Ophidian Paramyxovirus (OPMV)
Historically, Gram-negative microorganisms including Pseudomonas spp., Providencia spp., Proteus spp., Salmonella spp., Aeromonas hydrophila and Escherichia coli have been the most commonly isolated bacteria from the respiratory tract of reptiles with clinical signs of pneumonia. Prior to 1976, relatively few viral infections of reptiles had been reported, and virtually nothing was known about viral infections of the respiratory tract of reptiles. In 1972, a respiratory epizootic spread through a collection of fer-de-lance (Bothrops atrox) at a serpentarium in Switzerland. Initially Pseudomonas and Aeromonas were isolated from the respiratory tract of dead snakes and the disease was originally thought to be bacterial in origin. Eventually a virus with morphological and biochemical properties of certain myxoviruses was isolated and was tentatively placed in the paramyxovirus subgroup.
Since its first description, ophidian paramyxovirus (OPMV) has surfaced as an extremely important pathogen of viperid snakes. In 1979 the first outbreak was reported in a private collection in the United States, and since that time numerous outbreaks have been seen in collections of viperid snakes in the United States and Mexico. A similar virus has been isolated from a black mamba (Dendroospis polylepis) and multiple species of rat snakes including corn snakes (Elapha guttata), beauty snakes (E. taeniurus), Moelendorff’s rat snakes (E. moellendorffi), and tiger rat snakes (Spilotes pullaties). In the Federal Republic of Germany, myxovirus-like agents were recently recovered from a red-tailed rat snake (Elaphe oxycephala), diamond python (Morelia orgus) and rhinoceros viper (Bitis nosicornis).
All viperid species should be considered susceptible to infection. There also have been reports of infection in colubrid, boid, and elapid snakes.
Worldwide in zoological and private collections including United States, Mexico, Argentina, and Germany.
Both juveniles and adults. There are no reports of infection in neonates.
Members of the virus family Paramyxoviridae contain single-stranded RNA, are 100 to 200 nm in diameter, are enveloped, and have a molecular weight of 5,000,000 x to 8,000,000 daltons. Virus replication occurs mainly in the cytoplasm with maturation by budding from the cell membrane. The family includes the 4 genera: 1) Paramyxovirus (parainfluenza); 2) Morbillivirus (measles-distemper-rinderpest group); 3) Pneumovirus (respiratory syncytial virus and pneumonia virus of mice); 4) Rubulavirus (mumps). Fer-de-lance virus (FDLV), isolated from lance-headed vipers which died during the outbreak in Switzerland, was found to have ultrastructural properties similar to the myxoviruses (ortho- para- and meta-; Clark et al, 1979). Virions were pleomorphic, ranging from spheroidal to filamentous and budded from plasma membranes. The size of released particles ranged from 146 to 321 nm in diameter, depending upon the host cell system and the cell culture incubation temperature. That FDLV is a paramyxovirus was indicated by demonstrating it possessed a single-stranded RNA genome and had a sedimentation value of 50S. Clark and Lunger (1981) found that FDLV was serologically distinct from mammalian and avian paramyxoviruses. A similar virus isolated from an ottoman viper (Vipera xanthina xanthina) was related to parainfluenza. Protein patterns of an isolate of a myxovirus from a gaboon viper in Germany showed best correlation with that of respiratory syncytial virus proteins (Ahne and Neubert, 1989). Recent studies with isolates from a Neotropical rattlesnake (Crotalus durissus), Auba Island rattlesnake (Crotalus unicolor) and Bush viper (Atheris sp.) revealed the presence of 6 major proteins, and were considered analogous to those of mammalian paramyxoviruses (Richter et al, 1996). Attempts to establish the antigenic relatedness of the 3 isolates to a wide variety of avian and mammalian paramyxoviruses was unsuccessful. The Neotropical virus and bush viper virus have been deposited as VR-1408 and VR-1409 at American Type Culture Collection (ATCC)
In the original epizootic involving fer-de-lance, clinical signs lasted 5 to 12 days and progressed through 4 stages. During stage 1 there was a loss of muscle tone, with affected snakes exhibiting a “stretched out” linear posture with the head slightly elevated. During stage 2, which lasted 1 to 2 days, snakes showed abnormal activity. Affected snakes crawled about restlessly and kept their mouths partially opened. Their tongues were incompletely withdrawn into the sheathes and their pupils were extremely dilated. Stage 3 was seen from several hours to one day preceding death. The mouth was kept completely open and the snakes expelled a purulent material from the glottis. Stage 4 was seen from several minutes to one hour preceding death. The mouth was kept fully opened, the pupils were dilated, and animals were excessively active.
In an epizootic involving rock rattlesnakes (Crotalus lepidus), a new breeder male was introduced into an established collection without being quarantined. Ultimately, this snake was in contact with 8 other rock rattlesnakes, 7 of which died. On day 3 following introduction the new snake developed head tremors and loss of equilibrium (Figure 1); it died on day 14. Over the next 2 1/2 months, 4 females and 3 males died after manifesting clinical signs. Only one rattlesnake remained healthy and survived.
In those snakes seen in the terminal stages of the disease, immediately preceding death, these animals generally manifest a convulsive behavior. This should not be confused with primary central nervous system disease described in rock rattlesnakes. These are agonal signs and are rather non-specific. Snakes may twist around, become flaccid and quiet for a period of time, and initiate these death-throws all over again.
In many of the outbreaks on OPMV infection, minimal or no clinical signs are noted by the keeper/owner. Often snakes will be found dead in their cage early in the morning, having died the night before. Many of these snakes appear to be in good health with good weight and normal behavior prior to death. Clinical signs can be subtle or non-specific such as off feed for one to two weeks. Although clinical signs in the earlier stages of the disease are often subtle, abnormal respiratory sounds are audible when ill snakes are manually restrained. If the oral cavity is examined, exudate may be seen within the glottal opening. Some snakes die with blood expelled from the glottis and filling the oral cavity (Figure 2). In a group of Siamese cobras (Naja naja kaouthia), the major consistent clinical signs was polyuria (increased urination). These snakes became ill during a die-off of rattlesnakes in the same room; paramyxovirus was isolated from dead rattlesnakes.
It is the respiratory system that appears to be targeted by OPMV infections. Gross changes ranged from diffuse hemorrhage of the lung and air sac system to diffuse to focal accumulations of caseous necrotic cellular debris (Figure 3). Other organs which may be involved on a gross level are the pancreas and liver. Pancreatic hyperplasia is not uncommonly encountered in infected crotalid snakes. The authors have seen this quite commonly in timber rattlesnakes (Crotalus horridus). In the liver, areas of necrosis and formation of multifocal firm nodules (granulomas) may be seen.
The lung of normal snakes consists of relatively thin septae which are lined by capillaries and alveolar type I and Type II cells (Figure 4). Histologic examination of lung tissue from snakes which have died of OPMV infection revealed a moderate to profuse amount of cellular debris and exudate filling both the major and minor passageways (Figure 5; Figure 6; Figure 7). Macrophages and gram-negative microorganisms are often seen within this material. Alveolar type II cells lining the primitive alveoli undergo hypertrophy, hyperplasia, and metaplasia. Characteristically, hyperplastic epithelial cells completely proliferate over capillary beds with normally are at the surface of the primitive alveoli. Often these epithelial cells are vacuolated. Intracytoplasmic inclusions are occasionally seen within these cells. The pulmonary septa are often thickened with edema fluid and infiltrated with mixed inflammatory cells including macrophages, lymphocytes, and plasma cells; giant cells are occasionally seen. Well-organized granulomas are rarely seen.
Microscopically, in those cases having an enlarged pancreas, there is hyperplasia of acinar cells and terminal ductile epithelium. This is considered by the authors to be a direct result of the virus, similar to the epithelial proliferation seen in the respiratory tract.
In the liver, lesions range from areas of caseation necrosis to granuloma formation. By special staining, gram-negative microorganisms are often demonstrated in these lesions. A variety of bacterial organisms have been isolated from these lesions with Pseudomonas spp. being the most common isolates. Bacterial organisms can invade the liver either from showering of bacteria from the gastrointestinal tract or from secondary bacterial invaders in the respiratory tract. Paramyxoviruses in mammals are known to have immunosuppressive effects and most likely results in a compromised immune system in snakes. Thus, it is not surprising that these snakes often succumb to secondary bacterial pathogens.
Occasionally, OPMV infection may be manifested as an encephalitis. In a rock rattlesnake, demyelination and some degeneration of axon fibers with moderate ballooning of axon sheaths were seen in the brainstem and upper spinal cord (Figure 8). However, signs of central nervous system disease are not typically seen. When seen in a boa or python, consider inclusion body disease in the differential.
Transmission and Epidemiology
According to reports in the literature and our recent experience with epizootics in private collections, once snakes start dying of OPMV infection, the mortality within a collection generally progresses fairly rapidly and peaks at about one month following initial deaths. It then declines through 2-3 months. In each epizootic we have investigated, although several species may comprise the collection, the virus seems to target a particular species. In some die-offs, the disease may result in the death of a large number of snakes over a more prolonged period time.
Although OPMV infections have occurred throughout the year, in many cases, epizootics have been experienced from January to May. Replication of the vitrus in vitro has been demonstrated to be temperature-dependent with an optimum temperature for growth at 30°C and a range of temperature for growth of 23°C to 32°C. Thus, possibly a latent infection may become activated if snakes are kept at suboptimal environmental temperatures. This may account for many of the epizootics occurring during cooler times of the year or following hibernation.
Transmission most likely occurs by virus being expelled into the air as droplets from the respiratory system. Virus gaining access to water bowls and pools of water may persist for considerable periods of time. Transmission of virus via the digestive tract through feces is also a possibility. Although transovarian or transuterine transmission has not been firmly established, this may also be involved in the spread of the virus.
The natural host for OPMV is unknown. Since rat snakes have been found to harbor a similar agent, possibly a non-viperid snake could be the source of infection. Although we have isolated this virus from recently imported snakes, there have been no isolates from snakes in the field. Snakes are an extremely mobile group of animals in the pet and zoo trade and this probably has accounted for this virus being introduced into many species of naive snakes.
Presumptive diagnosis of OPMV infection can be made upon finding characteristic light microscopic changes in lung. Since lesions in the lungs can be segmented, sections of cranial, mid, and caudal lung should be examined. Recently, an avidin-biotin immunoperoxidase staining technique (Figure 9) for demonstration of viral antigen in lung tissue has been used (Homer et al, 1995). An immunofluorescent technique also has been used (Figure 10). Since long term storage of tissue in 10% neutral buffered formalin seems to adversely affect staining of antigen, tissues should be transferred to 70% ethanol after 48 hours of fixation in formalins. Specific diagnosis will depend upon isolation of the virus in cell culture and ultrastructural characterization.
OMPV has been isolated in a wide variety of cell types of reptilian and mammalian origin including gecko embryo, rattlesnake fibroma, rattlesnake spleen, viper heart, and baby hamster kidney cells. We typically use either viper heart cells (Figure 11) or Vero cells. OPMV will result in giant cell formation in cultured cells (Figure 12). By electron microscopy, virus can be seen budding from the cytoplasmic membrane of infected cells (Figure13). Virus will also replicate in snake eggs and chicken eggs. Replication in vitro (and presumably in vivo) is temperature-dependent with the highest titers achieved at incubation temperatures of approximately 23°C to 32 °C. The upper temperature limit for virus replication was in all cases less than 37°C.
A hemagglutination-inhibition test (Figure 14) has been developed to determine the presence of specific antibodies to OPMV in plasma/sera of exposed snakes. Blood samples are easily obtainable by cardiac puncture. See submission of samples below.
As in mammals, a positive titer is simply indicative of exposure to OPMV. Based upon a single sample, it would be impossible to make a statement about presence of virus and shedding status. If 2 samples are obtained form the same animal at a 2-4 week interval, and a rising titer can be demonstrated, this would be supportive evidence for recent OPMV infection.
Submission of Samples
There is no specific treatment for snakes showing clinical signs of OPMV infection. Since most affected snakes die with severe gram-negative respiratory tract infections, treating ill snakes with appropriate antibiotics is indicated. 2 months should lapse following the last death from OPMV before introducing new animals. The aminoglycosides, gentamin and amikacin, in combination with a cephalosporin such as ceftazidime, are the drugs of choice. Cages of ill snakes should be cleaned and completely disinfected with a solution of 0.15% sodium hypochlorite. Chlorox is 5.25% sodium hypochlorite; a 1:33 dilution can be used. Cages should remain empty for at least 2 weeks before introduction new animals. Additionally, as a rule, new snakes should not be introduced into a colony of snakes in which there is active OPMV infection. Minimally, 2 months should lapse following the last death from OPMV before introducing new animals. Needless to say, ill snakes should be removed from the collection and placed in a quarantine room.
Currently, there is no vaccine available for protecting snakes against OPMV. We have recently developed a killed vaccine utilizing two different adjuvants and have completed a one-year study in naive western diamondback rattlesnakes. Although there was an antibody response to vaccination, the response was both variable and transient between the various experimental groups. Over the next year a modified live vaccine will be evaluated.
Current and Future Research
- Sequencing of conserved genes to look at phylogenetic relationships between various isolates.
- Transmission and pathogenesis studies.
- Development of an immunofluorescent assay.
- Comparison of viral proteins using Western Blots.
For Further Information Contact
Dr. Elliott Jacobson
Department of Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida
Gainesville, Florida 32610-0126
Submission of samples and contact info
2015 SW 16th AV, Bldg 1017, Room V2-238
Gainesville, FL 32608