Circulating markers of endothelial activation in canine parvoviral enteritis.

ABSTRACT: Canine parvovirus (CPV) is a common cause of enteritis, immune suppression and systemic inflammation in young dogs. Endothelial markers, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and molecules that upregulate their expression, such as high mobility group box 1 protein (HMGB-1), provide insight into the state of the endothelium during inflammation. This study aimed to determine if circulating concentrations of ICAM-1, VCAM-1 and HMGB-1 were altered in CPV enteritis compared to healthy controls, and whether a correlation existed between these molecules and the degree of inflammation. Thirty dogs with naturally occurring CPV enteritis and ten control dogs were included. Physical examinations, complete blood count and C-reactive protein (CRP) measurements were performed on all dogs at presentation. The concentrations of ICAM-1, VCAM-1 and HMGB-1 were measured using commercially available canine-specific enzyme-linked immunosorbent assay (ELISA) kits. In dogs with CPV enteritis, ICAM-1 concentrations were significantly lower (median: 5.9 [IQR: 4.3-8.3]) and CRP higher (134 [IQR: 85-195]) compared to controls (8.0 [IQR: 6.9-10.3], p = 0.008; 1 [IQR: 0-7], p < 0.001). No significant difference was found for VCAM- 1 and HMGB-1. A strong correlation was identified between VCAM-1 and segmented neutrophil count (r = 0.612, p < 0.001). Despite the presence of systemic inflammation in CPV enteritis, evidenced by high CRP concentrations, our results suggest circulating concentrations of ICAM-1, VCAM-1 and HMGB-1 failed to show an increase. Endothelial activation with subsequent leukocyte adhesion and transmigration through the endothelium may be affected in CPV enteritis and these findings require further investigation.

Increased survival in puppies affected by Canine Parvovirus type II using an immunomodulator as a therapeutic aid.

Canine parvovirus type II (CPV-2) infection induces canine parvoviral enteritis (CPE), which in turn promotes sepsis and systemic inflammatory response syndrome (SIRS). Mortality in this disease is usually registered within 48-72 h post-hospitalization, the critical period of the illness. It has been recently described that the use of an immunomodulator, whose major component is monomeric ubiquitin (mUb) without the last two glycine residues (Ub∆GG), in pediatric human patients with sepsis augments survival. It is known that CXCR4 is the cell receptor of extracellular ubiquitin in humans. This work aimed to explore the effect of one immunomodulator (human Dialyzable Leukocyte Extract-hDLE) as a therapeutic auxiliary in puppies with sepsis and SIRS induced by CPE. We studied two groups of puppies with CPV-2 infection confirmed by polymerase chain reaction. The first group received conventional treatment (CT) and vehicle (V), while the second group received CT plus the immunomodulator (I). We assessed both groups' survival, clinical condition, number of erythrocytes, neutrophils, and lymphocytes during the hospitalization period. In addition, hematocrit, hemoglobin, plasma proteins and cortisol values, as well as norepinephrine/epinephrine and serotonin concentration were determined. Puppies treated with CT + I showed 81% survival, mild clinical signs, and a significant decrease in circulating neutrophils and lymphocytes in the critical period of the treatment. In contrast, the CT + V group presented a survival of 42%, severe clinical status, and no improvement of the parameters evaluated in the critical period of the disease. We determined in silico that human Ub∆GG can bind to dog CXCR4. In conclusion, the administration of a human immunomodulator (0.5 mg/day × 5 days) to puppies with CPE under six months of age reduces the severity of clinical signs, increases survival, and modulates inflammatory cell parameters. Further studies are necessary to take full advantage of these clinical findings, which might be mediated by the human Ub∆GG to canine CXCR4 interaction.

Canine parvovirus is shed infrequently by cats without diarrhoea in multi-cat environments.

Whether subclinical shedding of canine parvovirus (CPV) by cats might contribute to the epidemiology of canine CPV infections, particularly in facilities housing both cats and dogs, requires clarification. Conflicting results are reported to date. Using conventional PCR (cPCR) to amplify the VP2 gene, shedding of the CPV variants (CPV-2a, 2b, 2c) by healthy cats in multi-cat environments was reportedly common in Europe but rare in Australia. The aim of this study was to determine whether low-level faecal CPV shedding occurs in multi-cat environments in Australia and Italy using a TaqMan real-time PCR to detect Carnivore protoparvovirus 1 (CPV and feline parvovirus, FPV) DNA, and minor-groove binder probe real-time PCR assay to differentiate FPV and CPV types and to characterize CPV variants. In total, 741 non-diarrhoeic faecal samples from shelters in Australia (n = 263) and from shelters or cat colonies in Italy (n = 478) were tested. Overall, Carnivore protoparvovirus 1 DNA was detected in 49 of 741 (6.61 %) samples. Differentiation was possible for 31 positive samples. FPV was most common among positive samples (28/31, 90.3 %). CPV was detected in 4/31 samples (12.9 %) including CPV-2a in one sample, CPV-2b in another and co-infections of FPV/CPV-2b and CPV-2a/CPV-2b in the remaining two samples. A high rate of subclinical FPV infection was detected in one shelter during an outbreak of feline panleukopenia, during which 21 of 22 asymptomatic cats (95.5 %) sampled were shedding FPV. Faecal shedding of CPV by cats in multi-cat environments is uncommon suggesting that domestic cats are not significant reservoirs of CPV.

High-resolution asymmetric structure of a Fab-virus complex reveals overlap with the receptor binding site.

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab-virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab-virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

Molecular epidemiology and genetic evolution of canine parvovirus in East China, during 2018-2020.

Canine parvovirus type 2 (CPV-2) emerged in the late 1970s, which caused high rates of morbidity and mortality in dogs. In last decade, five genetic variants (CPV-2a, CPV-2b, CPV-2c, New CPV-2a, and New CPV-2b) were frequently reported in the dog population, and replaced the original CPV-2, rising widespread concerns. However, little is known about their recent genetic diversity and evolution. The aim of this study was to analyze the characteristics of the CPV-2 strains collected in East China from 2018 to 2020. The 57 CPV-2 strains were isolated from rectal swab samples (n=140). They belong to three different genotypes, based on VP2 protein amino acid sequence. The results revealed a high prevalence of CPV-2c (77.19%) compared to the New CPV-2a (5.26%) and New CPV-2b (17.54%) strains. Further analysis showed that nucleotide homology of the VP2 gene among the 57 CPV strains was 98.9%~100%, and the homology with 24 reference strains from different countries and regions was 98.1%~100%. The phylogenetic tree of VP2 gene sequence showed that 44 CPV-2c strains were distantly related to CPV-2, CPV-2a, CPV-2b, New CPV-2a, New CPV-2b and European/American CPV-2c strains, and were closely related to Asian CPV-2c strains. The results showed that these Asian CPV-2c strains had become the dominant strain, which renewed the knowledge of CPV-2 molecular epidemiology in East China.

Haemostatic changes associated with fluid resuscitation in canine parvoviral enteritis.

The haemostatic status of dogs with canine parvovirus (CPV) enteritis, within 24 h of admission after initial fluid administration, has been described previously, but the haemostatic status at admission and after standard fluid resuscitation, as well as after initial fluid redistribution, has not been investigated previously. The objective of this study was to characterise the haemostatic status at admission and describe the effect of crystalloid fluid resuscitation on haemostatic variables in dogs with CPV enteritis. Twenty-seven client-owned, hospitalised dogs with confirmed natural CPV infection and 15 healthy age-matched controls were included in a prospective, observational clinical study. The volume of resuscitation fluid, haematocrit (HCT), platelet count, thromboelastography (TEG) variables, antithrombin (AT) activity, fibrinogen- and C-reactive protein (CRP) concentrations were measured in all dogs at admission, after fluid resuscitation and, in 10 dogs, after receiving an additional 3 hours of maintenance-rate crystalloid fluids. For the CPV group at admission, the median TEG reaction time (R) and maximum amplitude (MA) or clot strength, as well as the median HCT, fibrinogen and CRP concentrations, were significantly increased compared to the controls. After fluid resuscitation, median R was significantly shorter, MA significantly increased and HCT and AT activity significantly decreased compared to admission values. The haemostatic variables remained unchanged after 3 h of maintenance-rate crystalloid therapy. The increased clot strength present in dogs with CPV enteritis at admission was exacerbated after fluid resuscitation and persisted for hours after large-volume crystalloid fluid administration.

New genotype classification and molecular characterization of canine and feline parvoviruses.

BACKGROUND: Canine parvovirus (CPV) and feline panleukopenia (FPV) cause severe intestinal disease and leukopenia. OBJECTIVES: In Korea, there have been a few studies on Korean FPV and CPV-2 strains. We attempted to investigate several genetic properties of FPV and CPV-2. METHODS: Several FPV and CPV sequences from around world were analyzed by Bayesian phylo-geographical analysis. RESULTS: The parvoviruses strains were newly classified into FPV, CPV 2-I, CPV 2-II, and CPV 2-III genotypes. In the strains isolated in this study, Gigucheon, Rara and Jun belong to the FPV, while Rachi strain belong to CPV 2-III. With respect to CPV type 2, the new genotypes are inconsistent with the previous genotype classifications (CPV-2a, -2b, and -2c). The root of CPV-I strains were inferred to be originated from a USA strain, while the CPV-II and III were derived from Italy strains that originated in the USA. Based on VP2 protein analysis, CPV 2-I included CPV-2a-like isolates only, as differentiated by the change in residue S297A/N. Almost CPV-2a isolates were classified into CPV 2-III, and a large portion of CPV-2c isolates was classified into CPV 2-II. Two residue substitutions F267Y and Y324I of the VP2 protein were characterized in the isolates of CPV 2-III only. CONCLUSIONS: We provided an updated insight on FPV and CPV-2 genotypes by molecular-based and our findings demonstrate the genetic characterization according to the new genotypes.

Comparison and validation of different models and variable selection methods for predicting survival after canine parvovirus infection.

BACKGROUND: Canine parvovirus (CPV) represents one of the major infections in dogs. While supportive therapy significantly reduces mortality, other approaches have been reported to provide significant benefits. Unfortunately, the high cost of these treatments is typically a limiting factor. Consequently, a reliable prognostic tool allowing for an informed therapeutic approach would be of great interest. However, current methods are essentially based on 'a priori' selection of predictive variables, which could limit their predictive potential. METHODS: In the present study, the predictive performances in terms of CPV enteritis survival likelihood of an operator-validated logistic regression were compared with those of more flexible methods featured by automatic variable selection. Several anamnestic, clinical, haematological and biochemical parameters were collected from 134 dogs at admission in a veterinary practice. Animal status was monitored until dismissal or death (mortality=21.6%). RESULTS: The best automatic variable selection method (random forest) showed excellent discriminatory capabilities (AUC=0.997, sensitivity=0.941 and specificity=1) compared with the logistic regression model (AUC=0.831, sensitivity=0.882 and specificity=0.652), when evaluated on a fully independent test data set. The implemented approaches allowed to identify antithrombin, serum aspartate aminotransferase, serum lipase, monocyte and lymphocyte count as the clinical parameter combination with the highest predictive capability, thus limiting the panel of required tests. CONCLUSION: The model validated in the present study allows prompt prediction of disease severity at admission and provides objective and reliable criteria to support the clinician in selection of the therapeutic approach.

Outbreak of canine parvovirus 2b and Clostridium difficile infection in Asian small-clawed otters.

A concurrent outbreak of infection by canine parvovirus 2b (CPV-2b) and Clostridium difficile producing A and/or B toxins occurred in Asian small-clawed otters (Amblonyx cinereus). The 5 clinically affected otters were 6- to 24-mo-old intact females that had severe diarrhea, dehydration, were acutely comatose, and died 1-4 d after the onset of clinical signs. Postmortem examination was performed in 3 of 7 otters. Macroscopically, the small intestine was diffusely reddened and contained red-to-brown, malodorous, watery digesta without formed feces (3 of 3). Histologic examination identified loss of enterocytes and necrosis of crypt epithelial cells. Denuded villi were often covered by mixed bacterial colonies with a predominance of gram-positive cocci to short rods in addition to larger gram-positive and -negative rods. There was also splenic lymphoid follicle depletion (2 of 3). Immunofluorescence assay revealed CPV antigen in enterocytes (2 of 3), mesenteric lymph nodes (3 of 3), and spleen (1 of 3). Immunohistochemistry revealed CPV antigen in enterocytes, lymphocytes, and dendritic cells of the Peyer patches and spleen (3 of 3), and lingual epithelial cells (1 of 2). CPV was isolated from tissues from 2 of 3 otters, and DNA sequencing identified CPV-2b for the 1 isolate tested. C. difficile producing A and/or B toxins were identified in the intestinal content by ELISA (3 of 3). To our knowledge, an outbreak of CPV-2b infection and C. difficile with clinically significant gastrointestinal disease has not been described previously in otters. The source of the viral infections remains unknown; however, these agents should be considered in otters and other mesocarnivores with similar clinical and pathologic findings.

Socioeconomic, geographic and climatic risk factors for canine parvovirus infection and euthanasia in Australia.

Infection of canids with canine parvovirus (CPV) can result in severe, often fatal disease. This study aimed to examine climatic, socioeconomic and geographic risk factors for CPV infection and CPV-associated euthanasia in Australia. Australian veterinary hospital responses (534; 23.5 %) to a national veterinary survey of CPV case occurrences and euthanasias in 2016 were used. Severe caseloads (>40 cases per annum) were reported by 26 (11 %) hospitals (median 60 cases; IQR 50-110). Case reporting, case numbers, and without-treatment euthanasia were significantly associated with disadvantage across all Socio-Economic Index for Areas quintiles (p < 0.0001) - the greater the disadvantage, the more reports. Strong negative correlations were found between case numbers and the Index of Relative Socioeconomic Disadvantage (rSP = -0.3357, p < 0.0001) and also between euthanasia and the Index of Education and Occupation (rSP = -0.3762, p < 0.0001). Hospitals in more remote areas were also more likely to report cases and to euthanize without treatment (p < 0.0001). Of the climate variables, temperature of the hottest month was most strongly positively correlated with case numbers (rSP = 0.421, p < 0.0001), and lower annual rainfall was associated with more case-reporting hospitals (p < 0.0001). These results confirm that socioeconomic disadvantage is a significant risk-factor for CPV infection and outcome, and high temperature may also contribute to risk.

Transferrin receptor binds virus capsid with dynamic motion.

Canine parvovirus (CPV) is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Cross-species transmission of CPV occurs as a result of mutations on the viral capsid surface that alter the species-specific binding to the host receptor, transferrin receptor type-1 (TfR). The interaction between CPV and TfR has been extensively studied, and previous analyses have suggested that the CPV-TfR complex is asymmetric. To enhance the understanding of the underlying molecular mechanisms, we determined the CPV-TfR interaction using cryo-electron microscopy to solve the icosahedral (3.0-Å resolution) and asymmetric (5.0-Å resolution) complex structures. Structural analyses revealed conformational variations of the TfR molecules relative to the binding site, which translated into dynamic molecular interactions between CPV and TfR. The precise footprint of the receptor on the virus capsid was identified, along with the identity of the amino acid residues in the virus-receptor interface. Our "rock-and-roll" model provides an explanation for previous findings and gives insights into species jumping and the variation in host ranges associated with new pandemics in dogs.

Onward transmission of viruses: how do viruses emerge to cause epidemics after spillover?

The critical step in the emergence of a new epidemic or pandemic viral pathogen occurs after it infects the initial spillover host and then is successfully transmitted onwards, causing an outbreak chain of transmission within that new host population. Crossing these choke points sets a pathogen on the pathway to epidemic emergence. While many viruses spill over to infect new or alternative hosts, only a few accomplish this transition-and the reasons for the success of those pathogens are still unclear. Here, we consider this issue related to the emergence of animal viruses, where factors involved likely include the ability to efficiently infect the new animal host, the demographic features of the initial population that favour onward transmission, the level of shedding and degree of susceptibility of individuals of that population, along with pathogen evolution favouring increased replication and more efficient transmission among the new host individuals. A related form of emergence involves mutations that increased spread or virulence of an already-known virus within its usual host. In all of these cases, emergence may be due to altered viral properties, changes in the size or structure of the host populations, ease of transport, climate change or, in the case of arboviruses, to the expansion of the arthropod vectors. Here, we focus on three examples of viruses that have gained efficient onward transmission after spillover: influenza A viruses that are respiratory transmitted, HIV, a retrovirus, that is mostly blood or mucosal transmitted, and canine parvovirus that is faecal:oral transmitted. We describe our current understanding of the changes in the viruses that allowed them to overcome the barriers that prevented efficient replication and spread in their new hosts. We also briefly outline how we could gain a better understanding of the mechanisms and variability in order to better anticipate these events in the future. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells.

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs-Nitazoxanide, Closantel Sodium, and Closantel-with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

Intestinal Microbial Dysbiosis in Beagles Naturally Infected with Canine Parvovirus.

Canine parvoviral enteritis (PVE) is an important intestinal disease of the puppies; however, the potential impact of the canine parvovirus (CPV) on the gut microbiota has not been investigated. Therefore, the aim of this study was to evaluate the gut microbial shifts in puppies naturally infected with CPV. Fecal samples were collected from healthy dogs and those diagnosed with PVE at 4, 6, 8, and 12 weeks of age. The distal gut microbiota of dogs was characterized using Illumina MiSeq sequencing of the bacterial 16S rRNA genes. The sequence data were analyzed using QIIME with an Operational Taxonomic Unit definition at a similarity cutoff of 97%. Our results showed that the CPV was associated with significant microbial dysbiosis of the intestinal microbiota. Alpha diversity and species richness and evenness in dogs with PVE decreased compared to those of healthy dogs. At the phylum level, the proportion of Proteobacteria was significantly enriched in dogs with PVE while Bacteroidetes was significantly more abundant in healthy dogs (p < 0.05). In dogs with PVE, Enterobacteriaceae was the most abundant bacterial family accounting for 36.44% of the total bacterial population compared to only 0.21% in healthy puppies. The two most abundant genera in healthy dogs were Prevotella and Lactobacillus and their abundance was significantly higher compared to that of dogs with PVE (p < 0.05). These observations suggest that disturbances of gut microbial communities were associated with PVE in young dogs. Evaluation of the roles of these bacterial groups in the pathophysiology of PVE warrants further studies.

Carnivore Parvovirus Ecology in the Serengeti Ecosystem: Vaccine Strains Circulating and New Host Species Identified.

Carnivore parvoviruses infect wild and domestic carnivores, and cross-species transmission is believed to occur. However, viral dynamics are not well understood, nor are the consequences for wild carnivore populations of the introduction of new strains into wild ecosystems. To clarify the ecology of these viruses in a multihost system such as the Serengeti ecosystem and identify potential threats for wildlife conservation, we analyzed, through real-time PCR, 152 samples belonging to 14 wild carnivore species and 62 samples from healthy domestic dogs. We detected parvovirus DNA in several wildlife tissues. Of the wild carnivore and domestic dog samples tested, 13% and 43%, respectively, were positive for carnivore parvovirus infection, but little evidence of transmission between the wild and domestic carnivores was detected. Instead, we describe two different epidemiological scenarios with separate routes of transmission: first, an endemic feline parvovirus (FPV) route of transmission maintained by wild carnivores inside the Serengeti National Park (SNP) and, second, a canine parvovirus (CPV) route of transmission among domestic dogs living around the periphery of the SNP. Twelve FPV sequences were characterized; new host-virus associations involving wild dogs, jackals, and hyenas were discovered; and our results suggest that mutations in the fragment of the vp2 gene were not required for infection of different carnivore species. In domestic dogs, 6 sequences belonged to the CPV-2a strain, while 11 belonged to the CPV-2 vaccine-derived strain. This is the first description of a vaccine-derived parvovirus strain being transmitted naturally.IMPORTANCE Carnivore parvoviruses are widespread among wild and domestic carnivores, which are vulnerable to severe disease under certain circumstances. This study furthers the understanding of carnivore parvovirus epidemiology, suggesting that feline parvoviruses are endemic in wild carnivores in the Serengeti National Park (SNP), with new host species identified, and that canine parvoviruses are present in the dog population living around the SNP. Little evidence of transmission of canine parvoviruses into wild carnivore species was found; however, the detection of vaccine-derived virus (described here for the first time to be circulating naturally in domestic dogs) highlights the importance of performing epidemiological research in the region.

Transmission ecology of canine parvovirus in a multi-host, multi-pathogen system.

Understanding multi-host pathogen maintenance and transmission dynamics is critical for disease control. However, transmission dynamics remain enigmatic largely because they are difficult to observe directly, particularly in wildlife. Here, we investigate the transmission dynamics of canine parvovirus (CPV) using state-space modelling of 20 years of CPV serology data from domestic dogs and African lions in the Serengeti ecosystem. We show that, although vaccination reduces the probability of infection in dogs, and despite indirect enhancement of population seropositivity as a result of vaccine shedding, the vaccination coverage achieved has been insufficient to prevent CPV from becoming widespread. CPV is maintained by the dog population and has become endemic with approximately 3.5-year cycles and prevalence reaching approximately 80%. While the estimated prevalence in lions is lower, peaks of infection consistently follow those in dogs. Dogs exposed to CPV are also more likely to become infected with a second multi-host pathogen, canine distemper virus. However, vaccination can weaken this coupling, raising questions about the value of monovalent versus polyvalent vaccines against these two pathogens. Our findings highlight the need to consider both pathogen- and host-level community interactions when seeking to understand the dynamics of multi-host pathogens and their implications for conservation, disease surveillance and control programmes.

The geographic distribution and financial impact of canine parvovirus in Australia.

Canine parvovirus (CPV) is an important cause of serious and often fatal disease in dogs worldwide, however, a national survey of CPV cases in Australia has not been conducted since 1982. For this study we surveyed the entire Australian veterinary clinic population and achieved a response rate of 23.5% (534 unique veterinary clinics). Respondents reported 4,451 CPV cases in 2015 and 4,219 cases in 2016; the estimated total CPV case load across Australia was 20,661 in 2015 and 20,110 in 2016. The overall reported euthanasia rate was 41%. Geospatial analysis revealed large numbers of CPV cases in rural and remote areas of Australia. Where cases occurred in capital city areas, these were found in peri-urban areas, away from the inner city. The median cost to treat CPV cases was $A1,500 per patient. A significant difference in the cost of treating cases was found between Australian states; Western Australia (median $A2,500) was the most expensive state. There was a strong correlation between cost of treatment and rate of euthanasia without treatment reflecting the important role of affordability in disease-related euthanasia. These findings highlight the considerable impact of the evolving CPV situation in Australia, particularly in regional and rural areas. This survey is the most comprehensive epidemiological investigation of canine parvoviral-related disease, to date, globally and provides a process for national disease surveillance.

ABSENCE OF PARVOVIRUS SHEDDING IN FECES OF THREATENED CARNIVORES FROM MISIONES, ARGENTINA.

Since its emergence in the 1970s, canine parvovirus (CPV) has spread worldwide and infects a wide variety of mammalian hosts, including domestic and nondomestic carnivores. Today it is one of the most important pathogenic viruses associated with high morbidity and mortality in domestic dogs ( Canis familiaris). In South America, the range of wild hosts has been scarcely studied and the epidemiology of CPV in wildlife is still unclear. In 2011, feces from five wild carnivores (bush dog [ Speothos venaticus] , jaguar [ Panthera onca], puma [ Puma concolor], oncilla [ Leopardus guttulus], and ocelot [ Leopardus pardalis]) were collected in Misiones, Argentina, using a detection dog. Of the 289 feces collected, 209 (72.3%) had sufficient sample remaining to be used in this study and the majority of these were genetically confirmed to individual (81.3%) and sex (78.4%) level. In fact, these samples represent a minimum of 115 individuals (10 jaguars, 13 pumas, 33 ocelots, 38 oncillas, and 21 bush dogs). Through polymerase chain reaction, a 583-bp fragment in the VP2 gene of CPV was amplified in these samples. While no samples showed evidence of infection, this does not exclude the occurrence of CPV in wild carnivores in the area, as intermittent viral shedding could condition the diagnosis of CPV in feces of infected wild mammals. Locally, it is recommended that long-term monitoring of parvovirus be continued in wildlife and expanded to domestic carnivores. Internationally, this study provides a useful contribution to the approach to the sylvatic cycle of parvovirus in wild carnivores.

A multiplex PCR method for the simultaneous detection of three viruses associated with canine viral enteric infections.

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 104 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Long-term effects of canine parvovirus infection in dogs.

BACKGROUND: Canine parvovirus (CPV) is the most important viral cause of acute canine enteritis leading to severe damage of the intestinal barrier. It has been speculated that dogs might develop chronic disorders after surviving CPV infection. However, no studies regarding the long-term implications of CPV infection have been published to date. The aim of this study was to evaluate whether dogs that have survived CPV infection will have an increased risk for developing chronic gastroenteritis, atopic dermatitis, or cardiac disease. METHODOLOGY/PRINCIPAL FINDINGS: Dogs that had been treated at the Clinic of Small Animal Medicine, LMU Munich, for CPV infection for which a follow-up of at least 12 months was available, were included in the study. Owners completed a questionnaire on the presence of chronic gastrointestinal and cutaneous signs, cardiac disease, and other potential disorders. An identical questionnaire was sent to owners of matched control dogs during the same time period. Seventy-one questionnaires of dogs with CPV infection and 67 of control dogs were analyzed. Significantly more CPV-infected dogs (30/71) compared to control dogs (8/67) had developed chronic gastrointestinal signs later in their lives (P < 0.001). No significant differences were observed regarding skin diseases (P = 1), cardiac problems (P = 0.160), or any other diseases (P = 0.173) later in life. CONCLUSIONS: Results of this study suggest that dogs that survive CPV infection have a significantly higher risk (odds ratio = 5.33) for developing a chronic gastrointestinal disease. Further prospective studies to identify the trigger for the development of chronic diarrhoea and possible targeted treatment strategies are needed.