Fabrizio Passamonti

Epidemiological survey on equine cryptosporidium and giardia infections in Italy and molecular characterization of isolates.

Cryptosporidium and Giardia are two of the most common enteric pathogens of domestic and wild animals and humans. However, little is known on the prevalence, clinical manifestations and economic and zoonotic significance of these infections in horses. This study was undertaken to investigate the prevalence, excretion patterns and risk factors related to the faecal shedding of Cryptosporidium oocysts and Giardia cysts in horses and the zoonotic potential of species/genotypes isolated. The survey was performed on 120 foals and 30 broodmares reared in five Italian farms. Foals were divided in four homogeneous groups of 30 animals each (age classes: 0–2, 2–4, 4–8, >8 weeks). Three sequential faecal samples were collected from each animal and analysed by three techniques: direct fluorescent antibody test (DFA), faecal flotation (FF) and stained faecal smears (SFS). The DFA results showed a prevalence of 8% for Cryptosporidium and of 13.33% for Giardia; the prevalence values obtained by FF and SFS were lower and in poor agreement with DFA results. Giardia and Cryptosporidium infections were more common in foals (23.33% and 26.66% respectively) and higher excretions were observed in the youngest foals. Distribution of Cryptosporidium prevalence was statistically related to farms (P < 0.01), age of animals (P < 0.01), but was unrelated to the presence of diarrhoea. In the case of Giardia, the prevalence was only related to age (P < 0.01). Pattern sheddings were related to intestinal diseases and horse age (P < 0.01). Risk factors for shedding included residence farms and age older than 8 weeks for both parasites. All DFA‐positive faecal samples were submitted to DNA extraction and PCR to determine Giardia and Cryptosporidium species/genotypes. Sequence analysis of the COWP gene of Cryptosporidium and of the SSU‐rRNA gene of Giardia revealed that they were identical to each other and identified Cryptosporidium parvum and Giardia duodenalis assemblage E. The potential role of infected horses in zoonotic transmission of Cryptosporidium was supported by the findings of this study.

Molecular Detection, Epidemiology, and Genetic Characterization of Novel European Field Isolates of Equine Infectious Anemia Virus

ABSTRACT The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the “clades” described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.

Anaplasma phagocytophilum in horses and ticks: a preliminary survey of Central Italy.

Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, affects several species of wild and domesticated mammals, including horses. In this work we compared direct and indirect methods to evaluate A. phagocytophilum presence in Central Italy: 135 sera were screened by IFA for A. phagocytophilum and other haemopathogens (Theileria equi and Babesia caballi). Each horse was also tested for A. phagocytophilum 16S rRNA with a nested-PCR technique. In order to examine the risk of A. phagocytophilum transmission, 114 ticks were examined for the presence of A. phagocytophilum by PCR targeting the 16S rRNA. The seroprevalence against A. phagocytophilum was 17.03% and 11 horses (8.14%) showed positive PCR results. The concordance rate of A. phagocytophilum detection between IFAT and PCR had a K value of 0.34.

Quantification of Equid herpesvirus 5 DNA in clinical and necropsy specimens collected from a horse with equine multinodular pulmonary fibrosis

A 15-year-old Belgian gelding was referred for fever, depression, and respiratory distress. Lung biopsy revealed interstitial fibrosis consistent with chronic interstitial pneumonia. Equid herpesvirus 5 (EHV-5) DNA was detected by polymerase chain reaction (PCR) in bronchoalveolar lavage and biopsy specimens. A presumptive diagnosis of equine multinodular pulmonary fibrosis (EMPF) was made, and the horse was administered a systemic treatment with corticosteroids and antiviral drugs. Despite initial clinical improvement, 4 weeks later, the condition of the horse rapidly deteriorated, and the animal was euthanized. Postmortem examination confirmed the presumptive diagnosis of EMPF. The EHV-5 DNA load in different tissues was estimated using a quantitative real-time PCR. Lung had a remarkable viral load, higher than in other organs, especially within the pulmonary fibrotic nodules, and a linkage between high viral burden and the most severely affected tissues was observed. The results suggest that the quantitative real-time PCR is a useful tool to quantify the EHV-5 load in different organs and to understand the relationship between EHV-5 and EMPF. The bronchoalveolar lavage was determined to be a good clinical sample to estimate the EHV-5 load in lung.

Efficacy and safety of an infectious bursal disease virus intermediate vaccine in ovo.

The study was divided into two experiments. In the first experiment, the efficacy of in ovo intermediate vaccine against infectious bursal disease virus (IBDV) was determined by challenge at 21 days of age with virulent IBDV in specific-pathogen-free (SPF) and commercial chickens. This vaccine was able to induce active immunity and to protect SPF chickens to challenge; protection was not complete in commercial chickens, as testified by bursal lesions, bursal index after challenge, and vaccine immunoresponse. In order to detect field and vaccinal viruses, immunoperoxidase staining, enzyme-linked immunosorbent assay, capture, and reverse transcriptase-polymerase chain reaction (RT-PCR) were tested; the RT-PCR was more effective at detecting both kind of viruses. In the second experiment, the immunosuppressive effect of in ovo vaccination was determined by evaluating the immunoresponse against Newcastle disease virus (NDV) vaccination effected at 10 days in both SPF and commercial chickens vaccinated in ovo. The in ovo vaccine causes a reduction of NDV immunoresponse, as testified by lowest geometric mean titer in group I (SPF chickens vaccinated against IBDV in ovo and against NDV at 11 days). In commercial chickens, immunoresponse to NDV vaccination was not influenced by in ovo vaccination.

Geographic structuring of global EIAV isolates: a single origin for New World strains?

Equine infectious anaemia virus (EIAV) is classified within the Retroviridae and, like other lentivirus, has the propensity for considerable antigenic variation. An extensive phylogenetic analysis in Bayesian fashion, with significant amounts of new EIAV gag sequence information, revealed a strong geographic compartmentalization clearly related to the phylogeographic history of modern horses, pointing out that New World EIAV strains form a distinct group with a potentially common origin. This evidence suggests that a single founder event may have occurred during the reintroduction of horses to the Americas by European colonists in the 15th century, a possibility that raises many interesting scenarios with implications for all evolutionary and ecological studies.

Occurrence of Borrelia lusitaniae infection in horses.

The aim of the study was to investigate Borrelia burgdorferi sensu lato (s.l.) infection in horses exposed to heavy tick infestations. Blood samples of 98 healthy horses from 5 stud farms were examined by SNAP(®) 4D× and PCR to detect antibodies against B. burgdorferi s.l. and Borrelia DNA, respectively. Ten samples (15.3%) were antibody positive and 5 samples (5.1%) were both antibody and PCR positive. Sequence analysis showed the highest homology with the B. lusitaniae genospecies. No differences were found between sexes and stud farms, while age was significantly related to seropositivity (p<0.05). Our data confirms the presence of B. lusitaniae infection in horses, previously not clearly demonstrated.

Insertion sequence IS256 in canine pyoderma isolates of Staphylococcus pseudintermedius associated with antibiotic resistance

Staphylococcus pseudintermedius is the most frequent staphylococcal species isolated from canine pyoderma. The control of S. pseudintermedius infection is often difficult due to the expanded antimicrobial resistance phenotypes. Antibiotic resistance in staphylococcal pathogens is often associated to mobile genetic elements such as the insertion sequence IS256 that was first described as a part of the transposon Tn4001, which confers aminoglycoside resistance in Staphylococcus aureus and in Staphylococcus epidermidis. In this study a collection of 70 S. pseudintermedius isolates from canine pyoderma was used to investigate antimicrobial susceptibility to 15 antibiotics and the presence of IS256, not revealed in S. pseudintermedius yet. Antibiotic resistance profiling demonstrated that all S. pseudintermedius isolates had a multi-drug resistance phenotype, exhibiting simultaneous resistance to at least five antibiotics; indeed methicillin resistant S. pseudintermedius isolates were simultaneously resistant to at least nine antibiotics and all were also gentamicin resistant. PCR analyses revealed the presence of IS256 in 43/70 S. pseudintemedius isolates. The association between the presence of IS256 and the resistance was particularly significant for certain antibiotics: cefovecin, amikacin, gentamicin and oxacillin (χ(2)p-value<0.05). However, there was a striking result in frequency of strains resistant to gentamicin and oxacillin, suggesting a specific association between the presence of the IS256 element and the determinants for the resistance to these antibiotics. To the best of our knowledge, this is the first report showing the detection of IS256 in S. pseudintermedius isolates and its association with antibiotic resistance. Our findings suggest that S. pseudintermedius may acquire antibiotic resistance genes through mobile genetic elements which may play a predominant role in the dissemination of multi-drug resistance.

Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals

Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (ehv-1) and equine herpesvirus type 4 (ehv-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by pcr and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the PCR for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to ehv-1 and/or ehv-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.

Ultrasonographic findings in 38 horses with septic arthritis/tenosynovitis.

Septic arthritis/tenosynovitis in the horse can have life-threatening consequences. The purpose of this cross-sectional retrospective study was to describe ultrasound characteristics of septic arthritis/tenosynovitis in a group of horses. Diagnosis of septic arthritis/tenosynovitis was based on historical and clinical findings as well as the results of the synovial fluid analysis and/or positive synovial culture. Ultrasonographic findings recorded were degree of joint/sheath effusion, degree of synovial membrane thickening, echogenicity of the synovial fluid, and presence of hyperechogenic spots and fibrinous loculations. Ultrasonographic findings were tested for dependence on the cause of sepsis, time between admission and beginning of clinical signs, and the white blood cell counts in the synovial fluid. Thirty-eight horses with confirmed septic arthritis/tenosynovitis of 43 joints/sheaths were included. Degree of effusion was marked in 81.4% of cases, mild in 16.3%, and absent in 2.3%. Synovial thickening was mild in 30.9% of cases and moderate/severe in 69.1%. Synovial fluid was anechogenic in 45.2% of cases and echogenic in 54.8%. Hyperechogenic spots were identified in 32.5% of structures and fibrinous loculations in 64.3%. Relationships between the degree of synovial effusion, degree of the synovial thickening, presence of fibrinous loculations, and the time between admission and beginning of clinical signs were identified, as well as between the presence of fibrinous loculations and the cause of sepsis (P ≤ 0.05). Findings indicated that ultrasonographic findings of septic arthritis/tenosynovitis may vary in horses, and may be influenced by time between admission and beginning of clinical signs.