Viral hepatitis in horses (we used to call it Theiler disease

Caution: Do not exceed the maximum IV dose for Bupivicaine in any patient (I do not exceed 1 mg/kg). If maximum IV dose is exceeded, it may potentiate arrhythmias and seizures. Delayed wound healing has been reported as a complication with large volumes; however I have not found this to be a problem with the volume of drug I administer to my ophthalmic patients.

PHARMACOLOGY REVIEW Local anesthetics are classified as esters and amides. The local anesthetics used in veterinary practice (lidocaine, bupivicaine, mepivicaine) are amides. Local anaesthetics are predominantly metabolised by the liver.

Agent lidocaine mepivicaine bupivicaine ropivicaine Recommended dose (mg/kg)

5 5 2 2 Toxic dose (mg/kg) 10 – 20

3.5 – 4.5 4.9 Onset time (mins) 10 – 15 15 - 20 20 - 30 20 - 30 Duration (hours) 1 – 2 1.5 - 3 2.5 - 6 2.5 - 6

AMINO ESTERS – procaine, 2-chloroprocaine, tetracaine AMINO AMIDES – lidocaine, mepivicaine, ropivicaine, bupivicaine

Clinical differences between the ester and amide LA’s involve their potential for producing adverse effects and the mechanisms by which they are metabolised Half-life of esters is only a few minutes, and of amides is a few hours Esters are metabolised by para-amino benzoic acid (PABA). Patients with reduced cholinesterase activity (newborn and pregnant) may have increased potential for toxicity from ester LA’s. Amides are mainly metabolised by the liver – so risk with severe hepatic disease Block nerve conduction through two mechanisms – by impairing propagation of the action potential in the axon; by interacting directly with specific receptors on the sodium channel, inhibiting sodium ion influx

Esters and amides differ with respect to their chemical stability, biodegradation and allergenicity. Esters have short shelf lives and are degraded in plasma by pseudocholinesterases. Amides are very stable and are metabolised by hepatic microsomes. The potential allergenicity of the

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ester LA’s is a result of production of PABA as a primary product of biotransformation. Amides aren’t broken down to PABA, so that allergic reactions are very rare.

KEY LEARNING POINTS: • The sub-conjunctival enucleation can be used in dogs, cats, and horses and affords several advantages over the trans-palpebral approach. • After an enucleation and as clots within the orbit break down, bloody fluid may drain via the nasolacrimal duct and appear at the nostril (usually on the third to fifth day after surgery; most noticeable in horses). • The emotional resistance of the owner to enucleation should be evaluated prior to surgery, and, when appropriate, globe sparing alternatives such as evisceration and intrascleral prosthesis may be considered. • All enucleated globes should be submitted for histopathologic examination to establish a definitive diagnosis that may alter management of the remaining eye (glaucoma, lens luxation) and to rule out an unsuspected disease process such as neoplasia, systemic mycoses, septic endophthalmitis, etc.

Additional references available upon request

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MU-CVM Lectures

Philip Johnson, DVM ACVIM

Professor of Veterinary Medicine and Surgery University of Missouri College of Veterinary Medicine Columbia, Mo.

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Viral hepatitis in horses (we used to call it Theiler disease)

Philip J. Johnson

At the outset of the twentieth century, Sir Arnold Theiler originally described outbreaks of an acute fatal liver disease in horses following administration of antisera, being developed for protection of horses against African Horse Sickness. He also reported that in-contact horses also developed severe liver disease, albeit that those horses had not received the antiserum. Theiler was a Swiss veterinarian working in what is now South Africa. Prior to his significant observations regarding the risk of ‘serum hepatitis’ in horses treated with antiserum, he had been celebrated for success in the production of a vaccine to combat an outbreak of smallpox that afflicted miners in the Witwatersrand. He was appointed as the state veterinarian for the Zuid-Afrikaansche, where he worked throughout the Anglo-Boer war (1899-1902). He also developed a vaccine against the contagious bovine disease, rinderpest. Acute fatal hepatitis following administration of antiserum (any disease) has subsequently been known as Theiler’s disease.

Fatal hepatitis (Theiler disease) outbreaks have arisen in the context of administration of antisera (and other equine biological treatments) since the time of Theiler. Deployment of antisera against Western Equine Encephalitis (WEE) to protect horses in the western USA (1930s) also led to substantial death from severe hepatic disease. Antisera against various other diseases (EEE, anthrax, tetanus, botulism, clostridiosis, strangles, EHV1, influenza) have also been implicated. The use of allogeneic stem cell therapy for orthopaedic injuries and the administration of plasma/serum products to horses have also resulted in Theiler disease (it is not restricted to the use of antiserum products per se). Moreover, the fact that in-contact but untreated horses also sometimes develop hepatitis has been well known. Therefore, the ‘idea’ that Theiler disease results from an infectious contagion has long been suspected. Attempts to cultivate viral pathogens using traditional virus isolation methods from the liver of affected individuals failed and the ‘etiology’ of Theiler disease has, for so many years, been consigned to that ‘last chapter’ in veterinary medical textbooks dedicated to diseases of unknown etiology.

Theiler’s disease in horses is an example of a situation in which molecular tools have enhanced our understanding of etiology. Using PCR-based massively parallel sequencing technology with subsequent nucleic acid-based screening, Chandriani and coworkers (2013) first described a previously unknown and highly divergent member of the Flaviviridae family in horses that developed Theiler’s disease after being treated with botulism antiserum. This candidate virus was designated “Theiler’s disease-associated virus” (TDAV) and could be demonstrated in both blood obtained from affected horses and from the equine antiserum product that had been administered to the horses. Only RNA viral metagenomic analysis was performed in that study, resulting in limited results. TDAV (flavivirus) is no longer thought to cause Theiler disease.

In further pursuit of a putative viral etiology for Theiler’s disease, the employment of modern molecular PCR-based diagnostic methods (unbiased amplification and high-throughput sequencing of serum and tissue samples) led to the identification of three additional candidate viruses that infect horses (another two RNA viruses and one DNA virus) since 2013. Of importance, two of these novel viruses have been shown to cause liver disease. One is a non-primate hepacivirus (NPHV) also called equine hepacivirus or hepacivirus A, the closest homologue to hepatitis C virus (HCV) infecting humans, and the other has been designated equine parvovirus-hepatitis (EqPV-H). Results of a prospective study showed that EqPV-H virus could be identified in the blood and/or liver of 18 cases of Theiler’s disease that occurred 4–12 weeks following administration of an equine-origin biologic product (usually tetanus antiserum). In many of those cases, presence of EqPV-H was also demonstrated in antiserum that had been administered to affected horses. Subsequent experimental EqPV-H infections showed that biochemical evidence for hepatitis correlated with peak viremia and initial EqPV-H antibody detection. Interestingly, retrospective testing of archived frozen samples (blood and antisera) from the first Theiler’s disease outbreak, in which TDAV was discovered, showed that all of those samples were also positive for EqPVH.

Presently, increasing evidence points to EqPV-H as being the responsible etiological agent in Theiler’s disease. The equine hepacivirus (NPHV) is now more strongly associated with the syndrome that had

previously been characterized by its histopathological features, chronic active hepatitis. There have been some reports in which both EqPV-H and NPHV have been identified in cases of Theiler’s disease, suggesting that co-infection may play a role, at least in some cases, but further work is needed to better explore the relationship between these viruses in the face of liver disease in horses. Infection with EqPVH has also been demonstrated in horses affected with Theiler’s disease but that did not receive treatment with an equine-origin biologic product.

In the past, veterinarians had to acknowledge risk of potentially fatal Theiler’s disease whenever there was a call for administration of equine-origin biological products, especially if antiserum was needed to provide protection against tetanus in wounded horses that had not been actively vaccinated and/or boosted. It is now possible to screen donor horses for both of these hepatitis viruses, thus significantly reducing the risk of both Theiler disease and chronic active hepatitis. PCR testing of blood for both EqPVH and NPHV is now available to veterinary diagnosticians at the Animal Health Diagnostic Center at Cornell University. Moreover, the USDA Center for Veterinary Biologics now requires that all licensed equine blood products must test negative for EqPV-H. Modern PCR technology has facilitated identification of two potential viral etiologies for serum hepatitis and, secondly, has enabled screening for these viruses in candidate horses potentially used to produce equine-origin biological products thus significantly impacting equine veterinary practice.

Of further note, an additional three viral agents (equine kirkovirus, equine hepatitis B virus, equine circovirus) have been more recently identified in the context of hepatic disease in horses. It remains to be seen whether these viruses represent important considerations in the USA context or whether they are rare/uncommon findings. That said, it is clear that both the parvovirus and the hepacivirus are endemic in the USA.

Viral hepatitis: parvovirus (EqPV-H):

Infection with EqPV-H (DNA virus) may result in mild or subclinical hepatitis or massive liver necrosis with attending signs of liver organ failure. This parvovirus (EqPV-H) has been identified in the blood of a substantial number of unaffected (ostensibly healthy) horses around the world. It is not known why some horses develop severe disease (classic Theiler disease) following infection with EqPV-H and others do not. It is likely that, as with most infectious diseases, contributory factors include the size of the infecting inoculum, the state of the host’s immune system, and differences in viral genotype/antigen characteristics. Signs of acute hepatitis include inappetence/anorexia, lethargy, jaundice, photodermatitis, edema, colic, encephalopathy and death. The case fatality rate for severe Theiler disease exceeds 80%. Milder forms of hepatitis may include inappetence/anorexia, lethargy, and biochemical evidence of hepatic disease. Fever is not typically reported. The DNA prevalence of EqPV-H has been estimated at 3.2-17% in the USA (seroprevalence 15-34.7%). Many healthy horses are positive for this virus in the blood. It has been suggested that 98% of infections occur subclinically (no clinical signs and biochemical evidence not investigated absent clinical disease). Early work suggests that there is no biochemical evidence of hepatitis following experimental exposure in approximately 10-15% of cases. The route of transmission is not completely understood but is believed to be via fecal-oral or respiratory routes (no longer believed to be via an insect vector). The number of ‘classic’ cases of Theiler disease has dropped very significantly since the USDA imposed viral screening for commercial products (such as tetanus antiserum). Infection with EqPV-H tends to cause individual hepatocyte necrosis with lymphocytic clustering. Some larger hepatocytes may be observed, making the histopathological characterization potentially similar to that for pyrrolizidine alkaloid toxicosis (megalocytic hepatopathy). Viral specificity at (hepatic) histopathology can be assured via both immunohistochemistry and in situ hybridization. Following infection, liver damage (as detected through plasma biochemistry) appears to coincide with the point of immune control (seroconversion), suggesting that it is the horse’s immune response to the virus that determines the severity of clinical disease. When provoked, evidence for hepatitis (example elevated GGT), even when mild, lasts up to 12 weeks. Detectable viremia (even at low levels) can last months and years. Chronic disease (fibrosis, cirrhosis) has NOT been reported with EqPV-H infections. Diagnosis of EqPV-H hepatitis cannot be based simply on demonstration of the virus in clinical cases as there are too many healthy horses in which the virus circulates, absent signs of disease. Diagnosis of EqPV-H viral hepatitis is based on demonstration of liver disease (plasma biochemistry), viremia (high viral titer), and

histopathology (biopsy, necropsy). Presence of EqPV-H in the context of hepatic lesions is important (quantitative PCR, ISH, IHC) in these regards. There is some evidence that disease severity is related to viral load in liver tissue, more so than the level of viremia. Disease resulting from EqPV-H can be fatal or self-resolving. There are no presently recommended anti-viral drugs for this infection. Questions arise regarding the biosecurity implications for this virus. It is generally recommended that isolation of affected horses is impractical. It is suggested that a vaccine against EqPV-H may be possible in the future.

Viral hepatitis: hepacivirus (EqHV): Also, worldwide distribution for this RNA virus (flaviviridae). Low genetic variability. RNA prevalence 1.4 –41.2% Seroprevalence up to 80%. Mode of transmission is being actively studied; EqHV can be transmitted vertically. As with parvoviral hepatitis, subclinical infections appear to be much more common than infection that results in disease. Disease associated with EqHV appears to be significantly less common than disease resulting from EqPV-H. Current thinking suggests that EqHV may be the etiological agent for ‘chronic active hepatitis’, a disease of the equine liver that has traditionally been regarded as ‘idiopathic’. ‘Chronic active hepatitis’ is diagnosed when clinical signs of hepatitis have been associated with specific hepatic histopathological features, that include fibrosis. Clinical signs associated with EqHV hepatitis are nonspecific and may include inappetence, weight loss, photodermatitis, HE, and (sometimes, but not commonly) fever. Clinical hepatitis associated with EqHV infection appears to be less common than that associated with EqPV-H. There have been instances in which both EqPV-H and EqHV are identified in the context of hepatitis. As with the parvovirus, the onset of clinical evidence of disease (including circulating biomarkers) appears to coincide with the time of seroconversion. Hepacivirus also causes hepatitis with lymphocytes but there is also a chronic component to this disease (fibrosis). Most (~80%) of infected horses can ‘clear’ the virus but some (~20%) develop hepatitis that, in some cases, can lead to cirrhosis eventually (slowly progressive disease with a diffuse distribution of lesions, including micronodular fibrosis). If infected during foal hood, it is more likely that viral tolerance will occur, and the animal will remain infected into the long term (years). There is currently no USDA ruling about screening donors for purposes of sale of equine-derived biologicals. Although there is one report of disease in a 4-year-old horse, most EqHV infections that progress with disease have been seen in teenage horses; fewer females are affected. This equine EqHV is, in many respects, similar to human hepatitis C. There is some anticipation that research regarding the equine virus may lead to vaccine development for HEP C-affected human patients (there is not presently a vaccine for the human hepatitis C virus as it mutates too quickly; interestingly, the equine EqHV virus is much slower in that regard). Similar to parvoviral hepatitis, diagnostic confirmation of EqHV hepatitis cannot be based simply on demonstration of the virus in clinical cases as there are too many healthy horses in which the virus circulates, absent signs of disease. Diagnosis requires demonstration of liver disease (plasma biochemistry), viremia (higher viral titers are more significant), and histopathology (biopsy, necropsy). Presence of EqHV in the context of hepatic lesions is important (quantitative PCR, IHC) in these regards but in situ hybridization is not (presently) available for this pathogen. The prognosis for EqHV hepatitis is difficult to gauge. Prognosis should be based on repeated clinical examinations (including hepatobiliary biomarkers, viral titers). If the plasma GGT activity continues to increase, it is possible that ‘bridging fibrosis’ is occurring; ‘bridging fibrosis’ is always considered a sign of an unfavorable prognosis in hepatobiliary disease, regardless of the cause. Histopathological conclusions for EqHV hepatitis can (confusingly) include all of the following: cholangiohepatitis, toxic insult, viral hepatitis, or even sometimes as suspected pyrrolizidine alkaloid toxicity. Co-infection with EqPV-H is not uncommon and in situ hybridization at Cornell AHDC can clarify if parvovirus is the primary pathogen.

Treatment considerations for viral hepatitis: Supportive treatments for liver function; control abnormal behavior (alpha2 agonists; avoid benzodiazepines) Reduce dietary protein, increase carbohydrate (sorghum, milo, beet pulp are good sources of branch chain amino acids); add molasses to promote palatability Decrease GIT ammoniagenesis (oral neomycin (or metronidazole), lactulose, mineral oil) IV glucose to support liver function Plasma for clotting, if needed Vitamin E (RRR-stereoisomer)(natural form) Anti-fibrotics (colchicine, pentoxifylline, benazepril (minimal equine evidence for efficacy)

Corticosteroids? Controversial (use in later stages, palliative) Anti-oxidants: S-Adenosyl-L-methionine (also called S-adenosyl methionine, S-adenosylmethionine, SAMe, or SAM-e in the United States or ademetionine in Europe, and also often abbreviated as SAM and AdoMet. Milk thistle extract. Silybin™. Good reputation in human medicine (not much studied in horses, poor oral bioavailability) Hepatalyte™: B-Vitamins B1, B2, B6, B12, and vitamin E, generous levels of Milk Thistle Sofosbuvir for EqHV? (in silico evidence) Ursodiol™ Bile acid formulation with anti-inflammatory properties (very positive reputation) – more recommended for cholangiohepatitis

References available upon request

MU-CVM Lectures

Pamela Adkins, MS, DVM, PhD, DACVIM

Assistant Professor of Food Animal Medicine and Surgery University of Missouri College of Veterinary Medicine Columbia, Mo.

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