Stefania Lauzi

Extended Genetic Diversity of Bovine Viral Diarrhea Virus and Frequency of Genotypes and Subtypes in Cattle in Italy between 1995 and 2013

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5′ UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction.

Molecular detection of Anaplasma platys, Ehrlichia canis, Hepatozoon canis and Rickettsia monacensis in dogs from Maio Island of Cape Verde archipelago

Tick-borne diseases are emerging worldwide and have an important zoonotic relevance. Dogs play an important role in the epidemiology of several zoonotic tick-borne pathogens acting as sentinels and/or reservoirs. This study focused on the molecular identification of tick-borne pathogens in blood samples of 153 autochthonous asymptomatic dogs in Maio Island, Cape Verde archipelago. Eighty-four (54.9%) dogs were positive for one or more pathogens. Fifty-five (35.9%) dogs were infected with Hepatozoon canis, 53 (34.6%) with Anaplasma platys, five (3.3%) with Ehrlichia canis and Rickettsia monacensis, an emerging human pathogen, was also identified in a single dog (0.7%). The former three pathogens cause important canine tick-borne diseases that are transmitted or potentially transmitted by Rhipicephalus sanguineus s.l., the only hard tick identified in Cape Verde. Furthermore, Wolbachia spp. was amplified from the blood of one dog. None of the dogs were positive for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Midichloria mitochondrii, Bartonella spp., Babesia spp. or Theileria spp. Fifty-four (35.3%) animals showed single infections and 30 (19.6%) co-infections, with A. platys and H. canis co-infection being the most frequent (28 dogs, 18.3%). The frequency of E. canis infection was statistically different among age groups (P=0.017), being higher among dogs older than 4 years compared to younger dogs. Infection by A. platys was also statistically different among age groups (P=0.031), being higher in dogs younger than 2 years compared to older dogs. The statistical analyses showed no significant association of PCR positivity with gender or location. The frequency of tick-borne pathogens detected in dogs in Maio Island, including R. monacensis, highlights the need to improve diagnosis and control in order to prevent the risk of transmission of these pathogens among dogs and humans living in or travelling to this touristic island.

Relationship between paraoxonase 1 activity and high density lipoprotein concentration during naturally occurring babesiosis in dogs

Paraoxonase 1 (PON1) is a negative acute phase protein bound to high density lipoproteins (HDL) and during the acute phase response (APR) protects HDL from peroxidation. The aim of this study was to assess the relationship between PON1 and HDL in canine babesiosis, a disease characterized by oxidative damages and by an APR. PON1, HDL and C-reactive protein (CRP), were measured in blood collected from 15 controls and 29 dogs with babesiosis sampled at admission, and on days 1 and 7 after treatment. At admission, PON1 and HDL were significantly lower in affected dogs. HDL concentration increased at day 1 while PON1 increased and CRP decreased at day 7. This suggests that the decrease of PON1 at admission is in part due to an increased consumption, the decreased HDL may depend on lipid peroxidation and its rapid increase after treatment may depend on the antioxidant activity of PON1.

Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

Abstract The Feline coronavirus (FCoV) is the etiological agent of feline infectious peritonitis (FIP), a lethal disease of felids. The role of molecular methods is controversial for the diagnosis of FIP, while essential for the identification of the shedders. Thus, a fast and inexpensive method for the detection of FCoV could be beneficial, especially in multicat environments. A reverse transcription loop mediated isothermal amplification (RT-LAMP) assay was developed. RNA extraction and RT-nPCR for FCoV were performed on thirty-two samples (11 faeces, 9 blood, 8 effusions, and 4 lymph nodes) collected from 27 cats. Six RT-LAMP primers were designed from the same conserved region of RT-nPCR, and the assay was run at 63°C for one hour. Results were evaluated through both agarose gel run and hydroxynapthol blue (HNB) dye and then compared with RT-nPCR results for the assessment of sensitivity and specificity. The overall specificity was 100%, but the sensitivity was 50% and 54.5% for agarose gel and HNB respectively. Therefore, RT-LAMP seems optimal to confirm the presence of the virus, but not applicable to exclude it.

Buffy coat smear or Knott's test: which to choose for canine microfilaria screening in field studies?

BACKGROUND In recent years, an increasing number of cases of canine dirofilariasis have been reported worldwide. However, the rate of infection in dogs is largely unknown in many remote areas, and the importance of field studies for determination of the prevalence of canine dirofilariasis in such areas is well recognized. The detection of microfilariae by the modified Knotts test (MKT) is a recommended screening method for canine dirofilariasis. OBJECTIVES The purposes of this study were to compare the diagnostic sensitivity of the MKT with the buffy coat smear method (BCS), and to evaluate the utility of these 2 methods under field study conditions. METHODS One hundred and fifty dogs of the Maio Island of Republic of Cabo Verde, were screened for microfilariae using MKT and BCS. The results of the 2 methods were generated in a blinded manner and statistically compared. RESULTS The detection rate was 4.67% with the MKT and 5.33% with BCS, which is statistically not different. The latter allowed a morphologic identification of Dirofilaria repens (later confirmed by molecular biology methods) and an estimation of parasite load, which varied from 15 to 185 microfilariae/mL. CONCLUSIONS The methods MKT and BCS were comparable in terms of diagnostic sensitivity. However, the BCS was technically less demanding and produced permanent preparations, in which co-infection with other hematologic pathogens can easily be assessed. Overall, this method is well suited to assess microfilariae in a large number of animals, and it could replace the MKT in studies devoted to dirofilariasis.

The occurrence of the filarial nematode Dirofilaria repens in canine hosts from Maio Island, Cape Verde.

The prevalence of canine Dirofilaria infection in Maio Island (Cape Verde) was analysed by serology, morphological and molecular identification of the parasite species. Blood and sera were collected from 150 dogs and 80 cats aged over 6 months from various localities of the island. DNA was extracted from blood and samples were screened by polymerase chain reaction (PCR) using microfilaria-specific primers. No Dirofilaria immitis was found in dogs while D. repens microfilariae were found in 5.3% of dogs and 6% were positive by PCR. The species identity was confirmed by sequencing of PCR products, which showed almost 100% homology with D. repens European sequences published in GenBank. No difference in Dirofilaria infection was observed between males and females or in dogs with different weights. However, older dogs and those from the western part of Maio Island were more frequently infected. No Dirofilaria was found in cats. This study represents the first evidence of D. repens in Cape Verde (West Africa) and highlights the need for implementing control measures and for a better surveillance of dirofilariosis in Africa.

Comparison of three blood transfusion guidelines applied to 31 feline donors to minimise the risk of transfusion-transmissible infections

Objectives The increased demand for animal blood transfusions creates the need for an adequate number of donors. At the same time, a high level of blood safety must be guaranteed and different guidelines (GLs) deal with this topic. The aim of this study was to evaluate the appropriateness of different GLs in preventing transfusion-transmissible infections (TTI) in Italian feline blood donors. Methods Blood samples were collected from 31 cats enrolled as blood donors by the owners’ voluntary choice over a period of approximately 1 year. Possible risk factors for TTI were recorded. Based on Italian, European and American GLs, specific TTI, including haemoplasmas, feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), Anaplasma phagocytophilum, Ehrlichia species, Bartonella species, Babesia species, Theileria species, Cytauxzoon species, Leishmania donovani sensu lato and feline coronavirus (FCoV), were screened. Rapid antigen and serological tests and biomolecular investigations (PCR) were used. Several PCR protocols for haemoplasma and FeLV DNA were compared. Results The presence of at least one recognised risk factor for TTI was reported in all cats. Results for FIV and FeLV infections were negative using rapid tests, whereas five (16.1%) cats were positive for FCoV antibodies. Four (12.9%) cats were PCR positive for haemoplasma DNA and one (3.2%) for FeLV provirus, the latter being positive only using the most sensitive PCR protocol applied. Other TTI were not detected using PCR. Conclusions and relevance Blood safety increases by combining the recommendations of different GLs. To reduce the risk of TTI, sensitive tests are needed and the choice of the best protocol is a critical step in improving blood safety. The cost and time of the screening procedures may be reduced if appropriate tests are selected. To this end, the GLs should include appropriate recruitment protocols and questionnaire-based risk profiles to identify suitable donors.

Comparison of the performance of laboratory tests in the diagnosis of feline infectious peritonitis

We compared the performance of clinicopathologic and molecular tests used in the antemortem diagnosis of feline infectious peritonitis (FIP). From 16 FIP and 14 non-FIP cats, we evaluated retrospectively the sensitivity, specificity, and likelihood ratios (LRs) of serum protein electrophoresis, α1-acid glycoprotein (AGP) on peripheral blood, screening reverse-transcription nested PCR (RT-nPCR) on the 3’–untranslated region (3’-UTR), and spike (S) gene sequencing on peripheral blood, body cavity effusions, and tissue, as well as body cavity cytology and delta total nucleated cell count (ΔTNC). Any of these tests on blood, and especially the molecular tests, may support or confirm a clinical diagnosis of FIP. A negative result does not exclude the disease except for AGP. Cytology, 3’-UTR PCR, and ΔTNC may confirm a clinical diagnosis on effusions; cytology or 3’-UTR PCR may exclude FIP. Conversely, S gene sequencing is not recommended based on the LRs. On tissues, S gene sequencing is preferable when histology is highly consistent with FIP, and 3’-UTR PCR when FIP is unlikely. Combining one test with high LR+ with one with low LR− (e.g., molecular tests and AGP on blood, ΔTNC and cytology in effusions) may improve the diagnostic power of the most used laboratory tests.

A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

Loop-mediated isothermal amplification (LAMP) is a molecular method that amplifies DNA under isothermal conditions. It relies on the use of 4 different primers recognizing 6 regions of the template sequence and on the use of a DNA polymerase with strand displacement activity (Notomi et al., 2000). The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002). The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensive detection of feline Coronavirus (FCoV). Six primers binding the conserved 3’UTR region of the FCoV were designed with the Primer Explorer software. Thirty-two samples of RNA (11 feces, 8 effusions, 9 blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (RT-PCR) for the 3’UTR region was performed were used. The reaction was carried out in 25μL reaction volume and the mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMP products were visualized under UV after electrophoresis migration on a 1.5% agarose gel stained with ethidium bromide, where they produce a ladder-like pattern if positive. Results where compared with those obtained on standard PCR. Sensitivity and specificity were respectively 60% and 100% on feces, 40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. The overall sensitivity and specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method, except for tissues.

Comparison between the diagnostic accuracy of clinico-pathological and molecular tests for feline infectious peritonitis (FIP)

The aim of this study was to compare the diagnostic accuracy for feline infectious peritonitis (FIP) of conventional clinic-pathological tests with that of molecular tests such as routine PCR and PCR followed by the sequencing of the Spike (S) gene. Blood, effusion and tissues specimens were collected from 21 FIP suspected cats. In vivo examination consisted of CBC, serum protein electrophoresis, AGP measurement, cytological and biochemical examination and the evaluation of the ΔTNC on effusions, and of molecular tests such the screening PCR (target: 3’UTR region) and the PCR directed towards the S gene followed by the amplification products sequencing in order to detect the aminoacidic substitution recently considered diagnostic for FIP 1 . These molecular techniques were applied to tissues collected during necropsy, which also allowed forming an FIP group (13 cats) and a non-FIP group (5 cats) based on histology and immunohistochemistry. The best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening PCR suffered of low specificity (spec: 33.3%) and the S gene sequencing showed low sensitivity (sens: 69.2%).On effusions, the best tests resulted screening PCR and cytology (sens and spec: 100%) in comparison with the ΔTNC measurement (sens: 85.7 %; spec: 100%) and the S gene sequencing (sens: 42.8%; spec: 100%).On blood, the best test resulted AGP measurement (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%). Screening PCR (sens: 55.6%; spec: 100%) and S gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy.