducing the use of synthetic acaricides. Results need to be validated under field conditions to provide a sustainable strategy for controlling red mite infestations.

Marek’s disease is an infectious disease in poultry that usually appears in neural and visceral tumors. This disease is caused by Gallid alphaherpesvirus 2 infection in lymphocytes, and its meq gene is commonly used in virulent studies for coding the key protein functional in oncogenic transformation of the lymphocytes. Although vaccines have been introduced in many countries to control its spread and are proven to be efficient, recent records show a decline of such efficiency due to viral evolution. In this study, we reviewed the outbreak of Marek’s disease in Asia for the last 10 years, together with associated meq sequences, finding a total of 36 studies recording outbreaks with 132 viral strains in 12 countries. The visceral type is the most common (13 in 16 studies) form of Marek’s disease, but additional unobserved neural changes may exist. MD induces liver lymphoma most frequently (11 in 14 studies), and tumors were also found in spleen, kidney, heart, gizzard, skin, intestine, lung, and sciatic nerve. Twelve viral strains distributed in China have been reported to escape the CVI988 vaccine, reaching a mortality rate of more than 30%. Phylogenetic analyses show the internal connection between the Middle East (Turkey, Iraq, Iran, Saudi Arabia), South Asia (India, Indonesia), and East Asia (China and Japan), while external viral communications might occasionally occur. In 18 strains with both sequential and mortality data, amino acid alignment showed several point substitutions that may be related to its virulence. We suggest more behavioral monitoring in Marek’s disease-endemic regions and further studies on strain virulence, together with its Meq protein structural changes.

The study describes the finding of an abnormal blue-tinged color found on rabbit carcasses in the refrigeration cell of two butcher shops in Apulia Region. The carcasses were from an industrial rabbitry for production of meat with a regularly authorized slaughterhouse. Pseudomonas azotoformans, a microorganism included in Pseudomonas fluorescens group, was isolated from samples collected by the altered carcasses, showing the growth of uniform bacterial colonies with fluorescent pigmentation. The bacterium was also isolated from an additional water sample and from the labelling gun collected in the slaughterhouse, whilst the knives used for slaughtering resulted negative. Chromatic alteration was experimentally reproduced on new carcasses using a 108 cfu/mL bacterial suspension prepared with the isolated strain. Due to their resistance characteristics, members of P. fluorescens group are very difficult to eradicate once introduced into the production environment. Therefore, their presence, even if not considered a public health problem, should be monitored by food industry operators in self-control plans.

Colibacillosis, caused by E. coli, is responsible for economic losses in the poultry industry due to mortality, decreased production, and the cost of antibiotic treatments. Prevention of colibacillosis is based on improved biosecurity measures and the use of the vaccine performed with O78 E. coli strains, which is responsible for most cases of colibacillosis. Recently, there has been increased interest in other infection control methods, such as the use of natural compounds. The aim of this study was to evaluate the antimicrobial efficacy of cinnamon essential oil (CEO) against E. coli strains isolated from poultry. The MIC50 and MIC90 of CEO were determined by testing 117 strains belonging to serogroups O78, O2, O128, O139, isolated from laying hens (91 strains), broilers (10 strains), and turkeys (16 strains). The bacterial strains were tested at cell densities of 108 and 106 CFU/mL. At the cell density of 108 CFU/mL, MIC50 and MIC90 were 0.4 and 0.5 µL/mL for most of the tested strains, while they corresponded to 0.5 µL/mL for all strains isolated from broilers and for strains belonging to serogroup O139. At the cell density of 106 CFU/mL, MIC50 and MIC90 were 0.3 and 0.4 µL/mL, regardless of bird species of origin and for strains belonging to serogroups O78 and O2. In addition, a concentration of 0.04 µL/mL of CEO corresponded both to MIC50 and MIC90 for strains belonging to serogroups O139 and O128. Based on these results, cinnamon essential oil showed an effective antibacterial activity against E. coli strains from poultry and could find field application for the prevention of colibacillosis.

Pasteurellosis, a disease caused by Pasteurella multocida, is responsible for economic losses in rabbit industrial farms due to rhinitis, conjunctivitis, pneumonia, metritis, mastitis, orchitis, subcutaneous abscesses, otitis, encephalitis, and septicaemic forms. Although the occurrence of the disease is conditioned by predisposing factors that affect the rabbit immune response, the strains of P. multocida involved in the infection may have a different pathogenic ability. Therefore, typing of strains spread among the rabbits is important to assess their pathogenic potential. The aim of this study is to investigate the P. multocida strains responsible for disease in rabbit industrial farms. A total of 114 strains identified from different lesions were serotyped. Additionally, the presence of virulence-associated genes was investigated using three PCR (polymerase chain reaction) protocols. Capsular type A was prevalently found in strains from respiratory lesions while types D and F in those from metritis, mastitis, and other lesions. Different associations between some virulence-associated genes and both capsular type and lesions found in rabbits were detected. The presence of 8 virulence-associated genes seems to increase the occurrence of metritis. In addition, strains belonging to capsular type A and responsible for respiratory disorders especially, were found equipped with 10 and 11 virulence-associated genes. Nevertheless, the presence of strains responsible only for rhinitis was also detected among the latter, suggesting that the pathogenic ability of the bacteria depends on the expression rather than the presence of a gene.