Canine Parvovirus-2: An Emerging Threat to Young Pets

Abstract

Canine parvovirus-2 (CPV-2) is a highly contagious and key enteropathogen affecting the canine population around the globe by causing canine parvoviral enteritis (CPVE) and vomition. CPVE is one of the the leading causes of morbidity and mortality in puppies and young dogs. Over the years, five distinct antigenic variants of CPV-2, namely CPV-2a, CPV-2b, new CPV-2a, new CPV-2b, and CPV-2c, have emerged throughout the world. CPV-2 infects a diverse range of wild animals, and the newer variants of CPV-2 have expanded their host range to include felines. Despite the availability of highly specific diagnostics and efficacious vaccines, CPV-2 outbreaks have been reported globally due to the emergence of newer antigenic variants, expansion of the viral host range, and vaccination failures. The present chapter describes the latest information pertaining to virus properties and replication, disease manifestations in animals, and an additional recent updates on diagnostic, prevention and control strategies of CPV-2.

1. Introduction

Canine parvovirus (CPV-2) is a member of the Parvoviridae family, Parvovirinae subfamily, and Protoparvovirus genus. It causes severe, acute hemorrhagic gastroenteritis and myocarditis infection in dogs [1]. It is the most important enteric virus affecting domestic and wild carnivores throughout the globe [2]. CPV-2 is a non-enveloped virus with a single-stranded negative-sense DNA genome [3]. The genetic diversity of CPV-2 resulted in the emergence of 5 distinct antigenic variants such as CPV-2a, CPV-2b, new CPV-2a, new CPV-2b, and CPV-2c with amino acid differences mainly restricted to the capsid VP2 protein [4]. CPV-2 are ubiquitous and sturdy viruses that remain viable for more than one year in the favorable environment [5, 6] and are transmitted usually by the faeco-oral route [7, 8].

CPV-2 causes 100 percent morbidity and mortality rate of 10 percent and 91 percent in adult and young dogs respectively [9]. However, a mortality of 91 percent was reported in experimentally infected dogs that were not treated [10]. CPV-2 affects predominately the younger dogs between 6 weeks and 6 months [8] with an increased susceptibility to puppies less than 6 months. In dogs over the age of 6 months, sexually intact males are more likely (twice) to develop canine parvovirus enteritis (CPVE) in comparison to intact females [11]. The CPV-2 antibody titer transmitted to the newborn via absorbed colostral antibody is 50–60% of the mother’s titer. The half-life of paroviral maternal antibodies is around 10 days [12]. Therefore, puppies are highly susceptible to the CPV-2 infection as the maternal antibody titres start declining. CPVE affects dogs of all ages, although it is more severe in puppies. Puppies can succumb to shock and die within two days after being sick. The most striking symptom of CPV-2 myocarditis is the abrupt mortality in young puppies, generally around the age of 4 weeks [13].

In recent years, CPVE outbreaks caused by multiple CPV-2 variants have been recorded in diverse geographical locations throughout the world. Previously, CPV-2, which could not infect cats, has been replaced by CPV-2 variants that can now infect cats, suggesting that CPV-2 may be capable of spreading between species [14]. Since CPV-2 infects a wide range of wild animals in the order Carnivora, subclinical infection appears to be prevalent. As a result, significant CPV-2 reservoirs in wildlife appear to exist, and transmission of virus between domestic dogs and wildlife appears to be common and bidirectional [15]. Despite the availability of a wide range of immunoprophylactic and antiviral agents to control CPV-2 infections in dogs, many outbreaks have been reported throughout the world, and the disease has remained a major veterinary and economic concern due to the presence of unvaccinated dogs, intervention of active immunization by maternally derived antibodies, and the emergence of a different antigenic variants of CPV-2.

2. Virology of CPV-2

Canine parvovirus infection is caused by Carnivore protoparvovirus-1 which is characterized under the genus Protoparvovirus, family Parvoviridae. CPV-2 is a member of the Parvoviridae family, which includes two subfamilies: Parvovirinae (infects vertebrates) and Densovirinae (infects invertebrates) Parvovirus, Erythrovirus, Dependovirus, Amdovirus, Bocavirus, and other unclassified vertebrate parvoviruses are all the genus which comes under Parvovirinae (Figure 1) [16]. CPV-2 genome is a single stranded, negative sense, linear DNA of about 5 kb [17] contained by two ORFs translated into 4 proteins through alternative splicing [18, 19]. One ORF is associated with the non-structural proteins NS1 and NS2, which are mainly related to the viral replication and the second ORF is related with the viral capsid constituents VP1 and VP2. After the cleavage of VP2, VP3 is formed due to the involvement of host proteases. The capsid has 60 protein subunits, 90% of which are VP2 (67 kDa) and 10% are VP1 (83 kDa) [20].

Figure 1.

Schematic representation of Parvovirdae taxonomy.

The virus is nonenveloped having icosahedral symmetry and is 25 nm in diameter. The CPV virus is made up of the sixty protein subunits containing VP1 (5–6 units) and VP2 (54–55 units). The protein structure is made up of antiparallel β-barrel (8-stranded) capsid. The viral replication occurs inside the nucleus of multiplying cells and therefore the intranuclear inclusion bodies are formed during the infection. The viral capsid structure is made up of spike at the three-fold axes of the icosahedral unit, a 15-Å depression around the five-fold axes and two-fold axes is formed. Antigenic determinant regions have been plotted to the three-fold protrusion and the two-fold depression are related to the host cell features [17]. The surface of the capsid is composed of four loops inserted between the strands, resulting in spike-like protrusions around threefold axes of approximately 22 Å. The antigen neutralization site, also known as epitope A, is composed of loops 1 and 2 of one VP2 and loop 4 of a threefold related molecule [21]. The molecular weight (MW) is around 5.5 to 6.2 × 10⁶ Da. There is an equal ratio of protein to nucleic acid.

NS1 is the largest non structural protein in CPV-2, and it is primarily involved in viral replication and pathogenicity [22]. NS1 is a key mediator of cytotoxicity of CPV and can selectively cause tumor cell lysis by inducing an antitumor immune response in different tumor models [23]. A recent study demonstrated the amino acid residues of T598 and T601 in the C-terminal phosphorylation sites of NS1 protein, involved in replication and pathogenicity of CPV-2 [24].

In the 1970s, CPV-2 emerged as a novel pathogen in dogs. Since then, CPVE has been reported across all the continents [25, 26]. Other related viruses such as Feline panleukopenia virus (FPV), Mink enteritis virus (MEV), Raccoon parvovirus (RPV) are closely related to the CPV-2 [27]. Mutations in the canine transferrin receptor (TfR) type-1 lead to adaptation of CPV-2 in different species 2 [28, 29]. There is more than 98% genome homology reported in the CPV and FPV nonetheless infect different species and have typical antigenic capsid and haemagglutination (HA) properties [28, 30]. The mutations in different amino acid positions have led to the effective adaptation in the new hosts [30]. There are over five to six mutations in the VP2 residue of the CPV-2 and FPV and also 375 and 323 amino acid position regulates the pH functionality of HA [31, 32]. CPV-2a (Asn CPV-2a) replaced CPV 2 in 1980s in the USA and various European countries. CPV 2a can infect the cats which was not a feature of CPV 2. CPV 2a has been displaced by the CPV-2b (426Asp) which was first reported in USA in 1982 and CPV-2c (426Glu) variant in Italy [31, 33]. Although two variants, CPV-2a and 2b had been identified much earlier, however, the third variant CPV-2c had been recognized in early 2000 [33]. Thereafter it has been reported frequently from many different countries. In addition, new CPV-2a and new CPV-2b have also been documented due to non-synonymous substitution at 297 residues (Ser to Ala) of VP2 protein [34]. In India, CPV-2a has recently become the most prevailing antigenic type among all variants. Recent emergence of new antigenic variants that differ significantly from the current vaccine strains is a matter of concern for efficacy of vaccine [35].

3. Canine parvovirus enteritis (CPVE)

The CPV is of two types: CPV-1, commonly known as minute virus of canine and accountable for gastrointestinal and respiratory infection of dogs whereas, CPV-2, most pathogenic type and is responsible for severe gastroenteritis/hemorrhagic gastroenteritis, in young puppies as well as adult dogs.

It is quite difficult to distinguish the clinical diseases caused by CPV-2 variants owing to its overlapping nature of signs and symptoms. These variants are believed to produce similar pathogenicity however; some studies showed that severity of clinical manifestations is influenced by variants of CPV-2 based on clinical, hematological, serological and histopathological examinations [36, 37].

Although puppies under 6 months of age are highly susceptible, adult dogs with insufficient immunity are also considered as high risk to the CPVE. CPV-2 can persist in the environment for more than a year, enabling susceptible dogs to pick up infection from CVP-2 contaminated feces, vomitus, or fomites. Although the feco-oral route is considered as primary path of disease transmission, infection through the oro-nasal route is also common in naive or under-immunized dogs due to ingestion of viruses shed in the vomitus or feces of CPV-2-infected animals [38]. However, direct contact or environmental contamination may also play a role [39]. Breed predisposition and seasonal prevalence of the disease are subject to considerable variations in wide geographical areas [40, 41].

Doberman, Rottweiler, and German shepherd (GS) dogs have been reported to be more susceptible to CPVE than other breeds [42]. Due to inherited immunodeficiency, the exotic breeds, German Sphered and Doberman, are more susceptible than the other breeds [43]. German shepherd has the highest CPV infection rate (70%) followed by the Doberman (55%) [44]. A cytokine bioassay revealed that the magnitude of TNF-α production by peripheral blood monocytes was greatest in dogs with a breed-related risk for CPVE. When compared to mixed breeds, highly susceptible breeds such as Rottweiler and Doberman Pinscher produce more TNF-α in response to LPS stimulation [45]. Increased TNF activity is predictive of mortality in naturally occurring CPVE infection in veterinary medicine [46]. Therefore, it has been hypothesized that dogs with a breed-related risk of developing CPVE, a disease associated with sepsis, would have a greater pro-inflammatory cytokine response to endotoxin [45].

The incubation period of CPV-2 infection ranges from 4 to 14 days. The infected dogs start to shed virus few days prior to the visible clinical signs and shedding of virus gradually declines 3–4 weeks postexposure [47]. Following entry into the body, the CPV-2 rapidly multiply in oropharyngeal lymph node, thymus and mesenteric lymph node, resulting in viremia within one week of exposure. After that, the virus attacks rapidly multiplying cells of crypts of intestine, epithelium of the tongue, oral cavity, bone marrow, and cardiac myocytes, besides lung, spleen, liver, and kidneys [48]. The key pathogenic event in CPV-2 infection is the virus-induced destruction of enterocyte, leading to mucosal barrier disruption, and villous atrophy. This causes profuse vomiting and hemorrhagic diarrhea, nutrient malabsorption, dehydration/hypovolemia, metabolic acidosis and/or alkalosis. The disruption of mucosal barrier allows bacterial translocation from intestinal compartment to systemic circulation, resulting in septicemia, endotoxemia, systemic inflammatory response syndrome as well as hypercoagulability [49]. The CPV-2 infection in the thymus and bone marrow precursor cells results in loss of thymic cortex and profound leucopenia, respectively [48]. Death may occur due to multi-organ failure when the affected dogs remain unattended [40, 49]. Previously, myocarditis was thought to be the acute cause of death in young puppies however, this form nowadays occurs rarely because of widespread CPV vaccination of dogs. The concurrent infections with parasitic, virus, or bacterial intestinal pathogens or stressors may aggravate the disease [50, 51, 52].

The degree of clinical manifestations may vary with age, breed, and immune status, duration of illness and virulence of virus. The clinical signs of dogs with CPV infection are nonspecific in nature and resembles to gastritis and enteritis. The most notable clinical signs of CPVE are lethargy, depression, weakness, lack of appetence, bouts of vomiting, and diarrhea. The diarrhea is characterized by foul-smell and mucoid to purely hemorrhagic because slugging of intestinal mucosa and bleeding. The excessive loss of fluid during vomiting and diarrhea causes marked dehydration that results in development of hypovolemic shock. Occasionally, intussusception occurs due to intestinal dysmotility. Neurologic signs in puppies with CPVE may result from hypoxia secondary to myocarditis, hypoglycemia, or intracranial thrombosis or hemorrhages [52]. The bacterial translocation from intestine to systemic circulation can cause fever, systemic inflammatory response syndrome and septic shock with hypotension and organ failure [40, 48]. Apart from diarrhea, respiratory distress, pulmonary congestion and edema, alveolar and bronchiolar hemorrhage and convulsions are also occasionally manifested due to hypovolemia, endotoxic and septicemic shock [8, 53]. The malabsorbtion of nutrients and inadequate storage of glycogen in muscle and liver result in hypoglycemic encephalopathy which leads to seizures. On hospital admission, the prognosis is poor in CPVE dogs with intussusception, systemic inflammatory response syndrome and severe leucopenia.

4. Diagnostic approaches

4.1 Virus isolation

Virus isolation is considered as a gold standard for any viral disease diagnosis. In case of CPV-2 different cell lines like CRFK (Crandell Rees feline kidney), MDCK (Madin-Darby canine kidney) and A-72 are used for the isolation and propagation of the virus. The adapted virus causes distinct cytopathic effect in infected cell lines as cell rounding, aggregation, and necrosis of the affected cells. This requires the presence of special laboratory and is laborious [54].

4.2 Electron microscopy

It is an expensive technique for the detection of the virions by negative staining in the stool samples or culture isolated virus. Immunoelectron microscopy can also be done by using CPV-specific antibodies. The need of expensive electron microscope makes it out of reach for regular usage [55].

4.3 Haemagglutination test (HA)

The property of the CPV to cause agglutination of the pig, cat or rhesus monkey red blood cells at 4°C is used for detection of the CPV. The reciprocal of the maximum dilution of virus exhibiting ample agglutination of erythrocytes (mat formation) is designated as HA titer. The HA titer of more than 1:32 is usually considered as specific for CPV-2 [56].

4.4 Counter-immunoelectrophoresis (CIEP)

The use of electric current allows the rapid movement of antigen and antibody towards each other resulting into the formation of precipitation line quicker than simple diffusion reaction. This technique is not commonly used but have been utilized for the prevalence of CPV infection in clinically suspected dogs [57].

4.5 Fluorescent antibody test (FAT)

In this test, an antibody tagged with fluorescent dye is employed for detection of specific CPV antigen. Mostly it is used as direct FAT for the diagnosis of CPVE but is not used routinely for diagnostic purpose [58].

4.6 Latex agglutination test (LAT)

This is a commonly used test utilizing antigen–antibody interactions employing specific antigen or antibody and is mostly useful under the field conditions. Here, the property of agglutination of polystyrene beads coated with either specific antigen or antibody on their surface is used with anti-CPV monoclonal and polyclonal antibody to detect CPV-2 in the stool samples. Earlier it has been used for both qualitative and quantitative evaluation of CPV in suspected dog feces. Also, a recombinant VP2 protein-based LAT for determination of immune status in dogs against CPV-2. Besides LAT, a slide agglutination inhibition test has been used to detect the presence of CPV-specific antibodies by utilizing the agglutination property of CPV-2 [59].

4.7 Slide inhibition test-slide agglutination test (SIT-SAT)

This method is developed for the detection of CPV-2. SIT is an antibody typing system based on the ability of viral antibodies to bind with the virus and prevents the virus from binding to RBC. SAT is used for antigen detection by serially diluting the clinical sample and then incubating it with a fixed amount of RBC containing virus surface receptors. The virus particles in the sample bind to the RBC and form a lattice that can be seen visually [60].

4.8 Enzyme-linked immunosorbent assay (ELISA)

It is an enzyme-based immunoassay involving antigen–antibody interactions to screen a large number of samples at a time. Recombinant VP2 protein-based indirect ELISAs has been developed to detect and quantify antibodies against CPV-2. Novel polyclonal antibody-based antigen capture ELISA using rabbit anti-CPV hyperimmune sera as capture antibody and guinea pig anti-CPV hyperimmune sera as detector antibody has been also developed. IgY-based ELISA comprising of the chicken egg yolk-derived has been developed for the detection of both antigen and antibodies. Different commercial ELISA kits are currently available for CPV-2 antigen and antibody detection [61].

4.9 Immunochromatographic (IC) assays

IC assays or Lateral flow assays are strip-based devices utilized for the detection of a target analyte in test samples. Colloidal gold nanoparticles are commonly used in synthesis of the probe (conjugate) in majority of these strip-based points of care assays. Different components used are the sample pad, conjugate pad, nitrocellulose membrane, absorbent pad and a plastic cassette. These tests are now used routinely for the parvovirus diagnosis in affected dogs. A number of lateral flow assay-based commercial kits are available for rapid detection of both CPV-2 antigen in feces and antibodies in serum, which are also available in the market. These are helpful in the field and gives rapid results within 10–15 mins. Recombinant VP2 protein based immunochromatography tests has also been developed based on the rapid detection of CPV-2 [62].

4.10 Dot blot/dot-ELISA

It is an immunological test which uses charging of test antigen on to a nitrocellulose or PVDF membrane followed by detection using specific antibody against the antigen and an enzyme labeled secondary antibody which forms a color on addition of an insoluble substrate. It is helpful as on the spot assay for CPV diagnoses. It has been developed for detection of CPV-2 using hyperimmune sera raised against the whole virus/recombinant VP2 protein. Commercial dot ELISA kits are also available for evaluating IgM response against CPV-2 after vaccination or infection [63].

4.11 Polymerase chain reaction (PCR)

PCR is a molecular diagnostic assay which is used for the detection of viral nucleic acid and is relatively more sensitive than other conventional tests. Diverse antigenic types of the CPV can be distinguished by employing strain-specific primer or nested PCR or restriction enzyme analysis of the PCR. Also strain differentiation may be carried out with the help of oligonucleotide sequencing of the amplified gene [64].

4.12 Nucleic acid hybridization/dot blot

This has also been reported for the detection of CPV nucleic acid. Here hybridization with CPV-specific biotin or radiolabelled probe is carried out onto the CPV nucleic acid charged nitrocellulose paper or nylon membrane from suspected samples and then formation of color and band in the radiograph indicates the presence of the virus [65].

4.13 In situ hybridization assay

It uses an isotopic-labeled probe for both the detection and tracking of CPV nucleic acid in affected morbid tissue specimens thus,using more incubation time for development of the positive reaction [66].

4.14 Real-time polymerase chain reaction (qPCR)

This technique can be employed to quantitate CPV-2 in samples using either TaqMan probe technology or SYBR Green method. It is used for strain differentiation of concurrent infection using Multiplex Real-time PCR; and also, to differentiate vaccine strain from wild CPV strains. Different multiplex assays real-time PCR has been validated for the presence of CPV, FPV and PPV [67].

4.15 Amplification refractory mutation system PCR (ARMS-PCR)

It is used for the detection and typing of the known point mutations/single nucleotide polymorphism based on variable size of PCR-amplified products specific to a particular allele. In this PCR basically 2 pairs of primers are used (2 inner and 2 outer specific primers matching to individual allele type) in a single PCR tube and there are no post-PCR protocols used as restriction enzyme digestion (PCR-RFLP) and sequencing therefore they provide an economical confirmation. ARMS-PCR is a well-known technique frequently employed for phenotypic association and single nucleotide polymorphism (SNP) studies. This has been used for CPV detection and its antigenic typing [54].

4.16 Peptide nucleic acid-based (PNA) Array

It contains a stable electrically neutral peptide backbone and the PNA-DNA hybridization assay are relatively more sensitive and specific than TaqMan-based real-time PCR for CPV differentiation [68].

4.17 Loop-mediated isothermal amplification assay (LAMP assay)

The assay is a sensitive and rapid technique used for amplification of DNA and thereby pathogen detection in an hour by using the DNA polymerase by autocycling strand displacement action by boiling at persistent temperature (60–65°C) in water bath. Usually, 2 sets of primers bind to 4 to 6 different regions of target viral DNA. LAMP has field application as there is no need for any thermocycler to carry out the target gene amplification. The amplification of VP2 gene of CPV-2 by LAMP assay has been developed. LAMP assay along with lateral flow dipstick (LFD) and LAMP-ELISA are also used for CPV DNA detection [69].

4.18 Insulated isothermal PCR method

It is a convection-based method using a hydrolysis probe for detection of CPV-2 and its antigenic variants. The reaction mixture is sequentially allowed to pass in an automatic manner through variable temperature zones in a capillary tube which undergoes thermocyclic phase to amplify the DNA and the probe hydrolysis produces optical output providing the result within an hour [70].

4.19 Polymerase spiral reaction (PSR)

This technique makes use of both conventional PCR and isothermal amplification as in LAMP and is completed within one and a half hour. Here mostly an exogenous sequence from an unrelated species or of botanical origin is incorporated at the 5′ end into the primer sequences used in PSR if a human or veterinary pathogen is targeted. PSR has been successfully used to detect all CPV antigenic variants with ten-fold higher sensitivity than traditional PCR [71].

4.20 Fluorescence melting curve analysis (FMCA)

It is a probe-based assay that uses melting curve analysis to detect and differentiate between CPV-2 variants. This assay consists of 2 TaqMan probes namely FAM labeled and HEX labeled. The FAM-labeled probe sequence is perfectly complementary to CPV-2a, with a 1 bp mismatch to CPV-2b and a 2 bp mismatch to CPV-2c. The HEX-labeled probe has complete complementarity with the original CPV-2 and a 1-bp mismatch with the other variants. This method is also capable of detecting samples containing more than one variant without sequencing [72].

4.21 DNA aptamers

Aptamers emerged as a good alternative to antibodies as affinity reagents. Recently, ssDNA aptamers that specifically bind with the recombinant VP2 (rVP2) protein of CPV-2 with affinity in the nanomolar range have been reported. The ssDNA aptamers specific to CPV-2 (rVP-2) were selected by the Systematic evolution of ligands through exponential enrichment (SELEX) method and their target binding was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA). Aptamers with high binding affinity and specificity against rVP-2 could be employed in diagnostics for rapid detection of CPV-2 [73].

4.22 Nucleotide sequencing

It is primarily used for most viral genome identification and confirmation. Thus, considered as a gold standard for the antigenic typing of CPV variants. The amplified PCR product is either directly sequenced or cloned which is sequenced in a sequencer utilizing apt primers. The sequence data is analyzed using the appropriate bioinformatics database. Either nucleotide or amino acid sequence data or even both could be employed to recognize the evolutionary analysis of CPV-2 isolates from different geographical sites [74].

4.23 Biosensor

It is an analytical device which detects the DNA/RNA/protein/enzymes and alters it to the detectable electrical signals. A biosensor for CPV detection has been established by means of quartz crystal microbalance biosensor and ProLinker B [75]. Summary of different types of diagnostic assays are listed in the Table 1.

Diagnosis	Specimen	Diagnostic assay used	Feature	Remarks
CPV antigen	Feces or rectal swab	ELISA	High specificity Low sensitivity	Feces or rectal swab
		Haemagglutination assay	Low-cost and rapid.	Sensitivity and specificity vary
Tissues or morbid samples	Necropsy specimens	Histopathology	Different histopathological techniques and IHC may be used.	Differential diagnosis with other enteric infections
Viral DNA	Feces or rectal swab or any tissue	Polymerase chain reaction (PCR);qPCR	Efficient in diagnosing even minute amount of viral genome, can be quantified; Antigenic typing	Sensitivity and specificity vary. Vaccine virus shedding occurs upto weeks after immunization leading to false positives results. Inhibitory components may lead to false negative results.
Virus	Feces or rectal swab or any tissue	Virus isolation	Confirmatory diagnosis	Requires special facility
Virus particles	Feces or rectal swab or any tissue	Electron microscopy	Confirmatory diagnosis	Requires special facility, expensive

Table 1.

Summary of the different types of diagnostic assays for CPVE diagnosis.

4.24 Commercially available kits

Commercially available kits are mostly based on antigen–antibody reactions, such as ELISA, dot ELISA, and immunochromatographic strip-based assays (Table 2).

Sl. No.	Test	Company	Principle	Reference
1	SNAP parvo antigen test	IDEXX, United States	ELISA	[76]
2.	Rapid Immunochromatographic (IC) strip test	ADDBIO, Korea	Immunochromatography test	[43, 77, 78]
3.	Witness Parvo Test Kit	Zoetis, United states	Rapid Immuno Migration (RIM™) technology.	[79]
4.	Fassisi® Parvo	Fassisi, Gottingen, Germany	Lateral flow immunoassays	[80]
5.	FASTest parvo card	Vet lab, UK	Lateral flow immunoassays	[55]
6.	4 CPV Antigen Rapid Test Kit	Ubio Biotechnology systems Pvt. Ltd., India	Lateral flow immunoassays	[79]
7.	Anigen Rapid CPV Ag Test Kit®	Bionote, Dongtan, South Korea	Lateral flow immunoassays	[80]
8.	ImmunoRun CPV antigen detection kit	Biogal- Galed labs, Israel	Immunochromatographic assay	[79]
9.	Primagnost® Parvo H + K	Dechra, Aulendorf, Germany	Lateral flow immunoassays	[80]
10.	Canine Parvovirus & Distemper IgMAntibody Test Kit	Biogal Galed Laboratories Acs Ltd., Israel	Immunocomb	[79]
11.	Vetexpert Rapid Test CPV Ag®	Vetexpert, Vienna, Austria	Lateral flow immunoassays	[80]

Table 2.

List of commericially available kits for CPV-2 detection.

5. Treatment of CPV-2 infection

5.1 Recent advances in therapeutic management of CPVE

In absence of effective and appropriate antiviral drugs, the most universal therapeutic regimen for CPVE is supportive and symptomatic care until vomiting and diarrhea have resolved. Because of long-term illness of CPVE infected dogs, the challenges faced by the pet owners are cost of treatment and hospitalization. In private practice settings, the treatment cost may be huge, indicating that financial constraints may be a factor in disease-related euthanasia [81]. Therefore, fatality of CPVE is documented more in socioeconomically underprivileged areas, where level of education and financial opportunity for care and vaccination are not adequate [82]. Although the survival rate of CPVE in hospitalized and outpatient dogs is debatable, a recent prospective, randomized trial found no significant differences in survival (90% vs. 80%, P = 0.66) or duration of hospitalization (4.6d vs. 3.8d, P = 0.20) between inpatient and outpatient dogs [83]. However, given the possible risks of long-term hypoglycemia and leukopenia, aspiration pneumonia, edema, and intussusception in CPVE dogs, hospitalization appears to be the better option over outpatient treatment [84].

The principal components of supportive and symptomatic therapy include 1) fluid therapy and oncotic support, 2) antibiotics, 3) antiemetics, and 4) nutritional support. A wide range of other treatment measures including, though not limited to, antiviral treatments and pain management have been assessed in the past or are currently under investigation regarding their potential utility in CPVE.

5.2 Management of fluid and electrolyte imbalance and oncotic support

The development of severe hypovolemia is the first impact of pathophysiology in dogs with CPVE, hence re-establishment of the circulating volume is the utmost need [85]. The hypokalemia, hypochloremic metabolic alkalosis, hypoglycemia, hypoproteinemia and loss of oncotic pressure in circulation are the major fluid and electrolyte abnormalities during episode of diarrhea and vomition in acute CPVE [86]. The most aggressive therapies consisting of administration of intravenous (IV) fluids to restore intravascular fluid volume status, replenish interstitial fluid losses, maintenance of hydration and oncotic support. A balanced isotonic crystalloid solution (eg, Lactated Ringers) should be used for initial restoration of intravascular volume and rehydration, with a rate titrated to improve perfusion parameters such as capillary refill time, mucosal color, pulse character, and mean arterial pressure or lactate concentrations. Apart from fluid administration, potassium need to be supplemented in hypokalemic patients whereas, 25% dextrose at the dose rate of 1-2 mL/Kg body weight followed by addition of 2.5–5% dextrose in the crystalloid fluids will be required for hypoglycemic patients with blood glucose level < 60 mg/dL. Initially, the fluid is administered at the dose rate of 80–90 mL/kg with a boluses of 15–20 mL/kg over 15–20 minutes to counter the hypovolemic shock and, to improve the fluid perfusion. After that, the maintenance dose for daily fluid depends on the body weight (kg) and percent of dehydration. The volume (L) required to correct the daily fluid loss is calculated as body weight (Kg) × % dehydration. Generally, 40–60 mL fluid for each kg body weight is considered as ideal maintenance dose. Since fluid absorption through subcutaneous route is impaired in hypovolemic patients, intravenous access is considered as choice of fluid treatment. However, intraosseous or jugular catheter are considered as appropriate option in severe hypovolemic or interstitially dehydrated patients [87].

In CPVE, protein loosing enteropathy attributes to pronounced hypoalbuminemia (<2 g/dL) and/or hypoproteinemia (<4 g/dL) resulting in peripheral edema, pleural or abdominal effusions [88]. In that case, provision of oncotic support in the form of either natural or synthetic colloids are very important to minimize the morbidity and mortality of patients [89]. For correction of hypoalbuminemia, fresh plasma (20 mL/kg) or fresh-frozen plasma (6.6–11 mL/kg IV or 3 doses administered intraperitoneally 12 hours apart) and canine-specific albumin concentrate are used [90]. The concentrated human albumin products can also be used but the risk of immune reaction is the major limitation. If further oncotic support is required, hydroxyethyl starch (20–30 mL/kg/d) can be given, depending on clinician choice [6]. Sometimes, administrations of whole blood (20 mL/kg, within 4 hours) or packed RBCs are needed in severe anemic dogs with CPVE.

5.3 Antiemetic treatment

Apart from fluid and electrolyte imbalance, emesis is another clinical manifestation in CPVE. So, antiemetic treatment is warranted in CPVE otherwise persistent vomition may enhance the duration of hospital stay and further aggravates the condition of patient. The clinical efficacy of number of antiemetics in CPVE had been investigated with varying degree of results. The earlier studies showed that metoclopramide, a dopaminergic antagonist, was found to be effective in reducing episode of vomition by exerting a prokinetic effect in the upper intestinal tract and blocking the chemoreceptor trigger zone when administered as a bolus or as a constant-rate infusion in dogs. The ondasetron or dolasetron, the serotonin receptor antagonists, are also found effective in reducing the number of vomiting events [85]. Recently, a substantial antiemetic effect of maropitant, an antagonist of neurokinin1 receptors, by stimulation of either central or peripheral emetic pathways has been reported in dogs however, the efficacy of maropitant in CPVE has yet to be thoroughly investigated [91]. The administration of maropitant once daily, singly or in combination with metoclopramide, is very effective in reducing vomition in CPVE [5].

5.4 Antimicrobial treatment

Translocation of bacteria from intestinal compartment to systemic circulation is very common in CPVE because of villous collapse and disruption of the mucosal barrier. The translocation with concurrent marked neutropenia leads to a high risk of septicemia and endotoxemia. Additionally, hypotension from fluid loss and sepsis make dogs with CPVE at high risk of developing acute kidney injury. Therefore, parenteral administration of broad-spectrum bactericidal antibiotics is necessary in dogs with CPVE. Ampicillin and cefoxitin as single-agent treatments or in combination with enrofloxacin are the choice antimicrobials against Gram-positive and negative bacteria [85]. Aminoglycosides may also be considered in well-hydrated animals otherwise it may be avoided due to its inherent risk of nephrotoxicity. Puppies with CPVE often have comorbidities, including gastrointestinal parasitism. Hence, antiparasite therapy should be initiated once the puppy can tolerate oral therapies [6].

5.5 Nutritional support and pain management

Restoration of early mucosal integrity and prevention of bacterial translocation from gut compartment to systemic circulation are very important for faster recovery of dogs with CPVE. Enteral feeding is reported to improve the mucosal integrity and faster repair, resulting in lower possibilities for bacterial translocation [8]. In earlier study, it was demonstrated that early enteral nutrition via nasoesophageal catheter starting 12 hours post-admission led to clinical improvement, significant weight gain, and improved gut barrier function was more early as compared to withholding of the traditional food until cessation of vomiting for 12 hours [92].

Severe vomition, enteritis, and or concurrent intussusception in CPVE are the possible reasons for abdominal pain. Hence, analgesic treatment to reduce visceral pain is one the important aspect in therapeutic management in CPVE. Partial mu-agonists such as buprenorphine (0.01–0.02 mg/kg IV every 8 hours) or an agonist–antagonist such as butorphanol (0.1–0.2 mg/kg/h) are the preferred analgesics over the pure mu agonists as opioid analgesics can promote ileus and vomiting. The α-2 agonists that promote extreme vasoconstriction and limit gastrointestinal perfusion, and non-steroidal anti-inflammatory drugs that impair gastrointestinal and renal perfusion, both are not indicated [93].

5.6 Antiviral drugs

Like other viral infections, prophylaxis is the cornerstone for prevention of CPV in dogs. Although, an adequate number of killed and live CPV vaccines are marketed by pharmaceuticals but vaccines sometimes fail to protect completely due to poorly responding breeds (Rottweilers and Doberman pinschers), variation in genetic makeup of field and vaccine viruses, interference by presence of maternal antibodies and adjunct factors [94]. Therefore, development of some suitable antiviral drugs is utmost important for effective management of the CPVE in its acute illness stage. Till now, only few antiviral drugs have been evaluated for its clinical efficacy against CPVE. In an earlier placebo-control study, the therapeutic efficacy of Oseltamivir, a neuraminidase inhibitor, in CPVE had been evaluated and noted that Oseltamivir did not produce any additional benefit in terms of reduction of mortality or duration of hospitalization except some improvements in body weight and hemogram in dogs with CPV-illness [95]. In another study on naturally infected dogs, a promising anti-CPV activity of recombinant feline interferon-ω (rFeIFN-ω) has been recorded as compared to placebo-group. The intravenous administration of rFeIFN-ω at the dose rate of 2.5 mU/kg daily for consecutive three days remarkably reduced the clinical symptoms and mortality [96, 97]. Although the drug is currently available for use in Europe and Australia, the high price and frequent non-availability are major limitations. Recently, another antivital drug, Acyclovir, guanine analogue commonly used to treat herpes simplex virus infection, have been shown to improve the disease conditions [98]. Further, an in-vitro study on A72 cell line showed that 9-(2- hydroxyethomethyl) guanine phosphoromonomorpholidate (ACV PMMPD), a phosphorimidate analogue of acyclovir inhibits CPV-2 replication with exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range (50 μM) [99]. Recently, broad-spectrum anti-CPV activity of some anti-parasitic drugs such as Nitazoxanide, Closantel Sodium, and Closantel have also been shown using F81 cells [100].

5.7 Passive immunotherapy

Passive immunization with specific antibodies against enteric viral infections in animals confers significant protection, reduces diarrhea and virus shedding and increase survival rates [101]. Thus, the of immunotherapeutics in viral infections is promising treatment approach because of lower adverse effects as well as no chance of any resistance as in antiviral drugs. The passive immunization by means of oral or intravenous administration of IgY specific for CPV-2 shows the protective effect in dogs challenged with the virus [102]. The reduction of clinical scores, duration of symptoms and mortality and improvement of body weight gain has been reported by anti-CPV-2 IgY therapy in experimentally produced CPVE [103]. Recent study reported that chicken IgY- single chain fragment variables (scFv) generated against the virus capsid protein could be a promising therapeutic target against CPV [104, 105]. Aside from IgY, the neutralization of CPV by anti-feline panleukopenia virus antibodies is also reported from an in-vitro study [106]. However, the prospective, randomized, placebo-controlled, double-blinded study on CPVE did not produce substantial benefits when compared with placebo group [86]. Apart from above therapeutics, plasma therapy is also another option. Although the administration of CPV-hyperimmune plasma is reported to decrease the clinical signs and improve the survival rate in dogs under experimental conditions, however the findings remain inconclusive in natural cases [107]. Another study showed that lyophilized IgG treatment reduced clinical signs and duration of hospitalization of dogs naturally infected with CPV [108].

5.8 Immunomodulators

The key physiopathological alterations of CPVE are destruction of intestinal crypts, neutropenia, secondary bacterial translocation, immunosuppression due to thymus atrophy, sepsis and systemic inflammatory response syndrome in puppies [6, 109]. Therefore, immunomodulators could be an option to enhance therapeutic efficacy of supportive treatment. A recent study demonstrated that subcutaneous administration of human dialyzable leukocyte extract-h (hDLE) along with supportive therapy in puppies with CPVE significantly increased the leukogram and reduced the clinical score, duration of hospitalization, mortality as compared to supportive therapy alone [110].

5.9 Cytokines based therapeutics

5.9.1 Granulocyte colony-stimulating factor

Leukopenia is one of the most important prognostic indicators of mortality in dogs with CPVE. Hence, stimulation of bone marrow and improvement of leukogram in peripheral circulation are considered as strategic approaches to reduce the CPVE associated mortality. Enhancement of endogenous canine G-CSF (cG-CSF) concentrations by exogenous administration of human G-CSF (hGCSF) and cG-CSF is reported to stimulate bone marrow, resulting in improvement of neutrophil counts in puppies with CPV infection [111]. However, the use of hG-CSF and cG-CSF may not necessarily improve survival [112, 113].

5.9.2 Interferons and biological response modifiers

The interferon (IFN)-ω, a type I IFN (similar to IFN-α), is known for its antiviral, anti-proliferation, and antitumor activities. A notable therapeutic effect of rIFN-ω on CPV-infected dogs is reported [114]. Additionally, the promising therapeutic potential of other type I (IFN-α, IFN-β, IFN-ε, and IFN-κ) and III (IFN-λ) IFNs in CPVE has also been reported [115].

Recently, anti-CPV activity of the serum derived transfer factors (TFs), low molecular weight (<5000 daltons) biological response modifiers has been documented. It imparts therapeutic benefit in CPVE by altering the cytokine response of the host [116].

5.10 Probiotics

Probiotics, primarily comprised of live microorganisms in fermented foods, protect gut from acute diarrhea through adherence and colonization on gut mucosa [117]. Therapeutic efficacy of probiotics has been verified in dogs with CPV associated illnesses [118]. In an earlier study, oral administration of probiotic preparations as an adjunct therapy to young dogs with CPVE has shown faster resolution of clinical signs, improved leukogram and decreased mortality as compared to supportive treatment alone [119]; whereas, no benefit with respect to length of hospital stay or case fatality was recorded in other study [120].

5.11 Antioxidants

The disturbance in oxidant/antioxidant equilibrium is evident in CPV-gastroenteritis and oxidative stress is believed to link with pathogenesis of CPVE [121]. Hence, addition of antioxidants in supportive therapy has emerged as a promising therapeutic option to improve the response of treatment in viral diseases. Treatment with N-acetylcysteine (NAC), a precursor to glutathione and the body’s primary cellular antioxidant, along with supportive therapy markedly improved the leukogram in dogs with CPVE when compared with supportive therapy alone [122].

5.12 Herbals

An interest in natural products including herbs, plants and their extracts/metabolites as antiviral drug candidates has increased in the last few decades especially due to rising emergence of antimicrobial resistance globally and potential side-effects of many antimicrobials [123]. Very recently, anti-parvoviral activity of propolis, a traditional Chinese medicine, prepared from honeybee hives has been documented [124]. The in vitro study on PK-15 cells showed that ferulic acid (FA), an important component of propolis attenuates the replication of porcine parvovirus by blocking proapoptotic factors (Bid, Bcl-2 and Mcl-1), and inhibiting the mitochondria-mediated response by hindering the activation of the Bid-related signaling pathway. The FA may serve as potential antiviral against CPV [124].

5.13 Fecal microbiota transplantation

Alteration in the gut microbiome is reported in enteric viral diseases including CPVE and other gastrointestinal diseases in dogs [125]. The disruption of gut microbiota leads to impediment in the enterocyte nutrition, immune regulation, protective barrier function, and gastrointestinal motility [126]. Therefore, restoration or re-establishment of the microbiota could have a good interest therapeutically. Recently, a randomized clinical trial showed that administration of fecal microbiota (10 g feces diluted in 10 mL of sterile 0.9% saline) obtained from healthy donor rectally at 6–12 hours post-admission caused faster resolution of diarrhea, shortened the duration of hospitalization and reduced the mortality in young dogs with CPVE when compared with standard therapy alone [126].

6. Prevention

A modified live virus (MLV) and an inactivated vaccine are the two types of CPV-2 vaccines currently available [94]. Administration of the vaccine should start at 6 to 8 weeks of age and then every 2–4 weeks until 16 weeks of age or older. For dogs that are 16 weeks or older, 2 doses of vaccination are recommended with an interval of 2–4 weeks [127]. A recombinant vaccine based on virus-like particles (VLPs) is being developed, which has the advantage of becoming highly immunogenic and safe [128]. Peptide vaccines containing major antigen neutralizing region N terminal of VP2 are also under developmental stage [129]. A single-dose vaccination of Vaccinia virus encoding CPV2-VP2 elicited substantial antibody responses and provided comparable protection for dogs with attenuated CPV2 vaccine. This vaccine could be used as a promising vaccine candidate to prevent CPV-2 infection in dogs [130].

7. Conclusion

CPV-2 is one of the most significant viral enteropathogens of canines causing high morbidity and mortality and manifested by vomition and severe acute haemorhagic gastroenteritis. Prompt symptomatic therapy will increase survivability of infected puppies but vaccination is best way to prevent the disease in dogs. Despite the pups are protected through vaccination from the pregnant bitch, it is more vulunerable to CPV-2 infection as maternal antibody titers started declining. Despite the availability of high sensitive and specific diagnostic approaches and the effective prophylactics such as modified live virus and inactivated vaccines, a large number of outbreaks are still reported in wide geographical areas across the globe in both vaccinated and unvaccinated dogs. The future studies should be taken up towards vaccination failures, occurrence of CPV-2 in different canine species and the emergence of antigenic variants of the CPV-2 involved in the outbreaks.

Acknowledgments

All the authors acknowledge and thank to their Institute.

Conflict of interest

The authors declare no conflict of interest.

Funding

This compilation is a book chapter written by its authors and required no substantial funding to be stated.