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.

Development of molecular assays for the analysis of genetic relationships of Mycoplasma iowae.

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson's diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results.

Detection of Mycoplasma columbinasale in Cases of Respiratory Disease in Domestic Pigeons (Columba livia var. domestica).

In 2017, respiratory disease and low mortality were reported in domestic flying pigeons (Columba livia var. domestica) trained as hunting live bait in a breeding farm in Umbria, Italy. Clinically, open beak breathing, dropped wings, and pharyngeal and laryngeal hyperaemia were observed. Three birds were submitted for necropsy. Gross pathological evaluation revealed in all cases diffuses hyperaemia of the tracheal mucosa in association with mild emaciation and multiorgan congestion. Microscopically, diffuse epithelial hyperplasia of the trachea (n = 3) and diffuse lymphocytic infiltration of the lamina propria (n = 3) were observed. No lesions were reported in other organs. Based on reported clinical signs and lesions, Mycoplasma spp. were suspected, and molecular detection was performed on tracheal specimens leading to the identification of Mycoplasma columbinasale. Immunohistochemistry was subsequently performed to localize the microorganism within tissue lesions. Immunohistochemistry confirmed the presence of Mycoplasma species on the tracheal epithelial cells of all birds. Following tylosin administration, complete resolution of the clinical condition and lack of recurrence of clinical signs were reported in the breeding farm. These findings suggest that M. columbinasale could potentially have a role in the respiratory disease and low mortality in domestic pigeons.

Genomic Diversity of a Globally Used, Live Attenuated Mycoplasma Vaccine.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

The Monitoring of Mycoplasma gallisepticum Minimum Inhibitory Concentrations during the Last Decade (2010-2020) Seems to Reveal a Comeback of Susceptibility to Macrolides, Tiamulin, and Lincomycin.

Mycoplasma gallisepticum (Mg) is a highly contagious avian pathogen responsible for significant economic losses for the poultry industry. In some circumstances, antimicrobial treatment is useful to contain clinical signs of Mg infection in birds. However, antimicrobial resistance emergence is now common among animal pathogens, becoming a worldwide health concern. The collection of minimum inhibitory concentration (MIC) data is fundamental for an appropriate antimicrobial use and for fighting antimicrobial resistance emergence. However, MIC data can only be generated in specialized laboratories, and therefore they are not regularly available. MICs of 67 non-vaccine-derived Mg isolates collected in Italy between 2010 and 2020 were obtained. Although 79.1% of the Mg isolates showed enrofloxacin MICs ≥ 8 µg/mL, a statistically significant trend toward low MICs of erythromycin, tylosin, tilmicosin, spiramycin, tiamulin, and lincomycin was observed, indicating a comeback to susceptibility of Mg toward these drugs. Doxycycline proved to be slightly more effective than oxytetracycline. The present study shows that Mg changed its susceptibility toward many of the drugs most commonly used for its containment over a ten-year period.

Silent Infection of Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b in a Commercial Chicken Broiler Flock in Italy.

From October 2021 to January 2022, different incursions of clade 2.3.4.4b H5N1 HPAIV (Highly Pathogenic Avian Influenza Virus) occurred in several Italian regions with its main diffusion in Densely Poultry Populated Areas (DPPAs) of north-eastern Italy. Monitoring and control activities applied in the affected area clearly evidenced that turkeys and broilers were the most affected species, although several flocks of broilers at times resulted HPAIV H5N1 infected in absence of increased mortality and/or clinical signs. Thus, an approach based on sampling dead birds was adopted in the broiler sector to improve the early detection of infection; this protocol allowed us to confirm that 15 farms were HPAIV-infected with birds ready to be delivered to the slaughterhouse. The aim of this report is to describe the results of the diagnostic activities carried out in one HPAIV H5N1-infected broiler farm, three days after laboratory confirmation during the pre-movement testing without showing increased mortality or clinical signs. Thus, clinical signs, daily cumulative mortality rate (CMR), virus shedding, seroconversion, pathobiology of clade 2.3.4.4b H5N1 HPAIV as well as Avian Influenza Viruses (AIVs) environmental contamination were thoroughly examined in the infected holding. Such in-depth investigation demonstrated low infection prevalence in live birds, low environmental contamination, no seroconversion for AIVs, gross and microscopic findings compatible with systemic infection with peracute death in H5N1 HPAIV-infected birds.

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).

Isolation of Mycoplasma iowae in turkey flocks with skeletal abnormalities: a retrospective study.

Mycoplasma iowae, a pathogen affecting the turkey industry, is commonly associated with decreased hatchability and leg abnormalities in young progeny. This Mycoplasma was in the spotlight more in the past than today since its prevalence has been decreasing over time. Reports of M. iowae in turkeys showing reduced growth performances, leg problems and skeletal abnormalities are scarce although there is no report whether this pathogen has been completely eradicated in commercial turkeys. Additionally, there are no comprehensive epidemiological data available on M. iowae prevalence in any country. Therefore, we carried out a retrospective study to evaluate the prevalence of the infection and any correlation between necropsy findings and M. iowae presence in Italian turkeys between 2011 and 2012. Necropsy was performed on 101 dead turkey submissions presented for diagnostic purposes. Fifty-six submissions (55.4%) tested positive for M. iowae, most of which (69.6%) were between 4 and 7 weeks of age. Skeletal abnormalities were observed in 36 cases (35.6%). The logistic regression analysis revealed that the probability of finding a M. iowae-positive submission was four times higher if the animals showed skeletal abnormalities (OR = 4.48, IC 95%: 1.66-12.15). This is the first retrospective, cross-sectional study on M. iowae field outbreaks in commercial turkeys. These results suggest that M. iowae should be considered as a differential diagnosis when skeletal abnormalities are observed. RESEARCH HIGHLIGHTSM. iowae was found in more than half of the turkey groups analysed.M. iowae was likely to be detected if skeletal abnormalities were present in the studied turkeys.

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.

Evaluation of Minimum Inhibitory Concentrations for 154 Mycoplasma synoviae isolates from Italy collected during 2012-2017.

Mycoplasma synoviae (MS) is a highly prevalent bacterial species in poultry causing disease and severe economic losses. Antibiotic treatment is one of the control strategies that can be applied to contain clinical outbreaks in MS-free flocks, especially because this bacterium can be transmitted in ovo. It becomes, then, very important for veterinarians to know the antibiotic susceptibility of the circulating strains in order to choose the most appropriate first-line antibiotic molecule as a proactive role in fighting antibiotic resistance. We evaluated the Minimum Inhibitory Concentrations (MICs) of enrofloxacin, oxytetracycline, doxycycline, erythromycin, tylosin, tilmicosin, spiramycin, tiamulin, florfenicol and lincomycin for MS isolates collected between 2012 and 2017 in Italy. A total of 154 MS isolates from different poultry commercial categories (broiler, layer, and turkey sectors) was tested using commercial MIC plates. All MS isolates showed very high MIC values of erythromycin (MIC90 ≥8 µg/mL) and enrofloxacin (MIC90 ≥16 µg/mL). MIC values of doxycycline and oxytetracycline obtained were superimposable to each other with only a one-fold dilution difference. Discrepancies between MIC values of tylosin and tilmicosin were observed. Interestingly, seven isolates showed very high MIC values of lincomycin and tilmicosin, but not all of them showed very high MIC values of tylosin. Most of the MS isolates showed low MIC values of spiramycin, but seven strains showed a MIC ≥16 µg/mL. In the observation period, the frequency of the different MIC classes varied dependently on the tested antibiotic. Interestingly, tilmicosin MICs clearly showed a time-dependent progressive shift towards high-concentration classes, indicative of an on-going selection process among MS isolates. Until standardized breakpoints become available to facilitate data interpretation, it will be fundamental to continue studying MIC value fluctuations in the meantime in order to create a significant database that would facilitate veterinarians in selecting the proper drug for treating this impactful Mycoplasma.

First molecular detection of ball python nidovirus in Italy - Short communication.

A retrospective study was conducted to investigate the presence of ferlavirus, ball python nidovirus and bacteria in 32 tracheobronchial lavages from ball pythons raised in captivity and affected by respiratory disease. A touchdown reverse transcription polymerase reaction (RT-PCR) was performed to detect ball python nidovirus RNA targeting a 260-bp portion of the ORF1a gene, while a nested RT-PCR was applied to identify RNA targeting the 518-bp ferlavirus partial L gene. RT-PCR positive products were submitted for Sanger's sequencing and phylogeny reconstruction. Bacteriological examinations were performed to diagnose a possible bacterial involvement. BLAST analysis revealed that the nucleotide sequences of the six (18.8%) RT-PCR positive amplicons were 90-97% identical to the partial sequence of the ORF1a gene of the recently described ball python nidovirus. All tested snakes were negative for ferlavirus. Thirteen out of 32 samples (40.6%) were bacteriologically positive. Respiratory tract diseases can be a substantial problem for snake breeders, considering the rapid transmission of respiratory pathogens. The results and published studies show that ball python nidovirus is circulating in python collections and could be linked to suboptimal management practices. Surveillance programs are desirable as part of the routine snake health assessment. Tracheobronchial lavage is a fast, practical, cost-effective procedure for sample collection.

Antimicrobial susceptibility survey on bacterial agents of canine and feline urinary tract infections: Weight of the empirical treatment.

OBJECTIVES: This work characterised the antimicrobial susceptibility of uropathogens isolated from empirically treated dogs and cats. Within-household transmission of uropathogens can involve humans and companion animals. Knowledge on the prevalence and susceptibility pattern of isolates from canine and feline urine samples and the impact of prior antimicrobial treatment is important to prevent the dissemination of antimicrobial resistance. METHODS: A retrospective study was conducted selecting antibiotic-treated companion animals. Urine samples were collected by cystocentesis and were submitted to an Italian diagnostic laboratory over a 2-year period (2013-2015). The antimicrobial susceptibility of the isolates was analysed both using Clinical and Laboratory Standards Institute (CLSI) guidelines and a formula to help select rational antimicrobial therapy. RESULTS: Gram-negative bacteria were clearly prevalent. Gentamicin had the highest impact factors. Amoxicillin/clavulanic acid and doxycycline appeared to be the most effective compounds against Gram-positive infections, whilst marbofloxacin may be a useful option against Gram-negative urinary tract infections (UTIs) as well as doxycycline and trimethoprim/sulfamethoxazole in cats and dogs, respectively. Consulting published studies, a comparable overall trend regarding bacterial species incriminated in canine and feline UTIs and their susceptibilities seems likely, despite different circumstances where the studies were conducted. CONCLUSIONS: Companion animals are potential reservoirs of drug-resistant uropathogens. Judicious use of antibiotics is necessary to maintain the efficacy of antimicrobials in human and veterinary medicine. Antimicrobial susceptibility monitoring programmes are therefore essential to facilitate the choice of antimicrobial agent that is most likely to be effective, particularly in cases of prior antimicrobial treatment.

DNA microarray assay and real-time PCR as useful tools for studying the respiratory tract Mycoplasma populations in young dairy calves.

PURPOSE: With more than 120 species, the genus Mycoplasma is one of the largest taxa in the class Mollicutes, a group of micro-organisms that are characterized by apparent simplicity and to which important animal pathogens belong. Mycoplasmabovis is the most frequently identified pathogenic Mycoplasma in cattle; however, the prevalence of other Mycoplasma species living in calves' airways is poorly understood. The aim of this work was to characterize the respiratory tract mycoplasma populations in calves on one of the largest dairy farms in Italy using a real-time PCR assay and a DNA microarray assay. METHODOLOGY: A total of 49 nasal swabs and 49 trans-tracheal aspirations from non-vaccinated veal calves were analysed. Genomic DNA was extracted from the samples and then tested using a real-time PCR targeting the oppD gene of M. bovis and a DNA microarray that was able to identify more than 70 Mycoplasma species. RESULTS: Forty-two out of 49 calves tested positive for Mycoplasma spp. (85.7 %). None of the samples tested positive for M. bovis. A majority (73.5 %) of the 98 samples tested positive for M. dispar, while 8 samples tested positive for M. bovirhinis (8.2 %). CONCLUSION: Our results expand our knowledge regarding the diversity of Mycoplasma populations in the respiratory airways of very young veal calves and add data regarding M. bovis prevalence in the Italian cattle population. However, the importance of these species in the respiratory diseases of calves still remains to be determined.

Serological and biomolecular survey on canine herpesvirus-1 infection in a dog breeding kennel.

Canine herpesvirus-1 (CaHV-1) is a globally distributed pathogen causing reproductive, respiratory, ocular and neurological disorders in adult dogs and neonatal death in puppies. This pathogen is considered poorly immunogenic, and neutralizing antibodies are found for only a short time following exposure. Further, seroprevalence can be affected by several epidemiological factors. A virological survey was conducted in a high-density population breeding kennel in Central Italy. There were several factors predisposing animals to CaHV-1 infection, such as age, number of pregnancies, experience with mating and dog shows, cases of abortion, management and environmental hygiene. Serum neutralization (SN) and nested PCR assays were used to estimate prevalence of CaHV-1. None of the submitted samples tested positive for nested PCR, and none of the sera tested CaHV-1 positive. No association was observed between antibody titers and risk factors, and no sign of viral reactivation was detected in either males or females. These results suggest that CaHV-1 is not circulating within this kennel and that further studies are needed in order to better understand the distribution of the virus within Italy.