Rabies is a viral disease that affects the central nervous system and is almost always fatal once symptoms appear. It is primarily transmitted through the bite or scratch of an infected animal, most often a dog. Rabies causes tens of thousands of deaths per year, mostly in Asia and Africa. Testing for rabies is critical for both human and animal health, as it helps determine if post-exposure treatment is needed.
The current standard testing methods require euthanizing the animal and examining its brain tissue. A non-lethal saliva or blood test would be preferable, as it would spare the animal’s life and eliminate delays in obtaining results. There is promising research into non-lethal rabies testing methods which may become viable alternatives.
Current Standard Testing Methods
The direct fluorescent antibody test (dFA) is the standard method for diagnosing rabies in animals (CDC, 2022). This test detects rabies virus antigens in brain tissue using a fluorescent microscope (CDC, 2022).
To perform the dFA test, samples are taken from at least two locations in the deceased animal’s brain (brain stem and cerebellum). The samples are then frozen and examined under a fluorescent microscope to detect rabies virus antigens (CDC, 2022).
If rabies virus antigens are detected, the test is positive. The dFA test is highly accurate when performed correctly (WHO, 2022). However, a limitation is that the test can only be performed post-mortem on brain tissue samples from deceased animals.
Currently, the direct fluorescent antibody test is the only approved method for officially diagnosing rabies in animals (CDC, 2022). This means euthanasia followed by brain testing is required, as rabies confirmation in live animals is not possible with current approved techniques.
Drawbacks of Current Testing
The current standard method for diagnosing rabies requires euthanizing the animal and testing brain tissue samples [1]. This is done either by fluorescent antibody testing, histopathology, immunohistochemistry or polymerase chain reaction [2]. Unfortunately, this means the animal must be killed first before a definitive diagnosis can be made.
A non-lethal diagnostic test for rabies would be highly desirable. It would prevent unnecessary euthanasia of animals that may not actually have the disease. It would also enable testing and observation of animals over time. Additionally, it would align with animal welfare interests to develop a humane approach that avoids killing animals during the diagnostic process whenever possible.
Given the potential impacts of rabies on both animal and human health, improved non-lethal testing methods are needed. Researchers continue investigating new options that can accurately detect rabies infection while allowing the animal to live.
Research into Non-Lethal Testing
In recent years, there has been increasing interest in developing non-lethal methods for testing animals for rabies. The current standard testing requires euthanizing the animal and examining its brain tissue, which is effective but involves killing the animal (CDC, 2023). Researchers have explored alternative approaches that could potentially detect rabies infection in living animals.
One area of research has focused on testing blood samples. Studies have shown promise in detecting rabies virus neutralizing antibodies in the blood of infected but asymptomatic dogs and cats (Botros, 1979). However, antibody levels can be variable, so blood tests alone may not provide definitive diagnosis. More research is still needed to determine the accuracy and reliability of blood testing.
Testing saliva samples has also been investigated. One recent study found that a paper-based device could detect rabies viral RNA in the saliva of infected dogs with high sensitivity and specificity, comparable to current gold standard tests (Nature, 2022). The device is faster, cheaper and easier to use than current methods. While further validation is still needed, saliva testing shows potential for efficient rabies detection in dogs in low-resource settings.
Other approaches undergoing exploration include testing tears, urine and cerebrospinal fluid. Research is still in early stages and no validated non-lethal methods have become standard practice yet. Overcoming limitations around diagnostic accuracy and logistical challenges remain important goals for ongoing research.
Blood Testing
The standard diagnostic test for rabies requires euthanizing the animal and testing brain tissue using the direct fluorescent antibody (DFA) test (CDC, https://www.cdc.gov/rabies/diagnosis/animals-humans.html). Researchers have explored alternative methods that avoid euthanasia by testing rabies antibodies in blood rather than directly in brain tissue.
One approach is detecting rabies virus neutralizing antibodies (rVNA) in blood serum. Studies have shown promising results in detecting rVNA in infected dogs, suggesting blood tests could replace brain testing. However, antibodies may not appear until late stages of infection, limiting early diagnostic sensitivity. More research is still needed to validate the accuracy of blood-based antibody tests (Singh et al., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094513/).
Overall, blood testing remains an area of active research. While not yet ready to fully replace direct brain testing, blood-based assays may eventually enable non-lethal rabies diagnosis in animals. Improving sensitivity in early infection stages is a key challenge still being investigated.
Saliva Testing
Researchers have investigated detecting rabies virus in saliva samples as a potential method for non-lethal rabies testing in dogs. One key study by researchers at Mahidol University in Thailand found that rabies virus antigen could be detected in dog saliva using latex particles coated with gamma globulin in an agglutination test on a glass slide (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC87197/). The rabies viral antigens in saliva samples interacted with the antibodies coated on the latex particles, causing visible clumping that could be observed. This provided proof-of-concept that rabies virus could be detected in saliva without euthanizing the animal.
However, the sensitivity of saliva testing was found to be lower than in brain tissue samples from deceased animals. Researchers concluded that saliva testing should not fully replace post-mortem brain testing at this time. Nonetheless, saliva testing remains a promising area of research for the future. If the sensitivity could be improved, it could allow non-lethal screening for rabies in dogs. This would be a major advancement in preventing unnecessary euthanasia.
Urine Testing
Some research has been done on testing urine samples for rabies antibodies and viral RNA. In 2003, a study published in Clinical Infectious Diseases looked at infectivity of blood and urine samples from rabid dogs https://academic.oup.com/cid/article/37/10/1399/452418. The researchers found that urine and blood from dogs infected with rabies was not infectious at the time of death. However, rabies viral RNA was predominantly detected in the urine samples.
While the study showed promise for urine-based testing, more research is still needed. The detection of viral RNA does not necessarily mean live virus is present that could infect another animal. Additional studies are required to determine if urine testing is a reliable option for diagnosing rabies infection in live dogs.
Other Approaches
There are some other promising non-lethal approaches being explored for rabies testing in dogs that do not involve blood, saliva or urine samples.
One method is detecting rabies virus RNA sequences using reverse transcriptase polymerase chain reaction (RT-PCR) on samples obtained through non-invasive means like nasal swabs. This approach has shown potential for accurate rabies detection without euthanizing the animal, but more research is still needed to validate it as a standard diagnostic tool (CDC, 2010).
Another emerging technique is using portable devices to detect rabies antibodies in samples of skin tissue obtained through a minimally invasive procedure. One example is the Rapid Fluorescent Focus Inhibition Test (RFFIT) assay performed on such samples. While promising, this method also requires further research and development (The Nature In Us, 2009).
Overall, developing accurate non-lethal rabies diagnostic tests for dogs remains an active area of study. But overcoming issues like test sensitivity and specificity in live animals is still a work in progress.
Challenges and Limitations
Developing an effective non-lethal rabies test comes with many challenges and limitations. The gold standard fluorescent antibody test requires brain tissue samples, which means euthanasia of the animal (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402924/). Attempts at developing non-lethal methods have faced difficulties in accuracy and practicality.
Blood testing is an area of promise, but rabies antibodies can be difficult to detect in blood, especially in the early stages of infection (https://www.nature.com/articles/d42473-022-00154-6). More research is still needed to improve sensitivity of blood tests.
Saliva testing is another option, but saliva viral loads can vary between infected animals. Collection methods can also be impractical for aggressive or wild animals (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916782/). Better saliva collection devices need to be developed.
Urine samples are easier to collect but have very low viral loads, making detection extremely challenging. The small number of rabies virus particles in urine reduces test sensitivity (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916782/).
In summary, developing a non-lethal rabies diagnostic test requires overcoming issues like low viral loads in certain sample types, impractical sample collection methods, and difficulty detecting antibodies consistently. More research and technological innovation is required to create an accurate and effective test that does not require euthanasia.
Conclusion
As discussed, there is an urgent need for a non-lethal rabies diagnostic test. The current standard direct fluorescent antibody test requires euthanizing the animal and examining brain tissue. This is traumatic for pet owners and prevents testing of rare or endangered species. Researchers are actively investigating alternative approaches like blood, saliva, and urine tests. These show promise but are not yet approved for routine diagnostic use. While there are still challenges to overcome, the growing focus on developing a non-lethal rabies test provides hope that humane and effective testing methods may become a reality in the not-too-distant future.
