Trost 10 GT 1

Julia Trost
10 GT/2
Independent Research I
Ms. Mary Jane Sasser
February 18, 2020

A Needle in a Haystack: Methods to Ensure an Accurate Diagnosis of Feline

Infectious Peritonitis (FIP).

According to the Humane Society, approximately 4 million cats and dogs are adopted

from shelters each year, providing everlasting joy to countless families. By adopting pets,

families are adding a new member to their homes with the hopes of their pet living a long life.

However, one rare disease is fatal in cats below the age of two; Feline Infectious Peritonitis (FIP)

takes away the lives of young cats and kittens across the United States. Along with there being

no legal cure available in the United States, there is also no definitive diagnostic test, making it

hard for veterinarians to diagnose this disease. Two antiviral drugs, GC-376 and GS-441524,

have been reported successful in treating FIP in numerous field studies, but are pending approval

to be released to the public. In order to guarantee a proper diagnosis of FIP, it is important

to conduct a clinical assessment, analyze the cerebrospinal fluid, and observe the cat’s

immunohistochemistry.

FIP kills 1.4% of cats around the world and is caused by a mutant coronavirus, otherwise

known as Feline Enteric Coronavirus (FECV). FECV is insignificant as an intestinal pathogen,

but “commonly mutates in vivo and at least one mutant form (i.e, biotype) causes a highly fatal

disease known as FIP” (Poland et al.). This single-stranded RNA virus undergoes mutations to

escape cells lining the intestine and infect the macrophage. The initial macrophage infection is
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eliminated in all but 0.3-1.4% of cats; in this small percentage of cats, FECV initiates a string of

mutations that causes FIP.

There are two different forms of FIP: the wet form and the dry form. The wet form is

characterized by a buildup of fluid in the abdomen, which causes difficulty in breathing. Cats

with the dry form develop neurological symptoms such as seizures and trouble walking. About

two thirds of cats suffer from the wet form, but the dry form is harder to treat and in most cases

is more severe, as it affects the major organs. In the dry form of FIP, also known as the

neurological and ocular form, the blood brain barrier presents challenges, as it doesn’t allow the

proper level of drugs needed to penetrate into the brain and treat the virus (Felten, et al).

Researchers are working on methods to infiltrate the blood brain barrier and effectively treat the

dry form of FIP.

One of the emerging treatments for humans targeting Ebola is GS-5734. This is an

antiviral drug that attacks proteins involved in RNA virus replication. Researchers from UC

Davis used a related drug called GS-441524 in a field trial for cats with FIP. Along with

GS-441524, another treatment called GC-376 is currently being researched, but clinical trials

show that GS-441524 may be more effective. Thirty-one cats were involved in a study at UC

Davis investigating the effectiveness of GS-441524, but five died from natural causes. For the

twenty-six cats that survived, the results were amazing; their fever was gone in one to two days,

and they experienced a full recovery afterward. Although some cats suffered relapses, they were

successfully treated with a higher dose of treatment. The results provide a hopeful glimpse into

what the future may look like with a cure.

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Despite encouraging results, the company that makes GS-441524, Gilead Sciences, has

not granted permission for veterinary use of the drug. GS-441524 is patented by Gilead

Sciences, Incorporated, but “they’re not allowing veterinary use for that drug so although the

research demonstrated that it was effective in treating cats with FIP, there’s kind of a roadblock

in allowing that to be used for cats. [And they are] going around to find another method in

identifying other antivirals that are effective in treating FIP in cats” (Murphy). Although

treatments have been proven effective, they will not be available for legal purchase in the United

States until Gilead Sciences, Inc. allows veterinary use or researchers develop a new compound.

Due to the rising pet market in China, black markets have formed to supply GS-441524

and GC-376. These drugs have been proven successful in some cats, but there are concerns about

whether these drugs are safe, authentic, or administered in the right dosages. Veterinarians would

have liked these drugs to be legalized first: “I must make it clear that I would have preferred

these drugs to be approved and commercialized in the normal manner. I am certain that this will

happen within the next few years, and as it does, the black-market demand for drugs like GS and

GC will wane” (Pedersen). At the same time, the FIP drugs supplied by the black markets are

helping thousands of cats, owners, and veterinarians around the world. In spite of the fact that

GS-441524 and GC-376 are valuable in treating cats for FIP, it is essential that they are legalized

first so owners do not have to break the law in order to obtain them.

Although GS-441524 and GC-376 serve as an established cure, they are not legal in the

U.S., leaving owners with few options in regard to saving their cats. A small percentage of cats

may live one to two years after being diagnosed with FIP, but others may deteriorate within

weeks; in 95% of cases, FIP is fatal (Cat FIP). In terms of treating FIP, owners may decide to
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put their cat on steroids “such as prednisone or cyclophosphamide [that] may slow disease

progression but do not produce a cure” (Hartmann, et al.). The vast majority of owners who have

a cat with FIP ultimately end up euthanizing their cat due to the devastating prognosis along with

the fact that they do not want them to suffer anymore.

Given FIP is highly fatal, it is important that veterinarians accurately and promptly

diagnose FIP, which can commonly be confused with treatable diseases such as toxoplasmosis,

fungal infections or lymphoma (Pedersen). ​Conducting a clinical analysis of the patient is an

important first step that will provide necessary background information and context for a

diagnosis. FIP is “commonly reported in cats < 2 years of age, although it may arise in older cats

from shelter environments” (Cook, et al.). Most cases are reported in adult cats and kittens three

months to five years and older cats aged 10-14 years. Very young kittens below 16 months are

the most vulnerable due to their incomplete immune system and brain development. Identifying

the cat’s age is an essential aspect to the path of a diagnosis, as the age factor may rule out other

diseases.

Since there are two different forms of FIP, it is necessary to distinguish between which

form the cat may have. Symptoms of both forms “include fever that doesn’t respond to

antibiotics, anorexia, weight loss, and lethargy” (Cat FIP). The wet form is characterized by an

accumulation of fluid in the abdomen or chest. In the dry form, “uveitis or chorioretinitis may be

noted in cats, and a careful ophthalmologic examination should be performed in any cat with an

unexplained fever” (Cook). Cats with the dry form commonly develop problems in their eyes,

such as a detached retina, and inflamed lesions on their kidneys, liver, and nervous system

(Lowe). It is important to designate whether the cat has the wet form or dry form to locate the
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source of the problem. From here, veterinarians can look into short-term treatment options and

determine a potential prognosis.

Assessing the cat’s risk factors will help to establish potential connections to FIP.

Outdoor cats and cats “that live in catteries and multi-cat households are at greater risk for FIP

than solitary, indoor animals. Cats in actively breeding households are also considered to be at

risk for FIP” (FIP Risk Factors). Along with these risk factors, others include stress, poor

physical condition, or the presence of Feline Leukemia Virus (FeLV) or Feline

Immunodeficiency Virus (FIV). Overall, the “incidence of FIP in a cat population can be as high

as 10 percent or as low as zero—and can fluctuate” (Burns). Kittens are already at high risk, but

if they come from crowded shelters or catteries, they can be at an even higher risk. A cat’s

medical history allows veterinarians to gather more information about the cat, such as their risk

factors and their life at home, making it easier for them to make an accurate diagnosis.

When looking at risk factors, it is also important to consider a cat’s risk of contracting

FECV, which can cause FIP in the first place. Other risk factors for FIP “included individual cat

age, individual cat coronavirus titer, overall frequency of fecal coronavirus shedding, and the

proportion of cats in the cattery that were chronic coronavirus shedders” (Foley, JE et al.). If

there is a cat with FECV in a shelter who transmits the virus through a fecal-based vector, then

there is a greater chance of the other cats developing FECV and contracting FIP as a result.

Fortunately, cats "do not transmit FIP virus to each other; the macrophage pathogen is present

only in diseased tissues and no longer replicates in the gut” (Burns). If a cat develops FECV, it

does not necessarily mean they will contract FIP; it all depends on their immune system’s

response. However, cats with FECV are more at risk for contracting FIP than those who do not
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have FECV. Thus, if a sick cat comes in who has been living with FECV, this may suggest a

diagnosis of FIP.

In summary, a clinical analysis of the patient will allow veterinarians to examine every

physical aspect of the cat and begin to connect the symptoms. When treating the cat, it is first

important to note the age. FIP is highly rare, meaning veterinarians do not see it as much as they

see other diseases. Since this disease commonly affects very young cats, age will be a clear sign

that the cat may have FIP, especially noting the smaller chance that young cats have of getting

sick compared to older cats. Then, it is necessary to ask the owner about specific details relating

to the cat’s home, eating habits, and where the cat came from originally. This will communicate

possible risk factors to the veterinarian, hinting that the cat may potentially be at higher risk for

FIP than other cats. In any medical diagnosis, it is highly important to gather background

information in order to confirm suspicions before moving on to tests and even a final diagnosis.

Testing for FIP is challenging: although there are countless tests that veterinarians can

conduct to assist them in their diagnosis of FIP, many are either ineffective or invasive. It is

important to note that the hardest decision is choosing which kind of tests are needed based on

the symptoms. There are both indirect tests, which “increase the odds that the clinical signs are

caused by FIP” (Update on FIP), and direct tests, which provide direct evidence that can be used

to support a diagnosis. However, “there is no single test that can diagnose every case of FIP with

complete sensitivity and specificity” (Wogan). This paper focuses on the effectiveness of direct

tests, although some indirect tests such as a blood count, analysis of effusions, or observing

feline coronavirus antibody titers may help bring veterinarians one step closer to diagnosing FIP.
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By assessing a cat’s cerebrospinal fluid (CSF), veterinarians can specifically detect

the RNA of FECV, which is the initial cause of FIP. A cat with FIP must have evidence of

FECV RNA in their tissue. CSF in a cat is a colorless body fluid found in the brain and spinal

cord produced by the ventricles in a cell (Doenges, et al.). Requiring a general anesthetic, CSF

can be collected from two different sites: the cerebellomedullary cistern, near the back of the

head and the lumbar cistern, which is near the pelvis (Ruotsalo, et al.). Depending on the cat’s

symptoms, the veterinarian will choose which site they will take CSF from, using a needle to do

so.

Veterinarians can use CSF as a factor in diagnosing FIP by analyzing the cells in the fluid

for real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) which can

detect FECV RNA in the cerebrospinal fluid of cats. Real-time RT-PCR “can be considered a

reliable specific tool for the diagnosis of FIP. Real-time RT-PCR of CSF showed a specificity of

100% in diagnosing FIP” (Doenges, et al.). Additionally, “all studies evaluating RT-PCR for the

detection of FCoV RNA in CSF have reported specificities of 100%, meaning that FCoV RNA

could only be detected in the CSF of cats with FIP (with and without neurological signs) but not

control cats (Felten, et al.).” RNA of Feline Coronavirus was found in the CSF of strictly cats

who had FIP. This study shows how real-time RT-PCR in CSF is effective in correctly

identifying FECV RNA, providing necessary context for a diagnosis.

There may be an exception for cats with the neurological form of the disease when

looking at CSF. ​Many cats with neurologic signs “have normal CSF. In one study, typical CSF

findings in cats with FIP were a protein concentration>200 mg/dL and a WBC count of >100

cells/uL, which consisted predominantly of neutrophils” (Felten, et al.). CSF may not help to
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differentiate FIP from other diseases in cats with the neurological form. In normal cases, the

analysis of CSF would reveal an increase in proteins. The blood to brain barrier may play a role

in why the CSF findings do not show anything in cats with the neurological form. In general,

“various infectious and inflammatory diseases of the central nervous system lead to the release of

cytokines, adhesion molecules, metalloproteases and other mediators, which can damage tight

junctions, basal membranes, and cerebrovascular endothelium. This enables the leakage of cells

and pathogens into the CSF across the blood-brain barrier [163]” (Felten, et al). Thus, excess

molecules that leak into the CSF can interfere with the test, and as a result, make it harder to

diagnose FIP in cats with the neurological form. There is still ongoing research involving the

blood to brain barrier, which not only makes the neurological form harder to diagnose but also

harder to treat.

Nevertheless, analyzing a cat’s CSF is a helpful approach when attempting to make a

definitive diagnosis. As shown by numerous studies, RT-PCR can be used to effectively detect

FECV in the CSF of cats infected with FIP. Detection of FECV can be incredibly helpful, as

“about one in 10 cats exposed to this mutant virus will go on to develop FIP after a subclinical

period ranging from days to a year or more” (Pedersen). Additionally, extracting CSF comes

with little risks and is not highly invasive unlike other methods. Although early studies suggest

that CSF may not serve as a benefit to cats with the neurological form of FIP, there is still

ongoing research investigating many unknowns. Overall, CSF is a useful tool for locating FECV

RNA, which will then confirm suspicions or rule out FIP.

Another method of staining cells can be used to detect FIP infection in the macrophage

and possibly other infected tissues. As a direct test, immunohistochemistry has been proven
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successful in aiding a diagnosis of FIP. Immunohistochemistry is characterized by the staining of

tissue to examine cells. This technique detects a specific antigen in tissue by using antibodies

that bind to that antigen (Overview of Immunohistochemistry). The interaction between the

antigen and antibody can be seen by using a fluorescent dye and viewing the tissue through a

microscope, or due to a color-changing reaction, where an enzyme binds to an antibody and

produces a chemical reaction (Day, et al.).

Immunohistochemistry allows veterinarians to inspect the possible area of

infection. ​For example, “Immunohistochemistry combines anatomical, immunological and

biochemical techniques to image discrete components in tissues by using appropriately-labeled

antibodies to bind specifically to their target antigens” (Overview of Immunohistochemistry). In

immunohistochemistry, specific tissues are stained to observe antigens, or foreign substances, in

which certain antibodies bind to. This helps to locate specific proteins that may not be working

and other causes of the disease, such as another virus or cancer. Thus, “these identifiable

substrates offer documentation of localization that can be seen through the microscope at the

cellular level. In this way, individual cells or groups of cells can be monitored for the presence of

the specific antigen” (Immunohistochemistry). After the antibodies are selected, veterinarians

can locate the antigens in diseased tissue. This allows veterinarians to look closely at tissues such

as the macrophage that are commonly infected in FIP.

Both histopathology and immunohistochemistry have to do with studying changes in

tissue. However, “histopathology is considered the gold standard test but immunohistochemistry

(IHC) is mandatory to confirm/exclude the disease” ​(Giori, et al.). Immunohistochemistry is

needed to either support or deny a diagnosis of this disease and is “considered to be accurate for
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the definitive diagnosis of FIP” (Giori, et al.). Additionally, when it comes to dealing with tissue,

“it is important to note that the quality of reagents used, tissues sampled, and qualifications of the

microscopist need to be accounted for when evaluating the reliability of immunohistochemistry”

(Pedersen). To guarantee the accuracy of immunohistochemistry, proper laboratory conditions

and preparations need to be followed.

In a study investigating the diagnostic features of FIP, researchers concluded that

“positive FIPV staining was detected mostly in macrophages in intensely inflamed areas of the

ependyma and choroid plexus. Macrophages free within the ventricular lumen often were

strongly positive, suggesting that immunohistochemical staining of cells from CSF samples may

provide valuable antemortem diagnostic information” (Foley, et al.). FECV affects the

macrophage, explaining why the macrophage is used for the staining of cells. There is a

relationship between CSF and immunohistochemistry, as CSF is needed to accurately determine

if a cat has FIP. As communicated by the results of the study, immunohistochemistry offers vital

information in regard to the overall condition of the macrophage.

Without a doubt, immunohistochemistry is a reliable tool for diagnosing FIP in cats. This

method allows veterinarians to take direct samples of diseased tissue in order to look for foreign

molecules. If completed properly, immunohistochemistry permits veterinarians to make a direct

diagnosis in comparison to an indirect diagnosis which may not be accurate and can take more

time. Similar to analyzing the cerebrospinal fluid, immunohistochemistry will serve as an

important indicator of what exactly is going on in both the cat’s macrophage and body, providing

essential information on whether they may have FIP.

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A diagnosis of Feline Infectious Peritonitis is both shocking and crushing at the same

time, as there is no cure available in the United States. Although veterinarians cannot know for

sure that a cat has FIP, due to the absence of definitive tests, there are several methods that

veterinarians can employ to rule out other similar diseases. Analyzing the cat’s physical features

allows veterinarians to gather background information on the cat and potential risk factors, which

are highly important when making a diagnosis. Observing a cat’s cerebrospinal fluid and

immunohistochemistry gives the veterinarian a detailed review of the cells in the cat’s tissues,

providing information on whether the cat may have FIP or another disease such as cancer.

Although these methods need to be executed appropriately in order to guarantee accurate results,

they provide much needed information when it comes to making the right diagnosis. Hopefully,

within the next couple of years, a cure will be released to the public, saving the precious lives of

young kittens and cats who deserve long lives ahead of them.
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Glossary

Adhesion Molecules​: Located on cell surface, involved in binding with other cells (Guangwen)

Antemortem​: Performed before death

Basal Membranes​: The layer of extracellular matrix (Felten, et al.)

Cattery: ​A place where cats are actively bred

Cerebrovascular Endothelium​: The cell layer on the inside of vessels (Felten, et al.)

Choroid Plexus​: Produces the cerebrospinal fluid (Kinaan, et al.)

Chorioretinitis​: The inflammation of choroid and retina of the eye (Cook)

Cytokines​: The number of substances secreted by certain cells that have an effect on other cells

(Felten, et al.)

Ebola: ​A deadly disease characterized by internal bleeding with occasional outbreaks (What is

Ebola Virus Disease?)

Ependyma​: The central canal of spinal cord (Brat)

Feline Immunodeficiency Virus (FIV)​: A lentivirus (slow virus) that is very similar to HIV

Feline Leukemia Virus (FLV)​: The leading cause of death in cats, transmitted by saliva

Fungal infection​: Fungi common in the environment infect a cat’s immune system

In vivo​: Takes place in a living organism

Lymphoma​: Cancer that begins in lymphocyte cells in immune system

Macrophage​: Either found as large white blood cell or large cell in the tissue

Metalloproteases​: A protease enzyme involving a metal (Felten, et al.)

Substrate​: A molecule that an enzyme binds to

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Toxoplasmosis​: Infection due to a parasite; occurs from eating undercooked contaminated meat

(Pedersen)

Uveitis​: Eye inflammation that affects the uvea (Cook)

Ventricular Lumen​: The inside artery of ventricle in the heart (Arteries)

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References

"Arteries." ​Dartmouth University,​ www.dartmouth.edu

Brat, Daniel J. "Ependyma-An Overview." ​Science Direct,​ 2018

Cook, et al. “Feline Infectious Peritonitis: Strategies for Diagnosing and Treating This Deadly
Disease in Cats." DVM 360, 1 Sept. 2013

Guangwen, Ren, and Arthur Roberts. "Adhesion Molecules." ​National Center for Biotechnology
Information​, 1 Jan. 2011

Kinaan, Javed, and Lui Forshing. "Neuroanatomy, Choroid Plexus." ​National Center for
Biotechnology Information

Pedersen, Niels. “A Review of Feline Infectious Peritonitis Virus Infection: 1963-2008." ​Sage
Pub Journals​, 1 Apr. 2009

"What Is Ebola Virus Disease?" ​Centers for Disease Control and Prevention
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Works Cited

Burns, Katie. "A Glimmer of Hope for a Fatal Feline Disease." ​American Veterinary Medical
Association,​ 29 Nov. 2017

"Cat FIP (Feline Infectious Peritonitis)." ​WebMD

Cook, et al. “Feline Infectious Peritonitis: Strategies for Diagnosing and Treating This Deadly
Disease in Cats." DVM 360, 1 Sept. 2013

Day, Michael, et al. "Immunohistochemistry (IHC)." ​Vet Stream

Doenges, Stephen, et al. "Detection of Feline Coronavirus in Cerebrospinal Fluid for Diagnosis
of Feline Infectious Peritonitis in Cats with and without Neurological Signs." Sage
Journals, vol. 18, no. 2, 3 Mar. 2015, pp. 104-09

"Feline Infectious Peritonitis (FIP) Risk Factors, Causes of FIP." Remedy's Health Communities,
1 Oct. 2001, Accessed Dec. 2014.

Felten, et al. "Detection of Feline Coronavirus Spike Gene Mutations as a Tool to Diagnose
Feline Infectious Peritonitis." ​U.S. National Library of Medicine National Institute of
Health,​ 10 July 2016

Foley, Janet, et al. "Diagnostic Features of Clinical Neurologic Feline Infectious Peritonitis."
Wiley Online Library

Foley, J.E., et al. "Risk Factors for Feline Infectious Peritonitis among Cats in Multiple-Cat
Environments with Endemic Feline Enteric Coronavirus." National Center for
Biotechnology Information, U.S. National Library of 
Medicine

Giori, L., et al. "Performances of Different Diagnostic Tests for Feline Infectious Peritonitis in
Challenging Clinical Cases." National Center for Biotechnology Information, U.S.
National Library of Medicine

Hartmann, K., and S. Ritz. "Treatment of Cats with Feline Infectious Peritonitis." ​PubMed

"How to Recognize the Signs of FIP in Shelter Cats." ​Maddie's Fund.​

"Human Antiviral 'GS-441524' Shows Great Promise against Infectious Disease

in Cats." ​Science Daily​, 19 Feb. 2019.

"Legal Treatment for Cat Disease Known as FIP Still Years Away." ​VinNews
Service​, 22 Aug. 2019.
Levy, Julie K., and Staci Hutsell. "Overview of Feline Infectious Peritonitis." MERK Manual
Veterinary Manual
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Lowe, Amy. "FIP." ​Cat Friendly Homes

Murphy, Brian. Telephone interview. 23 Dec. 2019.

"Neurological and Ocular FIP." ​UC Davis Veterinary School of Medicine​, 29 Apr. 2019

"Overview of Immunohistochemistry (IHC)." ThermoScientific

Pedersen, Niels. “A Review of Feline Infectious Peritonitis Virus Infection: 1963-2008." ​Sage
Pub Journals​, 1 Apr. 2009

"Pets by the Numbers." The Humane Society of the United States.

Ruotsalo, Kristina, and Margo Tant. "Cerebrospinal Fluid Collection and Examination." ​VCA

"An Update on Feline Infectious Peritonitis: Diagnostics and Therapeutics." The Veterinary
Journal, vol. 201, no. 2, 27 Apr. 2014, pp. 133-141