Andrea Balboni

Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats

Parvoviruses of carnivores include three closely related autonomous parvoviruses: canine parvovirus (CPV), feline panleukopenia virus (FPV) and mink enteritis virus (MEV). These viruses cause a variety of serious diseases, especially in young patients, since they have a remarkable predilection for replication in rapidly dividing cells. FPV is not the only parvovirus species which infects cats; in addition to MEV, the new variants of canine parvovirus, CPV-2a, 2b and 2c have also penetrated the feline host-range, and they are able to infect and replicate in cats, causing diseases indistinguishable from feline panleukopenia. Furthermore, as cats are susceptible to both CPV-2 and FPV viruses, superinfection and co-infection with multiple parvovirus strains may occur, potentially facilitating recombination and high genetic heterogeneity. In the light of the importance of cats as a potential source of genetic diversity for parvoviruses and, since feline panleukopenia virus has re-emerged as a major cause of mortality in felines, the present study has explored the molecular characteristics of parvovirus strains circulating in cat populations. The most significant findings reported in this study were (a) the detection of mixed infection FPV/CPV with the presence of one parvovirus variant which is a true intermediate between FPV/CPV and (b) the quasispecies cloud size of one CPV sample variant 2c. In conclusion, this study provides new important results about the evolutionary dynamics of CPV infections in cats, showing that CPV has presumably started a new process of readaptation in feline hosts.

Genetic diversity and molecular epidemiology of Anaplasma

Anaplasma are obligate intracellular bacteria of cells of haematopoietic origin and are aetiological agents of tick-borne diseases of both veterinary and medical interest common in both tropical and temperate regions. The recent disclosure of their zoonotic potential has greatly increased interest in the study of these bacteria, leading to the recent reorganisation of Rickettsia taxonomy and to the possible discovery of new species belonging to the genus Anaplasma. This review is particularly focused on the common and unique characteristics of Anaplasma marginale and Anaplasma phagocytophilum, with an emphasis on genetic diversity and evolution, and the main distinguishing features of the diseases caused by the different Anaplasma spp. are described as well.

Detection of a virus related to betacoronaviruses in Italian greater horseshoe bats.

The association between coronaviruses and bats is a worldwide phenomenon and bats belonging to genus Rhinolophus are the reservoir host for several coronaviruses, including a large number of viruses closely related genetically to severe acute respiratory syndrome-coronavirus (SARS-CoV). We carried out a survey in colonies of Italian bats (Rhinolophus ferrumequinum) for the presence of coronaviruses. Two of 52 R. ferrumequinum captured from different Italian areas tested positive by reverse transcription-PCR for a fragment of RNA-dependent RNA polymerase (RdRp) gene of viruses related to Coronavirus. Phylogenetic analysis revealed close correlations between one of the positive samples and SARS-related CoV belonging to the genus Betacoronavirus.

Tetracycline Susceptibility in Chlamydia suis Pig Isolates

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

Antibody-neutralizing activity against all urogenital Chlamydia trachomatis serovars in Chlamydia suis-infected pigs

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.

Molecular analysis of carnivore Protoparvovirus detected in white blood cells of naturally infected cats

BackgroundCats are susceptible to feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants 2a, 2b and 2c. Detection of FPV and CPV variants in apparently healthy cats and their persistence in white blood cells (WBC) and other tissues when neutralising antibodies are simultaneously present, suggest that parvovirus may persist long-term in the tissues of cats post-infection without causing clinical signs. The aim of this study was to screen a population of 54 cats from Sardinia (Italy) for the presence of both FPV and CPV DNA within buffy coat samples using polymerase chain reaction (PCR). The DNA viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the positive cats.ResultsCarnivore protoparvovirus 1 DNA was detected in nine cats (16.7%). Viral DNA was reassembled to FPV in four cats and to CPV (CPV-2b and 2c) in four cats; one subject showed an unusually high genetic complexity with mixed infection involving FPV and CPV-2c. Antibodies against parvovirus were detected in all subjects which tested positive to DNA parvoviruses.ConclusionsThe identification of FPV and CPV DNA in the WBC of asymptomatic cats, despite the presence of specific antibodies against parvoviruses, and the high genetic heterogeneity detected in one sample, confirmed the relevant epidemiological role of cats in parvovirus infection.

A60 Molecular epidemiology of canine parvovirus type 2 (CPV-2) in Italy

Lastly, we describe the nearly complete sequence of the Wuhan Tick Virus-like (WTV-like), which is comprised of one negativesense single stranded RNA molecule, with 11,208 nucleotides. This virus was classified into Chuviridae family and encodes the polymerase, glycoprotein, nucleoprotein, and VP4. Interestingly, the tick pools from all the four sites of collection were positive for MGTV, LT2V-like, and WTV-like viruses, indicating that these viruses can be found in the tick population of a large area of the Brazilian territory, which is an important cattle producing region in the country and one of the leading regions in animal produce exportation. On the other hand, only the MGTV was simultaneously detected in 19.4 per cent (7/36) serum cattle, indicating viremia in these animals. In summary, we have identified three potentially novel tick-borne viruses with broad distributions in South of Brazil, which include potential novel pathogens for cattle.

Prospective evaluation of rapid point-of-care tests for the diagnosis of acute leptospirosis in dogs

The early diagnosis of acute leptospirosis is still a major challenge in dogs. The aim of this prospective study was to evaluate the suitability of two in-clinic tests detecting anti-leptospiral IgM and IgG antibodies in diagnosing canine leptospirosis. The performances of the two rapid tests were compared to the microscopic agglutination test (MAT) carried out on acute sera and to diagnostic criteria adopted in this study to confirm leptospirosis infection (MAT upon admission, convalescent MAT and quantitative real-time PCR on blood and/or urine). The dogs were enrolled on the basis of reported exposure to known risk factors and clinical presentation (acute kidney injury and/or systemic inflammatory response syndrome with multi-organ damage). Eighty-nine dogs included in the study were sub-grouped on the basis of the results of the diagnostic criteria adopted: (1) confirmed leptospirosis cases (42/89 dogs); (2) negative leptospirosis cases (36/89 dogs); and (3) unconfirmed leptospirosis cases (11/89 dogs). The results supported the usefulness of the two rapid diagnostic tests as a first in-clinic screening tool for suspected leptospirosis; positive results in the in-clinic tests in dogs with suggestive clinical and laboratory signs strongly indicated acute leptospirosis, while negative results required additional diagnostic investigation to exclude the infection. Confirmatory tests recommended for canine leptospirosis are still necessary in addition to the use of rapid in-clinic tests.

Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission

A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (−29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23-fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS.

A screening for canine distemper virus, canine adenovirus and carnivore protoparvoviruses in Arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) from Arctic and sub-Arctic regions of Norway

ABSTRACT Canine distemper virus (CDV), canine adenovirus (CAdV) and canine parvovirus type 2 (CPV-2) cause disease in dogs (Canis familiaris). These, or closely related viruses, may also infect wild carnivores. The aim of this study was to investigate exposure to CDV, CAdV and CPV-2 among fox populations in Norway. Arctic foxes (n = 178) from High-Arctic Svalbard were investigated for antibodies against CDV. Arctic foxes (n = 301) from Svalbard and red foxes from Low-Arctic (n = 326) and sub-Arctic (n = 74) regions in Finnmark County, Norway, were investigated for antibodies against CAdV and for the presence of carnivore protoparvovirus DNA in spleen and mesenteric lymph nodes using polymerase chain reaction. Seroprevalence against CDV in Arctic foxes decreased from 25% (1995/96) to 6% (2001/02), whereas the seroprevalence against CAdV increased from 25–40% during the seasons 1995/96 to 2001/02 to 68% for the last study year (2002/03). In red foxes, the seroprevalence against CAdV varied between 31% and 67% for the seasons 2004/05 to 2007/08, increasing to 80% for the last study year. Carnivore protoparvovirus DNA was not detected in any of the 301 Arctic foxes and the 265 red foxes investigated. These results show that CDV and CAdV are enzootic in the Arctic fox population (Svalbard), and that CAdV is enzootic in both the Low-Arctic and sub-Arctic red fox populations (Finnmark). Further studies are needed to better understand the infection biology and the impact of CDV and CAdV in these fox populations, and if viruses may be shared between foxes and other carnivores, including dogs.