Canine parvovirus detected from a serval (Leptailurus serval) in South Africa

Canine parvovirus first emerged in domestic dogs (Canis familiaris), most likely as a variant of the feline panleucopaenia virus. Relatively recently, canine parvovirus-2a and canine parvovirus-2b infections have been identified in both symptomatic and asymptomatic domestic cats, while canine parvovirus infections have also been demonstrated in wild felids. This report documents the first known case of canine parvovirus-2b detected in unvaccinated serval (Leptailurus serval) from South Africa. The serval presented with clinical signs of vomiting, anorexia and diarrhoea that responded to symptomatic treatment. Two weeks later, severe leucopaenia, thrombocytopenia and death occurred. Typical enteric histological lesions of parvovirus infection were not observed on histopathological examination of the small intestine; however, histological lesions consistent with septicaemia were present. Canine parvovirus was detected in formalin-fixed paraffin-embedded small intestine using polymerase chain reaction. Phylogenetic analysis of the sequence of the canine parvovirus viral capsid protein gene showed similarities between the sample from the serval and canine parvovirus-2b isolates from domestic dogs in Argentina and South Africa. A case of canine parvovirus-2b in a domestic dog from South Africa in 2012 that fell within the same clade as the serval sample appears distantly related because of the long branch length. The significance of these findings is explored. More extensive surveys of canine parvovirus in domestic and wild felids and canids are needed to understand the epidemiology of canine parvovirus in non-domestic felids in South Africa.


Introduction
Parvoviruses (family Parvoviridae) and their associated diseases affect various carnivores including felids and canids (Siegl et al. 1985). Feline panleucopaenia virus (FPLV) infection was one of the first viral diseases identified in domestic cats (Felis catus), during the 1930s and 1940s, and also infects non-domestic felids (Parrish 1990). At roughly the same time (~1947), parvoviral gastroenteritis was identified in farmed mink (Mustela vison), and within 15 years, it was observed on mink ranches throughout Canada, the United States, Europe and Scandinavia (Parrish 1990;Pearson & Gorham 1987); it is currently known as mink enteritis virus (MEV) (Barker & Parrish 2001).
During the late 1970s, canine parvovirus (CPV) first emerged in domestic dogs (Canis familiaris), most likely as a variant of FPLV or a closely related parvovirus. Although the exact mechanism of emergence is unclear, amplification of parvovirus DNA sequences intermediate between FPLV and CPV from red foxes (Vulpes vulpes) suggests that wildlife may have played a role in the adaptation of the virus in a new host (Parrish 1995;Truyen et al. 1996Truyen et al. , 1998. The original strain (CPV type 2) spread worldwide rapidly, shortly thereafter being replaced by type 2a and a few years later by type 2b. These two variants, however, differ very little from the original strain (Parrish 1995;Truyen et al. 1996). Strain CPV type 2c was diagnosed much later (~2000), from domestic dogs in Italy, and has been proven to induce disease in cats as well (Buonavoglia et al. 2001;Nakamura et al. 2001).
Host range seems to be complex, as even within affected families only certain genera or species have been reported to be susceptible (Barker & Parrish 2001). The natural hosts of CPV (dogs), FPLV (cats) and MEV (mink) have been identified; however, the natural host ranges of feline subgroup parvoviruses are poorly defined (Barker & Parrish 2001). Wild felids that have been reported to be susceptible to FPLV include cheetah (Acinonyx jubatus), Siberian tiger (Panthera tigris altaica), African wildcat (Felis lybica), puma (Puma concolor), ocelot (Leopardus pardalis) and spotted cat Canine parvovirus first emerged in domestic dogs (Canis familiaris), most likely as a variant of the feline panleucopaenia virus. Relatively recently, canine parvovirus-2a and canine parvovirus-2b infections have been identified in both symptomatic and asymptomatic domestic cats, while canine parvovirus infections have also been demonstrated in wild felids. This report documents the first known case of canine parvovirus-2b detected in unvaccinated serval (Leptailurus serval) from South Africa. The serval presented with clinical signs of vomiting, anorexia and diarrhoea that responded to symptomatic treatment. Two weeks later, severe leucopaenia, thrombocytopenia and death occurred. Typical enteric histological lesions of parvovirus infection were not observed on histopathological examination of the small intestine; however, histological lesions consistent with septicaemia were present. Canine parvovirus was detected in formalin-fixed paraffin-embedded small intestine using polymerase chain reaction. Phylogenetic analysis of the sequence of the canine parvovirus viral capsid protein gene showed similarities between the sample from the serval and canine parvovirus-2b isolates from domestic dogs in Argentina and South Africa. A case of canine parvovirus-2b in a domestic dog from South Africa in 2012 that fell within the same clade as the serval sample appears distantly related because of the long branch length. The significance of these findings is explored. More extensive surveys of canine parvovirus in domestic and wild felids and canids are needed to understand the epidemiology of canine parvovirus in non-domestic felids in South Africa.
Genetic material from FPLV has been detected in a serval (Leptailurus serval) with histological lesions typical for parvoviral disease and in another with salmonellosis (Lane et al. 2016). This case explores the significance of the detection of CPV-2b in a serval from South Africa with bacterial septicaemia.

Case presentation
A captive adult male serval that presented with inappetence, anorexia, vomiting and diarrhoea was hospitalised at a veterinary practice in Nelspruit, Mpumalanga. The animal reacted well to symptomatic treatment and regained its appetite. Fifteen days later, however, it presented with epistaxis, mild icterus, anaemia (haematocrit = 28), severe leucopaenia and thrombocytopenia (subjectively evident on a blood smear). Treatment was initiated but the animal died the following day. No evidence of warfarin or other anticoagulant exposure was reported. No further clinical tests or culture were performed, and no bone marrow was submitted for evaluation or further analysis.
A necropsy showed that the serval was in good condition with mild icterus, a slightly enlarged liver and mild fluid accumulation in the body cavities. The cause of death was suspected to be because of aspiration of blood from the epistaxis. Selected tissue samples preserved in 10% buffered formalin were processed routinely and stained with haematoxylin-eosin stain. The histopathological examination confirmed septicaemia (multifocal hepatic and pulmonary necrosis associated with fine bacterial bacilli, and fibrin deposition in the splenic red pulp sinuses). Hepatic macrophages contained bile pigment in the cytoplasm. The urinary bladder mucosa showed multifocal mucosal ulceration and haemorrhage with mild submucosal lymphoplasmacytic interstitial infiltration and fibroplasia. An unspecified lymph node was mildly active and cortical follicular structures contained central hyalinisation of the stroma and possible fibrin deposition. The small intestine did not reveal any enteric lesions including changes characteristic of parvovirusinduced necrosis.
The molecular diagnostics and phylogenetic analyses formed part of a larger project at the National Zoological Garden, South African National Biodiversity Institute (NZG, SANBI), which investigates the molecular identification and genetic diversity of FPLV in both wild and domestic felids (Lane et al. 2016 Molecular analysis of parvovirus from the isolate was achieved by amplification of a ~1400 bp region of the viral capsid protein (VP2) gene (Table 2) using DreamTaq™ Green PCR (polymerase chain reaction) master mix (ThermoFischer Scientific) and previously published primers (Horiuchi et al. 1996;Meers et al. 2007;Steinel et al. 2000;Wasieri et al. 2009). A standard polymerase chain reaction (PCR) setup was used consisting of 8.5 µL nuclease-free water (supplied with DreamTaq™), 12.5 µL DreamTaq master mix, 1 µL of each primer (Forward and Reverse; 10 pmol) and 2 µL DNA sample standardised to a concentration of 50 ng/µL. Cycling conditions for tissue isolates using primer sets 2 to 5 were as follows: initial denaturation (94 °C for 5 minutes), 30 cycles of denaturation (94 °C for 30 seconds), annealing (58 °C for 50 s) and extension (72 °C for 1 min). A final extension step (72 °C for 10 min) concluded the cycling. Cycling conditions for primer sets 1 and 6 were identical except annealing occurred at 54 °C. Conditions were adjusted for FFPE isolates by decreasing the annealing temperatures to 54 °C for primer sets 2 to 5, and 50 °C for primer sets 1 and 6. Polymerase chain reaction setup was performed in a DNA-free hood and a DNA negative control was included.
All positive amplicons were purified using an exonuclease I and alkaline phosphatase PCR purification protocol, sequenced using BigDye V3.1 (Applied Biosystems) chemistry and analysed on an ABI3500 genetic analyser. Sequences were aligned using the ClustalX function incorporated in MEGA6 (Tamura et al. 2011) and phylogenetically analysed using neighbour-joining (NJ) in MEGA6. The Tamura 3-parameter model with gamma distribution (T92+G; G = 0.12) was determined to be the best-fit model of sequence evolution under the Akaike Information Criterion (AIC) in jModeltest (Posada 2008

Ethical considerations
Ethical approval was obtained from the NZG Research Ethics and Scientific Committee (NZG/RES/P/16/10).   FIGURE 1: Evolutionary relationships of detected parvoviral taxa using trimmed nucleotide sequences and the neighbour-joining method. The tree was generated using the Tamura 3-parameter model with Gamma distribution and 1000 bootstrap replicates. Cases from the NZG wildlife disease database and the study by Lane et al. (2016) are prefixed with laboratory numbers (PV), vaccine strains are prefixed with the vaccine name, and reference strains collected from GenBank are prefixed with the accession number. Rooted with CPV as the out-group.

Results
Results from the NJ analysis (1000 bootstrap replicates) illustrate a clear separation between FPLV and CPV isolates, as indicated in Figure 1. Additionally, the CPV clade consists of four distinct clusters representing CPV-2, -2a, -2b and -2c. The serval isolate from this study (PV19) grouped with the CPV-2b cluster with a domestic dog sample from the NZG, SANBI database (PV35), and with previously reported NCBI reference sequences from domestic dogs in Argentina (Gallo Calderón et al. 2012) and South Africa (Dogonyaro et al. 2013). Isolate PV19, however, appears to be distantly related to these isolates as indicated by the long branch length. The Namibian domestic dog isolate (PV31) from our database grouped with the CPV-2c cluster on a separate branch. Domestic dog isolates PV36, PV38 and PV40 grouped with the CPV-2a cluster also on separate branches. The majority of the South African felid samples grouped with the FPLV Clade I, including a sample from a serval with salmonellosis (PV10) (Lane et al. 2016). The South African Clade II contained one serval with histological lesions of FPLV (PV15) with a seemingly unique viral strain (Lane et al. 2016) that is more similar to a Felocell vaccine strain than to Clade I FPLV.

Discussion
This study reports the first detection of CPV-2b in a captive serval from South Africa, while the study by Lane et al. (2016) previously detected FPLV in two serval isolates. Although CPV infection has previously been detected in wild felids, such as cheetah and Siberian tiger , as well as domestic cats, the CPV-2b strain detected in the serval corresponds more closely with those detected in domestic dogs from South Africa (Dogonyaro et al. 2013) and Argentina (Gallo Calderón et al. 2012).
The significance of the presence of CPV and FPLV in tissues from servals with septicaemia is uncertain. Parvovirus is known to cause lymphoid necrosis and atrophy in lymphoid organs such as the thymus, gut-associated lymphoid tissue and lymph nodes as well as intestinal epithelial necrosis. The resultant immunosuppression and breach of intestinal integrity make affected animals susceptible to secondary systemic bacterial infection. Typically, domestic dogs and cats that die as a result of septicaemia secondary to parvoviral infection show histological lesions of intestinal epithelial necrosis. Detection of FPLV in a serval (PV15) with typical intestinal necrosis suggests that the disease, in FPLV at least, may show a similar course in servals. However, as domestic cats have been shown to be asymptomatic carriers of CPV-2a, -2b and -2c (Buonavoglia et al. 2001;Clegg et al. 2012;Nakamura et al. 2001), we cannot rule out the possibility that servals may be asymptomatic carriers of both FPLV and CPV-2b. Alternatively, servals may at times show mild transient intestinal infections with either virus that result in immune suppression, secondary bacterial enteritis and septicaemia. This could possibly explain the 2-week period between the initial gastrointestinal signs and the terminal septicaemia in this case (PV19). It is unclear what role the damaged bladder mucosa played in this case; it may have been the route of bacterial infection.
The presence of CPV-2a and CPV-2b in the samples collected from domestic dogs from South Africa 2 years before the serval case indicates the existence of these strains in the domestic dog population in the country, and this may also indicate a likely source of infection for the serval (PV19). Information on the contact or not between domestic dogs and this serval was not available. Further investigation of samples PV36, PV38 and PV40 may reveal whether or not these isolates represent unique variants of CPV-2a. The case of CPV-2c isolated from a domestic dog sample collected in Namibia is most likely an artefact of shortening the sequences to the shortest sequence in the data set, as this virus variant has not been reported in Africa or Australia (Decaro et al. 2005;Dogonyaro et al. 2013;Meers et al. 2007;Sykes 2013). Additionally, the results of the NCBI website based Basic Local Alignment Search Tool (BLAST) which compares an input sequence to the GenBank database of sequences, identified the full length sequence of this virus as CPV-2b.
Canine parvoviruses have spread widely across the globe since the 1970s because of contact with infected animals, inanimate fomites, flies or other reservoirs (Bagshaw et al. 2014;Barker & Parrish 2001). Over the last 15 years (2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016), however, very little information about the genetic diversity of parvoviruses and possible variants in South African domestic and non-domestic carnivores has been published. The epidemiology of these viruses is therefore largely unknown, including the transmission between domestic and non-domestic species and whether or not vaccination with live virus in either group affects the epidemiology. Natural recombination has been reported in porcine, mink and rodent parvoviruses (Shackelton & Hoelzer 2007). Similarly, recombination between FPLV and CPV-2, CPV-2a and CPV-2c, as well as vaccine and field strains has been reported but the exact mechanism is unknown (Mochizuki et al. 2008;Ohshima & Mochizuki 2009;Pérez et al. 2014). Therefore, recombination remains an important factor to consider when studying the evolution and genetic diversity of parvoviruses. Extensive surveys of parvoviral strains present in wild South African carnivores, as well as domestic dogs and cats, will help determine the strain diversity, geographic and host species distribution as well as possible sources of infection. How this recombination mechanism affects vaccine efficacy is largely unknown. As it has been previously shown that domestic cats, cheetah, tiger and serval (at present) are susceptible to both CPV and FPLV, the revision of currently accepted vaccination strategies, which primarily involves vaccinating felids against FPLV only, is required.