Empowering veterinary clinical diagnosis in industrial poultry production by ambient mass spectrometry and chemiometrics: a new approach for precise poultry farming.

Untargeted metabolomic profiling, by ambient mass spectrometry and chemometric tools, has made a dramatic impact on human disease detection. In a similar vein, this study attempted the translation of this clinical human disease experience to farmed poultry for precise veterinary diagnosis. As a proof of principle, in this diagnostic/prognostic study, direct analysis in real-time high resolution mass spectrometry (DART-HRMS) was used in an untargeted manner to analyze fresh tissues (abdominal fat, leg skin, liver, and leg muscle) of pigmented and non-pigmented broilers to investigate the causes of lack of pigmentation in an industrial poultry farm. Afterwards, statistical analysis was applied to the DART-HRMS data to retrieve the molecular features that codified for 2 broiler groups, that is, properly pigmented and non-pigmented broilers. Higher abundance of oxidized lipids, high abundance of oxidized bile derivatives, and lower levels of tocopherol isomers (Vitamin E) and retinol (Vitamin A) were captured in nonpigmented than in pigmented broilers. In addition, conventional rapid analyses were used: 1) color parameters of the tissues of pigmented and non-pigmented broilers were measured to rationalize the color differences in abdominal fat, leg skin and leg muscle, and 2) macronutrients were determined in broiler leg muscle, to capture a detailed picture of the pathology and exclude other possible causes. In this study, the DART-HRMS system performed well in retrieving valuable chemical information from broilers that explained the differences between the 2 groups of broilers in absorption of xanthophylls and the subsequent lack of proper broiler pigmentation in affected broilers. The results suggest this technology could be useful in providing near real-time feedback to aid in veterinary decision-making in poultry farming.

Pathogenic avian mycoplasmas show phenotypic differences in their biofilm forming ability compared to non-pathogenic species in vitro.

Mycoplasmas are known as the minimalist microorganisms in the microbes' world. Their minimalist nature makes them highly sensitive to the environmental conditions and limits their ability to survive for extended periods outside their animal host. Nevertheless, there are documented instances of mycoplasma transmission over significant distances and this phenomenon may be linked to relatively unexplored abilities of mycoplasmas, such as their capacity to synthesize biofilm-the predominant mode of bacterial growth in nature. The authors decided to establish a method aimed at inducing the clustering of mycoplasma planktonic cells within a biofilm in vitro and subsequently assess the capacity of certain avian mycoplasmas to synthesize a biofilm. A total of 299 avian mycoplasma isolates were included in the study, encompassing both pathogenic (Mycoplasma gallisepticum, M. synoviae, M. meleagridis, M. iowae) and non-pathogenic species (M. gallinaceum, M. gallinarum, M. iners and M. pullorum). The authors successfully demonstrated the feasibility of inducing avian mycoplasmas to synthetize in vitro a biofilm, which can be visually quantified. The only species that did not produce any biofilm was M. iowae. In general, the pathogenic mycoplasmas produced greater quantities of biofilm compared to the non-pathogenic ones. Furthermore, it was observed that the ability to produce biofilm appeared to vary, both qualitatively and quantitatively, not only among different species but also among isolates of a single species. Future studies will be necessary to determine whether biofilm production plays a pivotal epidemiological role for the pathogenic avian mycoplasmas.

DART-HRMS allows the detection of toxic alkaloids in animal autopsy specimens and guides the selection of confirmatory methods in accidental plant poisoning.

BACKGROUND: In cases of suspected animal poisonings or intoxications, there is the need for high-throughput, rapid and accurate analytical tools capable of giving rapid answers and, thus, speeding up the early stages of investigations. Conventional analyses are very precise, but do not meet the need for rapid answers capable of orienting the decisions and the choice of appropriate countermeasures. In this context, the use of ambient mass spectrometry (AMS) screening methods in toxicology laboratories could satisfy the requests of forensic toxicology veterinarians in a timely manner. RESULTS: As a proof of principle, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was applied to a veterinary forensic case in which 12 of a group of 27 sheep and goats died with an acute neurological onset. Because of evidence in the rumen contents, the veterinarians hypothesized an accidental intoxication after ingestion of vegetable materials. The DART-HRMS results showed abundant signals of the alkaloids calycanthine, folicanthidine and calycanthidine, both in the rumen content and at the liver level. The DART-HRMS phytochemical fingerprinting of detached Chimonanthus praecox seeds was also compared with those acquired from the autopsy specimens. Liver, rumen content and seed extracts were then subjected to LC-HRMS/MS analysis to gather additional insights and confirm the putative assignment of calycanthine anticipated by DART-HRMS. HPLC-HRMS/MS confirmed the presence of calycanthine in both rumen contents and liver specimens and allowed its quantification, ranging from 21.3 to 46.9 mg kg-1 in the latter. This is the first report detailing the quantification of calycanthine in liver after a deadly intoxication event. SIGNIFICANCE AND NOVELTY: Our study illustrates the potential of DART-HRMS to offer a rapid and complementary alternative to guide the selection of confirmatory chromatography-MSn strategies in the analysis of autopsy specimens from animals with suspected alkaloid intoxication. This method offers the consequent saving of time and resources over those needed for other methods.

Antimicrobial susceptibility profiles of Mycoplasma hyorhinis strains isolated from five European countries between 2019 and 2021.

Mycoplasma hyorhinis is an emerging swine pathogen bacterium causing polyserositis and polyarthritis in weaners and finishers. The pathogen is distributed world-wide, generating significant economic losses. No commercially available vaccine is available in Europe. Therefore, besides improving the housing conditions for prevention, antimicrobial therapy of the diseased animals is the only option to control the infection. Our aim was to determine the minimal inhibitory concentrations (MIC) of ten antimicrobials potentially used against M. hyorhinis infection. The antibiotic susceptibility of 76 M. hyorhinis isolates from Belgium, Germany, Hungary, Italy and Poland collected between 2019 and 2021 was determined by broth micro-dilution method and mismatch amplification mutation assay (MAMA). Low concentrations of tiamulin (MIC90 0.312 µg/ml), doxycycline (MIC90 0.078 µg/ml), oxytetracycline (MIC90 0.25 µg/ml), florfenicol (MIC90 2 µg/ml) and moderate concentrations of enrofloxacin (MIC90 1.25 µg/ml) inhibited the growth of the isolates. For the tested macrolides and lincomycin, a bimodal MIC pattern was observed (MIC90 >64 µg/ml for lincomycin, tulathromycin, tylosin and tilmicosin and 5 µg/ml for tylvalosin). The results of the MAMA assay were in line with the conventional method with three exceptions. Based on our statistical analyses, significant differences in MIC values of tiamulin and doxycycline were observed between certain countries. Our results show various levels of antimicrobial susceptibility among M. hyorhinis isolates to the tested antibiotics. The data underline the importance of susceptibility monitoring on pan-European level and provides essential information for proper antibiotic choice in therapy.

Herpetic Pneumonia in Indian Ringneck Parrots (Psittacula krameri): First Report of Novel Psittacid Alphaherpesvirus-5 Infection in Europe.

The first two European outbreaks of herpetic pneumonia caused by Psittacid alphaherpesvirus-5 were diagnosed based on gross pathology findings, histological examination, transmission electron microscopy visualization and genome sequencing. The outbreaks, characterized by high morbidity and high mortality rates, involved two parrot species, namely the Indian ringneck parrot (Psittacula krameri) and the Alexandrine parakeet (Psittacula eupatria). Clinical signs observed were ruffled feathers, dyspnea, tail bobbing, open wings while breathing, depression and anorexia. Necropsy was performed on Indian ringneck parrots only, and the most evident and serious gross lesion found in all the birds was a diffuse marked consolidation of the lungs associated with parenchyma congestion and oedema. Histological examination confirmed the existence of bronchopneumonia characterized by the presence of syncytial cells with intranuclear inclusion bodies. In one bird, fibrinous airsacculitis was observed as well. Lung tissue inspection through electron microscopy revealed the presence of virus particles resembling herpesviruses. Viral DNA was extracted, amplified using primers for Alloherpesviridae DNA polymerase gene detection, and then sequenced. BLAST analysis showed a 100% identity with the only previously reported sequence of PsHV-5 (MK955929.1).

The pathogen Mycoplasma dispar Shows High Minimum Inhibitory Concentrations for Antimicrobials Commonly Used for Bovine Respiratory Disease.

Mycoplasma dispar is an overlooked pathogen often involved in bovine respiratory disease (BRD), which affects cattle around the world. BRD results in lost production and high treatment and prevention costs. Additionally, chronic therapies with multiple antimicrobials may lead to antimicrobial resistance. Data on antimicrobial susceptibility to M. dispar is limited so minimum inhibitory concentrations (MIC) of a range of antimicrobials routinely used in BRD were evaluated using a broth microdilution technique for 41 M. dispar isolates collected in Italy between 2011-2019. While all isolates had low MIC values for florfenicol (<1 µg/mL), many showed high MIC values for erythromycin (MIC90 ≥8 µg/mL). Tilmicosin MIC values were higher (MIC50 = 32 µg/mL) than those for tylosin (MIC50 = 0.25 µg/mL). Seven isolates had high MIC values for lincomycin, tilmicosin and tylosin (≥32 µg/mL). More, alarmingly, results showed more than half the strains had high MICs for enrofloxacin, a member of the fluoroquinolone class considered critically important in human health. A time-dependent progressive drift of enrofloxacin MICs towards high-concentration values was observed, indicative of an on-going selection process among the isolates.

Infection Dynamics of Mycoplasma bovis and Other Respiratory Mycoplasmas in Newly Imported Bulls on Italian Fattening Farms.

Italian beef production is mainly based on a feedlot system where calves are housed with mixed aged cattle often in conditions favourable to bovine respiratory disease (BRD). In Veneto, an indoor system is also used for imported bulls around 300-350 kg. Mycoplasmas, in particular Mycoplasma bovis and Mycoplasma dispar, contribute to BRD in young calves, but their role in the disease in older cattle has not been investigated. In this study, ten heads of cattle were selected from each of the 24 groups kept in 13 different farms. Bulls were sampled by nasal swabbing at 0, 15, and 60 days after arrival for Mycoplasma isolation. Identification was carried out by 16S-rDNA PCR followed by denaturing gradient gel electrophoresis. M. bovis, M. dispar, and M. bovirhinis were identified, and prevalence was analysed by mixed-effects logistic regression models. This showed that most bulls arrived free of M. bovis, but within two weeks, approximately 40% became infected, decreasing to 13% by the last sampling. In contrast, the prevalence of M. dispar was not dependent on time or seasonality, while M. bovirhinis only showed a seasonality-dependent trend. The Italian fattening system creates an ideal environment for infection with M. bovis, probably originating from previously stabled animals.

Detection of a New Genetic Cluster of Influenza D Virus in Italian Cattle.

Influenza D virus (IDV) has been increasingly reported all over the world. Cattle are considered the major viral reservoir. Based on the hemagglutinin-esterase (HEF) gene, three main genetic and antigenic clusters have been identified: D/OK distributed worldwide, D/660 detected only in the USA and D/Japan in Japan. Up to 2017, all the Italian IDV isolates belonged to the D/OK genetic cluster. From January 2018 to May 2019, we performed virological surveillance for IDV from respiratory outbreaks in 725 bovine farms in Northern Italy by RT-PCR. Seventy-four farms were positive for IDV. A full or partial genome sequence was obtained from 29 samples. Unexpectedly, a phylogenetic analysis of the HEF gene showed the presence of 12 strains belonging to the D/660 cluster, previously unreported in Europe. The earliest D/660 strain was collected in March 2018 from cattle imported from France. Moreover, we detected one viral strain with a reassortant genetic pattern (PB2, PB1, P42, HEF and NP segments in the D/660 cluster, whilst P3 and NS segments in the D/OK cluster). These results confirm the circulation of IDV in the Italian cattle population and highlight the need to monitor the development of the spreading of this influenza virus in order to get more information about the epidemiology and the ecology of IDV viruses.

Influenza D in Italy: towards a better understanding of an emerging viral infection in swine.

Influenza D virus (IDV), a new member of the Orthomyxoviridae family, was first reported in 2011 in swine in Oklahoma, and consequently found in cattle across North America and Eurasia. To investigate the circulation of IDV among pigs in Italy, in the period between June 2015 and May 2016, biomolecular and virological tests were performed on 845 clinical samples collected from 448 pig farms affected by respiratory distress located in the Po Valley. Serological tests were conducted on 3698 swine sera, including archive sera collected in 2009, as well as samples collected in 2015 from the same region. Viral genome was detected in 21 (2.3%) samples from 9 herds (2%), while virus was successfully isolated from 3 samples. Genetic analysis highlighted that Italian swine IDVs are closely related to the D/swine/Oklahoma/1334/2011 cluster. Sera collected in 2015 showed a high prevalence of IDV antibody titers (11.7%), while archive sera from 2009 showed statistically significant lower positivity rates (0.6%). Our results indicate an increasing epidemiological relevance of the pathogen and the need for in-depth investigations towards understanding its pathogenesis, epidemiology and possible zoonotic potential of this emerging virus.

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2.

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200µl for avian-like H1N1, 1×10(0) TCID(50)/200µl for human-like H1N2 and 1×10(1) TCID(50)/200µl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies.

Antimicrobial resistance of Actinobacillus pleuropneumoniae isolated from swine.

The aim of this retrospective study was to evaluate the antimicrobial resistance rates and the trend in resistance of Actinobacillus pleuropneumoniae isolated from pigs in Italy from 1994 to 2009. A total of 992 A. pleuropneumoniae isolates were tested for their susceptibility to a panel of antimicrobial agents in a disk diffusion method. Resistance to 7 drugs (amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, cotrimoxazole, penicillin G and tilmicosin) showed a significant increasing trend over the time, while for 2 drugs (gentamycin and marbofloxacin) a significant decrease was observed. Resistance to the remaining 14 antimicrobial agents tested did not change significantly over the study period. Most of the isolates retained high susceptibility to antimicrobials usually effective against A. pleuropneumoniae such as amphenicols, fluoroquinolones and ceftiofur. However, high rates of resistance were observed for potentiated sulfa drugs, tetracyclines and penicillins which are currently recommended antimicrobials for pig pleuropneumonia therapy. Our results suggest the importance of continued monitoring of A. pleuropneumoniae clinical isolates in order to choose the most appropriate treatment of infections and to control the increase of resistance to currently used antimicrobials.