FTA Cards as a Rapid Tool for Collection and Transport of Infective Samples: Experience with Foot-and-Mouth Disease Virus in Libya.

Foot-and-mouth disease (FMD) is a viral disease, widespread and highly contagious, that mainly affects cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to the reduction in animal production such as a drop in milk production, loss of body weight, and a high mortality rate in young ruminants. Sixteen samples were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Flinders Technology Associates (FTA) cards impressed with blood, swabs, or vesicular epithelium samples were shipped to the WOAH FMD reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards, and molecular testing based on real-time RT-PCR assays was carried out, of which one was specifically designed for the detection of the FMD virus of serotype O, topotype O/East Africa-3 (O/EA-3), that was further confirmed by a sequence analysis of the VP1 gene. The phylogenetic analysis of the VP1 gene showed a nucleotide identity of more than 99% between the virus circulating in Libya and the FMD virus strains isolated in Algeria in 2019.

Investigation of the "Antigen Hook Effect" in Lateral Flow Sandwich Immunoassay: The Case of Lumpy Skin Disease Virus Detection.

Lumpy skin disease (LSD) is an infectious disease affecting bovine with severe symptomatology. The implementation of effective control strategies to prevent infection outbreak requires rapid diagnostic tools. Two monoclonal antibodies (mAbs), targeting different epitopes of the LSDV structural protein p32, and gold nanoparticles (AuNPs) were used to set up a colorimetric sandwich-type lateral flow immunoassay (LFIA). Combinations including one or two mAbs, used either as the capture or detection reagent, were explored to investigate the hook effect due to antigen saturation by the detector antibody. The mAb-AuNP preparations were optimized by a full-factorial design of experiment to achieve maximum sensitivity. Opposite optimal conditions were selected when one Mab was used for capture and detection instead of two mAbs; thus, two rational routes for developing a highly sensitive LFIA according to Mab availability were outlined. The optimal LFIA for LSDV showed a low limit of detection (103.4 TCID50/mL), high inter- and intra-assay repeatability (CV% < 5.3%), and specificity (no cross-reaction towards 12 other viruses was observed), thus proving to be a good candidate as a useful tool for the point-of-need diagnosis of LSD.

SARS-CoV-2 in a Mink Farm in Italy: Case Description, Molecular and Serological Diagnosis by Comparing Different Tests.

This study described a SARS-CoV-2 infection in minks on an Italian farm. Surveillance was performed based on clinical examination and a collection of 1879 swabs and 74 sera from dead and live animals. The farm was placed under surveillance for 4.5 months, from the end of July 2020, when a man working on the farm tested positive by RT-PCR, till mid-December 2020 when all the animals were sacrificed. Clinical examination revealed no clinical signs or increased mortality rates attributable to SARS-CoV-2, while diagnostic tests detected only four weak PCR-positive samples, but 100% of sera were positive for SARS-CoV-2 anti-S antibodies. The phylogenetic analysis of two SARS-CoV-2 sequences from two minks and the sequence of the worker showed that they belonged to different clades. It could be therefore assumed that two distinct introductions of the virus occurred on the farm, and that the first introduction probably occurred before the start of the surveillance period. From the data collected, and especially from the detection of specific antibodies through the combination of different tests, it can be postulated that syndromic surveillance combined with genome detection by PCR may not be sufficient to achieve a diagnosis in asymptomatic animals. In particular, the serological approach, especially when using tests directed towards the S protein, may be useful for improving the traceability of virus circulation in similar environments.

Identification of diffusion routes of O/EA-3 topotype of foot-and-mouth disease virus in Africa and Western Asia between 1974 and 2019 - a phylogeographic analysis.

Foot-and-mouth disease (FMD) affects the livestock industry and socioeconomic sustainability of many African countries. The success of FMD control programs in Africa depends largely on understanding the dynamics of FMD virus (FMDV) spread. In light of the recent outbreaks of FMD that affected the North-Western African countries in 2018 and 2019, we investigated the evolutionary phylodynamics of the causative serotype O viral strains all belonging to the East-Africa 3 topotype (O/EA-3). We analyzed a total of 489 sequences encoding the FMDV VP1 genome region generated from samples collected from 25 African and Western Asian countries between 1974 and 2019. Using Bayesian evolutionary models on genomic and epidemiological data, we inferred the routes of introduction and migration of the FMDV O/EA-3 topotype at the inter-regional scale. We inferred a mean substitution rate of 6.64 × 10-3  nt/site/year and we predicted that the most recent common ancestor for our panel of samples circulated between February 1967 and November 1973 in Yemen, likely reflecting the epidemiological situation in under sampled cattle-exporting East African countries. Our study also reinforces the role previously described of Sudan and South Sudan as a frequent source of FMDVs spread. In particular, we identified two transboundary routes of O/EA-3 diffusion: the first from Sudan to North-East Africa, and from the latter into Israel and Palestine AT; a second from Sudan to Nigeria, Cameroon, and from there to further into West and North-West Africa. This study highlights the necessity to reinforce surveillance at an inter-regional scale in Africa and Western Asia, in particular along the identified migration routes for the implementation of efficient control measures in the fight against FMD.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV).

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1-660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens' immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394-608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394-608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461-544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

Combining Multiple Assays Improves Detection and Serotyping of Foot-and-Mouth Disease Virus. A Practical Example with Field Samples from East Africa.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012-2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVß6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

Retrospective Characterization of the 2006-2007 Swine Vesicular Disease Epidemic in Northern Italy by Whole Genome Sequence Analysis.

Advances in the epidemiological tracing of pathogen transmission have been largely driven by the increasing characterisation of whole-genome sequence data obtained at a finer resolution from infectious disease outbreaks. Dynamic models that integrate genomic and epidemiological data further enhance inference on the evolutionary history and transmission dynamics of epidemic outbreaks by reconstructing the network of 'who-infected-whom'. Swine Vesicular Disease (SVD) was present in Italy from 1966 until 2015, and since the mid-1990s, it has mainly been circulating within Italy's central-southern regions with sporadic incursions to the north of the country. However, a recrudescence of SVD in northern Italy was recorded between November 2006 and October 2007, leading to a large-scale epidemic that significantly affected the intensive pig industry of the Lombardy region. In this study, by using whole-genome sequence data in combination with epidemiological information on disease occurrences, we report a retrospective epidemiological investigation of the 2006-2007 SVD epidemic, providing new insights into the transmission dynamics and evolutionary mode of the two phases that characterised the epidemic event. Our analyses support evidence of undetected premises likely missed in the chain of observed infections, of which the role as the link between the two phases is reinforced by the tempo of SVD virus evolution. These silent transmissions, likely resulting from the gradual loss of a clear SVD clinical manifestation linked to sub-clinical infections, may pose a risk of failure in the early detection of new cases. This study emphasises the power of joint inference schemes based on genomic and epidemiological data integration to inform the transmission dynamics of disease epidemics, ultimately aimed at better disease control.

Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system.

The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.

Diagnostic Performances of Different Genome Amplification Assays for the Detection of Swine Vesicular Disease Virus in Relation to Genomic Lineages That Circulated in Italy.

During the last 25 years, swine vesicular disease (SVD) has occurred in Italy mostly sub-clinically. Therefore, regular testing of fecal samples from suspected holdings and high turnover premises was fundamental to identifying virus circulation and to achieve SVD eradication. In this study, we evaluated diagnostic performances of six genomic amplification methods, using positive fecal samples from 78 different outbreaks (1997-2014), which included different lineages. Comparison of three RT-PCRs, designed to amplify the same 154 nt portion of the gene 3D, demonstrated that a conventional and a real-time based on SYBR Green detection assay showed the highest diagnostic sensitivity, detecting all samples, while a real-time TaqMan-based test missed three cases, owing to two mismatches in the probe target sequence. Diagnostic and analytical specificities were optimal, as 300 negative field samples and other enteroviruses reacted negative. Three further evaluated tests, previously described, were a 3D-targeted reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and two real-time RT-PCRs targeted on the 5'UTR region. Here, the presence of multiple mismatches in probe and primers reduced the diagnostic performances, and two of the assays were unable to detect viruses from one sub-lineage. These results highlight that the choice of tests using less nucleotide targets significantly contributed to the success of the SVD eradication plan.

Cross-Reactivity Antibody Response after Vaccination with Modified Live and Killed Bovine Viral Diarrhoea Virus (BVD) Vaccines.

Pestivirus A or bovine viral diarrhoea virus (BVDV) type 1 is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. The objective of the present study was to verify whether animals immunised with four commercial vaccines also developed a protective humoral immunity against other viral subgenotypes than those contained in each vaccine. Four groups of 25 bovines each were formed and vaccinated according to the manufacturer's instructions of the commercial vaccines. On sera collected 28 days after the last vaccination, virus neutralisation tests (VNT) were performed using homologous and heterologous viruses and enzyme-linked immunosorbent assay (ELISA) methods. Finally, the VNT results were comparatively evaluated through a statistical analysis. Serological results highlighted that, although with a different degree of efficiency, the four vaccines resulted in not developing a solid antibody-mediated cross-immunity against all the strains used.

Development and validation of a simplified serotyping ELISA based on monoclonal antibodies for the diagnosis of foot-and-mouth disease virus serotypes O, A, C and Asia 1.

This study describes the development and validation of a simplified enzyme-linked immunosorbent assay (ELISA) for the detection and discrimination of foot-and-mouth disease virus (FMDV) serotypes O, A, C and Asia 1. The multiplex ELISA was designed using selected, type-specific monoclonal antibodies (MAbs) coated onto ELISA plates as catching antibodies and a unique pan-FMDV MAb (1F10) as detector conjugate. Capture MAbs with the broadest intratypic reactivity were selected for each of the four FMDV serotypes by screening large panels of candidate MAbs with a wide spectrum of representative FMDV isolates. An additional pan-FMDV ELISA using 1F10 MAb for both capture and detection was used to complement the specific typing ELISAs to detect virus isolates, which might escape binding to the selected serotype-specific MAbs. This multiplex ELISA was prepared in a stabilized format, with immunoplates pre-coated with six MAbs and positive antigen controls already trapped by the relevant MAb, with the view to make available a diagnostic kit. Diagnostic performance of the MAbs-multiplex ELISA was analysed using 299 FMDV-positive epithelial suspensions representative of the antigenic and genomic variability within each serotype. Overall, the results provided evidence that the diagnostic performance of this assay platform is improved compared to the more complex polyclonal-based antigen detection ELISA; combining data from different serotypes and referring to the gold standard tests (i.e. virus isolation and/or RT-PCR), the MAbs-multiplex ELISA showed a sensitivity of 79% for the serotype-specific ELISA, compared to 72% for the polyclonal ELISA. The absence of reactivity of a minority of FMDV strains using the MAbs-multiplex ELISA can largely be attributed to deteriorated or low antigen concentration in the sample. This multiplex ELISA is simple, rapid and stable. FMDV antigenic diversity was adequately covered by the selected MAbs. Therefore, it can be used to replace existing polyclonal ELISAs for FMDV detection and serotyping.

Foot-and-mouth disease outbreaks due to an exotic virus serotype A lineage (A/AFRICA/G-IV) in Algeria in 2017.

This study describes the genetic characterization of serotype A viruses collected during outbreaks of foot-and-mouth disease (FMD) that occurred in Algeria in 2017. These are the first reports of clinical cases due to this serotype in the country since 1977. One complete genomic sequence (comprising 8,119 nucleotides) and three additional near-complete genomic sequences were generated. Phylogenetic analyses demonstrated that these viruses were classified within the A/AFRICA/G-IV lineage, most closely related to viruses circulating in Nigeria between 2009 and 2015. These unexpected results motivate further studies to define the precise pathways by which this viral lineage has been introduced into North Africa in order to understand risks of future disease incursions into the region.

Reconstructing the evolutionary history of pandemic foot-and-mouth disease viruses: the impact of recombination within the emerging O/ME-SA/Ind-2001 lineage.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock affecting animal production and trade throughout Asia and Africa. Understanding FMD virus (FMDV) global movements and evolution can help to reconstruct the disease spread between endemic regions and predict the risks of incursion into FMD-free countries. Global expansion of a single FMDV lineage is rare but can result in severe economic consequences. Using extensive sequence data we have reconstructed the global space-time transmission history of the O/ME-SA/Ind-2001 lineage (which normally circulates in the Indian sub-continent) providing evidence of at least 15 independent escapes during 2013-2017 that have led to outbreaks in North Africa, the Middle East, Southeast Asia, the Far East and the FMD-free islands of Mauritius. We demonstrated that sequence heterogeneity of this emerging FMDV lineage is accommodated within two co-evolving divergent sublineages and that recombination by exchange of capsid-coding sequences can impact upon the reconstructed evolutionary histories. Thus, we recommend that only sequences encoding the outer capsid proteins should be used for broad-scale phylogeographical reconstruction. These data emphasise the importance of the Indian subcontinent as a source of FMDV that can spread across large distances and illustrates the impact of FMDV genome recombination on FMDV molecular epidemiology.

Differential Biological Activities of Swine Interferon-α Subtypes.

Interferons (IFNs) play a crucial role in the host's immune response and other homeostatic control actions. Three IFN types and several IFN families within the types allow for a plethora of regulatory actions. The number of distinct IFN molecules is highest among type I IFNs and, in particular, within the IFN-α family. In pigs, there are 17 IFN-α subtypes with different antiviral activities and different expression profiles; however, no data are available about biological properties other than the antiviral effector activities. Therefore, 16 porcine IFN-α genes were cloned, expressed in mammalian Chinese hamster ovary cells, and characterized for antiviral, anti-inflammatory, and MHC-modulating activities at a pre-established level of 10 IU/mL. Antiviral activity: IFN-α2, -α5, -α9, and -α10 showed the highest level of activity in a pseudorabies virus yield reduction assay. On the contrary, little, if any, activity was shown by IFN-α3, -α7, -α13, -α4, and -α15. Anti-inflammatory activity: With the exception of IFNs-α2, -α7, -α9, and -α11, all IFN-α subtypes had significant anti-inflammatory control activity in an interleukin-8 (IL-8) yield reduction assay. Gene expression analyses showed that some IFN-α subtypes can significantly downregulate the expression of IL-8, tumor necrosis factor α (TNF-α), IL-6, Toll-like receptor 4 (TLR4), ßD1, and nuclear factor-κB (NF-kB) genes, while maintaining or upregulating the expression of ßD4. Immunomodulation: A significant upregulation of class I and/or class II MHC was induced by all the IFNs under study, with the exception of IFNs-α11, -α15, and -α16, which instead significantly downregulated class I MHC. Our results indicate that gene duplications in the porcine IFN-α family underlie diverse effector and regulatory activities, being therefore instrumental in host survival and environmental adaptation. This role of IFN-α could be founded on fine-tuning and regulation of pro- and anti-inflammatory control actions after exposure to both infectious and noninfectious environmental stressors.

Comparison of three in-house ELISAs for the detection of hepatitis E virus infection in pigs under field conditions.

Hepatitis E virus (HEV) is a RNA non-enveloped virus that comprises four genotypes. The genome of HEV is organized into three Open Reading Frames (ORFs), and the ORF2 is responsible for encoding capsid proteins. HEV can infect a wide range of hosts, and pigs are considered the main reservoir. HEV infection is considered a zoonosis and it is responsible for acute hepatitis in humans, especially in developing countries. The development of a blocking ELISA would be of high value for screening purpose, because there is no need of species specific reagents. The present study was conducted to assess three in-house ELISAs for the detection of HEV infection in 779 sera collected from breeding and fattening farms under field conditions. Two assays were indirect ELISAs, while the third was a blocking ELISA. Two different recombinant antigens were generated from specific sequences of the HEV-ORF2, and a Latent Class approach in a Bayesian framework was used to evaluate the diagnostic accuracy of each ELISA. Because the three ELISAs cannot be thought of as independent, all possible dependence structures were modelled starting from the general case of conditional independence to the most complex situation of three mutually dependent assays. Results showed that none of the three ELISAs was significantly superior to the others in terms of sensitivity (posterior median value ranging from 89% to 94%, all 95% posterior credible intervals (95%PCI) overlapped). In terms of specificity, one of the indirect ELISAs was superior to blocking ELISA (posterior median indirect ELISA: 99%, 95%PCI: 98-100%; blocking ELISA: 90%; 95%PCI: 86-94%). However, this difference could be due to the potential wider spectrum of antibodies that blocking ELISA can actually detect.

Recombinant ELISA using baculovirus-expressed VP2 for detection of antibodies against canine parvovirus.

The gene encoding the VP2 protein of canine parvovirus type 2 was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination, Western blotting and hemagglutination inhibition test, using Canine parvovirus type-2 (CPV-2) positive sera. An enzyme-linked immunosorbent assay (ELISA) using the rVP2 was used for testing CPV-2 positive and negative sera from dogs and for determining the threshold of maternally derived antibodies interfering with successful vaccination of pups against CPV-2.