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Mycoplasma Control Explored at AVMA Convention

16 November 2013

Recent advances in the control of Mycoplasma synoviae and Mycoplasma gallisepticum were presented in the poultry sessions that were included in the 150th annual convention of the American Veterinary Medical Association (AVMA), held in July 2013 in Chicago. The session also included a paper on a method to distinguish antibody Responses to Influenza A and Newcastle Disease viruses.

Impact of In-feed Chlortetracycline on Prevention of Infection and Horizontal Transmission of Mycoplasma synoviae

Mycoplasma infections are a source of substantial economic loss in commercial poultry production. Representing the University of Georgia, Elise A. Myers said that, although antibiotics have been used to control the spread of mycoplasma infections as well as to treat infected flocks, there have been no recent objective investigations to evaluate the effectiveness of preventive treatment.

In their study, the ability of antibiotic treatment to prevent infection and slow transmission of a Mycoplasma synoviae (MS) strain (S-6 genotype), isolated from a recent outbreak in broiler breeder flocks in Northeast Georgia, was evaluated in two bird trials.

An infectious dose trial was conducted using specific pathogen-free laying hens to evaluate the ability of in feed chlortetracycline at 400g per tonnes to increase the infectious dose of MS.

This trial demonstrated that providing chlortetracycline in the feed at the above inclusion level increased the dose of MS required for infection by at least 1-log in comparison to the group receiving non-medicated feed.

A transmission trial was also conducted, using 59 week-old broiler breeders to evaluate the efficacy of in-feed chlortetracycline at varied concentrations (200g or 400g per tonne) to control the spread of the S-6 genotype of MS.

Myers reported that the inclusion of chlortetracycline in feed at 400g per tonne successfully prevented MS infection in hens exposed to MS-positive roosters over a period of four weeks.

Early Serological Detection of Mycoplasma synoviae in Challenged and Contact Exposed Chickens Using ELISA

Early detection of new MS infections through monitoring programmes is essential in controlling/eradicating MS infections, according to Gwenllyan F. Slacum of BioChek USA Corp. Historically, monitoring programs for MS have been based on the use of rapid plate agglutination (RPA), haemagglutination inhibition (HI) and enzyme-linked immunosorbent assays (ELISAs). However, opinions vary regarding the sensitivity of the RPA and ELISA. Previous studies in which the ELISA showed lower sensitivity were conducted with commercially available MS ELISAs produced using whole cell antigen.

Recently, a new MS ELISA based on a recombinant antigen with excellent specificity (more than 98 per cent) has been approved for use in the US.

In this study, the sensitivity of the recently approved MS ELISA was compared to MS RPA for the detection of MS from seven to 21 days post-challenge (dpc). The sensitivity of the ELISA (BioChek) and RPA was tested using different lots of each assay on the same samples derived from broiler breeders challenged at four weeks of age with MS strains WVU 1853 and K5664 and from comingled broiler breeders.

Slacum reported the results at the meeting. In summary, the ELISA used in this study detected positive birds starting at seven dpc, whereas, two of the three RPA lots showed positive birds at seven dpc.

Development of Fluorescent Antibody Test for the Detection of Mycoplasma synoviae in Chickens

Vijay Durairaj of the Poultry Diagnostic and Research Center at the University of Georgia reported that MS causes subclinical or clinical upper respiratory tract infections and systemic reactions in chickens affecting multiple organs such as synovial membranes of joints and tendon sheaths.

Vertical or horizontal transmission aids in spreading MS between chickens. Aerosol exposure is one of the common routes of horizontal transmission. Exposure to an aerosol antigen stimulates high levels of IgA antibody production associated with the mucosal immune system.

The main objective of this Athens study was to evaluate whether the locally produced specific IgA antibodies against MS could be used as a predictor for determining early infection of MS.

Three-week-old SPF chickens were challenged with MS by the intratracheal, intraocular and intranasal routes. The chickens were bled and choanal cleft and tracheal swabs (in PBS) were collected at 3, 5, 7, 9 and 11 days post infection. Serum was used for rapid plate agglutination test, hemagglutination inhibition (HI) test and commercial ELISA. Choanal cleft swabs were used for real time PCR and culture. Tracheal swabs (in PBS) were used for this fluorescent antibody test. MS cultures grown on modified Frey’s medium agar plate were used for this assay. Tracheal swab washes in PBS was used as a primary antibody followed by corresponding secondary antibody conjugated with FITC. Positive and negative samples were identified by the presence and absence of immunofluorescence, respectively.

Origins and Generation of Mycoplasma gallisepticum Diversity

Examples of phenotypic, and more recently genotypic, diversity of Mycoplasma gallisepticum (MG) strains (reference and vaccine) and field isolates have become well recognised and have important implications to disease impacts, diagnostics, intervention strategies and control programmes, according to David H. Ley from the College of Veterinary Medicine at NC State University.

Sequence-based genotyping and databases have recently enabled both improved and efficient strain/isolate identification for outbreak investigations and epidemiology, as well as appreciation for MG diversity and evolution.

The researchers' phylogeny (based on analyses of 8399 nucleotides from 13 loci/isolate) of 107 MG isolates from songbirds (mainly house finches) and domestic poultry showed that the house finch epidemic was derived from a much more diverse set of MG pathogens circulating in poultry.

However, they infer that MG has repeatedly jumped between these two groups of hosts but that to date, only a single lineage of MG has persisted and evolved in house finches. All house finch isolates from western North America were derived and slightly differentiated from a single colonisation of that region by an eastern North America source.

Phylogeny also indicated that genetic diversity of MG in a geographic area increases with the length of time that MG has been established in a population, so the house finch epidemic now consists of monophyletic eastern and western MG clades.

By extension, Ley asked, is it possible that discrete, endemically infected populations - islands of MG infection, such as the wide variety of non-commercial poultry flocks - are the most likely origins and generators of MG diversity that is usually only recognised after infections are transmitted to commercial poultry flocks?

Surveillance Studies of Mycoplasma gallisepticum in Georgia by Targeted Sequencing

Georgia ranks number one in poultry production in the US, and it accounts for 54 per cent of Georgia agriculture. It is important to the Georgia poultry industry to retain healthy, mycoplasma-free flocks. MG is both vertically and horizontally transmitted, making biosecurity of paramount importance in reducing disease transmission, according to Victoria A. Laibinis of the University of Georgia.

In 2006, an MG outbreak in Georgia was identified as wild-type ts-11/HF (house finch) by targeted sequencing of the mgc2/IGSR regions. The sequence from this wild-type was 100 per cent match to ts-11 vaccine strain in the mgc2 gene, and 100 per cent match to HF in the IGSR region.

Early in 2007 through 2011, ts-11 vaccine was approved in broiler breeders in Georgia, in an effort to control the disease.

In this survey, targeted sequencing was conducted on more than 150 cases from both commercial flocks and backyard birds from 38 counties in Georgia between 2006 and 2012.

Laibinis reported that the ts-11/HF wild-type persisted in the commercial population until 2009. In 2010, ts-11/HF was identified for the first time in backyard birds, and seen only once in 2011 in backyard birds, disappearing in all poultry by 2012. Sequences from all house finch samples collected from 2008-2009, remained consistent as HF/HF type.

Development of a Quadriplex Fluorescent Microsphere Immunoassay (FMIA) for the Detection of Antibody Responses to Influenza A and Newcastle Disease Viruses

Highly pathogenic avian Influenza (AIV) and Newcastle Disease (NDV) viruses cause serious illnesses in domestic poultry, both of which are reportable to the World Organization of Animal Health (OIE).

Mathieu M. Pinette of the University of Manitoba in Canada explained that the clinical presentation of AI and ND are difficult to differentiate, emphasising the importance of rapid and sensitive serologic assays that are able to distinguish them and at the same time able to subtype AIV infection to H5 or H7.

Currently, a variety of serological assays are used for the serologic diagnosis of both diseases but none of these assays is used in multiplex formats.

In this study, the researchers developed a quadriplex FMIA based on xMAP Luminex technology. The assay employs Luminex magnetic beads that are covalently coated with different recombinant AIV and NDV antigens (nucleoprotein and/or hemagglutinin) produced in a baculovirus insect cell expression system. The developed assay is able to detect AI antibodies against all existing haemagglutinin (H1-H16) subtypes and simultaneously subtype AI infection to H5 or H7 with high efficiency. In addition, the assay is able to detect antibodies against different strains of Avian Paramyxovirus type 1.

In the process of development, multiple protein purification techniques and different bead coupling conditions were compared. Assay validation was accomplished by using field and experimental sera and has a comparable or better detection capability than currently used assays in our laboratory.

Pinette added that the assay also has the capability of detecting serum antibodies from different domestic and wild birds and could be used for future surveillance and diagnostic testing.

Further Reading

Find out more information on the diseases mentioned here by clicking here.

November 2013

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