To build on the internal validation performed by VMRD, our Serum Amyloid A (SAA) test was independently evaluated by at the University of California-Davis. Blood samples were collected from horses with acute inflammation, and results were compared between sample types (blood and EDTA plasma) and to results from a reference assay. Assay linearity, repeatability, and reproducibility were also assessed. Full data will be included in a research manuscript that has been submitted for publication in a peer-reviewed journal, which will represent the first published validation of a point-of-care SAA test using whole blood. Findings of this study support the product performance claims of consistent clinical accuracy across the SAA concentration range of <20 to 3000 mg/mL with both blood and plasma, using recommended dilutions. Results from the VMRD SAA test exhibited a strong positive correlation and agreement with the Eiken LZ-SAA assay (performed at the University of Miami Acute Phase Protein Laboratory) used as a reference. The VMRD SAA test reliably measured SAA in both whole blood and plasma, with excellent overall agreement between paired blood and EDTA plasma samples. It is however still recommended that the same sample type be used for monitoring a clinical case over time, as exact values may vary to some degree with sample type. Based on serial dilution of a high SAA sample, linearity of the VMRD SAA test was solid across the entire range of dilutions, demonstrating the ability of this test to detect SAA reliably and accurately across a broad range of physiologically relevant concentrations. When tracked over time in individual horses, results from the VMRD SAA test increased and decreased as would be expected following an acute inflammatory stimulus, peaking at 2-4 days and gradually decreasing over time. Comparison of VMRD and Eiken results for these horses revealed clinically similar SAA kinetics with both tests. Evaluation of test precision revealed appropriate coefficients of variation ranging between 7.8-13.3% for repeatability (multiple tests performed on one day) and 5.7-12.0% for reproducibility (multiple tests performed on consecutive days), showing the consistency of results obtained with this test. The SAA kit comes with the appropriate components and instructions for performing tests with either fresh or anticoagulated blood. Serum or plasma can also be used but require a different sample dilution. If interested in using plasma or serum, please contact VMRD for an appropriate protocol. Additional studies are ongoing to further expand the capabilities of this assay – stay tuned for further updates!

To avoid unexpected SAA results it is critical to understand basic SAA biology and interpret results in light of other clinical data. Presenting complaint and duration of disease are particularly valuable to consider when a normal or low result is obtained, as this would be expected with localized, chronic, or allergic disease. Likewise, historical information should be evaluated to determine if a horse has any other factors that could cause increased SAA, as this may affect interpretation of values. Veterinarians are often most confused by negative or low results in animals that are clearly sick. Production of SAA is stimulated by acute, systemic inflammation; therefore localized, chronic, or allergic issues should not be expected to cause significant increase. These negative or low positive results can still be very useful as they help narrow down possible etiologies and indicate a low likelihood of systemic inflammation due to bacterial or viral infection. It is also important to note that although SAA increases very rapidly following an inflammatory insult, a horse can show peracute signs of illness before measurable levels of SAA have been produced. If this is the case, treatment should be based on clinical signs, and rechecking SAA in 12-24 hours will show an increase. SAA will increase from any acute inflammatory stimulus, which can include intramuscular administration of vaccines or other injections.(1,2) Maximum levels are reached at 2-4 days post-vaccination and should gradually return to normal by 7-10 days, depending on the peak value. In horses with a history of regular vaccination, concentrations typically stay below 500-1000 mg/mL However, some horses can reach over 3000 mg/mL while still appearing completely healthy, which may be more prevalent in younger or vaccine-naïve individuals. Increased SAA may also be seen in horses receiving other intramuscular injections, particularly large-volume antimicrobials. The exact level is dependent on the amount of inflammation stimulated by the injection(s). Surgery itself will also cause an increase, peaking at 48-72 hours post-op. However, persistent or re-emergent elevation should trigger investigation for underlying complications.(3,4) If no confounding inflammatory stimulus can be identified, elevated SAA should be considered an indicator of a subclinical process that could have significant disease potential, and further investigation is warranted. At a minimum, SAA should be rechecked 24 hours later. If an active inflammatory process is occurring, values should be even higher. Significant increases in SAA are not expected due strictly to stress, and physical exertion will cause minimal increase at most.(5) Potential issues with testing procedure can be considered if unexpected results are observed without other logical explanation. A control line should always be present in tests that develop normally, decreasing in intensity with increasing concentration of SAA. Absence of this line indicates an issue with the test. If suspicion is high that something went wrong, it can be repeated and the procedure evaluated to ensure all necessary steps are performed properly, including following all precautions. It can be helpful to retain an anticoagulated blood sample for testing in more controlled circumstances if necessary, such as back at the clinic or office. References 1 Andersen SA, Petersen HH, Ersbøll AK, et al. Vaccination elicits a prominent acute phase response in horses. Vet J 2012;191:199-202. 2 Smith M, Kristula M, Aaveto H, et al. Acute phase protein response in native and imported horses after routine combination vaccination protocol, in Proceedings. Am Assoc Equine Pract 2019;65:271-272. 3 Jacobsen S, Jensen JC, Frei S, et al. Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: a field study. Equine Vet J 2005;37(6):552-556. 4 Jacobsen S, Nielsen JV, Kjelgaard-Hansen M, et al. Acute phase response to surgery of varying intensity in horses: A preliminary study. Vet Surg 2009;38:762-769. 5 Nolen-Walston R. How to interpret serum amyloid A concentrations, in Proceedings. Am Assoc Equine Pract 2015;61:130-137.

The dynamic nature of SAA makes it an ideal marker for monitoring progression or resolution of illness, including response to treatment.(1-6) It is rapidly responsive to clinical changes and directly reflects the inflammatory status of the horse. Point-of-care testing with numerical results that can be compared and tracked over time facilitates this process. SAA may continue to increase for up to 4 days following an acute inflammatory insult, even in the face of treatment (Fig. 1). It is necessary to consider this when monitoring treatment response. Depending on the timing of initial examination, SAA may not yet have peaked when the horse is evaluated. This could result in a similar or possibly higher value being obtained later if follow up testing is not planned carefully. If SAA does not decrease appropriately, it likely indicates that the chosen treatment is not efficacious, and alternatives should be considered.(6) Ideally, patients should be tested during the initial exam and then be retested in 24-48 hours in case SAA increases further (Fig. 1). Identifying the peak value will help with interpretation of subsequent results. Different peak values could indicate different disease processes, and higher values will take more time to return to normal. To evaluate efficacy of antimicrobials or other treatments, follow up testing should be performed 3-4 days after the start of treatment, or 1-2 days after peak [SAA] has been identified. If treatment is effective and inflammation/infection is resolving, SAA should begin to decline (Fig. 1).(6)  Follow up assessments can be performed every 24-48 hours if desired to document continued clinical improvement. If at any time the SAA stops decreasing or starts to increase, treatment failure or additional complications should be considered.(6)  At a minimum, it is advisable to test SAA before discontinuing therapy and/or prior to discharge (if hospitalized) to verify that the horse has returned to normal. Monitoring is also useful for early identification of complications post-operatively or with significant medical conditions.(1-5,7-11) Surgery itself will also cause an increase but understanding the pattern and degree of this increase allows SAA to still be used as a very effective post-operative monitoring tool. For post-operative monitoring, SAA can be re-evaluated every 24-48 hours, depending on budget and patient proximity. SAA should peak at 2-4 days post-op (depending on procedure) and then gradually decline (Fig. 1).(7,12) If post-operative values remain high, or spike again later during recovery, complications are highly likely. (3,7-11) As early identification can be difficult, this type of monitoring can be invaluable, providing a trigger for more in-depth exploration of possible causes. It is important to remember that SAA measured at one specific time does not necessarily reflect the overall peak as animals may be transitioning from local to systemic inflammation or between chronic and acute states.(6) This underscores the particular value of SAA for monitoring patients. It mirrors clinical condition much more closely than other diagnostic tools such as WBC count or fibrinogen(1,13,14), and unlike body temperature should not be affected by NSAID therapy.(13) SAA does not answer all clinical questions but adds immediate objective input to help guide clinical decision-making. References 1 Belgrave RL, Dickey MM, Arheart KL. Assessment of serum amyloid A testing of horses and its clinical application in a specialized equine practice. J Am Vet Med Assoc 2013;243(1):113-119. 2 Nolen-Walston R. How to interpret serum amyloid A concentrations, in Proceedings. Am Assoc Equine Pract 2015;61:130-137. 3 Jacobsen S, Andersen PH. The acute phase protein serum amyloid A (SAA) as a marker of inflammation in horses. Equine Vet Educ 2007;19(1):38-46. 4 Radcliffe RM, Buchanan BR, Cook VL, et al. The clinical value of whole blood point-of-care biomarkers in large animal emergency and critical care medicine. J Vet Emerg Crit Care 2015;25(1):138-151. 5 Westerman TL, Tornquist SJ, Foster CM, et al. Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with inflammatory disease examined at a tertiary care hospital. Am J Vet Res 2015;76(10):882-888. 6 Pepys MB, Baltz ML, Tennet GA. Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response. Equine Vet J 1989;21(2):106-109. 7 Jacobsen S, Jensen JC, Frei S, et al. Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: a field study. Equine Vet J 2005;37(6):552-556. 8 Westerman TL, Foster CM, Tornquist SJ, et al. Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with colic. J Am Vet Med Assoc 2016;248(8):935-940. 9 Daniel AJ, Leise BS, Burgess BA, et al. Concentrations of serum amyloid A and plasma fibrinogen in horses undergoing emergency abdominal surgery. J Vet Emerg Crit Care 2016;26(3):344-351. 10 De Cozar M, Sherlock C, Knowles E, et al. Serum amyloid A and plasma fibrinogen concentrations in horses following emergency exploratory celiotomy. Equine Vet J 2020;52(1):59-66. 11 Aitken MR, Stefanovski D, Southwood LL. Serum amyloid A concentration in postoperative colic horses and its association with postoperative complications. Vet Surg 2018; 1–9. https://doi.org/10.1111/vsu.13133 12 Jacobsen S, Nielsen JV, Kjelgaard-Hansen M, et al. Acute phase response to surgery of varying intensity in horses: A preliminary study. Vet Surg 2009;38:762-769. 13 Hooijberg EH, van der Hoven R, Tichy A, et al. Diagnostic and predictive capability of routine laboratory tests for the diagnosis and staging of equine inflammatory disease. J Vet Intern Med 2014;38:1587-1593. 14 Hultén C, Demmers S. Serum amyloid A (SAA) as an aid in the management of infectious disease in the foal: comparison with total leucocyte count, neutrophil count and fibrinogen. Equine Vet J 2002;34(7):693-698.

In 1995, when we first tested the Clostridium chauvoei antiserum that serves as the raw material for our C. chauvoei conjugate, vehement expostulations proceeded from our darkened microscopy room that are probably not printable in this newsletter. The source of this angst was the fluorescence of not only rod-shaped C. chauvoei bacteria but also what appeared to be spirochetes.  As you can see from Figures 1 & 2, there is a certain resemblance between the spiral structure that our conjugate labeled and a typical spirochete.  We theorized that the C. chauvoei culture against which we had raised our antiserum (and from which we had made our slides) had been contaminated with a spirochete, resulting in the development of antibody not only to C. chauvoei but also to the mystery spirochete.  Though we were somewhat chagrined by this development we decided that, since spirochetes are easy to differentiate from rods, the antibody would still be useful for detecting C. chauvoei.  We conjugated the antibody, noted the apparent snafu on our Certificate of Analysis, and sold the conjugate. Fast-forward a decade to 2005 when a gentleman named Peter Wragg at Veterinary Laboratory Agencies in the UK observed what we had thought was cross-reactivity.  Being better informed than we, Peter knew that Clostridium chauvoei is peritrichous (has flagella uniformly distributed over its cell wall).  Peter realized that what we had thought to be spirochetes were actually quite probably detached flagella.  He was kind enough to share some photomicrographs of our conjugate staining a fresh culture of C. chauvoei that clearly show the flagella attached (Figure 3).  Following Peter’s revelation, we applied our conjugate to quite a few wells of our C. chauvoei slides, which are made with our killed C. chauvoei culture, and photographed a number of spiral structures (Fig.4).  We agree with Peter’s opinion of these structures and have modified our Certificate of Analysis accordingly. We thank Peter Wragg for pointing out our error and kindly sharing his photomicrographs.  We also thank Veterinary Laboratory Agencies for permitting the use of Peter’s findings and photos in this newsletter.

Watch how to run an FA test using VMRD reagents.

A highly sensitive and specific commercial cELISA for the diagnosis of Foot and Mouth Disease Foot and Mouth Disease Virus (FMDV) is a single-strand, positive-sense RNA virus in the Picornaviridae family. The resulting disease is exceedingly contagious, with high fever and vesicular lesions in cloven hoofed animals including domestic livestock such as cattle, pigs, sheep, and goats. It is of major international concern, with heavy impact on trade economics and extensive regulations governing control. Seven regionally specific, immunologically distinct serotypes have currently been identified. In order to address important challenges in FMD diagnosis, an effective assay must be broadly reactive across viral serotypes and host species, and be able to differentiate between infected and vaccinated animals (DIVA capable). A commercial competitive ELISA (cELISA) has been developed and validated by VMRD, Inc. in collaboration with the Institute for Infectious Animal Diseases and the Plum Island Animal Disease Center of the US Department of Homeland Security and is in the final stages of USDA licensure. This cELISA detects antibodies against the FMDV 3ABC nonstructural protein through inhibition of specific monoclonal antibody binding to a highly immunogenic yet highly conserved epitope within the target protein. This target protein enables the assay to be DIVA capable, as it is only present in replicating virus and not in purified vaccines. The cELISA format enhances specificity while offering the flexibility of multispecies use. Thus far, this assay has been validated for cattle, pigs, and sheep. An optimal cutoff of 40% inhibition was determined. Serum samples of known infection status were evaluated from 503 FMDV negative cattle of US origin (FMDV-free), 121 cattle experimentally infected with various FMDV isolates representing all 7 serotypes, 117 naturally infected cattle from Cameroon and South Africa, and 52 vaccinated cattle later challenged with live FMDV. A subset of these samples (n=386) was also run for comparison on another commercial non-structural protein ELISA that is currently utilized by the USDA and considered to be the most specific assay on the market at this time. Results demonstrate the broad serotype reactivity of the VMRD cELISA, with identification of antibody to all seven serotypes represented in experimental infections and five serotypes in naturally infected cattle. DIVA capability was confirmed by negative test results in vaccinated, unchallenged cattle that then became positive after challenge, with the VMRD cELISA detecting more positives than the other commercial assay in this challenge group. In unvaccinated, experimentally infected animals, the VMRD assay identified seroconversion in all by 15 days post-infection, with comparatively delayed detection of seroconversion by the other commercial assay. The broad reactivity, DIVA capability, and high sensitivity and specificity shown in this study demonstrate the ideal nature of this new assay for use in both US and international FMDV control programs.

Neospora caninum is one of the leading causes of abortion and decreased reproductive efficiency in cattle. (1,2) This protozoan parasite is insidious, often with abortion as the only obvious sign of disease. Median economic cost of Neospora abortion has been estimated at ~$12,000 per herd in the United States (3), with additional losses of $128 per infected cow due to decreased milk production4. Vaccine efficacy for this disease is questionable and no effective treatment exists, therefore diagnostic testing by serology is the mainstay of disease control. Positive serology indicates current infection, as there is no way to eliminate the parasite once an animal is infected. Fortunately, the solution for effective control in an infected herd is relatively simple and straightforward. Transmission of infection from dam to calf is extremely efficient (>80%). (1,3) Therefore, if all breeding females are tested, calves subsequently born to seropositive dams are presumed to be infected. These animals should not be used as replacement heifers as they will propagate infection in the herd and are at high risk of abortion if bred. (1) Purchased replacement heifers should also be tested prior to acquisition to ensure they are seronegative. (5) This focus on raising and purchasing negative replacement heifers should lead to a dramatic reduction in herd seroprevalence and associated financial loss within just a few years. (1) Serologic testing should also be utilized in cases of abortion, along with examination of aborted fetuses, in order to determine whether N. caninum has been introduced to the herd. (5) Although infection cannot be transmitted between cattle, the parasite’s natural life cycle involves horizontal transfer from infected canids to any one of several intermediate host species, including cattle. When this occurs in a naïve herd, an abortion storm can result. Therefore, wild and domestic canids should be prevented from accessing and potentially contaminating cattle food and water sources. All tissues associated with abortion should be disposed of promptly in order to prevent ingestion by canids, which can perpetuate the infection cycle. (5) Producers can explore other advanced management options by consulting with a veterinarian. Abortion is a serious issue for cattle producers, as their livelihood is based on successful bovine reproduction. N. caninum serology plays a pivotal role in control of this problem by identifying targets for appropriate management intervention. By investing in strategic serological surveillance, abortion risk can be greatly reduced, resulting in improved financial return. References 1 Larson RL et al. 2004. J Am Vet Med Assoc 224(10):1597-1604. 2 Wilson DJ et al. 2016. Vet Parasitol 218:46-51. 3 Reichel MP et al. 2013. Int J Parasitol 43:133-142. 4 Hernandez J et al. 2001. J Am Vet Med Assoc 219(5):632-635. 5 Dubey JP et al. 2007. Clin Microbiol Rev 20(2):323-367.

Many of our customers have considered and some have asked about published sensitivity and specificity data Bovine Leukemia Virus Antibody Test Kit, ELISA (Cat. No. 284-2; 2 Plate).  We publish our field validation data as required and reviewed by CVB-USDA.  The idea is to make sure that companies use third party field validation data for published specificity and sensitivity information rather than in-house data which might look more enticing to the buyer. The field validation data for our kit contained no false positives among the 280 BLV antibody negative samples tested in 3 different laboratories.  This led to jubilation, albeit temporary, on our part because we could (or had to) legitimately claim 100% specificity for the kit.  Temporary because about the time we had the sense to think it through we also had fielded a few technical service calls which indicated that some customers had encountered samples that were borderline positive with the ELISA and AGID negative. If there’s one thing we should have learned in 25+ years of manufacturing immunodiagnostic tests, it’s “there’s no such thing as 100% specificity, or sensitivity for that matter.”  Well, you could get something like 95% and round it up to 100%, or 51% and round up to 100%.  Or you could get lucky like we did with a specific sample set that had no false positives.  But in the real world there is no 100% sensitivity or specificity.  We have encountered false positives with this assay subsequent to the field validations and have modified our literature with the following proviso about our 100% specificity claim: “[100% specificity is] based on a specific sample set. However, no diagnostic test kit is always 100% specific on all sample populations. Since market introduction of our BLV kit, occasional false positives have been encountered. We therefore advise all users that when BLV prevalence is low, positive samples should be confirmed by some other method, particularly where valuable animals may be involved and/or when BLV status is used as the single criterion for disposition of animals. Whenever import restrictions do not prohibit it, VMRD will provide reference assay service for positives of high-value animals or for positives in low-prevalence situations. We are not capable of testing large numbers of samples, and therefore cannot provide this reference assay service for all positives found.” Since the realization of this problem, we have expended considerable research effort trying to improve the specificity of this assay.  In fact we have spent more time and money post introduction on this assay than on any other we have every produced. We continue to do so and have been able to make substantive progress with the current format.  We are thankful for the patience and help provided by our customers, especially, in providing problem samples for analysis.  The best way for us to make improvements is to work with the problem samples.  In doing so, we have been able to determine that anti-mouse IgG antibodies in bovine sera account for the majority of false positive reactions.  For those not familiar with the assay configuration, the gp51 antigen is captured with monoclonal mouse anti-gp51 antibody. In a recent analysis of data from one laboratory where 2682 samples were tested by the ELISA as it currently exists, the specificity was 97.5% against AGID as the referent assay.   There were 57 samples that were determined to be “false positives.”  Of these we were able to test 43 with an improved iteration of the ELISA which registered positive on only 4.  Extrapolating and assuming all other factors would remain the same, this would have improved the specificity in that sample set to 99.9%. We realize, as I’m sure others do, that there are no other USDA licensed BLV antibody test kits in ELISA format available for sale the United States at this time. We are working towards licensure of the improved BLV ELISA as quickly as possible. Our current kit is a good assay with only very rare occurrence of “false positives,” but thanks to the cooperation of our many customers and the hard work of our technicians we have an opportunity to turn a very good kit into an excellent one.  We would like to extend a “thank you” to everybody that has assisted us by providing the troublesome samples found in the field. If you would like to be notified when the new format BLV kit is available for commercial release please contact VMRD, Inc. at vmrd@vmrd.com

We all know that hog wash is to be eschewed, but we have recently encountered another type of wash that should be avoided as it can cause needless headaches—and possibly invalid results—with some of our ELISA assays. We have always enjoyed working with Bio-Tek Instruments, Inc. They make good equipment, their people are easy to work with, and they have good technical support. We have recently discovered, though, that many of their plate washers have a feature that, if used, is deleterious to the performance of our assays.  This feature is called “bottom wash.”  When bottom wash is enabled, a wash cycle consists of the machine’s dispensing a small amount of wash in the bottom of the wells, aspirating it out, and finally completely filling and aspirating the wells.  Because of the way our plates are coated, the use of bottom wash effectively doubles the amount of washing that the antigen-antibody complex receives.  In some of our assays, this does not matter too much, but in some, for instance the Anaplasma cELISA, bottom wash deprives the assay of much of its ability to differentiate positives from negatives.  As a rule our washing recommendations should be strictly observed.  More washing is not better.  Less washing is not better.  When we recommend three washes, you can be sure that we have tried one, two, three, four, and five washes, and three worked the best.  Doubtless there are applications where the Bio-Tek bottom wash feature is a great thing, but for VMRD assays, we highly recommend that bottom wash be turned off.

EIA is still clearly a threat; however the threat is less than in the 1970s. The overall percentage of EIA positive horses among those tested in the United States decreased from near 4% in 1972 to <0.01% in 20051. Prior to the mid-1990s, 92% of the positive horses were in a “hot zone” (see illustration) of states that included the South (including Texas and Oklahoma) and parts of the Midwest (1). Since the mid-1990s there have been clusters of EIA occurring in states outside the hot zone that can be seen on the website, which contains complete data and maps on EIA testing in the United States from 1972 to 2007. Notable increases in positive horses outside the hot zone states include those in Nevada in 2003; South Dakota in 2001; Utah in 2000; North Dakota in 1999; Utah, Oregon and Indiana in 1998; South Dakota in 1997; Idaho in 1996; and New York and Michigan in 1995. For 2006, the percent positive horses among those tested varied by state from zero in 27 states to 0.048% in Mississippi where 20 of 41,331 samples were positive. It seems that infected horses in the hot zone states continue transmission and that sometimes transmission occurs in states outside the hot zone. Infections in the states inside and outside the hot zone are due to transmission from infected horses, although the sources of the infected horses are not always known. A documented outbreak of EIA outside the United States demonstrates the threat of an infected horse. On June 15, 2006, the first case of EIA was reported in the Republic of Ireland (2) and by November 14, 2006, the number of cases in the Republic of Ireland was 263. All the 26 cases appeared to be epidemiologically linked. In August of 2006 the first case of EIA occurred in Northern Ireland and the case was epidemiologically linked to the outbreak in the Republic of Ireland (3). The source of the outbreak in the Republic of Ireland was thought to be due to the use of an unauthorized veterinary medical product (2). Regardless of the source of the initial infection with EIA virus, there was transmission to other horses. In this outbreak, the first horse infected was documented to be a threat to other horses. Transmission of EIA from an infected horse usually occurs by blood transfer to a non-infected horse. Blood transfer and transmission occurs naturally by biting insects such as horseflies and deerflies following interrupted feeding on an infected horse (1). Blood transfer and transmission also occurs from the use of blood-contaminated needles and equipment. Transmission with other body fluids may occur. Recent quantification of EIA virus RNA by RT-PCR demonstrated significant RNA copies in nasal, buccal and genital secretions obtained from swabs; however, the quantities were always less than in plasma (4). Transmission from horses in the carrier-state with low-level viremia is much less probable than from an acute case with high-level viremia. The practical problem in EIA control and in determining the threat from a particular EIA-infected horse is distinguishing whether the infected horse has a high- or low-level viremia. Further, depending on the horse and the duration of infection, periods of low-level viremia are interrupted with episodes of high-level viremia increasing the potential for transmission. References 1 https://www.aphis.usda.gov/vs/nahss/equine/eia/eia_info_sheet.pdf 2 Reynolds, DI. Equine infectious anaemia in Ireland. Veterinary Record 159:187, 2006. 3 Menzies F, Patterson T. Description of the first case of equine infectious anaemia in Northern Ireland. Veterinary Record 159:753-754, 2006. 4 Quinlivan M, Cook RF, Cullinane A. Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based sequence determination of the gag gene. Veterinary Record 160:611-618, 2007.

Our quality control lab was troubled with a spate of FAs and IFAs that had extreme autofluorescence–or so we thought. Negative cells ranged in color from dull orange to olive-drab with sufficient brightness to make the apple-green specific fluorescence difficult to observe. Particularly puzzling was that this supposed autofluorescence was intermittent in the QC lab and was not happening in our other labs. After much hair-pulling and tinkering, we discovered that it only happened when we used a red lab marker to mark the slides. Further, we discovered that it only happened if the writing in red ink on the frosted edge of the slide was submerged in the soaking buffer.  We presume that there is an orange-fluorescing dye in the red ink that rapidly and efficiently stains cells.  Apparently, only minute quantities of the dye are necessary to stain the cells as there was no visible leaching of the written ink.  We have traditionally used black lab markers to mark slides and have experienced no problems with these, regardless of whether the ink was submerged in the soaking buffer. We report this incident not only to warn our customers against using red lab markers on FA slides, but also to demonstrate the sort of minute factors that can affect any laboratory process.  A zany red lab pen put our QC lab in a quandary for several weeks.  When things stop working, it is necessary to look for any change in procedure, no matter how seemingly trivial.

We have received several claims that the VMRD Clostridium septicum direct FA conjugate does not bind certain cultures of Clostridium thought to be C. septicum. To investigate these claims, we obtained one of the putative C. septicum isolates.  The stench it produced in anaerobic culture supported its identity as a Clostridium species, and the swarming behavior on agar reported by our customer suggested C. septicum. A Gram stain showed Gram-positive rods with some spore formation.  This culture did not react with our C. septicum direct FA conjugate.  It also did not react with our C. chauvoei, C. novyi, or C. sordellii direct FA conjugates.  All that could be seen by FA microscopy were faint outlines of long rods.  We were prepared to develop a new C. septicum conjugate that would react with this ornery culture but first we had to be certain of its identity.  DNA analysis by PCR amplified a segment of the 16S ribosomal RNA gene.  The closest sequence match—homology was 100%—in GenBank was C. fallax.  An earlier clue suggesting that the sample was not C. septicum was that chopped meat broth turned partially black instead of pink after 48 hours of growth (1). The species name, fallax, meaning “deceptive” in Latin (2),  was most appropriate for this sample, which misled even the best bacteriologists to believe that it was C. septicum. References 1.  Sneath, Peter H. A.. “Bergey’s Manual of Systematic Bacteriology, Volume 2.” Maryland: Williams & Wilkin, 1986.  Page 1188 2.  Ibid.  Page 1167