2016 Veterinary Diagnostic Laboratory Report: a new director, PRRS, PEDV, and Senecavirus

The Veterinary Diagnostic Laboratory’s mission is to protect and promote animal and human health through early detection and monitoring of animal diseases.
The 2016 report was published last month and we are compiling here the highlights related to swine. We can also read the full 2016 UMN VDL report.

  • In April 2016, the VDL welcomed its new director Dr. Jerry Torrison.
  • More than 50% of the procedures  in the VDL were related to the porcine species last year.
  • A new multiplex PCR test that combines Porcine Epidemic Diarrhea Virus (PEDv), Porcine Deltacoronavirus (PDCoV) and Transmissible Gastroenteritis Virus (TGEV) into one assay was implemented into the Molecular Diagnostic clinical testing schedule effective October 31st, 2016. The new assay provides clients with timely, quality results for all three viruses at the same time. The VDL ran 40,131 PEDv and PDCoV Multiplex Real Time PCR tests and 5,238 Triplex (PEDv/TGE/PDCoV) RT-PCR tests.
    Additionally, the Serology lab conducted intensive testing in collaboration with Zoetis for validation of PED antibody test kit which they are planning to release on the market soon.
  • Seneca Valley Virus PCR was validated and is part of routine testing. 3,205 Senecavirus A EZ Real time RT-PCR tests were run. An ELISA test for antibodies to Seneca Valley Virus in pigs is also available.
  • The IHC lab participated in the 2016 AAVLD/NVSL Program for Inter-laboratory Comparison, and scored 100% in its detection of Porcine Circovirus type 2 in the test samples provided.

Science Page: Continued reporting on unusual CNS cases

This is our Friday rubric: every week a new Science Page from the Bob Morrison’s Swine Health Monitoring Project. The previous editions of the science page are available on our website.

This week, the Science Page focuses on unusual Central Nervous system (CNS) cases that have been emerging in the past few years. Etiological agents like porcine teschovirus (PTV), porcine enteroviruses (PEV), porcine sapelovirus (PSV), and atypical porcine pestivirus (APPV) have been implicated in those cases leading to the creation of a set of criteria to positively identify a CNS case.

Three criteria are therefore required: identifying the clinical signs, a positive PCR test for one or more of the viruses, and histological results consistent with viral encephalitis from spinal cord or brain tissue.

Key points from this week edition:

  • An apparent increase in the number of cases associate with atypical neurological signs have been observed over the last two years.
  • Since then, the Veterinary Diagnostic Laboratories (VDLs) have identified a set of criteria required to meet the CNS case definition.
  • MSHMP will continue reporting CNS cases diagnosed at the KSU, ISU, SDSU, and UMN VDLs.

Take a look at the number of cases recorded since September 2016.

The latest trends in PRRSV diagnostics: less serum samples, more oral fluids, and more 1-7-4 RFLP pattern

Today, Dr. Albert Rovira from the University of Minnesota, Veterinary Diagnostic Laboratory shares with us the trends he has observed in PRRSV diagnostics over the past years. The findings can be found in the slideshow below.

  • The use of tissue samples follows a seasonal pattern and represents clinical cases with a percent of positives of 30%
  • The number of oral fluid samples is increasing. Used for monitoring positive farms and more recently for surveillance in negative farms as well:: 15% of positive samples
  • The number of blood swabs, serum samples, and semen samples, typically used for surveillance in negative farms, is decreasing. Lowest percent of positive samples: 8%
  • RFLP patterns are changing over time. In the past years, 1-7-4 > 1-3-4 or 1-8-4 or 1-4-4
Reminder: what is a RFLP pattern?

RFLP stands for Restriction Fragment Length Polymorphism and is a technique used to detect nucleotide changes in a genetic sequence. The genetic material is put in contact with restriction enzymes which are very specific to a genetic sequence. If the enzyme recognizes the sequence pattern, it will cleave the DNA or RNA fragment. After that, a type is determined based on the number of fragments and its size.

For example with PRRSV, three enzymes are used and the number of fragments each of them produces makes up the numbers of the RFLP pattern. Currently, the RFLP type is not actually performed in the lab. Instead, it is predicted based on the ORF5 RNA sequence and the knowledge of the cutting capabilities of each enzyme.

Therefore, the RFLP pattern gives us a way to cluster PRRSV strains in groups but very little indication about how similar they are to each other.

Detecting Senecavirus A in tissues: development of a new diagnostic test at the University of Minnesota

Summary

In Canada and the USA alike, Senecavirus A is a challenge for producers and veterinarians because of its clinical similarity to Food and Mouth Disease (FMD). Indeed, Senecavirus A, is a causative agent of swine vesicular disease with lesions developing on the snout, around the mouth and on the coronary band of the feet. Therefore, being able to differentiate Senecavirus A infections from FMD rapidly is of utmost importance to be able to take the appropriate measures.

In the past months, several diagnostic tests have been developed at the University of Minnesota to detect antibodies against Senecavirus A. The difference between those tests and the in situ hybridization (ISH) described here is that ISH targets the genetic material included in the viral particle and marks it as a red spot as can be seen on the figure below. This advantage of this method is to be able to locate the virus and gives additional information to researcher wanting to study the behavior of Senecavirus A in the body of the pig.

in situ hybridization senecavirus A pigs
Red dots and clusters represent the presence of SVV mRNA within an erosive lesion on the tongue of a pig © 2017 Resende et al.

Abstract

Seneca Valley virus (SVV) is the causative agent of an emerging vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. In addition, SVV has been associated with neonatal mortality in piglets. While a commercial SVV qRT-PCR is available, commercial antibodies are lacking to diagnose SVV infections by immunohistochemistry (IHC). Thus, a novel in situ hybridization technique—RNAscope (ISH) was developed to detect SVVRNA in infected tissues. From a total of 78 samples evaluated, 30 were positive by qRT-PCR and ISH-RNA, including vesicular lesions of affected sows, ulcerative lesions in the tongue of piglets and various other tissues with no evidence of histological lesions. Nineteen samples were negative for SVV by qRT-PCR and ISH-RNA. The Ct values of the qRT-PCR from ISH-RNA positive tissues varied from 12.0 to 32.6 (5.12 x 106 to 5.31 RNA copies/g, respectively). The ISH-RNA technique is an important tool in diagnosing and investigating the pathogenesis of SVV and other emerging pathogens.

Link to the full article

Science page: M. hyopneumoniae outbreaks: what you need to know to aid in your investigation

This is our Friday rubric: every week a new Science Page from the Swine Health Monitoring Project. The previous editions of the science page are available on our website.

Key points from this week edition:

Molecular characterization tools such as p146 sequencing for Mycoplasma hyopneumoniae (M. hyopneumoniae) can provide insight towards investigating elimination failures or new introductions within swine herds.

Sample and diagnostic types for early detection of Mycoplasma hyopneumoniae

Summary:

Mycoplasma hyopneumoniae is the causative agent enzootic pneumonia, an economically significant disease in pigs. In this study published by Drs. Pieters and Rovira from the University of Minnesota, pigs experimentally inoculated with M.hyopneumoniae were sampled 0, 2, 5, 9, 14, 21, and 28 post-inoculation.

Different sample types were compared:

  • Nasal swabs
  • Laryngeal swabs
  • Tracheobronchal lavages
  • Oral fluids
  • Serum samples

Using different diagnostic tests:

  • PCR
  • ELISA IgG anti M.hyopneumoniae
  • ELISA Ig M anti M.hyopneumoniae
  • ELISA C-reactive protein

Laryngeal swab samples tested by PCR were highly sensitive for detection of Mycoplasma hyopneumoniae in live pigs.
Various commercial ELISA kits for detection of Mycoplasma hyopneumoniae antibodies showed similar sensitivity.
Oral fluids showed a low sensitivity for detection of Mycoplasma hyopneumoniae in experimentally infected pigs.

Pieters Mhyopneumoniae early detection test sample 2017

Abstract

Detection of Mycoplasma hyopneumoniae in live pigs during the early stages of infection is critical for timely implementation of control measures, but is technically challenging. This study compared the sensitivity of various sample types and diagnostic methods for detection of M. hyopneumoniae during the first 28 days after experimental exposure. Twenty-one 8-week old pigs were intra-tracheally inoculated on day 0 with M. hyopneumoniae strain 232. Two age matched pigs were mock inoculated and maintained as negative controls. On post-inoculation days 0, 2, 5, 9, 14, 21 and 28, nasal swabs, laryngeal swabs, tracheobronchial lavage fluid, and blood samples were obtained from each pig and oral fluid samples were obtained from each room in which pigs were housed. Serum samples were assayed by ELISA for IgM and IgG M. hyopneumoniae antibodies and C-reactive protein. All other samples were tested for M. hyopneumoniae DNA by species-specific real-time PCR. Serum antibodies (IgG) to M. hyopneumoniae were detected in challenge-inoculated pigs on days 21 and 28. M. hyopneumoniae DNA was detected in samples from experimentally inoculated pigs beginning at 5 days post-inoculation. Laryngeal swabs at all samplings beginning on day 5 showed the highest sensitivity for M. hyopneumoniae DNA Detection, while oral fluids showed the lowest sensitivity. Although laryngeal swabs are not considered the typical M. hyopneumoniae diagnostic sample, under the conditions of this study laryngeal swabs tested by PCR proved to be a practical and reliable diagnostic sample for M. hyopneumoniae detection in vivo during early-stage infection.

Link to the full-article

Development and validation of a competitive ELISA as a screening test for Senecavirus A

An article published in the Journal of Veterinary Diagnostic Investigation (JVDI) presents a competitive Enzyme-Linked ImmunoSorbent Assay (cELISA) and a virus neutralization test (VNT), both validated for the screening of Senecavirus A in a research setting, by the National Centre for Foreign Animal disease (NCFAD). The diagnostic specificity and sensitivity were 98.2% and 96.9% for the cELISA, and 99.6% (99.0–99.9%) and 98.2% (95.8–99.4%) for the VNT, respectively.

In Canada and the USA alike, Senecavirus A is a challenge for producers and veterinarians because of its clinical similarity to Food and Mouth Disease (FMD). Indeed, Senecavirus A, is a causative agent of swine vesicular disease with lesions developing on the snout, around the mouth and on the coronary band of the feet. Therefore, being able to differentiate Senecavirus A infections from FMD rapidly is of utmost importance to be able to take the appropriate measures.

The University of Minnesota, Veterinary Diagnostic Laboratory has developed an ImmunoFluorescence Assay (IFA) to detect antibodies against Senecavirus A. This test was used as a reference for the validation of the cELISA and VNT established by Drs. Goolia, Yang, Babiuk, and Nfon from NCFAD in collaboration with Drs. Vannucci and Patnayak from the UMN-VDL.

celisa-vnt-senecavirus-a-vannucci

Abstract: Senecavirus A (SVA; family Picornaviridae) is a nonenveloped, single-stranded RNA virus associated with idiopathic vesicular disease (IVD) in swine. SVA was detected in pigs with IVD in Brazil, United States, Canada, and China in 2015, triggering the need to develop and/or validate serologic assays for SVA. Our objective was to fully validate a previously developed competitive enzyme-linked immunosorbent assay (cELISA) as a screening test for antibodies to SVA. Additional objectives included the development and validation of a virus neutralization test (VNT) as a confirmatory test for SVA antibody detection, and the comparison of the cELISA, VNT, and an existing immunofluorescent antibody test (IFAT) for the detection of SVA antibodies in serial bleeds from SVA outbreaks. The diagnostic specificity and sensitivity were 98.2% (97.2–98.9%) and 96.9% (94.5–98.4%) for the cELISA, and 99.6% (99.0–99.9%) and 98.2% (95.8–99.4%) for the VNT, respectively. There was strong agreement among cELISA, VNT, and IFAT when compared based on kappa coefficient. Based on these performance characteristics, these tests are considered suitable for serologic detection of SVA in pigs.

Link to the entire article