1. Introduction
“CPV-2” typically refers to Canine Parvovirus-2, which is a highly contagious viral illness that affects dogs, especially puppies. It can cause severe gastrointestinal symptoms and can be life-threatening if not treated promptly period. It is caused by canine parvovirus, a single-stranded DNA virus categorized within the
Protoparvovirus genus, specifically the carnivore
Protoparvovirus 1 species [
1]. CPV-2 leads to hemorrhagic gastroenteritis in canines and various other species. The original CPV-2 strain emerged in the USA during a 1978 epidemic of gastroenteritis and subsequently spread globally. CPV-2 was swiftly succeeded by its variants: CPV-2a (1980), CPV-2b (1984), and CPV-2c (2000) [
2].
Research conducted in Morocco [
3] has confirmed the presence of canine parvovirus using the Snap Parvo-Ag
® rapid test (IDEXX, Westbrook, ME, USA), Hemagglutination test, and Hemagglutination inhibition. Sequencing work by Lafquih [
4] revealed the co-circulation of the original CPV-2 strain and the CPV-2c variant. Canine parvovirus was successfully isolated in cell culture from MDCK lines by Amrani [
5] in 2013. Additionally, Mellor [
6] developed an adjuvanted inactivated vaccine based on a Moroccan parvoviral strain, while Achaoui [
7] highlighted the coagglutination test for diagnosing canine parvovirus. Despite vaccination efforts, canine parvovirus remains a significant concern in Morocco due to the high number of vaccinated dogs succumbing to the disease. This is largely due to the lack of means for early and rapid diagnosis and uncertainty regarding the efficacy of vaccines used. Thus, there is a pressing need for cost-effective, rapid diagnostic tools to facilitate timely treatment and prevent fatal complications. Furthermore, the parvovirus vaccines currently used in Morocco are based on the original CPV-2 strain, with unproven efficacy.
Several authors have demonstrated limited cross-protection between CPV-2 variants [
8,
9] and suggested incorporating variant strains into vaccines. Therefore, it is essential to identify circulating variants in Morocco to assess cross-protection and develop more efficient, accessible, and affordable vaccines based on circulating strains.
This study aims to confirm and type all variants circulating in Morocco using specific real-time PCR on 11 samples collected in 2021. Subsequently, isolation on MDCK cells was attempted to select a candidate strain for producing and evaluating an inactivated vaccine derived from the Moroccan canine parvovirus strain.
2. Materials and Methods
2.1. Samples and Preparation
The eleven specimens utilized in this investigation were obtained from puppies exhibiting symptoms of gastroenteritis and suspected canine parvovirus infection during the month of May 2021. Rectal swabs were immersed in 2 mL of PBS (phosphate-buffered saline) and macerated for one hour before being homogenized.
2.2. DNA Extraction
DNA extraction from rectal swabs was conducted using the Venor GeM Sample Preparation kit (Minerva Biolabs, Berlin, Germany) following the manufacturer’s guidelines.
2.3. Parvovirus Detection by Real-Time PCR
Real-time polymerase chain reaction (PCR) was performed using the Superscript III Platinum One-Step qRT-PCR kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. A volume of 5 µL of DNA was utilized in a total reaction volume of 20 µL. The primers and probe employed in this study were designed by [
10]. The assay was conducted on AriaMx real-time PCR system (Agilent technologies, Santa Clara, CA, USA) with the following cycling profile: polymerase activation at 95 °C for 2 min, followed by 40 cycles of amplification (95 °C for 1 min, 60 °C for 1 min, and 72 °C for 20 s).
2.4. Genotyping by Real-Time PCR
Molecular typing was conducted using the Superscript III Platinum One-Step qRT-PCR kit (Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Specific primers and probes (
Table 1) targeting CPV-2a, CPV-2b, and CPV-2c were employed, as described by [
11]. A volume of 5 µL of DNA was utilized in a total reaction volume of 20 µL. The assay was performed on an AriaMx real-time PCR system (Agilent technologies, Santa Clara, CA, USA) with the following cycling profile: polymerase activation at 95 °C for 2 min, followed by 40 cycles of amplification (95 °C for 1 min, 60 °C for 1 min, and 72 °C for 20 s).
2.5. Isolation
The strain was initially isolated on a Crandell–Rees Feline Kidney (CRFK) cell and subsequently adapted on Madin–Darby Canine Kidney (MDCK) cells through a total of six passages; these passages constitute the primary viral strain.
In the isolation studies, Madin–Darby Canine Kidney (MDCK) cell lines were utilized. Samples exhibiting a high threshold cycle (Ct < 25) value in polymerase chain reaction (PCR) which indicated a significant amount of virus were filtered using a 0.22 µm membrane filter (Millipore, Bangalore, India), and the filtrates were then employed in the isolation studies. Each sample underwent five passages for virus isolation. The infected cell monolayers were harvested on the fourth day post-inoculation, regardless of cytopathic effects (CPE), through three cycles of alternate freezing and thawing. The virus suspension was clarified by centrifugation at 6000 rpm for 15 min in a refrigerated centrifuge, and the supernatant was subsequently frozen at −40 °C for further use [
12].
2.6. Inactivated Vaccine Production
The “Canivax-parvo” vaccine’s active ingredient is produced through propagation of the MDCK cell line in single-use Multitrays, followed by inoculation with the seed virus and incubation in roller flasks. After 24 h, the medium is changed, and the cells are left to incubate for 3 days. The viral harvest undergoes quality control sampling before being inactivated with beta-propiolactone overnight at 2–8 °C.
MDCK cells are meticulously examined to ensure their sterility from bacterial, fungal, and mycoplasma contaminants, as well as their virological purity. This comprehensive assessment involves testing for cytopathic and hemadsorbent exogenous viruses at various stages, including the initial stock (MCS), primary stock, and working stock (WCS), in accordance with European Pharmacopoeia monograph No. 50204. Furthermore, throughout the cell amplification phases required for active ingredient production, ongoing checks for bacterial and fungal sterility are conducted at each subculture level to maintain quality standards.
2.7. Evaluation Protocol
The inactivated vaccine is subjected to rigorous assessment to determine both its safety and efficacy.
2.7.1. Safety
The safety study for “Canivax-parvo” vaccine was carried out in accordance with European Pharmacopoeia monograph 0795, edition 10.0.
Eight healthy seven-week-old local breed puppies, dewormed and stabilized, were kept at BIOPHARMA pet store for two weeks. They tested negative for CPV-2 before vaccination. Prior to vaccination, a veterinarian confirmed their good health. The primary vaccination involved a 1 mL subcutaneous dose on the first day, followed by another dose three weeks later. Daily clinical examinations and temperature recordings were conducted for 14 days post-vaccination. Blood samples were taken every 4 days to check for leukopenia. Clinical examinations assessed general condition, behavior, injection site reaction, feces consistency, mucous membranes, breathing, cardiac function, dehydration, and any abnormal signs possibly due to vaccination. Monitoring was recorded on the registration form provided for this purpose.
2.7.2. Efficacy
Efficacy trials were conducted following the guidelines and recommendations outlined in the European Pharmacopoeia monograph 0795 edition 10.0.
Two trials were conducted: the vaccination-virulent test and the evaluation of seroconversion in vaccinated puppies and guinea pigs, adhering to the protocol outlined by the European Pharmacopoeia.
vaccination-virulent test
Seven puppies (eight weeks old) were divided into two groups: five received “Canivax-parvo” vaccine, while two served as controls. After vaccination, all puppies were monitored daily. After 21 days, both groups were challenged with the test virus and observed for 14 days. Blood samples and rectal swabs were collected for analysis. The vaccine’s effectiveness was determined based on the absence of clinical signs and low viral shedding compared to unvaccinated puppies.
Evaluation of post-vaccination seroconversion
In the target species, seroconversion was evaluated by detecting hemagglutination-inhibiting antibodies in the serum of five vaccinated puppies before the challenge on days 7–14 and day 21. The inhibition of hemagglutination was observed using pig red blood cells at a temperature of 4 °C.
Guinea pigs were chosen as the animal model to assess the serological response following vaccination. Eight guinea pigs received two subcutaneous injections spaced 14 days apart, each administered at half the recommended dose (0.5 mL). Blood samples were collected on the 14th day after the second injection. The serum was separated by centrifugation and stored at −20 °C until further use. Compliance of the vaccine is determined if the average hemagglutination-inhibiting antibody titer obtained from the eight guinea pigs is at least 1/80.
4. Discussion
The objectives of this article are to confirm the circulation of the CPV-2c variant despite the use of vaccines based on the original CPV-2 strain and to highlight the need for a new vaccine targeting the circulating genotypes (2-bc). We aim to develop a new vaccine from a selected Moroccan strain containing the 2-bc variant and to evaluate its safety and efficacy.
Canine parvovirus is extremely contagious, primarily spreading through direct contact with infected feces. Infected dogs can excrete the virus in their feces for several weeks to several months, even after symptoms have resolved [
13].
In Morocco, research has confirmed the presence of canine parvovirus (CPV-2) [
3,
4,
5] and identified the co-circulation of CPV-2b and CPV-2c by [
14,
15]. This finding raises important questions: does the existing vaccine fail to provide adequate protection, and is the development of a new vaccine necessary?
To answer these questions, we employed several approaches, including screening and genotyping canine parvovirus by using real-time PCR, and attempting to isolate the candidate strain to produce a vaccine suitable for the Moroccan context.
The results from the screening and genotyping tests using real-time PCR clearly indicate the presence of multiple CPV variants circulating in Morocco, including CPV-2b and CPV-2c the presence of co-infection has been detected, the co-infections with different CPV-2 variants were previously reported in Italy [
14,
15]. This indicates a diverse viral population contributing to the challenge of controlling the disease in the region. This has been reported in recent years [
14] and demonstrated in this current study
Followed by attempts to isolate the virus on MDCK cells [
16]. The “Canivax-parvo” vaccine was produced from an indigenous strain isolated from a symptomatic dog, emphasizing the importance of local strain selection for vaccine development.
Despite vaccination with the current vaccine (CPV-2-ab), the disease remains present, suggesting a lack of protection, mutations in the VP2 gene of CPV-2 variants can alter the virus’s antigenic profile, potentially reducing the efficacy of vaccines designed based on the original virus strain. This highlights the importance of developing new vaccines that target prevalent variants, such as CPV-2c, to ensure effective protection [
17].
The relationship between these mutations and vaccine efficacy is crucial. Vaccines function by prompting the immune system to identify and combat the virus based on its specific antigens, mainly the VP2 protein. When the VP2 protein mutates, the antigens presented by the virus can change, making it more difficult for the immune system to recognize the mutated virus. As a result, vaccines designed for the original CPV-2 strain might not offer sufficient protection against new variants with significant VP2 mutations, such as CPV-2b and CPV-2c [
17].
In addition, causes of vaccine-related failures include vaccine storage or administration errors, non-compliance with vaccine schedules, and failures in vaccine immunogenicity [
18,
19]. A 2017 survey of Australian veterinarians’ vaccination protocols found that nearly half of respondents did not comply with the recommended guideline to finish primary vaccination at or after 16 weeks of age [
20].
Moreover, the vaccine was produced following the standards and guidelines [
21] for good vaccine manufacturing practice using the ”CaPV M/3-2022” vaccine strain which is a CPV-2-bc. Various controls, including inactivation, physico-chemical, and biological assessments, confirmed the high quality of the production.
At the conclusion of the safety study conducted at BIOPHARMA’s animal facility to evaluate “Canivax-parvo”, an adjuvanted inactivated vaccine for preventing canine parvovirus, it was determined that administering two injections three weeks apart (primary vaccination) to eight-week-old puppies via the subcutaneous route did not cause any adverse effects or clinical manifestations attributable to the vaccine. The vaccinated puppies maintained excellent health throughout the observation period. “Canivax-parvo”complies with the safety requirements of the European Pharmacopoeia.
Additionally, the efficacy of the “Canivax-parvo” vaccine, an adjuvanted inactivated vaccine against canine parvovirus made from the Moroccan strain “M3/22”, fully complies with the requirements of the European Pharmacopoeia outlined in chapter 0795 “canine parvovirus vaccine (inactivated)”. The serological results and virulence test demonstrate its satisfactory effectiveness. The vaccine protects vaccinated subjects from the clinical signs of hemorrhagic gastroenteritis, significantly reduces virus excretion compared to unvaccinated controls, and produces a satisfactory serological response with reassuring antibody levels, indicating strong immunity. “Canivax-parvo” provides sufficient efficacy guarantees as required for an inactivated vaccine for the prophylaxis of canine parvovirus.
The vaccine developed from the local strain has demonstrated safety and efficacy in challenge trials. However, the comparison between “Canivax-parvo” and the current vaccines used in Morocco should be accomplished to provide more robust understanding of the cross protection between the autologous vaccines, the commercial vaccines formulated with heterologous strains and the circulating field strains.