Interactive Quantum Chemistry Enabled by Machine Learning, Graphical Processing Units, and Cloud Computing.

Modern quantum chemistry algorithms are increasingly able to accurately predict molecular properties that are useful for chemists in research and education. Despite this progress, performing such calculations is currently unattainable to the wider chemistry community, as they often require domain expertise, computer programming skills, and powerful computer hardware. In this review, we outline methods to eliminate these barriers using cutting-edge technologies. We discuss the ingredients needed to create accessible platforms that can compute quantum chemistry properties in real time, including graphical processing units-accelerated quantum chemistry in the cloud, artificial intelligence-driven natural molecule input methods, and extended reality visualization. We end by highlighting a series of exciting applications that assemble these components to create uniquely interactive platforms for computing and visualizing spectra, 3D structures, molecular orbitals, and many other chemical properties.

Evaluation of the role of clathrin and bacterial viability in the endocytosis of Lawsonia intracellularis.

Lawsonia intracellularis is an obligate intracellular bacterium and causative agent of proliferative enteropathy. The pathogenesis of L. intracellularis is not completely understood, including the endocytic mechanisms to access the host cell cytoplasm. In this study, we evaluated the mechanisms involved in endocytosis of L. intracellularis in vitro using intestinal porcine epithelial cells (IPEC-J2). Confocal microscopy was used to co-localize L. intracellularis and clathrin. Clathrin gene knockdown was then applied to verify whether L. intracellularis endocytosis is clathrin-dependent. Finally, internalization of viable and non-viable (bacteria were inactivated by heat) L. intracellularis organisms were assessed to study the role of the host cell during bacterial endocytosis. L. intracellularis organisms were observed co-localized with clathrin by confocal microscopy but the amount of L. intracellularis internalized in cells, with and without clathrin knockdown, did not differ statistically. The internalization of non-viable L. intracellularis showed a decrease in the internalization in cells with less clathrin synthesis (P<0.05). The present study is the first to elucidate the involvement of clathrin in the endocytosis of L. intracellularis. Clathrin-mediated endocytosis was shown to be an important, but not required, process for L. intracellularis internalization in porcine intestinal epithelial cells. Independence of bacterial viability for host cell internalization was also confirmed.

Senecavirus A seroprevalence and risk factors in United States pig farms.

Senecavirus A (SVA) is a non-enveloped, single-stranded, positive-sense RNA virus belonging to the Picornaviridae family. Senecavirus A is constantly associated with outbreaks of vesicular disease in pigs and has been reported in several countries since its first large-scale outbreak in 2014. Senecavirus A's clinical disease and lesions are indistinguishable from other vesicular foreign animal diseases (FAD). Therefore, an FAD investigation needs to be conducted for every SVA case. For this reason, SVA has been attributed as the cause of an alarming increase in the number of yearly FAD investigations performed by the United States Department of Agriculture (USDA). The objectives of this study were to estimate the seroprevalence of SVA antibodies in breeding and growing pig farms in the United States and to determine the farm-level risk factors associated with seropositivity. A total of 5,794 blood samples were collected from 98 and 95 breeding and growing pig farms in 17 states. A farm characteristics questionnaire was sent to all farms, to which 80% responded. The responses were used to conduct logistic regression analyses to assess the risk factors associated with SVA seropositivity. The estimated farm-level seroprevalences were 17.3% and 7.4% in breeding and growing pig farms, respectively. Breeding farms had 2.64 times higher odds of SVA seropositivity than growing pig farms. One key risk factor identified in breeding farms was the practice of rendering dead animal carcasses. However, the adoption of a higher number of farm biosecurity measures was associated with a protective effect against SVA seropositivity in breeding farms.

Eosinophilic Granulomatous Myocarditis in Two Pigs.

Eosinophilic myocarditis is a human condition that has been rarely documented in animals. We now report two unrelated porcine cases of idiopathic eosinophilic granulomatous myocarditis that resembled the human disease and which were associated with sudden death. The most relevant gross finding in both cases was marked cardiomegaly, accompanied by raised, multifocal to coalescent small white nodules (1-2 mm) and poorly demarcated multifocal pale areas in the epicardium. Histologically, there were multifocal to coalescent areas of cardiomyocyte loss with replacement by an intense inflammatory infiltrate of eosinophils and epithelioid macrophages, and proliferation of fibrous connective tissue. Immunohistochemistry for porcine circovirus type 2 (PCV2) and Toxoplasma gondii, in-situ hybridization and quantitative polymerase chain reaction tests for PCV2 and porcine circovirus type 3 and aerobic bacterial culture on myocardium samples were negative.

Persistence and shedding of senecavirus A in naturally infected boars.

Senecavirus A (SVA) infection in pigs causes vesicular disease and results in a short viremia and transient shedding of the virus, mainly in oral fluids and feces. Here we describe the consistent prolonged shedding of SVA in the semen of 2 boars, and persistence of SVA within the tonsils and testes of 3 adult boars. Two SVA-infected boars that were identified on a Minnesota sow farm in 2017 shed SVA RNA in semen for >3 mo after an outbreak of vesicular disease had occurred on the farm. SVA was isolated from 1 semen sample collected 9 d after clinical disease began on the farm. The third SVA-infected boar was identified on an Indiana sow farm in 2020. All boars had SVA RNA detected in the testes and tonsils by RT-rtPCR, with lower Ct values obtained for the testes than from the tonsils. All boars had multifocal lymphocytic orchitis with segmental degeneration and atrophy of the germinal epithelium within the seminiferous tubules. One boar also had areas of seminiferous tubule collapse and interstitial fibrosis within the testes. In all boars, in situ hybridization demonstrated the presence of SVA mRNA within cells located basally in the seminiferous tubules of the testes, and within the basal surface epithelial cells, crypt epithelial cells, and subepithelial and parafollicular lymphocytes and histiocytes of the tonsil.

Correction: De Conti et al. Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs. Animals 2021, 11, 1520.

The authors wish to make the following correction to this paper [...].

Oral Vaccination Reduces the Effects of Lawsonia intracellularis Challenge on the Swine Small and Large Intestine Microbiome.

Porcine proliferative enteropathy remains one of the most prevalent diseases in swine herds worldwide. This disease is caused by Lawsonia intracellularis, an intracellular bacterial pathogen that primarily colonizes the ileum. In this study, we evaluated changes to the microbiome of the ileal mucosa, ileal digesta, cecal digesta, and feces subsequent to challenge with L. intracellularis and to an oral live vaccine against L. intracellularis. Given that gut homogenates have been used since 1931 to study this disease, we also characterized the microbial composition of a gut homogenate from swine infected with L. intracellularis that was used as challenge material. The L. intracellularis challenge led to a dysbiosis of the microbiome of both the small and large intestine marked by an increase of pathobionts including Collinsella, Campylobacter, Chlamydia, and Fusobacterium. This microbiome response could play a role in favoring L. intracellularis colonization and disease as well as potentially predisposing to other diseases. Vaccination altered both small and large intestine microbiome community structure and led to a significant 3.03 log10 reduction in the amount of L. intracellularis shed by the challenged pigs. Vaccination also led to a significant decrease in the abundance of Collinsella, Fusobacterium, and Campylobacter among other microbial changes compared with non-vaccinated and challenged animals. These results indicate that L. intracellularis infection is associated with broad changes to microbiome composition in both the large and small intestine, many of which can be mitigated by vaccination.

Porcine circovirus 3 in North and South America: Epidemiology and genetic diversity.

Porcine circovirus 3 (PCV3) is a recently discovered virus that has been detected in the swine population worldwide. PCV3 infection has been associated with several signs, but its pathogenicity is currently uncertain. This review article aimed to analyse the PCV3 strains that circulate in different countries in North and South America. We demonstrated the main regions of polymorphisms in the capsid protein structure. Furthermore, we found that PCV3 has at least six different lineages circulating in the Americas. Additional studies are required to determine the role of PCV3 in different clinical syndromes and its epidemiology in swine herds in North and South American countries.

Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs.

Porcine circovirus type 3 (PCV3) has been recently described as a potential cause of abortions and systemic vasculitis in pigs. Although the virus has been detected by real-time PCR in several porcine tissues from countries worldwide, PCV3-associated diseases have not been satisfactorily clarified. The objective of this study was to investigate the association between the presence of PCV3 mRNA detected by in situ hybridization (ISH) within histological lesions and PCV3 DNA detected by real-time PCR in naturally infected pigs. A total of 25 PCV3 PCR-positive cases were analyzed. Formalin-fixed tissues from these cases were evaluated for histologic lesions and for ISH-RNA positive signals for PCV3. The most frequent tissue type with histopathologic lesions was heart, 76.2%, with lymphoplasmacytic myocarditis and epicarditis as the most frequent lesions observed. Lymphoplasmacytic interstitial pneumonia was also a frequent finding, 47.6%. There were also lesions in kidney, liver, spleen and lymph nodes. PCV3-ISH-RNA positive signals were mostly observed in association with lymphoplasmacytic inflammatory infiltrate in various tissues, including arteries. Based on our results, the minimum set of specimens to be submitted for histopathology and mRNA in situ hybridization to confirm or exclude a diagnosis of PCV3 are heart, lung and lymphoid tissues (i.e., spleen and lymph nodes), especially for differential diagnosis related with PCV2-associated diseases.

Pathogenicity and immune response against porcine circovirus type 3 infection in caesarean-derived, colostrum-deprived pigs.

Recently, a novel PCV species (PCV3) has been detected in cases associated with sow mortality, lesions consistent with porcine dermatitis and nephropathy syndrome, reproductive failure and multisystemic inflammation. The pathogenesis and clinical significance of PCV3 is still unclear. In this study, we investigated the immunopathogenesis of PCV3 in CD/CD pigs. Four treatment groups, PCV3 (n=6), PCV3-KLH (n=6), control (n=3) and control-KLH (n=3), were included with PCV3-positive tissue homogenate (gc=3.38×1012 ml-1 and gc=1.04×1011 ml-1), confirmed by quantitative PCR (qPCR) and next-generation sequencing. Clinical signs, viremia, viral shedding, systemic cytokines, humoral (IgG) and T-cellular response were evaluated for 42 days. At necropsy, tissues were collected for histological evaluation and PCV3 detection by qPCR and in situ hybridization. No significant clinical signs were observed through the study. Viremia was detected in both PCV3-inoculated groups from 3 days post-inoculation (p.i.) until the end of the study. Nasal shedding was detected from 3 to 28 days p.i. and faecal shedding was transient. PCV3 induced an early (7 days p.i.) and sustained (42 days p.i.) IgG response. No significant T-cell response was observed. Histological evaluation demonstrated lesions consistent with multisystemic inflammation and perivasculitis. All tissues evaluated were positive by qPCR and virus replication was confirmed by positive in situ hybridization. This study demonstrated the potential role of PCV3 in subclinical infection, producing a mild, multisystemic inflammatory response, prolonged viremia detectable for 42 days p.i., presence of IgG humoral response and viral shedding in nasal secretions. More research is required to understand and elucidate potential co-factors necessary in the manifestation and severity of clinical disease.

Genetic Determinants of Resistance to Extended-Spectrum Cephalosporin and Fluoroquinolone in Escherichia coli Isolated from Diseased Pigs in the United States.

Fluoroquinolones and cephalosporins are critically important antimicrobial classes for both human and veterinary medicine. We previously found a drastic increase in enrofloxacin resistance in clinical Escherichia coli isolates collected from diseased pigs from the United States over 10 years (2006 to 2016). However, the genetic determinants responsible for this increase have yet to be determined. The aim of the present study was to identify and characterize the genetic basis of resistance against fluoroquinolones (enrofloxacin) and extended-spectrum cephalosporins (ceftiofur) in swine E. coli isolates using whole-genome sequencing (WGS). blaCMY-2 (carried by IncA/C2, IncI1, and IncI2 plasmids), blaCTX-M (carried by IncF, IncHI2, and IncN plasmids), and blaSHV-12 (carried by IncHI2 plasmids) genes were present in 87 (82.1%), 19 (17.9%), and 3 (2.83%) of the 106 ceftiofur-resistant isolates, respectively. Of the 110 enrofloxacin-resistant isolates, 90 (81.8%) had chromosomal mutations in gyrA, gyrB, parA, and parC genes. Plasmid-mediated quinolone resistance genes [qnrB77, qnrB2, qnrS1, qnrS2, and aac-(6)-lb'-cr] borne on ColE, IncQ2, IncN, IncF, and IncHI2 plasmids were present in 24 (21.8%) of the enrofloxacin-resistant isolates. Virulent IncF plasmids present in swine E. coli isolates were highly similar to epidemic plasmids identified globally. High-risk E. coli clones, such as ST744, ST457, ST131, ST69, ST10, ST73, ST410, ST12, ST127, ST167, ST58, ST88, ST617, ST23, etc., were also found in the U.S. swine population. Additionally, the colistin resistance gene (mcr-9) was present in several isolates. This study adds valuable information regarding resistance to critical antimicrobials with implications for both animal and human health.IMPORTANCE Understanding the genetic mechanisms conferring resistance is critical to design informed control and preventive measures, particularly when involving critically important antimicrobial classes such as extended-spectrum cephalosporins and fluoroquinolones. The genetic determinants of extended-spectrum cephalosporin and fluoroquinolone resistance were highly diverse, with multiple plasmids, insertion sequences, and genes playing key roles in mediating resistance in swine Escherichia coli Plasmids assembled in this study are known to be disseminated globally in both human and animal populations and environmental samples, and E. coli in pigs might be part of a global reservoir of key antimicrobial resistance (AMR) elements. Virulent plasmids found in this study have been shown to confer fitness advantages to pathogenic E. coli strains. The presence of international, high-risk zoonotic clones provides worrisome evidence that resistance in swine isolates may have indirect public health implications, and the swine population as a reservoir for these high-risk clones should be continuously monitored.

Survival of Lawsonia intracellularis in porcine peripheral blood monocyte-derived macrophages.

Lawsonia intracellularis, an obligately intracellular enteric bacterium, infects intestinal epithelial cells, but may also be found within macrophages in the intestinal lamina propria of affected pigs. Macrophages play an important role in host defense against infectious agents, but the role of this cell in L. intracellularis infection is not well understood. The aim of this study was to evaluate the permissibility of macrophages to L. intracellularis infection in vitro. Pure culture of L. intracellularis was added to swine peripheral blood monocyte-derived macrophages. Viability of intracytoplasmic L. intracellularis was evaluated at different time points by transmission electron microscopy (TEM). Potential replication of L. intracellularis in macrophages was also evaluated by qPCR. By TEM, phagocytosis L. intracellularis within of phagolysosomes were observed 1-hour post-infection (hpi) and bacterial structures in binary fission at 48 hpi. The number of intracellular bacteria was determined at 1, 4, 24, 48, and 72 hpi by qPCR in infected macrophages and compared to the number of intracellular bacteria from culture in McCoy cells. In both cell lines, the amount of L. intracellularis was decreased at 4 hpiand increased at 24 hpi. The number of intracellular bacteria continued to increase in McCoy cells over time. This is the first study showing interaction, survival and propagation of L. intracellularis in macrophages. These findings are critical to establish an experimental model for future studies of the pathogenesis of porcine proliferative enteropathy and the potential persistence of L. intracellularis in macrophages during chronic infections.

Early-Life Intake of an Isotonic Protein Drink Improves the Gut Microbial Profile of Piglets.

A healthy microbial community in the gut of piglets is critical to minimize the negative performance consequences associated with dietary and environmental changes that occur at weaning. Tonisity Px, an isotonic protein drink, is a potential alternative to balance the gut microbiota as it contains key ingredients for nourishing the small intestine. In the present study, 16 litters comprising 161 piglets were randomly allocated to a group to which Tonisity Px was provided from days 2 to 8 of age (TPX group) or to a control group, to which no Tonisity Px was provided. The TPX group also received Tonisity Px in the 3 days before and after weaning. At days 9, 17, and 30 of age, fecal and ileum samples were collected from piglets belonging to both groups and analyzed using 16S rRNA gene sequencing, semiquantitative PCR of Rotavirus serogroups, and semiquantitative Escherichia coli culture. Overall, Tonisity Px increased the abundance of beneficial bacterial populations (Lactobacillus and Bacteroides species) and reduced potentially pathogenic bacterial populations (E. coli and Prevotellaceae), in both the pre-weaning and post-weaning periods.

Immune responses to porcine epidemic diarrhea virus (PEDV) in swine and protection against subsequent infection.

Understanding the immune responses against Porcine epidemic diarrhea virus (PEDV) is important to prevent infection and to design control strategies. We evaluated both systemic and mucosal immune responses to PEDV in pigs and assessed if prior exposure to virus protects against re-infection. Three-week-old pigs were infected with PEDV and immune response in blood, intestine, and mesenteric lymph node (MLN) was evaluated. At 30 dpi, virus exposed pigs were challenged with a field isolate of PEDV and immune response at 5 d post challenge was evaluated. We found that PEDV RNA persists in the intestine even after fecal shedding of the virus was stopped at 28 dpi and pigs previously exposed to PEDV are protected from virus shedding after re-infection. PEDV infection induced both humoral and cell mediated immune response with an increase in PEDV specific IgA and IgG antibodies in intestine and serum. Flow cytometry analysis showed a significantly higher frequency of B cells and lower frequency of T cells at 4 dpi. The frequency of CD4/CD8 double positive (DP) memory T cells was significantly increased in the MLN of challenged animals. These studies may provide further insights into understanding the mucosal immune response to PEDV and its role in protection against disease.

In situ hybridization of feline leukemia virus in a primary neural B-cell lymphoma.

An 8-y-old castrated male, outdoor European shorthair cat was presented with a history of hindlimb weakness and paralysis. Disease progression was continuous from the onset; deep algesia disappeared at the final stage. Radiography of the vertebral column was unremarkable; along with patient history and physical examination results, magnetic resonance imaging suggested inflammatory lesions in the spinal cord, although neoplasia could not be ruled out. Feline leukemia virus (FeLV) positivity was confirmed by a serum ELISA prior to euthanasia. Upon postmortem examination, hemorrhages were present in the spinal cord at the level of vertebrae T7-8. Histologic and immunohistochemical analysis revealed primary diffuse large B-cell lymphoma of the spinal cord with multifocal myelomalacia and hemorrhages. To determine the presence of a pathogen within the lesion, we developed a novel in situ hybridization protocol for FeLV (RNAscope). The reaction revealed large amounts of FeLV viral RNA in the tumor cells.

Isolation of PCV3 from Perinatal and Reproductive Cases of PCV3-Associated Disease and In Vivo Characterization of PCV3 Replication in CD/CD Growing Pigs.

Porcine circovirus 3 (PCV3) has been identified as a putative swine pathogen with a subset of infections resulting in stillborn and mummified fetuses, encephalitis and myocarditis in perinatal, and periarteritis in growing pigs. Three PCV3 isolates were isolated from weak-born piglets or elevated stillborn and mummified fetuses. Full-length genome sequences from different passages and isolates (PCV3a1 ISU27734, PCV3a2 ISU58312, PCV3c ISU44806) were determined using metagenomics sequencing. Virus production in cell culture was confirmed by qPCR, IFA, and in situ hybridization. In vivo replication of PCV3 was also demonstrated in CD/CD pigs (n = 8) under experimental conditions. Viremia, first detected at 7 dpi, was detected in all pigs by 28 dpi. IgM antibody response was detected between 7-14 dpi in 5/8 PCV3-inoculated pigs but no IgG seroconversion was detected throughout the study. Pigs presented histological lesion consistent with multi systemic inflammation characterized by myocarditis and systemic perivasculitis. Viral replication was confirmed in all tissues by in situ hybridization. Clinically, all animals were unremarkable throughout the study. Although the clinical relevance of PCV3 remains under debate, this is the first isolation of PCV3 from perinatal and reproductive cases of PCV3-associated disease and in vivo characterization of PCV3 infection in a CD/CD pig model.

Prevalence and trend analysis of antimicrobial resistance in clinical Escherichia coli isolates collected from diseased pigs in the USA between 2006 and 2016.

Antimicrobial resistance (AMR) is an emerging threat to both human and animal health. Antimicrobial use and resistance in food animal production, including swine, has received increased scrutiny as a source of resistant foodborne pathogens. Continuous surveillance of AMR in bacterial isolates of swine origin can guide in conservation of antimicrobials used in both human and swine medicine. The objective of this study was to evaluate the prevalence and trends of the phenotypic AMR in Escherichia coli of swine origin isolated from clinical samples at the Minnesota Veterinary Diagnostic laboratory between 2006 and 2016. The prevalence of resistance to ampicillin, tetracyclines and sulphadimethoxine remained greater than 50% throughout the period. There was a drastic change in enrofloxacin resistance, increasing from less than 1% to more than 20% between 2006 and 2016 (annual relative increase of 57% between 2006 and 2013 and 16% between 2013 and 2016). The prevalence of resistance to other antimicrobials remained constant (ceftiofur, oxytetracycline and chlortetracycline) or changed significantly (annual relative changes of less than 10%) for at least some time-period between 2006 and 2016 (ampicillin, florfenicol, gentamicin, neomycin, sulphadimethoxine, trimethoprim-sulphamethoxazole and spectinomycin). Rarefaction analysis revealed an increase in the number of unique combinations of AMRs per year. Network analysis was performed by estimating and plotting partial correlations between minimum inhibitory concentrations (MICs) of various antimicrobials. An increase in strength of these networks was observed, particularly in networks created after 2010, which can be indicative of increased multiple AMR in these isolates. These results provide valuable insight into the trends in AMR in E. coli of swine origin in the USA and act as supplementary information to the existing active AMR surveillance systems.

Evaluation of swine enteroids as in vitro models for Lawsonia intracellularis infection1,2.

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However, these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs, specifically, the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study, we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

Prevalence and time trend analysis of antimicrobial resistance in respiratory bacterial pathogens collected from diseased pigs in USA between 2006-2016.

Swine respiratory disease complex (SRDC) causes massive economic losses to the swine industry and is a major animal welfare concern. Antimicrobials are mainstay in treatment and control of SRDC. However, there is a lack of data on the prevalence and trends in resistance to antimicrobials in bacterial pathogens associated with SRDC. The objective of this study was to estimate the prevalence and changes in resistance to 13 antimicrobials in swine bacterial pathogens (Streptococcus suis, Pasteurella multocida, Actinobacillus suis and Haemophilus parasuis) in the U.S.A using data collected at University of Minnesota Veterinary Diagnostic Laboratory between 2006 and 2016. For antimicrobials for which breakpoints were available, prevalence of resistance remained below 10% except for tetracycline in S. suis and P. multocida isolates, and these prevalence estimates remained consistently low over the years despite statistical significance (p < .05) in trend analysis. For antimicrobial-bacterial combinations without available breakpoints, the odds of isolates being resistant increased by >10% annually for 7 and 1 antimicrobials in H. parasuis and S. suis isolates respectively, and decreased >10% annually for 4 and 1 antimicrobials in A. suis and H. parasuis isolates, respectively, according to the ordinal regression models. Clinical implications of changes in AMR for A. suis and H. parasuis should be interpreted cautiously due to the lack of interpretive criteria and challenges in antimicrobial susceptibility tests in the case of H. parasuis. Future studies should focus on surveillance of antimicrobial resistance and establishment of standardized susceptibility testing methodologies and interpretive criteria for these animal pathogens of critical importance.