Rathayibacter toxicus is primarily vectored by the nematode Anguina funesta. Other nematodes of the same genus can also vector the bacterium including: A. agrostis, A. australis, A. paludicola, and A. tritici.

If bacterial ooze is present on the plant surface, those seed heads should be prioritized for sampling. The seed can be examined visually for nematode and bacterial galls using a light box or tested for the nematode vector, Anguina funesta , by a soak-sieve method or randomly selected. Any seed that is positive for A. funesta should be tested further for the presence of Rathayibacter toxicus using the molecular methods in the Key Diagnostics section.

Clinical signs in livestock appear abruptly, usually following some external stimulation. They include tremors, ataxia, adoption of a wide-based stance, stumbling and falling over, nystagmus, convulsions while recumbent and death (Allen, 2012). Animals may appear to recover between episodes of ataxia and convulsions. Sheep often exhibit a high stepping gait with their forelimbs, while the head is held high. When forced to run, some sheep have a stiff-legged or "rocking horse" gait. Some cattle appear disorientated and wander aimlessly between episodes of convulsions. Signs may appear as soon as four days after introduction to toxic pasture or feed. Animals may continue to exhibit neurological signs and die for up to 10 days after removal from toxic feed. Ewes may abort (Allen, 2012).

Nematode-infected seeds are pointed, shorter than normal, and are black or yellow. A yellow coloration indicates that bacteria are present in the gall (Clarke and Nurse, 1999).

When introduced to a gall, Rathayibacter toxicus bacteria rapidly multiply soon after the gall is initiated, smother the nematode larvae, and colonize the structure to form a bacterial gall. If the bacteria are deposited outside the galls but within the developing inflorescence, under suitable environmental conditions they can multiply to form a yellow slime that will become apparent on the surface of the plant at the time of head emergence. With time, the slime hardens and turns orange and then brown. The slime does not always appear and may also be washed off by rain (Clarke and Nurse, 1999).

Rathayibacter toxicus can go undetected for long periods, making visual detection a serious challenge. Most of the infected plants do not show any visible symptoms. Thus, absence of visible slime (gummosis) does not necessarily mean that a pasture is free of R. toxicus. In many cases, bacteria and nematode infections go undetected or the disease is misidentified. The nematode vector and bacterium can survive in the dry state for many years (Murray et al., 2014).

Molecular: Under review: There are real-time PCR assays available to detect Rathayibacter toxicus in plants and nematodes and for identification of four species of Anguina (funesta, tritici, agrostis , and pacificae). Although work instructions are available from CPHST Beltsville for both assays, they are being evaluated and validated. Fully validated work instructions will be available by the end of fiscal year 2017.

Annual ryegrass toxicity (ARGT) can be confused with perennial ryegrass staggers (caused by a toxin produced by an endophytic fungus in the grass), botulism, and tetanus. However, animals affected by ARGT may have periods of apparent recovery, and this distinguishes ARGT from these other diseases (Kerr and Gibb, 1997).

Rathayibacter rathayi, a non-toxigenic species, causes bacterial head blight or Rathay"s disease in orchardgrass (Dactylus glomerata L.) in the United States. Symptoms look superficially similar to infection by R. toxicus, and molecular or serological confirmation of the causal agent is required to differentiate the two bacterial species (Putnam, 2009).

Rathayibacter tritici causes spike blight or gumming disease in wheat, but is not known to be present in the United States. Rathayibacter iranicus also causes a gumming disease of wheat but it has been documented only from Iran and Turkey (Putnam, 2009).

Anguina funesta was once synonymized with congeners A. agrostis and A. wevelli. There are now at least 11 valid species of Anguina, and Powers et al. (2001) provide a key to differentiate them using PCR-RFLP analysis. In addition to molecular differences, these nematodes are generally host specific.

Li et al. (2015) developed a real-time PCR which can detect Anguina funesta. Atha et al. (2015) have developed a real-time PCR which can detect R. toxicus. There is reportedly real-time PCR under development by Norman Schaad for detection of R. toxicus (Murray et al., 2014). This may be the same project as Atha et al., (2015) above. PCR-based assays for identification of Rathayibacter toxicus and the bacteriophage isolate NCPPB 3778 have also been described (Kowalski et al., 2007).

Masters et al. (2006) developed an Enzyme Linked Immunosorbent Assay (ELISA) to detect R. toxicus in feeds. This ELISA has since been improved (Masters et al., 2011) and can also be used to detect the presence of the organism in rumen contents and feces.

An ELISA that detects and quantifies the corynetoxins in feed has also been developed, but it is not as quick and is more costly than the ELISA for R. toxicus, and studies have shown a good correlation between the results from both ELISAs. Therefore, the ELISA for the bacterium is still the preferred test (Allen, 2012).

Note: The ELISA test is not currently commercially available.