Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Mycoplasma hyopneumoniae infection dynamics in naïve replacement gilts introduced to positive farms.

This study was designed to characterize the dynamics of infection of Mycoplasma hyopneumoniae in naïve replacement gilts after introduction to positive systems. Ninety-eight naïve gilts were monitored in three positive commercial farms (A, B, and C). The näive gilts were housed for 21 days in pens adjacently located to older gilt cohorts (named seeders), which have been naturally exposed to the positive farms. The infection dynamics was evaluated by PCR and ELISA, from laryngeal swabs and serum samples, respectively. Samples were collected at 150 (arrival), 165, 180, 210, 240, 270, 300 days of age (doa), and pre-farrowing. Infection occurred rapidly on farms A and B, taking 25.2 and 23.9 days for 95% of gilts to be PCR positive, respectively. There was no influence on the number of seeders at the time of exposure, but their absence (farm C) could explain the extended period it took for gilts to get infected (69.4 days). On average, it took 162.2 days after the first PCR detection for 85% of gilts to stop shedding the bacterium. The serology results were consistent with the herd infection curve. At pre-farrowing, 100% of gilts seroconverted and 36.7% remained PCR positive. A total of 1.33% of piglets were positive at weaning. Fifteen variants were detected among the three farms by MLVA. The acclimation protocol was efficient and easy to perform, and the presence of seeders was likely critical for early acclimation for M. hyopneumoniae.

Molecular characterization of Glaesserella parasuis strains circulating in North American swine production systems.

BACKGROUND: Glaesserella parasuis is the causative agent of Glässer's disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted. RESULTS: In silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency;  blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%).   CONCLUSION: This study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.

Predicting antimicrobial resistance of bacterial pathogens using time series analysis.

Antimicrobial resistance (AMR) is arguably one of the major health and economic challenges in our society. A key aspect of tackling AMR is rapid and accurate detection of the emergence and spread of AMR in food animal production, which requires routine AMR surveillance. However, AMR detection can be expensive and time-consuming considering the growth rate of the bacteria and the most commonly used analytical procedures, such as Minimum Inhibitory Concentration (MIC) testing. To mitigate this issue, we utilized machine learning to predict the future AMR burden of bacterial pathogens. We collected pathogen and antimicrobial data from >600 farms in the United States from 2010 to 2021 to generate AMR time series data. Our prediction focused on five bacterial pathogens (Escherichia coli, Streptococcus suis, Salmonella sp., Pasteurella multocida, and Bordetella bronchiseptica). We found that Seasonal Auto-Regressive Integrated Moving Average (SARIMA) outperformed five baselines, including Auto-Regressive Moving Average (ARMA) and Auto-Regressive Integrated Moving Average (ARIMA). We hope this study provides valuable tools to predict the AMR burden not only of the pathogens assessed in this study but also of other bacterial pathogens.

A tool to enhance antimicrobial stewardship using similarity networks to identify antimicrobial resistance patterns across farms.

Antimicrobial resistance (AMR) is one of the major challenges of the century and should be addressed with a One Health approach. This study aimed to develop a tool that can provide a better understanding of AMR patterns and improve management practices in swine production systems to reduce its spread between farms. We generated similarity networks based on the phenotypic AMR pattern for each farm with information on important bacterial pathogens for swine farming based on the Euclidean distance. We included seven pathogens: Actinobacillus suis, Bordetella bronchiseptica, Escherichia coli, Glaesserella parasuis, Pasteurella multocida, Salmonella spp., and Streptococcus suis; and up to seventeen antibiotics from ten classes. A threshold criterion was developed to reduce the density of the networks and generate communities based on their AMR profiles. A total of 479 farms were included in the study although not all bacteria information was available on each farm. We observed significant differences in the morphology, number of nodes and characteristics of pathogen networks, as well as in the number of communities and susceptibility profiles of the pathogens to different antimicrobial drugs. The methodology presented here could be a useful tool to improve health management, biosecurity measures and prioritize interventions to reduce AMR spread in swine farming.

Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples.

Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from - 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

Cough associated with the detection of Mycoplasma hyopneumoniae DNA in clinical and environmental specimens under controlled conditions.

BACKGROUND: The association of cough with Mycoplasma hyopneumoniae (MHP) DNA detection in specimens was evaluated under conditions in which the MHP status of inoculated and contact-infected pen mates was closely monitored for 59 days post-inoculation (DPI). METHODS: Seven-week-old pigs (n = 39) were allocated to five rooms (with one pen). Rooms contained 9 pigs each, with 1, 3, 6, or 9 MHP-inoculated pigs, respectively, except Room 5 (three sham-inoculated pigs). Cough data (2 × week) and specimens, tracheal swabs (2 × week), oral fluids (daily), drinker wipes (~ 1 × week), and air samples (3 × week) were collected. At 59 DPI, pigs were euthanized, and lung and trachea were evaluated for gross and microscopic lesions. Predictive cough value to MHP DNA detection in drinker and oral fluid samples were estimated using mixed logistic regression. RESULTS: Following inoculation, MHP DNA was first detected in tracheal swabs from inoculated pigs (DPI 3), then oral fluids (DPI 8), air samples (DPI 10), and drinker wipes (21 DPI). MHP DNA was detected in oral fluids in 17 of 59 (Room 1) to 43 of 59 (Room 3) samples, drinker wipes in 4 of 8 (Rooms 2 and 3) to 5 of 8 (Rooms 1 and 4) samples, and air samples in 5 of 26 (Room 2) or 3 of 26 (Room 4) samples. Logistic regression showed that the frequency of coughing pigs in a pen was associated with the probability of MHP DNA detection in oral fluids (P < 0.01) and nearly associated with drinker wipes (P = 0.08). Pathology data revealed an association between the period when infection was first detected and the severity of gross lung lesions. CONCLUSIONS: Dry, non-productive coughs suggest the presence of MHP, but laboratory testing and MHP DNA detection is required for confirmation. Based on the data from this study, oral fluids and drinker wipes may provide a convenient alternative for MHP DNA detection at the pen level when cough is present. This information may help practitioners in specimen selection for MHP surveillance.

Comparison of Mycoplasma hyopneumoniae response to infection by route of exposure.

Mycoplasma hyopneumoniae (MHP) is a concern both for pig well-being and producer economic viability. In the absence of fully protective health interventions, producers rely on controlled exposure to induce an immune response in pigs and minimize the clinical outcomes of MHP infection in pig populations. This study compared the effect of route of exposure on MHP infection, antibody response, clinical signs, and pathology. Six-week-old MHP-negative pigs (n = 78) were allocated to negative control (n = 6) or one of three MHP exposure routes: intratracheal (n = 24, feeding catheter), intranasal (n = 24, atomization device), and aerosol (n = 24, fogger). Body weight, cough indices, and samples (serum, oral fluid, tracheal) were collected weekly through 49 days post-exposure (DPE). Intratrachal exposure produced the highest proportion (24/24) of MHP DNA-positive pigs on DPE 7, as well as earlier and higher serum antibody response. Intranasal and aerosol exposures resulted in infection with MHP DNA detected in tracheal samples from 18/24 and 21/24 pigs on DPE 7, respectively. Aerosol exposure had the least impact on weight gain (0.64 kg/day). No difference was observed among treatment groups in coughing and lung lesions at necropsy. While intratracheal inoculation and seeder animals are frequently used in swine production settings, intranasal or aerosol exposure are viable alternatives to achieve MHP infection. Regardless of the route, steps should be taken to verify the purity of the inoculum and, in the case of aerosol exposure, avert the unintended exposure of personnel and animals to other pathogens.

Adapting a porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISA to routine surveillance.

Distinct from tests used in diagnostics, tests used in surveillance must provide for detection while avoiding false alarms, i.e., acceptable diagnostic sensitivity but high diagnostic specificity. In the case of the reproductive and respiratory syndrome virus (PRRSV), RNA detection meets these requirements during the period of viremia, but antibody detection better meets these requirements in the post-viremic stage of the infection. Using the manufacturer's recommended cut-off (S/P ≥ 0.4), the diagnostic specificity of a PRRSV oral fluid antibody ELISA (IDEXX Laboratories, Inc., Westbrook, ME, USA) evaluated in this study was previously reported as ≥ 97 %. The aim of this study was to improve its use in surveillance by identifying a cut-off that would increase diagnostic specificity yet minimally impact its diagnostic sensitivity. Three sample sets were used to achieve this goal: oral fluids (n = 596) from pigs vaccinated with a modified live PRRSV vaccine under experimental conditions, field oral fluids (n = 1574) from 94 production sites of known negative status, and field oral fluids (n = 1380) from 211 sites of unknown PRRSV status. Based on the analysis of samples of known status (experimental samples and field samples from negative sites), a cut-off of S/P ≥ 1.0 resulted in a diagnostic specificity of 99.2 (95 % CI: 98.8, 99.7) and a diagnostic sensitivity of 96.5 (95 % CI: 85.2, 99.2). Among 211 sites of unknown status, 81 sites were classified as antibody positive using the manufacturer's cut-off; 20 of which were reclassified as negative using a cut-off of S/P ≥ 1.0. Further analysis showed that these 20 sites had a small proportion of samples (18.0 %) with S/P values just exceeding the manufacturer's cut-off (x̄ = 0.5). Whereas the remainder of positive sites (n = 61) had a high proportion of samples (76.3 %) with high S/P values (x̄ = 6.6). Thus, the manufacturer's cut-off (S/P ≥ 0.4) is appropriate for diagnostic applications, but a cut-off of S/P ≥ 1.0 provided the higher specificity required for surveillance. A previously unreported finding in this study was a statistically significant association between unexpected reactors and specific production sites and animal ages or stages. While beyond the scope of this study, these data suggested that certain animal husbandry or production practices may be associated with non-specific reactions.

Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions.

Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Weekly serum and daily oral fluid samples were collected through 56 days postinoculation (dpi). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period. Analysis of ELISA performance at various cutoffs found that the manufacturers' recommended cutoffs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives. In study 2, the 3 best-performing ELISAs from study 1 were compared using serum samples generated under field conditions. Ten 8-week-old pigs were intratracheally inoculated with M. hyopneumoniae Matched serum and tracheal samples (to establish the true pig M. hyopneumoniae status) were collected at 7- to 14-day intervals through 98 dpi. Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current M. hyopneumoniae ELISAs and an understanding of their use in surveillance.

Molecular epidemiology of Mycoplasma hyorhinis porcine field isolates in the United States.

Mycoplasma hyorhinis is one of the causative agents of polyserositis and arthritis in post-weaning pigs. Here we describe the development of a multi-locus sequence typing (MLST) protocol for the characterization of M. hyorhinis field isolates. A total of 104 field isolates from different geographical locations, swine production systems, and clinical backgrounds, were analyzed. Twenty-seven genes, including housekeeping and those encoding surface proteins, were evaluated to index diversity. Genes encoding surface proteins were included to increase the discriminatory power of the MLST. Four target gene fragments were selected to be included in the final MLST-s (surface) protocol: pdhB, p95, mtlD and ung. Within each locus the nucleotide variation ranged from 1.4% to 20%. The 104 field isolates were classified into 39 distinct sequence types (STs). Multiple STs were found within the same production system and within the same pig. The majority of STs grouped strains from the same production system; however, cases existed where multiple systems shared a ST, indicating potential relationships between pig flows. The majority of the nucleotide changes observed in these genes generated synonymous changes, while non-synonymous changes were exclusively in the mtlD gene fragment, suggesting that this protein is undergoing selection. Molecular typing of M. hyorhinis will primarily aid swine practitioners with pig flow management and identifying sources of infection during outbreaks.

Genotyping of Mycoplasma hyorhinis using multiple-locus variable number tandem repeat analysis.

Mycoplasma hyorhinis (M. hyorhinis) has re-emerged as an important swine pathogen in recent years causing significant economic losses in post weaning pigs. Genetic variability of M. hyorhinis has been described based on different molecular methods that have limited resolution and reproducibility. The present study was undertaken to develop a molecular epidemiological typing tool for M. hyorhinis based on multiple loci of variable number of tandem repeats in its genome, termed MLVA. The typing method was designed on the basis of the number of repeats in two hypothetical proteins, MHR_0152 and MHR_0298. A total of 205 samples were analyzed, including field isolates, clinical specimens, and a reference strain. Analysis of the combination of the 2 loci revealed 16 MLVA types in 165 of the 205 samples. In the remaining forty samples only one locus could be amplified. The most frequent types obtained from the set of samples were 8-4 (36.9%), 8-3 (11.5%), 7-4 (11.5%), 9-4 (10.9%) and 10-4 (9.3%). The Simpson's diversity index for the assay was D=0.814 when the 165 samples were taken into account. No clustering was observed based on the geographical location, sample type, or year of isolation or sampling. The MLVA assay developed in this investigation showed to be a reproducible and portable assay which could be easily performed and transferred to other laboratories. The use of this technique will assist in epidemiological investigations and can be used to improve the understanding the molecular biology of M. hyorhinis variants.

Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis.

Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/µl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

Evaluation of CrystalTM Rapid Stool/Enteric ID System for Identification of Aerobic Gram-negative Bacilli.

OBJECTIVE: The evaluation of the new miniaturized CrystalTM Rapid Stool/Enteric System (Becton-Dickinson, USA) for identification of aerobic gram-negative bacilli. METHODS: a total of 154 clinical organisms (Enterobacteriaceae: 120 strains; oxidase-positive fermenters: 13 strains; non-fermenters: 21 strains) were tested. Results were compared with those obtained with the PASCOR system (Difco, USA) and divergent identifications were evaluated by standard biochemical tests. RESULTS: without additional testing, correct identification was obtained for 146 strains (Enterobacteriaceae: 95%; oxidase-positive fermenters 87%; non-fermenters 100%). For adequate identification of Yersinia enterocolitica strains, however, panels had to be incubated for 5 instead of 3 hours. CONCLUSIONS: the CrystalTM Rapid Stool/Enteric system offers a safe, accurate and rapid method for the identification of frequent isolates of the family Enterobacteriaceae and bacterial stool pathogens.