Diagnostic survey of analytical methods used to determine bone mineralization in pigs.

Pigs from 64 commercial sites across 14 production systems in the Midwest United States were evaluated for baseline biological measurements used to determine bone mineralization. There were three pigs selected from each commercial site representing: 1) a clinically normal pig (healthy), 2) a pig with evidence of clinical lameness (lame), and 3) a pig from a hospital pen that was assumed to have recent low feed intake (unhealthy). Pigs ranged in age from nursery to market weight, with the three pigs sampled from each site representing the same age or phase of production. Blood, urine, metacarpal, fibula, 2nd rib, and 10th rib were collected and analyzed. Each bone was measured for density and ash (defatted and non-defatted technique). A bone × pig type interaction (P < 0.001) was observed for defatted and non-defatted bone ash and density. For defatted bone ash, there were no differences among pig types for the fibulas, 2nd rib, and 10th rib (P > 0.10), but metacarpals from healthy pigs had greater (P < 0.05) percentage bone ash compared to unhealthy pigs, with the lame pigs intermediate. For non-defatted bone ash, there were no differences among pig types for metacarpals and fibulas (P > 0.10), but unhealthy pigs had greater (P < 0.05) non-defatted percentage bone ash for 2nd and 10th ribs compared to healthy pigs, with lame pigs intermediate. Healthy and lame pigs had greater (P < 0.05) bone density than unhealthy pigs for metacarpals and fibulas, with no difference observed for ribs (P > 0.10). Healthy pigs had greater (P < 0.05) serum Ca and 25(OH)D3 compared to unhealthy pigs, with lame pigs intermediate. Healthy pigs had greater (P < 0.05) serum P compared to unhealthy and lame pigs, with no differences between the unhealthy and lame pigs. Unhealthy pigs excreted significantly more (P < 0.05) P and creatinine in the urine compared to healthy pigs with lame pigs intermediate. In summary, there are differences in serum Ca, P, and vitamin D among healthy, lame, and unhealthy pigs. Differences in bone mineralization among pig types varied depending on the analytical procedure and bone, with a considerable range in values within pig type across the 14 production systems sampled.

There is little literature or data comparing bone diagnostic results for healthy, lame, and unhealthy pigs. Typically, diagnosticians assessing clinical lameness cases in pigs will measure bone mineralization along with histopathological evaluation to diagnose and assess the severity of metabolic bone disease. Bone ash is the primary method to determine bone mineralization, with the removal of the lipid in the bone (defatting) before the bone is ashed, compared to not removing the lipid before the ashing (non-defatted). Defatting the bone reduces the amount of variation across the bones compared to non-defatting. In this diagnostic survey, there was no difference among the healthy, lame, or unhealthy pigs when comparing defatted bone ash, however, unhealthy pigs had an increased bone ash percentage compared to the healthy and lame pigs when the bones were assessed using the non-defatted procedure. There was variation across production systems and pig types for serum vitamin D. When comparing the pig types, healthy pigs had increased serum Ca, P, and vitamin D [25(OH)D3] compared to the unhealthy pigs, with the lame pigs intermediate.

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.

Genomic characterization of Streptococcus equi subspecies zooepidemicus from a 2021 outbreak in Indiana with increased sow mortality.

IMPORTANCE: This study highlights a Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) strain isolated from an outbreak in Indiana, which resulted in mortality events among a swine herd in 2021. The Indiana outbreak strain was found to be genetically and phylogenetically distant to a strain isolated from the 2019 outbreaks in Ohio and Tennessee, which caused high swine mortality. We also discovered multiple unique genetic features in the Indiana outbreak strain, including distinct S. zooepidemicus genomic islands, and notable S. zooepidemicus virulence genes-many of which could serve as biomarkers for the diagnosis of this strain. These findings provide significant insights into monitoring and potentially preventing severe outbreaks caused by the Indiana outbreak strain in the future.

The effect of bone and analytical methods on the assessment of bone mineralization response to dietary phosphorus, phytase, and vitamin D in nursery pigs.

A total of 360 pigs (DNA 600 × 241, DNA; initially 11.9 ±â€…0.56 kg) were used in a 28-d trial to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to dietary P, vitamin D, and phytase in nursery pigs. Pens of pigs (six pigs per pen) were randomized to six dietary treatments in a randomized complete block design with 10 pens per treatment. Dietary treatments were designed to create differences in bone mineralization and included: (1) 0.19% standardized total tract digestibility (STTD) P (deficient), (2) 0.33% STTD P (NRC [2012] requirement) using monocalcium phosphate, (3) 0.33% STTD P including 0.14% release from phytase (Ronozyme HiPhos 2700, DSM Nutritional Products, Parsippany, NJ), (4) 0.44% STTD P using monocalcium phosphate, phytase, and no vitamin D, (5) diet 4 with vitamin D (1,653 IU/kg), and (6) diet 5 with an additional 50 µg/kg of 25(OH)D3 (HyD, DSM Nutritional Products, Parsippany, NJ) estimated to provide an additional 2,000 IU/kg of vitamin D3. After 28 d on feed, eight pigs per treatment were euthanized for bone (metacarpal, 2nd rib, 10th rib, and fibula), blood, and urine analysis. The response to treatment for bone density and ash was dependent upon the bone analyzed (treatment × bone interaction for bone density, P = 0.044; non-defatted bone ash, P = 0.060; defatted bone ash, P = 0.068). Thus, the response related to dietary treatment differed depending on which bone (metacarpal, fibula, 2nd rib, or 10th rib) was measured. Pigs fed 0.19% STTD P had decreased (P < 0.05) bone density and ash (non-defatted and defatted) for all bones compared to 0.44% STTD P, with 0.33% STTD P generally intermediate or similar to 0.44% STTD P. Pigs fed 0.44% STTD P with no vitamin D had greater (P < 0.05) non-defatted fibula ash compared to all treatments other than 0.44% STTD P with added 25(OH)D3. Pigs fed diets with 0.44% STTD P had greater (P < 0.05) defatted second rib ash compared to pigs fed 0.19% STTD P or 0.33% STTD P with no phytase. In summary, bone density and ash responses varied depending on bone analyzed. Differences in bone density and ash in response to P and vitamin D were most apparent with fibulas and second ribs. There were apparent differences in the bone ash percentage between defatted and non-defatted bone. However, differences between the treatments remain consistent regardless of the analytic procedure. For histopathology, 10th ribs were more sensitive than 2nd ribs or fibulas for the detection of lesions.

Lameness is defined as impaired movement or deviation from normal gait. There are many factors that can contribute to lameness, including but not limited to: infectious disease, genetic and conformational anomaly, and toxicity that affects the bone, muscle, and nervous systems. Metabolic bone disease is another cause of lameness in swine production and can be caused by inappropriate levels of essential vitamins or minerals. To understand and evaluate bone mineralization, it is important to understand the differences in diagnostic results between different bones and analytical techniques. Historically, percentage bone ash has been used as one of the procedures to assess metabolic bone disease as it measures the level of bone mineralization; however, procedures and results vary depending on the methodology and type of bone measured. Differences in bone density and ash in response to dietary P and vitamin D were most apparent in the fibulas and second ribs. There were apparent differences in the percentage of bone ash between defatted and non-defatted bone; however, the differences between the treatments remain consistent regardless of the analytic procedure. For histopathology, 10th ribs were more sensitive than 2nd ribs or fibulas for detection of lesions associated with metabolic bone disease.

Streptococcus gallolyticus and Bacterial Endocarditis in Swine, United States, 2015-2020.

To evaluate trends in bacterial causes of valvular endocarditis in swine, we retrospectively analyzed 321 cases diagnosed at Iowa State University Veterinary Diagnostic Laboratory (Ames, IA, USA) during May 2015--April 2020. Streptococcus gallolyticus was the causative agent for 7.59% of cases. This emerging infection in swine could aid study of endocarditis in humans.

Replication of Streptococcus equi subspecies zooepidemicus infection in swine.

Streptococcus equi subspecies zooepidemicus (SEZ) is a commensal bacterium of horses and causes infections in mammalian species, including humans. Historically, virulent strains of SEZ caused high mortality in pigs in China and Indonesia, while disease in the U.S. was infrequent. More recently, high mortality events in sows were attributed to SEZ in North America. The SEZ isolates from these mortality events have high genetic similarity to an isolate from an outbreak in China. Taken together, this may indicate SEZ is an emerging threat to swine health. To generate a disease model and evaluate the susceptibility of healthy, conventionally raised pigs to SEZ, we challenged sows and five-month-old pigs with an isolate from a 2019 mortality event. Pigs were challenged with a genetically similar guinea pig isolate or genetically distinct horse isolate to evaluate comparative virulence. The swine isolate caused severe systemic disease in challenged pigs with 100 % mortality. Disease manifestation in sows was similar to field reports: lethargy/depression, fever, reluctance to rise, and high mortality. The guinea pig isolate also caused severe systemic disease; however, most five-month-old pigs recovered. In contrast, the horse isolate did not cause disease and was readily cleared from the respiratory tract. In conclusion, we were able to replicate disease reported in the field. The results indicate differences in virulence between isolates, with the highest virulence associated with the swine isolate. Additionally, we generated a challenge model that can be used in future research to evaluate virulence factors and disease prevention strategies.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA.

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74-100% (94.79-100%), 91.9-100% (96.3-100%) and 90.71-100% (93.51-100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5'UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3'UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3' termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Streptococcus pneumoniae serotype 22F infection in respiratory syncytial virus infected neonatal lambs enhances morbidity.

Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection, especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting results. Moreover, little knowledge about the pathogenesis of emerging Spn serotype 22F, especially the co-pathologies between RSV and Spn, is known. Here, colostrum-deprived neonate lambs were divided into four groups. Two of the groups were nebulized with RSV M37, and the other two groups were mock nebulized. At day three post-RSV infection, one RSV group (RSV/Spn) and one mock-nebulized group (Spn only) were inoculated with Spn intratracheally. At day six post-RSV infection, bacterial/viral loads were assessed along with histopathology and correlated with clinical symptoms. Lambs dually infected with RSV/Spn trended with higher RSV titers, but lower Spn. Additionally, lung lesions were observed to be more frequent in the RSV/Spn group characterized by increased interalveolar wall thickness accompanied by neutrophil and lymphocyte infiltration and higher myeloperoxidase. Despite lower Spn in lungs, co-infected lambs had more significant morbidity and histopathology, which correlated with a different cytokine response. Thus, enhanced disease severity during dual infection may be due to lesion development and altered immune responses rather than bacterial counts.

Cases of high mortality in cull sows and feeder pigs associated with Streptococcus equi subsp. zooepidemicus septicemia.

Investigations of 2 cases of high mortality in cull sows and feeder pigs from a buying station in Ohio and cull sows at an abattoir in Tennessee were conducted at the Iowa State University Veterinary Diagnostic Laboratory. The animals were presented as weak, lethargic, and some with high fever. Rapidly escalating mortality was reported to be as high as 30-50% within groups at the buying station over 8-10 d, and 30-40% over 5-7 d at the abattoir. Splenomegaly and red lymph nodes were the most consistent macroscopic findings, with scant fibrinous polyserositis observed in one sow. The microscopic lesions of vasculitis, fibrin thrombi, fibrinosuppurative polyserositis, and intralesional bacteria were consistent with acute bacterial septicemia. Bacterial culture isolated Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) from multiple organs, including spleen, lung, and kidney. PCR tests were negative for African swine fever virus, classical swine fever virus, Erysipelothrix rhusiopathiae, porcine reproductive and respiratory syndrome virus, porcine circovirus 2, and Salmonella spp. Porcine circovirus 3 was inconsistently detected at low levels by PCR, with a lack of associated lesions. Next-generation sequencing identified S. zooepidemicus and porcine partetravirus in the serum sample of the feeder pig from the buying station. Phylogenetic analysis of the szP gene indicated that the S. zooepidemicus isolates from Ohio and Tennessee are in genotype VI. We conclude that the cause of these high mortality events in swine was S. zooepidemicus septicemia.

Genetic characterization of Streptococcus equi subspecies zooepidemicus associated with high swine mortality in the United States.

High mortality events due to Streptococcus equi subspecies zooepidemicus (Streptococcus zooepidemicus) in swine have not previously been reported in the United States. In September and October 2019, outbreaks with swine mortality up to 50% due to S. zooepidemicus septicaemia were reported in Ohio and Tennessee. Genomic epidemiological analysis revealed that the eight outbreak isolates were clustered together with ATCC 35246, a Chinese strain caused outbreaks with high mortality, also closely related to three isolates from human cases from Virginia, but significantly different from an outbreak-unrelated swine isolate from Arizona and most isolates from other animal species. Comparative genomic analysis on two outbreak isolates and another outbreak-unrelated isolate identified several genomic islands and virulence genes specifically in the outbreak isolates only, which are likely associated with the high mortality observed in the swine population. These findings have implications for understanding, tracking and possibly preventing diseases caused by S. zooepidemicus in swine.

A model of respiratory syncytial virus (RSV) infection of infants in newborn lambs.

Many animal models have been established for respiratory syncytial virus (RSV) infection of infants with the purpose of studying the pathogenesis, immunological response, and pharmaceutical testing and the objective of finding novel therapies and preventive measures. This review centers on a neonatal lamb model of RSV infection that has similarities to RSV infection of infants. It includes a comprehensive description of anatomical and immunological similarities between ovine and human lungs along with comparison of pulmonary changes and immune responses with RSV infection. These features make the newborn lamb an effective model for investigating key aspects of RSV infection in infants. The importance of RSV lamb model application in preclinical therapeutic trials and current updates on new studies with the RSV-infected neonatal lamb are also highlighted.

Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor.

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.

Protection of human influenza vaccines against a reassortant swine influenza virus of pandemic H1N1 origin using a pig model.

Since the pandemic H1N1 emergence in 2009 (pdmH1N1), many reassortant pdmH1N1 viruses emerged and found circulating in the pig population worldwide. Currently, commercial human subunit vaccines are used commonly to prevent the influenza symptom based on the WHO recommendation. In case of current reassortant swine influenza viruses transmitting from pigs to humans, the efficacy of current human influenza vaccines is of interest. In this study, influenza A negative pigs were vaccinated with selected commercial human subunit vaccines and challenged with rH3N2. All sera were tested with both HI and SN assays using four representative viruses from the surveillance data in 2012 (enH1N1, pdmH1N1, rH1N2 and rH3N2). The results showed no significant differences in clinical signs and macroscopic and microscopic findings among groups. However, all pig sera from vaccinated groups had protective HI titers to the enH1N1, pdmH1N1 and rH1N2 at 21DPV onward and had protective SN titers only to pdmH1N1and rH1N2 at 21DPV onward. SN test results appeared more specific than those of HI tests. All tested sera had no cross-reactivity against the rH3N2. Both studied human subunit vaccines failed to protect and to stop viral shedding with no evidence of serological reaction against rH3N2. SIV surveillance is essential for monitoring a novel SIV emergence potentially for zoonosis.

Detection of Actinobacillus Pleuropneumoniae ApxIV Toxin Antibody in Serum and Oral Fluid Specimens from Pigs Inoculated Under Experimental Conditions.

INTRODUCTION: The prevention and control of Actinobacillus pleuropneumoniae in commercial production settings is based on serological monitoring. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect specific antibodies against a variety of A. pleuropneumoniae antigens, including long-chain lipopolysaccharides (LPS) and the ApxIV toxin, a repeats-in-toxin (RTX) exotoxin unique to A. pleuropneumoniae and produced by all serovars. The objective of this study was to describe ApxIV antibody responses in serum and oral fluid of pigs. MATERIAL AND METHODS: Four groups of pigs (six pigs per group) were inoculated with A. pleuropneumoniae serovars 1, 5, 7, or 12. Weekly serum samples and daily oral fluid samples were collected from individual pigs for 56 days post inoculation (DPI) and tested by LPS and ApxIV ELISAs. The ApxIV ELISA was run in three formats to detect immunlgobulins M, G, and A (IgM, IgG and IgA) while the LPS ELISA detected only IgG. RESULTS: All pigs inoculated with A. pleuropneumoniae serovars 1 and 7 were LPS ELISA serum antibody positive from DPI 14 to 56. A transient and weak LPS ELISA antibody response was observed in pigs inoculated with serovar 5 and a single antibody positive pig was observed in serovar 12 at ≥35 DPI. Notably, ApxIV serum and oral fluid antibody responses in pig inoculated with serovars 1 and 7 reflected the patterns observed for LPS antibody, albeit with a 14 to 21 day delay. CONCLUSION: This work suggests that ELISAs based on ApxIV antibody detection in oral fluid samples could be effective in population monitoring for A. pleuropneumoniae.