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Allen D. Leman Swine Conference 2013

Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention

Published on: 4/21/2014
Author/s : Co-authored by Sarah Probst Miller (AgCreate Solutions) and Alejandro Ramirez (Iowa State University) Original case pathology and histology photographs by Kuldeep Singh (KS), University of Illinois (UICVM); Diagnostic plan coordinated with Paul Knoernschild and Everett Rosey from Zoetis, Monitoring diagnostics sponsored by Zoetis via STOMP and completed by IS- CVM
Review of Mycoplasma hyosynoviae as a pathogen (SPM)

Mycoplasma hyosynoviae is one of several agents capable of instigating arthritis in growing swine. This arthritis is typically noted as inflammation of the intra-articular tissue of one of more joints accompanied by an increased volume of intra-articular fluid along with other descriptors such as serous, fibrinous, purulent, macrophagic, lymphoplasmacytic, etc1. An increase in M. hyosynoviae instigated diagnosis has been noted by Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) in the Midwestern United States. From 2003 to 2010 Mycoplasma species accounted for 17% of cases at ISU-VDL while in 2010 Mycoplasma species accounted for up to 37% of cases.While the majority of animal afflicted with infectious arthritis may make it to commercial markets, the concern lies in the welfare of afflicted animals as well as potential impact on pig profitability.1 If a swine operation is involved in the business of raising and developing replacement females, the concern is threefold: 1) Reduction in percent sold from the gilt herd 2) Potential contagiousness of the disease to the sow farm 3) Effect of the infectious arthritis on the females’ longevity in the sow herd. This case study reports the process of diagnosing and treating a gilt herd afflicted with aesthetic and clinical lameness.

Background (SPM)

In September of 2013 a wean to market gilts site of 7200 gilts sourced from an 1800 multiplication unit complained of reduced selects sold due to “lameness.” Gilts are negative to PRRS, Mycoplasma hyopneumoniae, and APP. The reported “lameness” was purely aesthetic (Image 1) in many cases and clinical in others (Image Groups 2 and 3). Most females appeared to recover and not suffer lameness in the sow herd if they were in the aesthetic lameness category. Those farms receiving clinical lameness cases (Image Groups 2 and 3) reported improvement in some females and deterioration in others. An effort was made to weed out all clinical lameness cases during the selection process.

Past percent sold on the select market had ranged from 66 to 79% sold per group over the previous 6 months (Table 8 and 9). At the time of first assessment, the herd had reduced the percent sold per group to 51.9% in August 2012 followed by 38.6% in September 2012 of opportunity females (Table 8). This reduction was not due to demand but was due to true reduction in ability to select because of this condition.

Maternal barrows in a variety of wean-to market barns from this parent source also went through this “lameness.” However, the “lameness” did not impact pigs sold at the commercial level nor did it appear to impact growth. In fact, at the commercial level, the “lameness” was not
considered problematic just purely incidental. At the gilt grow out however, the height of the swelling and associated lameness occurred at the time when the majority of females were sold, 230 to 250 pounds, hence the strong impact on percent sold.

Clinical observations at first visit 09/19/2012 (SPM)

Clinically at the time of first visit, the observing veterinarian noted various levels of swelling surrounding the hock and/or shoulder region (See Image Groups 1, 2 and 3). Many females had one limb affected, however other females had two, three or all legs affected. Original prevalence by age is shown in Table 1. Varying levels of lameness was observed ranging from no effect on mobility, to mild aversion to weight on the affected limb, to pigs with muscular tremors walking in an eggshell-like fashion and going down in back. Initially two females were delivered live to the University of Illinois Veterinary Diagnostic Laboratory.
Diagnostics (SPM and KS)

Diagnostics on sacrificed females by UICVM pathologist on call reported the following:

Morphologic diagnosis:

1. Moderate bilateral subacute to chronic tibiotarsal and hock joints tenosynovitis
2. Marked chronic tenosynovitis of the left carpal and right tibiotarsal region
3. Moderate acute synovitis of the left stifle joint
Microscopic report:

Tendon sheaths were markedly thickened and expanded by mature fibrous connective tissue, as well as multiple, variably sized, occasionally coalescing foci with small to moderate numbers of reactive fibroblasts, newly formed blood vessels and small to moderate numbers of lymphocytes, plasma cells, and rare eosinophils and neutrophils. The synovial lining is multifocally thickened, eroded and partially replaced by proteinaceous fibrillar material fibrin. The underlying areas are multifocally to locally extensively infiltrated by small to moderate numbers of lymphocytes, plasma cells, few eosinophils and rare neutrophils. In other areas the synovial lining is multifocally thickened (hyperplastic). The tendon sheath lumen contains variably sized clumps of fibrin with minimal amounts of necrotic cellular debris. The synovial membrane lining is multifocally minimally thickened, forms rare, small papillary projections, and the underlying soft tissues are infiltrated a few aggregates of small numbers of lymphocytes, plasma cells, and rare eosinophils.

Microscopic summary:

1. A moderate to marked diffuse chronic lymphoplasmacytic tenosynovitis with fibrin formation and granulation  tissue proliferation (Image Groups 3 and 4)
2. Minimal to mild multifocal chronic lymphoplasmacytic synovitis (Image Group 5)
3. Moderate to severe reactive lymphadenopathy with lymphoid hyperplasia. (Image Group 6)
In regards to long bone articular cartilage, the pathologist noted some lesions within the articular cartilage and physis, but said that gross lesions and histopathology did not support OCD. He also reported that the observed lesions on the cartilage were likely secondary to expansion of inflammation centered on tendons and that nutritional disease seemed unlikely.

M. hyosynoviae was isolated from Pig B’s right tarsal tendon. Other affected tendons in Pig A were not positive but the pathologist said that lesions were typical of M. hyosynoviae. At a later date from subsequent diagnostic submissions, M. hyorhinis was isolated from affected
pigs (lungs) but not from leg lesion sites. Post treatment implementation there were no non-treated females on site. No further isolates occurred on subsequent submissions (6) likely do to treatment impact or efficacy. All lesions on submitted females looked the same on morphologic and microscopic examination and remained consistent with findings in the original case submitted.

After these diagnostics, the veterinarian was tasked with development of a diagnostic, treatment, preventative and monitoring plan. The goal was to reduce lesion prevalence and severity and increase the number of selects sold and to measure the effectiveness of the treatment/preventative plan.

First plan of action and results (SPM)

The first treatment program implemented had a dual preventative and treatment purpose. To accomplish this, a feed medication chlortetracycline (10 mg/lb.) pulsing program was initially instigated. Shortly after implementation the treatment/preventative plan, diagnostics showed the isolated M. hyosynoviae resistant to chlortetracycline, but sensitive to lincomycin. Lincomix® is not labeled for reduction or control of Mycoplasma hyosynoviae however it is labeled for control of ileitis.2 This herd also needed a control plan for ileitis. Switching the treatment plan to Lincomix® achieved this end goal as well and also had a secondary benefit of assisting with the Mycoplasma hyosynoviae prevention and treatment plan. Details of the plan implemented in late November of 2012 involved concentration of Lincomix® therapy at 100 g per ton from 3 to 8 weeks post entry, with pulses of Lincomix starting at 11 weeks and again at 15 weeks post entry. Table 1 a and 1 b show the prevalence results of lameness (Table 1) and diarrhea (Table 2) and Fallouts (Table 3) per age per monthly visit as we monitored the quantitative impact of this prevention/treatment program on diarrhea and fallout prevalence as well as lameness.
Simultaneously, our isolate was forwarded to laboratories to be poised for potential production of autogenous vaccine.

Case communication to customers (SPM)

The goal with preventative and treatment implementation was not to hide the situation, but rather be transparent and clear regarding our diagnostic findings and preventative/treatment approach. As the plan was implemented, vet-to-vets were completed with all customers and diagnostics and preventative/treatment plan discussed. During the vet-to-vet’s communication, we made it clear that this herd remained negative to Mycoplasma hyopneumoniae, but like many herds was positive to Mycoplasma hyosynoviae, and to the best of our knowledge was now experiencing clinical expression of Mycoplasma hyosynoviae. Vet-tovet’s information gathered reported that most of gilts merged well with the sow herds that were negative to PRRS and Mycoplasma hyopneumoniae, however if the herd converted to PRRS or Mycoplasma hyopneumoniae during acclimation, the severity of the lameness would increase vs. decrease. When Lincomix®2 was implemented at the time of PRRS and Mycoplasma hyopneumonaie conversion, this increase in severity was mitigated or eliminated. During and post communication customers were concerned, but expressed desire to remain on the same gilt source. They reported good performance of gilts in spite of the situation we were addressing.

Further diagnostics plan started (SPM)
A diagnostic and monitoring plan was implemented to further work to evaluate the impact of the prevention/treatment program and impact on M. hyosynoviae and to evaluate whether M. hyorhinis was impacting the signs as well. The veterinarian in charge, Sarah Probst Miller, coordinated with the Zoetis representatives, Drs. Paul Knoernschild and Everett Rosey from Zoetis as well as Drs. Alex Ramirez and Phil Gauger at ISU-CVM.

Background summary of diagnostic capabilities at the time of the plan development (AR)

Unfortunately not all swine diseases have well established validated diagnostic tools available. Such is the case for M. hyorhinis and M. hyosynoviae. Culture for Mycoplasma spp is not easy and is usually time consuming. Mycoplasmas tend to be overgrown by contaminants quite easily. There are several PCR assays available for both of these mycoplasmas. These PCRs are quite sensitive and specific but unfortunately difficult to quantify the amount of DNA present in the sample; especially for M. hyosynoviae. As such, sometimes we can rely on CT values as close proxy to help us compare amount of mycoplasma DNA between two samples. This is not a perfect science, especially when comparing CT values from different days, but with caution, CT values can provide some insight into possible pathogen load until quantitative PCR assays are developed and validated.

These PCR assays can be used on blood, tonsil scrapings as well as oral fluids. Past experience and research by ISU has shown that tonsil scrapings and oral fluids are the best samples for detecting M. hyosynoviae. Serologically we still have the problem on having a validated assay. Currently the ISU-VDL has a Tween-20 ELISA assay for both M. hyorhinis and M. hyosynoviae that is still under research development. Although cutoff values have not been well established for these assays, the trend in increased in corrected optical density (OD) values can be used to demonstrate exposure through increased values. As a test under research development, OD values between dates can vary. Currently, based on past experience, we believe that the immune response detected by this Tween-20 ELISA assay occurs much later than the traditional 2 weeks post exposure we are used to with many other pathogens. 
Serologically we still have the problem on having a validated assay. Currently the ISU-VDL has a Tween-20 ELISA assay for both M. hyorhinis and M. hyosynoviae that is still under research development. Although cutoff values have not been well established for these assays, the trend in increased in corrected optical density (OD) values can be used to demonstrate exposure through increased values. As a test under research development, OD values between dates can vary. Currently, based on past experience, we believe that the immune response detected by this Tween-20 ELISA assay occurs much later than the traditional 2 weeks post exposure we are used to with many other pathogens.
Diagnostic plan created (SPM)

The following diagnostic plan was created:

1. Quantitative assessments of prevalence and severity levels:

a. To monitor the prevalence rate and severity (ABCE ranking levels described in b.), whole barns of gilts were monitored monthly by choosing 4 pens of gilts per barn and assessing all gilts per pen for signs of lameness and associated severity.
b. In each pen, 4 gilts were tagged and their level of lameness was monitored over time. Gilts were given a severity ranking of A, B, C or E. (See Images 4, 5, 6 and 7), i. A pigs showed aesthetic swelling but would likely sell as sound due full mobility (Image 4).

ii. B pigs also showed swelling on limbs but it was more advanced. These hogs if sound might sell and if not fully weight bearing on all limbs would not sell as selects (Image 5).
iii. C pigs would for sure end up a market hog vs. a select due to level of swelling and clinical lameness (Image 6).
iv.E pigs were severely lame hogs that would either go on the cull/lite truck or be euthanized (Image 7).

2. To further assess presence and intensity of M. hyosynoviae and M. hyorhinis in the population, the following sampling methodology was completed:

a. Every month rope sampling was done in the selected pens
b. Every month serum sampling and tonsilar swabbing was done on the tagged gilts
Results (SPM and AR)

Quantitative results showing prevalence per symptoms of concern per month are reported in Tables 1, 2, and 3. Total prevalence does not reduce over time. In fact, it appears to increase. At first, only total prevalence of each symptom was assessed. We implemented ranking by severity in February. We believe by inclusion of the aesthetic lameness category of A where these animals had mild visual signs, but no actual mobility lameness; we were more willing to put females into this category thus increasing our prevalence totals. At this same time, as we started to have females sold that had been through the entire treatment program; our percent selects sold started to go up (Table 8 and 9). April 9, 2013 had our first group that had received 80% of the preventative treatment plan and gilts sold on 5/9/2013 through 7/9/2013 had received 90 to 100% of the preventative/treatment plan. Once females receiving the preventative/treatment plan for most of their life started to be sold the range of percent sold moved from 38 to 55% sold to 58 to 75% sold.

Quantitative results reporting the severity breakdown per prevalence are shown in Tables 4a and b through Tables 7a and b. These charts show the percentage of A, B, C and E pigs in the total prevalence reported. Since A pigs could be sold as selects, we also created a Linear Forecast Trend line showing the total pigs with symptoms minus the A pigs. Most of these forecasts in every age group are trending down indicating a trend towards more saleable females, except for the 15 to 18 week trend. We believe this 15 to 18 week trend may be kewed by potentially aberrant July results. At the time of the presentation we will have August quantitative numbers in and will be able to understand if this July spike is artifact or real. Of importance is that the trend line is headed in the right direction for animals in the 19 to 22 week range as this is the age that the majority of females are sold (Table 7b).

Serology results April, May and June 2013 testing results

In April, Tween 20 testing showed presence of both M. hyosynoviae and M. hyorhinis. Table 10a shows the average S/P ratio of M. hyosynoviae across age groups. Both pathogens peaked at 22 and 17 weeks respectively. Initial exposure Table 11 shows the average S/P ratios for pigs ranked as A,B or C and Normal (labeled as blank in table) pigs. Normal pigs had lowest S/P ratios while B (subacute) and C (chronic) had the highest.

In May and June we focused on just M. hyosynoviae to reduce total testing cost as M. hyorhinis was never isolated from a leg lesion and only from lung tissue. Also the height of symptoms matched better with the timing of typical M. hyosynoviae infections. May had similar results to April and June’s Tween 20 results are be redone to check test accuracy due to unexpected variation from April and May.

Rope PCR results

Rope PCR results are reported in Chart 1a-c. By pen females appear to arrive with some pens positive. As they start Lincomix® treatment, the majority of pens are negative.

As pigs move to non-medicated feed, pens start to move back positive again. This test lets us know that the Lincomix® program does appear to reduce the presence of the pathogen when under full treatment; but it does show that in any given pen, the pathogen does reappear
over time once treatment is discontinued. This would be discouraging if not for the results from the tonsilar swabs from the individual females.
Tonsilar swabs of individual females

In Chart 2a and b we have M. hyosynoviae results from tagged females. Results show a reduction in the number of pigs who are individually positive in those females who had received a more complete preventative/treatment course. With this test we speculate that by continuing the preventative/treatment Lincomix® plan we appear to be reducing pathogen load over time.

Next steps

Based on these results, a decision has been made to hold the current preventative/treatment course and work to move the Lincomix® program as close to arrival as possible. With this plan we hope to continue to reduce the pathogen load at this site. Due to a K88 diarrhea at arrival the herd is currently reluctant to move their Mecadox program in the first two weeks post arrival. Lincomix® will go in the feed 2 weeks post entry for most females.

At this time the herd is poised for potential autogenous production, but does not have immediate intentions of starting autogenous production. 

This report will be communicated to the sow farm and a possible pathogen reduction program at the sow farm level will be discussed.

Discussion and significance of the case study

M. hyosynoviae is hard to diagnose and prove correlation with lameness in certain circumstances. The novel rope tests for M. hyosynoviae involved in this case showed promise as a diagnostic tool for the industry to use in the future to help diagnose presence of M. hyosynoviae and treatment efficacy. In addition, tonsilar swabs of individual animals allowed us to better understand reduction in pathogen presence. When diagnostic testing is paired with regular intervals of quantitative assessment of prevalence and severity of symptom, the presence and absence of clinical signs during therapeutic intervention can be potentially assist with diagnostics interpretation.

This quantitative and diagnostic approach allowed us to achieve an early understanding of trends to know if we were on the right track.

Summary of what we learned about diagnostic capabilities from this case (AR)

One of the key points we have learned from this case study is that in working up a case like this using multiple diagnostic tools is essential; especially since we are dealing with tools that we are still learning how to use for this particular pathogens. If we just take one individual test, say tonsil scraping PCR vs. oral fluid PCR, we would arrive at different conclusions based on which test we would have chosen. In this flow, tonsil scrapings were more likely to be positive in younger animals while oral fluids tended to be more positive in older animals.Using multiple tools has allowed us to have a better picture on what may be going on in this flow. Also incorporating be big overall clinical picture as well as having a keen understanding of when and how pigs are being medicated is a critical skill that attending herd veterinarians are able to bring to the table to help interpret diagnostic results. This re-emphasizes the importance of the clinical perspective and understanding necessary to interpret any diagnostic results.

References

1. Gomes Neto JC, Gauger PC, Strait EL, et al. Mycoplasmaassociated arthritis: Critical points for diagnosis. J Swine Health Prod. 2012;20(2):82–86
2. Lincomix® Label https://online.zoetis.com/US/EN/products/Pages/Lincomix? Feed.aspx
Images 1-7:
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 1
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 2
 
 
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 3
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 4
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 5
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 6
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 7
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 8
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 9
Mycoplasma hyosynoviae A Case Study: Quantitative assessment of incidence and severity across time alongside a diagnostic monitoring plan and intervention - Image 10
 
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