Modern diagnostics in equine medicine–don't throw out the old (Proceedings)

Issues facing practitioners

Understanding the older and the newer formats for detection of infection

Table 1 is a listing of the basic definitions of PCR methods. Several previous presentations at the ACVIM and at other veterinary meetings have discussed PCR, and a recent paper indicates that a basic understanding of the different PCR test formats is needed for continuing education of practitioners and diplomates. While it is not necessary for the practitioner to be a molecular biologist, this table provides a short review of the definitions of molecular testing.

Proper procedures: collection, transport, storage

The main advantage of genomic strategies for testing is that a live organism is not necessarily needed for diagnosis. While the fact that diagnosis based on molecular techniques does not require live organism, DNA and RNA are subject to the same microbiological, biochemical and physical factors as live organism for degradation. Although in some situations, genomic DNA may be robust in its survival for forensic purposes, overgrowth and chemical contamination of a small microbiological sample will result in false negative reactions for any of the aforementioned molecular techniques. A sample collected using the correct media that is solely dedicated for PCR testing should be taken. For blood, EDTA or purple top tubes are best. Use of EDTA as an anticoagulant for testing of virus in plasma or buffy coat is applicable for most genomic techniques and viral culture techniques. However, if one wishes to culture plasma (such as that of a foal) for bacteria, EDTA is bacteriostatic and a blood culture bottle must also be collected. Anticoagulants such as heparin can inhibit PCR reactions especially when a kit extraction method is used which will likely the typical formats in most diagnostic laboratories.

For detection of nasal/respiratory pathogens, wooden swabs contain formalin which can inhibit PCR (fixed tissue methods are performed on paraffin and formalin has been removed in the paraffin process). Thus, plastic, polypropylene sticks with Dacron or rayon swabs, not those with calcium alginate, are essential for nasal swabbing. Furthermore, cotton allows bacteria and virus to become embedded in fibers, and frequently the extraction method calls for direct extraction off the swab. This step will inactivate most viruses and bacteria because of the detergent used in the first lysis step. Finally, use of a transport media is best for viruses because this will inhibit overgrowth of bacteria that may inactivate and break down nucleic acids needed for the successful detection of viruses. The proteins in the viral media will also assist in keep the virus in a biologically active state, so this is preferable for viral culture also.

Most testing has actually been validated on specimens collected under the best conditions without inhibitors and contamination and these samples have been stored appropriately. Specimens requiring storage before shipping to a laboratory should not be stored and the MOST optimal time for testing by PCR is less than 3 days in a sample stored consistently at 4°C. If one can not ship a sample within 3 days we recommend that the sample be allowed to sit in a refrigerator for 20 minutes and then the plasma drawn off and placed in a new tube without any anti-coagulants. For horses, do not centrifuge the sample! The white blood cells of horses settle in this time and do not form a buffy coat that is adhered to red blood cells. This allows collection of as few RBCs as possible.8 This is essential because as RBCs lyse in older samples, heavy protein contamination occurs and these particular proteins (iron) are toxic to many PCR reactions. Many laboratories historically have indicated shipment at either 4°C or not to exceed 75°C. The latter is not appropriate for blood or nasal specimens due to the potential for hemolysis and bacterial overgrowth, respectively. Ship all samples on ice packs (NO WET ICE) and then overnight for 24 hour arrival. Do not ship expecting successful Saturday delivery and it is our experience that samples become lost more frequently with weekend deliveries.

Interpretation of pcr results

So given, the problems with inappropriate sampling and handling and lack of standards for these new techniques, what can be said regarding interpretation of test results? There is no question that molecular medicine has dramatically revolutionized infectious disease diagnosis, treatment, and, especially biosecurity. In the long run molecular based assays are more efficient and allow for minimal exposure of laboratory personnel and veterinarians to many infectious agents. Nothing will ultimately take the place of isolation of an infectious agent as confirmation of active infection from a properly collected and handled sample, but the efficiency and accuracy with good sampling and laboratory standardization make PCR diagnostics how most infectious diseases will be tested in the future.

Given that sampling, handling and all quality assurance for a particular laboratory is reliable, one must understand what a positive or a negative genomic-based test means. For instance, a positive PCR test results means that nucleic acids that belong to the genome of that particular pathogen was detected in the sample. This agent may or may not be live, infectious or capable of replication in that sample. At least three different scenarios may be occurring in regards to the sample tested: 1) the pathogen is present and directly is causing the clinical signs observed, 2) the pathogen is present but is not responsible for the clinical signs observed or 3) the pathogen is not present but the reaction mixture is binding to some other target in the assay. By the same token, a negative sample has failed to detect the nucleic acids of the infectious agent. A negative result can reflect at least three different scenarios' in regards to the sample tested: 1) the pathogen was absent at the time of testing, 2) the pathogen is present but not detectable within the limits of sensitivity, and 3) there was some type of inhibition of the positive reaction. The mere results cannot be interpreted without understanding the context in which testing was performed in the first place unless this is used for regulatory purposes. In the absence of case criteria, the results by themselves are not confirmatory for disease causation. This is especially true for negative tests; hence repeated sampling is recommended should the case criteria create a high degree of suspicion for that disease. In the end, a comprehensive investigation using multiple samples utilizing different detection formats may be the only way to confirm disease causation in an outbreak or new emergence of disease in a group of animals. There is no magic bullet when it comes to testing.

Status of molecular testing for particular diseases

Vesicular diseases

Vesicular diseases are highly infectious agents that usually have very high morbidity with low mortality. However, their intensive infectiousness, and painful nature, when involved in outbreaks in hoofed livestock result in economic catastrophe. Investigatory laboratories around the world have developed several PCR assays that detect simultaneously (called a multiplex reaction) several vesicular viruses. These assays will likely revolutionize early detection of outbreak spread, but thus far have several issues. Both conventional and real-time formats are available and the OIE investigatory laboratories are working on some of these assays. The initial assays focused on detection FMDV and swine vesicular disease (SVD). No incorporation was made for VSV, a disease affecting cattle and horses of economic importance with disease activity in the U.S. The VSV PCR is still run as a separate assay format. In addition, many subtypes of FMDV exist and not all of the techniques available incorporate primers and probes that detects all subtypes. The most comprehensive assay in the literature is a "conventional" PCR format and this was validated by testing "spiked" samples and experimentally inoculated swine. Limited multiplex real-time PCR formats have been developed, but this was validated on a limited number of samples. Development of these tests is crucial for rapid disease surveillance. In experimental inoculation, virus detection (SVDV, VSV, and FMD) was possible even with multiplex conventional rt-PCR in either blood or serum by the 1st and 2nd day post-inoculation and before vesicular lesions. Testing of nasal swabs was ultimately more sensitive for VSV. Consisted with the OIE handbook, testing of vesicular lesions in clinical affected animals is the sample of choice for viral culture and conventional and real-time rtPCR.

Diarrheal diseases

PCR protocols are described in the literature for many diarrheal pathogens. Specifically diagnostics for the horse include Salmonella enterica, Clostridium perfringens A toxins, Lawsonia intracellularis, rotavirus, and several miscellaneous pathogens (Table 2). Regarding Salmonella PCR in the horse, its excellent sensitivity is most useful for identification of subclinical shedders and environmental contamination during an outbreak.14 Standard microbial culture methods are still required to obtain the isolates and confirm actual presence of the organism. A clinical horse that tests positive by PCR, but negative by repeated culture should be interpreted with caution. Given the ultra-sensitivity of this technique and the ability for bacterial elements to mobilize between fecal bacteria, only validated PCR techniques for Salmonella should be used. Unfortunately for horses, there is little standardization between the few laboratories that use PCR for detection of Salmonella.

Detection and typing of C. perfringens in the human field has gone beyond that of PCR and to microarray in order to elucidate the complexities involved in differentiation of possible Clostridial food and water-borne poisoning. In the horse, much attention has been given to the beta2 C. perfringens toxin as an important casus of diarrhea in adult horses and foals, however, only in the pig has active transcription of beta2 toxins in the positive strains and only in the pig has correlation been strong to disease. One study has provided a wider epidemiological correlation in horses. At the molecular level, although the toxin is present in C. perfringens type A isolates garnered from equine clinical cases, the expression of this toxin is extremely low compared to the pig. Well designed case control and molecular epidemiology studies are paramount for further analysis of this toxin in horses. The most commonly used technique is a conventional PCR protocol that detects the presence of the toxin genes (not activity or expression). Many AAVLD laboratories now offer this technique and it has largely supplanted biological assays which utilize rodents. This is usually performed on C. perfringens isolates rather than directly on fecal samples, although this is likely the more practical approach. Unfortunately interpretation is very important since Clostridium perfringens is a common component of fecal flora.

Respiratory

Molecular techniques have greatly altered the efficiency of diagnosis of equine respiratory pathogens. Nowhere is this more apparent then for the diagnosis of Streptococcus equi subs equi. Since Streptococcus equi equi is in many cases the notifiable pathogen and one that control is directly correlated to biocontainment practices, differentiation and early identification of S. equi subsp. equi in an outbreak of respiratory disease is crucial. Historically S. equi equi is differentiated from S. equi subsp zooepidemicus on the basis of sugar fermentation. Conventional PCR was first performed with the 16S ribosomal gene and sequencing or in terms of more easily differentiated, the superoxide dismutase (SOD) A gene. In addition, other genes such as the the SePE-I gene (pyrogenic mitogen) has been characterized and found present in S. equi but not S. zooepidemicus. Both of these genes have also been characterized using a real-time format. In this format, real-time PCR was able to detect and correctly identify all cultivable S. equi equi isolates. In addition, six additional samples meeting the case criteria were positive for S. equi equi, two of which were identified as S. equi equi and four were identified as S. zooepidemicus. This technique did not identify two S. equi zooepidemicus isolates. Sequencing demonstrated that the target gene had molecular differences not previously described for S. equi zooepidemicus. These results compare to previously reported results for conventional PCR. Isolation and identification of S. equi equi positive horses can be greatly enhanced by multiple sampling. Three consecutive obtained nasal swabs increase sensitivity of detection to 85% which is equal to a single guttural pouch flushing.

Influenza testing has remarkable efficiency for detection of Influenza A in human patients. With vaccination in the horse, the window of positive testing is restricted mainly to the period of clinical signs, although even vaccinated horses will shed virus during an outbreak. Since most outbreaks in the horse are currently caused by Equine-2 H3N8 influenza strains, the specificity of most viral testing is unquestioned. Virus isolation is considered the gold standard, but RT-PCR and antigen test kits are supplanting this very specialized culturing because it must be done in egg cultures (a real art). Real-time PCR was more sensitive than 5 antigen detection kits and viral isolation. In addition, viral detection using real-time was correlated with quantization by tissue culture techniques.

There are many different PCR techniques for detection of equine Herpesviruses. Equine herpesviruses 1 through 4 as a group are all detectable by conventional PCR. Real-time methods have been described for EHV 1 and EHV-4 and several simultaneous detection assays (multiplex) are in the literature. Those that target DNA of the polymerase gene of EHV appear to be most sensitive. A recent paper has described utilization of RNA targets to examine latency although a control population. The current concept is based on location of virus and amount for latent infections where in biopsy of pharyngeal tissues with conventional nested PCR (double round of PCR essentially) has currently defined latency. In vaccinated horses viral shedding of EHV-1 (non-neurotropic) and EHV-4 is extremely short and must be performed when horses are febrile if early identification of emergence is to be obtained.

CNS Pathogens

One of the most frustrating areas of diagnostic medicine is diagnosis of infectious encephalitis. The primary indigenous U.S. etiologies for encephalitis include West Nile virus (WNV), Western equine encephalomyelitis, Eastern Equine encephalomyelitis, various Bunyaviruses, EHV-1, rabies virus, various parasitic infections, and various fungal infections. Foreign animal diseases (FAD's) include Venezuelan equine encephalomyelitis virus, Murray Valley virus, Semliki Foris, Japanese encephalitis virus, Hendra virus, Nipah virus, Powassan virus, Kunjin virus and Borna virus. While various private laboratories and even AAVLD laboratories have published sequences on the detection of the U.S. arboviruses, the Centers for Disease control has recommended primers/probes for real-time PCR detection. For the purposes of standardization, these targets are utilized by the respective state Departments of Health for surveillance and communication of public health threat. Although a conventional nested PCR tested has been described and shown to be more sensitive for detection WNV in horse tissues, this technique is fraught with greater probability of false positive results. Also, use of the real time format is more amenable to automation (hence, more rapid results). Although one paper compared the nested PCR with the real-time format, many of the samples in the literature are not controlled for sampling site of tissue. In our laboratory with experimental inoculation and in studies where field specimens were evaluation, the highest viral load for WNV was seen in thalamus and pons/medulla. Should these sections of brain be consistently evaluated, real-time PCR is likely reliable in horse brain. There is no question that the use of the CDC primers for detection of Eastern equine encephalitis virus is sensitive and reliable. Horses have high viral load in thalamus, pons and medulla. Thus the same samples utilized for diagnostic testing for WNV can also be used for EEE and WEE testing. In arbovirus testing, plasma and serum are not appropriate for testing in neurological horses.

With neurotropic EHV-1, it is our experience that there is high amounts of nasal shedding of virus and a high viral load in the hindbrain of neurologically affected horses. Several real-time PCR techniques have been developed and validated OIE laboratories and investigators. Currently, the recommended protocol is to screen for EHV-1 using a generalized target for EHV-1 glycoprotein B gene. This gene is highly conserved and can be used to differentiate EHV-1 from EHV-4. In cases where in EHV-1 presence is questionable, the OIE nested protocol is considered the PCR gold standard. Once EHV-1 is identified, then a special PCR protocol (a single nucleotide polymorphism assay) is run for the differentiation of the strain associated with neurological disease from the other EHV-1 not usually associated with CNS disease. Unfortunately, the specific viral mutation (ORF 30) only accounts for approximately 80-85% of equine herpes myeloencephalitis cases. Irrespective field investigation, outbreak details, and recent molecular studies have indicated that horses affected with equine herpes myeloencephalitis shed high amounts of virus early in the course of disease, thus early quarantine and detection of nasal shedding within the exposed population is paramount to control. Thus automated, rapid molecular assays are essential for containment of outbreaks.

Miscellaneous PCR based assays of diagnostic utility

Cyathostomin Infection. One of the most exciting areas of diagnostic investigation with PCR techniques is for detection and differentiation of cyathostome infection in horses. There are approximately 43 cyathostomin species. Not only will PCR allow differentiation of these species but is being developed to detected egg and L3 and L4 stages of infection in horses feces. PCR protocols have also been used to detect helminth resistance. A PCR technique for detection of Anoplocephala has been described. This technique is sensitive and will likely aid in detection of another equine parasite that is notoriously difficult to identify. Furthermore this technique will likely contribute to our understanding of the relationship between acute abdominal disease in the horse and infection. Habronema infection can be extremely difficult to confirm in pre-mortem biopsy sections, especially in the Southeast U.S. where diagnosis is complicated by fungal infection. An extremely sensitive technique has been developed for detection in feces. This technique needs to be validated for peripheral tissue sections.

Molecular tests which detect pathogen fungi are also a much needed area for diagnosis of infection in the equine. Several PCR techniques have been developed for detection of Pneumocystis jirovecii in human HIV infection. Since Pneumocystis organisms are considered host-species specific, these techniques must be validated for equine infections. Likewise invasive Aspergillus infections are another area of interest for molecular detection formats. These techniques will likely be most useful for tissue invasion rather than detection of primary respiratory infection since Aspergillus can be a trans-tracheal wash contaminant. Candida infection occurs in the blood of equine neonates. These infections can be extremely hard to diagnose. Other pathogenic yeast for which PCR techniques are highly applicable include Cryptococcus neoformans, Coccidioides immitis, Hisotplasmosis, and Blastomyces dermititidis. Of extreme usefulness is the differentiation of Pythium insidiosum infection from Mucomycotic fungi in the horse because the former is highly resistant to treatment while the latter can under surgical excision/debulking and respond to anti-fungal therapies.

Faster, more discriminating identification of equine pathogens is possible through development of molecular assays. However standardization of molecular techniques between laboratories, validation with appropriate sampling, and use of appropriate controls for quality assurance is necessary for expert and quality results in this rapidly expanding service for stakeholders. Ultimately it is up to the equine practitioner to have a basic understanding of the methods and interpret the disease in the face of appropriate case criteria.