Generation of canine neutralizing antibodies against canine parvovirus by single B cell antibody technology.

Canine parvovirus (CPV) is a significant threat to canines and is widely distributed worldwide. While vaccination is currently the most effective preventive measure, existing vaccines are not able to offer comprehensive and dependable protection against CPV infection. Hence, there is a need to explore alternative or complementary strategies to tackle this problem. In this study, we present an approach for the efficient screening of canine antibodies targeting CPV using a single B cell antibody technique. We sorted single IgM- IgG+ CPV+ B cells from canine peripheral blood mononuclear cells using fluorescence-activated cell sorting (FACS) and obtained the variable region genes of heavy and light chains (VH and VL) by nested PCR amplification. Canine monoclonal antibodies were expressed in HEK293 cells, and a total of 60 antibodies were obtained, five of which demonstrated neutralizing activity against CPV. Those findings demonstrate the effectiveness of the method for obtaining canine monoclonal antibodies, which in turn aids in the identification and screening of neutralizing antibodies against various canine pathogens.

Canine parvovirus in North-East India: a phylogenetic and evolutionary analysis.

Canine parvovirus type 2 (CPV-2) infection in dogs is considered as one of the most common cause of morbidity and mortality in young dogs and continues to occur with high incidence worldwide. Despite a single-stranded DNA virus, CPV-2 possesses a high mutation rate which has led to the development of new variants from time to time. These variants are classically classified based on the amino acid markers present in the VP2 gene. In this study, we examined 20 different cases of CPV-2 infection from seven different states of the North East region (NER) of India. The near-complete genome sequences of all these isolates were subjected to phylodynamic and phylogeographic analysis to evaluate the genetic diversity and geographical spread of CPV-2 variants. Analysis of the deduced amino acid sequences revealed residues characteristic of the 'Asian CPV-2c lineage' in all the 20 sequences confirming it as the dominant strain circulating in NER, India. The phylogenetic analysis based on the whole genome showed that all 20 sequences formed a monophyletic clade together with other Asian CPV-2c sequences. Furthermore, phylogeographic analysis based on the VP2 gene showed the likely introduction of Asian CPV-2c strain to India from China. This study marks the first comprehensive report elucidating the molecular epidemiology of CPV-2 in India.

Genome Sequences of Canine Parvovirus Type 2c Prevalent in Western Mexico.

Canine parvovirus type 2 (CPV-2) is one of the main etiologies of viral gastroenteritis in dogs across the globe. This disease is mainly characterized by the presence of diarrhea, abdominal pain, vomiting, anorexia, and dehydration. This virus is responsible for high mortality and morbidity rates in unvaccinated dogs and those younger than three months. The monitoring of viral variants in our region has demonstrated that in the last seven years, variant CPV-2c has been circulating exclusively, which is unusual if we consider that in the rest of the world, at least two variants co-circulate among dog populations. To the best of our knowledge, no studies in Mexico have reported genomic sequences of CPV-2, which are relevant for population comparisons at the genetic level. Therefore, the present study aimed to sequence genomes associated with CPV-2c. To meet this objective, rectal swab samples were collected from dogs with suspected CPV-2 infection. Five positive cases diagnosed by lateral flow testing and polymerase chain reaction were selected for viral genome sequencing. Comparative analyses illustrated that the obtained genome sequences were > 99% homologous to those reported for CPV-2 in the GenBank. On the other hand, 52 nucleotide mutations were identified in the vp1/vp2 gene, out of which three impacted amino acid transition (T226S, F267Y, and A440T). Phylogenetic analysis of the vp1/vp2 gene demonstrated that the five sequences clustered in a clade called "III", pertaining to sequences from USA and Uruguay. To our knowledge, this was the first report of genomic sequences associated with CPV-2 in Mexico, which is of great relevance for the epidemiological-molecular understanding and evolution of the virus.

Isolation and genetic characterization of parvoviruses from domestic cats reveals emergence of CPV-2c in India: A first report.

The objective of the present study was to isolate and characterize the VP2 gene of parvoviruses from domestic cats in India. For that, 38 fecal samples were screened by PCR with 36.84% positivity. Sequence analysis of those isolates showed canine parvovirus type-2c (CPV-2c) as the predominant variant, followed by feline panleukopenia virus (FPV) and 2a. Phylogenetic analysis of the CPV-2c sequences revealed clustering with Singaporean, South Korean, Mongolian and Bangladeshi dog 2c sequences. Phylogenetic analysis of the 2a isolate (MZC 2) was found to be clustered with Indian, Thai and Singaporean dog 2a isolates. Similarly, all the four FPV sequences were ancestrally related to Indian dog and cat FPV sequences hinting towards interspecies transmission between dogs and cats. Both synonymous and non-synonymous mutations were evident in CPV-2c, 2a and FPV sequences indicative of active evolution. In cell culture medium, CPV-2 showed cytopathogenic effects at the third passage level. In conclusion, the study provided the first report of CPV-2c in cats from India, which demands for extensive epidemiological surveillance to monitor interspecies spread and to shed more light on viral phylogenomics, their distribution in the country and in the Southeast Asian region and usage of current vaccines.

The first evidence of Asian-like CPV-2b in Slovakia in a vaccinated dog with an acute fatal course of parvovirus infection: a case report.

This study provides a comprehensive description of the clinical course of a fatal parvovirus infection in a vaccinated dachshund puppy, along with the first identification of a new CPV-2 variant in Slovakia, elucidated through molecular amino acid analysis of the VP2 gene. The dog exhibited clinical signs such as apathy, vomiting, and bloody diarrhea. After confirming CPV-2 infection with a commercial snap test, intensive therapy was initiated. The dog succumbed within 48 h of admission. A rectal swab sample was collected, CPV-2 was examined using the PCR method, and sequenced. The virus detected in the patient was related to strains of CPV-2c of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (VP2: 5Gly, 267Tyr, 324Ile, 370Arg, and 440Thr). Phylogenetic analysis classified this strain as similar to Asian strains of CPV-2c. It is believed to be derived from an Asian strain similar to CPV-2c that acquired the 426Asp mutation. With this finding, we present the first evidence of an Asian-like CPV-2b strain in the territory of Slovakia.

Whole-genome sequence analysis of canine parvovirus reveals replacement with a novel CPV-2c strain throughout India.

Canine parvovirus (CPV) infection causes severe gastroenteritis in canines, with high mortality in puppies. This virus evolved from feline panleukopenia virus by altering its transferrin receptor (TfR), followed by the emergence of CPV-2 variants in subsequent years with altered immunodominant amino acid residues in the VP2 protein. While previous studies have focused on the VP2 gene, there have been fewer studies on non-structural protein (NS1 and NS2) genes. In the present study, CPV genome sequences from clinical samples collected from canines throughout India in 2023, previous Indian CPV isolates from 2009-2019, and the current Indian CPV vaccine strain were compared. The study showed that the CPV-2c (N426E) variant had almost completely replaced the previously dominant CPV-2a variant (N426) in India. The Q370R mutation of VP2 was the most common change in the recent CPV-2c strain (CPV-2c 370Arg variant). Phylogenetic analysis showed the existence of three clades among the recent CPV-2c strains, and sequence analysis identified several new sites of positive selection in the VP1 (N-terminus), VP2, NS1, and NS2 protein-encoding genes in recent CPV strains, indicating the emergence of new CPV-2c variants with varied antigenic and replication properties. The predominant 'CPV-2c 370Arg variants' were grouped with the Chinese and Nigerian CPV-2c strains but were separate from the CPV vaccine strain and earlier isolates from our repository. VP2 epitope analysis predicted nine amino acid variations (including two new variations) in four potential linear B-cell epitopes in the CPV-2c 370Arg variants that might make vaccine failure more likely. This pan-Indian study lays the foundation for further research concerning the dynamics of virus evolution and understanding genetic mutations.

In silico designing of multi-epitope vaccine against canine parvovirus using reverse vaccinology.

Canine parvovirus (CPV-2) is a highly contagious virus affecting dogs worldwide, posing a significant threat. The VP2 protein stands out as the predominant and highly immunogenic structural component of CPV-2. Soon after its emergence, CPV-2 was replaced by variants known as CPV-2a, 2b and 2c, marked by changes in amino acid residue 426 of VP2. Additional amino acid alterations have been identified within VP2, with certain modifications serving as signatures of emerging variants. In Brazil, CPV-2 outbreaks persist with diverse VP2 profiles. Vaccination is the main preventive measure against the virus. However, the emergence of substitutions presents challenges to conventional vaccine methods. Commercial vaccines are formulated with strains that usually do not match those currently circulating in the field. To address this, the study aimed to investigate CPV-2 variants in Brazil, predict epitopes, and design an in silico vaccine tailored to local variants employing reverse vaccinology. The methodology involved data collection, genetic sequence analysis, and amino acid comparison between field strains and vaccines, followed by the prediction of B and T cell epitope regions. The predicted epitopes were evaluated for antigenicity, allergenicity and toxicity. The final vaccine construct consisted of selected epitopes linked to an adjuvant and optimized for expression in Escherichia coli. Structural predictions confirmed the stability and antigenicity of the vaccine, while molecular docking demonstrated interaction with the canine toll-like receptor 4. Molecular dynamics simulations indicated a stable complex formation. In silico immune simulations demonstrated a progressive immune response post-vaccination, including increased antibody production and T-helper cell activity. The multi-epitope vaccine design targeted prevalent CPV-2 variants in Brazil and potentially other regions globally. However, experimental validation is essential to confirm our in silico findings.

First identification of canine parvovirus -2a/2b variant in unvaccinated domestic dogs with gastrointestinal signs in Türkiye.

BACKGROUND: Canine parvovirus type 2 (CPV-2) is the most common enteric virus that infects canids. CPV is the causative agent of a contagious disease defined mostly by clinical gastrointestinal signs in dogs. During the late 1970s, CPV-2 emerged as a new virus capable of infecting domestic dogs and growing across the world. The VP2 gene stands out as a key determinant in the pathogenicity, antigenicity, and host interactions of CPV-2. AIMS: The molecular characterization of the VP2 gene is crucial for understanding CPV evolution and epidemiology. MATERIALS & METHODS: Genes encoding the VP2 protein were sequenced and compared to reference strains worldwide. The maximum likelihood method was used to build a phylogenetic tree using CPV VP2 gene nucleotide sequences. RESULTS: Our phylogenetic analysis of the VP2 gene revealed that five strains were very similar and clustered together, and three strains were in the 2b clade, whereas the other two were in the 2a/2b clade. DISCUSSION: This paper reports the molecular characterization of two novel CPV-2a/2b subtypes in dogs with gastrointestinal symptoms. Genetic analysis was conducted on a CPV genomic region encompassing one of the open reading frames (ORFs) encoding the structural protein VP2. Sequence analysis indicates new and unreported sequence changes, mainly affecting the VP2 gene, which includes the mutations Ser297Ala and Leu87Met. This study represents the first evidence of a new CPV-2a/2b subtype in Türkiye. Due to VP2's crucial role in encoding the capsid protein of CPV-2 and its significant involvement in the host-virus interaction, it is critical to closely monitor its evolutionary changes and be cautious while searching for novel or pre-existing subtypes. CONCLUSION: This study highlights the significance of continuous molecular research for acquiring more insights on the circulation of novel CPV mutants.

A LAMP-CRISPR/Cas12b rapid detection platform for canine parvovirus detection.

Canine parvovirus (CPV) is one of the main pathogens causing toxic diarrhea in Chinese dogs, is the cause of large-scale epidemic of dogs, and poses a great threat to the dog industry in China. Rapid, sensitive, and specific CPV testing facilitates the timely diagnosis and treatment of sick dogs. The aim of this study was to build a LAMP-CRISPR/Cas12b platform for CPV detection. The loop mediated isothermal amplification (LAMP) technique was combined with CRISPR-Cas12b analysis to establish a "two-step" and "one-tube" CRISPR/Cas12b rapid CPV method, respectively. The detection system was constructed with specific LAMP primers and single guide RNA (sgRNA) for the highly conserved short fragment of the CPV gene, which could be detected within 1 h without cross-reaction with the other viruses causing canine diarrhea. The detection limits of both "two-step" and "one-tube" CRISPR/Cas12b reactions were 10-1 copies per µL, which was 100 times more sensitive than qPCR and LAMP. In order to achieve point-of-care testing (POCT) of CPV, a one-tube LAMP-CRISPR/Cas12b nucleic acid extraction and detection platform based on magnetic nanoparticle enrichment technology was established to achieve "sample in-result out". The results of this method for simulated samples were compared with those of quantitative real-time PCR; the results showed 100% consistency, and the time was shorter, which could be used to detect the diseased dogs earlier and provide a basis for clinical diagnosis. The LAMP-CRISPR/Cas12b method established in this study provides a sensitive and specific method for rapid detection of CPV, and provides technical support for rapid diagnosis of CPV.

Epidemiological, and molecular investigation of Canine parvovirus-2 infection in Egypt.

IMPORTANCE: Canine parvovirus enteritis (CPE) is a contagious viral disease of dogs caused by the canine parvovirus-2 (CPV-2) associated with high morbidity and mortality rates. CPV-2 has a high global evolutionary rate. Molecular characterization of CPV-2 and understanding its epidemiology are essential for controlling CPV-2 infections. OBJECTIVE: This study examined the risk factors and survival outcomes of dogs infected with CPV-2. Molecular characterization of CPV-2 genotypes circulating in Egypt was performed to determine the evolution of CPV-2 nationally and globally. METHODS: An age-matched case-control study was conducted on 47 control and 47 CPV-infected dogs. Conditional logistic regression analysis examined the association between the potential risk factors and CPE in dogs. Survival analysis was performed to determine the survival pattern of the infected dogs. Thirteen fecal samples from infected dogs were collected to confirm the CPV genotype by CPV-2 VP2 gene sequencing, assembly of nucleotide sequences, and phylogenic analysis. RESULTS: Unvaccinated and roamer dogs had eight and 2.3 times higher risks of CPV infection than vaccinated dogs and non-roamer dogs, respectively. The risk of death from CPE was high among dogs without routine visits to veterinary clinics and among non-roamer dogs. Molecular characterization of CPV-2 confirmed its genotype identity and relationship with the CPV-2 c and b clade types. CONCLUSIONS AND RELEVANCE: This study highlights the potential factors for CPE control, especially vaccination and preventing dogs from roaming freely outside houses. Isolated CPV genotypes are closely related to southern Asian genotypes, suggesting a substantial opportunity for global transmission.

Prognostic indicators for canine parvoviral enteritis in a Teaching Hospital in Italy: a retrospective study of 76 cases.

Potential prognostic indicators have been associated with decreased survival during canine parvoviral enteritis (CPE), such as body weight, sex, and clinicopathological parameters. Few studies reported the prognostic factors for CPE in Italy; therefore, the aim of this study was to identify prognostic factors associated with the survival of dogs admitted to the Veterinary Teaching Hospital of Perugia University, naturally infected with canine parvovirus. Seventy-six medical records of dogs with a definitive diagnosis of parvoviral infection admitted from 2017 to 2021 have been reviewed and included in the study. From medical records were extracted data on signalment, history, clinical examination, hematology, serum biochemistry, treatments, progression of clinical signs during hospitalization and outcome. The data have been subjected to univariate and multivariate statistical analysis. Our results showed winter season, male sex, dog ownership, small breed, normal sensory status, normal heart rate, normal hydration status, abdominal pain, increased capillary reperfusion time, and normal white blood cell count as positive prognostic factors. The survival model confirmed that parameters such as male sex, small breed, and ownership increased the survival rate during hospitalization. Data reported in the present study are partially in agreement with previous studies and added new information on the possible prognostic factors in dogs affected by CPE in Italy.

Comparative genomics of canine parvovirus in South America: Diversification patterns in local populations.

Canine parvovirus (CPV) is a significant pathogen in domestic dogs worldwide, causing a severe and often fatal disease. CPV comprises three antigenic variants (2a, 2b, and 2c) distributed unevenly among several phylogenetic groups. The present study compared genetic variability and evolutionary patterns in South American CPV populations. We collected samples from puppies suspected of CPV infection in the neighboring Argentina and Uruguay. Antigenic variants were preliminarily characterized using PCR-RFLP and partial vp2 sequencing. Samples collected in Argentina during 2008-2018 were mainly of the 2c variant. In the Uruguayan strains (2012-2019), the 2a variant wholly replaced the 2c from 2014. Full-length coding genome and vp2 sequences were compared with global strains. The 2c and 2a strains fell by phylogenetic analysis into two phylogroups (Europe I and Asia I). The 2c strains from Argentina and Uruguay clustered in the Europe I group, with strains from America, Europe, Asia, and Oceania. Europe I is widely distributed in South America in the dog population and is also being detected in the wildlife population. The 2a strains from Uruguay formed the distinct Asia I group with strains from Asia, Africa, America, and Oceania. This Asia I group is increasing its distribution in South America and worldwide. Our research reveals high genetic variability in adjacent synchronic samples and different evolutionary patterns in South American CPV. We also highlight the importance of ancestral migrations and local diversification in the evolution of global CPV strains.

Detection of canine parvovirus type 2c (CPV-2c) in Palestine.

INTRODUCTION: The objective of the present study was to report, for the first time, the presence of canine parvovirus type 2c (CPV-2c) in domesticated dogs with acute gastroenteritis and to characterize the antigenic variants circulating in Palestine. METHODOLOGY: A veterinary clinical-based epidemiological study was carried out between December 2022 and April 2023. Fifty fecal samples were collected from dogs with gastroenteritis and screened for CPV-2 infection by polymerase chain reaction. The distribution of positive cases according to various epidemiological factors was studied. Partial sequencing of the viral protein 2 (VP2) gene was performed for the analysis of CPV-2 variants. RESULTS: Most of the investigated samples (60%; n = 50) during the study period were found positive for CPV-2 infection. There was no difference in the distribution of positive cases of CPV-2 infection based on age group, gender, location, and vaccination status. The analysis of nucleotide and amino acid sequences from amplified products, as well as phylogenetic analysis, revealed the presence of CPV-2c clustered with Asian CPV-2c variants. CONCLUSIONS: In summary, this study represents the initial genetic analysis of CPV-2 present in Palestinian dogs with gastroenteritis and provides evidence that confirms the existence of the CPV-2c variants. To determine the prevailing CPV-2 variant associated with the infection, it is crucial to conduct further sequence analysis using large populations of both domestic and wild canines.

Viral pathogens in domestic cats in southern Italy: A retrospective analysis in Sicily, 2020-2022.

A retrospective study was carried out on selected feline viral pathogens detected in domestic cat in Sicily, southern Italy. Samples from 64 cats, collected from 2020 to 2022, were analysed for the presence of feline panleukopenia virus, canine parvovirus type 2 (CPV-2), feline coronavirus (FCoV), feline calicivirus (FCV), feline herpesvirus type 1, norovirus (NoV), and rotavirus (RoV). Single (45 %) or mixed (38 %) viral infections were detected. FPV, related with other Italian FPV strains, remains the main viral cause of infection (66 %). CPV-2c Asian lineage strains (3 %) were detected for the first time in domestic cats in Europe. FCoV (29.6 %), either enteric or systemic, and systemic FCV (18.7 %) infections were detected in positive cats. Less commonly reported viruses (GIV.2/GVI.2 NoVs, RoV), potentially related to the animal/human interface, were detected at lower rates as well (5 %). The present epidemiological data suggest the need to improve disease prevention, immunization, and biosecurity strategies.

Molecular characterization of full-length VP2 gene of canine parvovirus type 2 strains circulating in Egypt 2019-2021.

Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over time lead to the emergence of three antigenic variants CPV-2a, CPV-2b, and CPV-2c. VP2 is the major capsid protein that determines virus antigenicity, and host range. Although the three CPV-2 variants were previously identified in Egypt, most reports covered a restricted geographic region and/or time period, and only analyzed partial fragments of VP2 gene. Therefore, this study was designed to test 100 rectal swabs collected from 7 Egyptian governorates between 2019 and 2021 for CPV-2 using PCR. A total of 65 positive samples were identified, mostly in pure dog breeds of young age. The three variants co-circulated in 2019, while CPV-2b was not detected in 2020 and 2021. The frequency of CPV-2b and CPV-2c was higher in 2019 and 2021, respectively. Analysis of CPV-2 full-length VP2 gene sequence from 19/65 positive samples has identified four common amino acid substitutions F267Y, S297A, A300G, Y324I, which are characteristic for the new CPV-2 variants currently circulating worldwide. Unique substitutions including A5G, G36R, V38E, Q370R, and G392V were recognized in certain samples, and appears to have distinct effect on receptor binding, nuclear translocation, and inter-species transmission. Phylogenetic analysis showed separation of CPV-2 strains into two clades. All strains of this study were classified in clade I with Asian strains. In conclusion, this study provides updated comprehensive molecular analysis of CPV-2 variants in Egypt.

Development and characterization of a novel specific monoclonal antibody against mink enteritis virus and its antigen epitope analysis.

This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 106 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (378YAFGR382) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.

Global distribution, cross-species transmission, and receptor binding of canine parvovirus-2: Risks and implications for humans.

For canine parvovirus -2 (CPV-2), a zoonotic virus capable of cross-species transmission in animals, the amino acid changes of capsid protein VP2 are key factors when binding to other species' transferrin receptors (TfR). CPV-2 variants can spread from felines and canines, for example, to Carnivora, Artiodactyla, and Pholidota species, and CPV-2c variants are essential to spread from Carnivora to Artiodactyla and Pholidota species in particular. In our study, a CPV-2a variant maintained a relatively stable trend, and the proportion of CPV-2c gradually rose from 1980 to 2021. The VP2 amino acid sequence analysis showed that five amino acid mutations at 426E/D, 305H/D, and 297S may be necessary for the virus to bind to different host receptors. Meanwhile, receptor-binding loop regions and amino acid sites 87 L, 93 N, 232I, and 305Y were associated with CPV-2 cross-species transmission. The homology of TfRs in different hosts infected with CPV-2 ranged from 77.2 % to 99.0 %, and from pig to feline, canine, and humans was 80.7 %, 80.4 %, and 77.2 %, respectively. The amino acid residues of TfRs involved in the viral binding in those hosts are highly conserved, which suggests that CPV-2 may be capable of pig-to-human transmission. Our analysis of the origin, evolutionary trend, cross-species transmission dynamics, and genetic characteristics of CPV-2 when binding to host receptors provides a theoretical basis for further research on CPV-2's mechanism of cross-species transmission and for establishing an early warning and monitoring mechanism for the possible threat of CPV-2 to animal-human public security.

Development of a time-resolved fluorescence immunoassay kit for detecting canine coronavirus and parvovirus through double labeling.

OBJECTIVE: Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases. METHODS: A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated. RESULTS: A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than - 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%. CONCLUSION: In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Detection and genetic characterization of circulating canine parvovirus from stray dogs in Shanghai, China.

Canine parvovirus (CPV) is the main cause of viral diarrhea in dogs. CPV became a global disease in 1978 and was endemic all over the world. CPV-2 was the first strain to be identified, but with genetic mutations, new genotypes such as CPV-2a/2b/2c/new-2a/new-2b have emerged. In this study, 128 fecal samples of stray dogs suspected of CPV-2 infection were collected from January to March 2021 in Shanghai, China. All samples were screened by PCR and further analyzed by VP2 gene. The positive rate of CPV-2 was 9.4% (12/128), of which 6 CPV-2 isolates were successfully isolated. Phylogenetic tree analysis showed that 4 isolates were CPV-2c genotype and 2 were new-CPV-2b genotype. VP-2 is a key protein that determines the antigenic properties, host range and receptor binding of cpv-2. The results of VP2 amino acid sequence analysis in this study showed that the CPV-2c isolated strain was the same as the previous strains reported in China, including F267Y, Y324I, Q370R and A5G mutations in addition to the typical N426E mutations. Similarly, in addition to the conventional N426D, S297A, F267Y and Y324I mutations, the new CPV-2b isolate also had a new mutation of T440A. This study further confirmed the prevalence of CPV-2c and new-CPV-2b in Shanghai, and also found a new mutation site of new-CPV-2c, which provided a theoretical basis for further enriching the epidemiological data of CPV-2 in Shanghai, as well as the development of vaccines and the prevention and control of the disease.

Epidemiological survey of infectious agents in free-ranging maned wolves (Chrysocyon brachyurus) in Northeastern Brazil.

Infectious diseases are one of the most concerning threats to maned wolves (Chrysocyon brachyurus) due to the potential impact on free-ranging populations. The species is currently classified as vulnerable according to the national list of threatened species and occurs mainly in open habitats, such as the Cerrado, a tropical savannah, which comprises its main distribution area in Brazil. In the northeastern region, it occurs in the Cerrado of Bahia, Piauí, Maranhão, and Tocantins states. Therefore, this study aimed to investigate the occurrence of infectious agents in Chrysocyon brachyurus through an epidemiological assessment of free-ranging individuals in western Bahia, specifically in the Barreiras microregion, a Cerrado area intensely fragmented and anthropized by agricultural activity. Eleven specimens were evaluated for serological titration, antigen research, and genetic material research for canine distemper virus (CDV), canine parvovirus (CPV), adenovirus-canine-type 1 (CAdV-1), canine coronavirus (CCoV), Leptospira interrogans and Toxoplasma gondii from 2020 to 2022. In addition to maned wolves, domestic dogs were also evaluated and tested. All maned wolves (100%) evaluated by the dot-ELISA technique exhibited immunoglobulin M (IgM) and seven (64%) exhibited immunoglobulin G (IgG) against CDV and CPV, while 100% exhibited IgG against CDV when using the immunochromatographic technique. Regarding CAdV-1, 90% were seropositive for IgG, while 64% exhibited IgG against T. gondii. Nine dogs from the region were also sampled, and all (100%) exhibited IgM and IgG against CDV and CPV. For IgG against T. gondii and against CAdV-1, 90% of the animals were seropositive. Molecular evaluation yielded negative results for all maned wolves and dogs assessed for CAdV-1, CDV, and T. gondii, as well as the CCoV antigen. These data indicate the occurrence of viral agents and Toxoplasma gondii in maned wolves and dogs, suggesting circulation in both populations.