Occurrence and molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife: A systematic review and meta-analysis.

INTRODUCTION: Tick-borne pathogens (TBPs) constitute an emerging threat to public and animal health especially in the African continent, where land-use change, and wildlife loss are creating new opportunities for disease transmission. A review of TBPs with a focus on ticks determined the epidemiology of Rhipicephalus ticks in heartwater and the affinity of each Rickettsia species for different tick genera. We conducted a systematic review and meta-analysis to collect, map and estimate the molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife. MATERIALS AND METHODS: Relevant scientific articles were retrieved from five databases: PubMed, ScienceDirect, Scopus, Ovid and OAIster. Publications were selected according to pre-determined exclusion criteria and evaluated for risk of bias using the appraisal tool for cross-sectional studies (AXIS). We conducted an initial descriptive analysis followed by a meta-analysis to estimate the molecular prevalence of each pathogen. Subgroup analysis and meta-regression models were employed to unravel associations with disease determinants. Finally, the quality of evidence of every estimate was finally assessed. RESULTS: Out of 577 retrieved papers, a total of 41 papers were included in the qualitative analysis and 27 in the meta-analysis. We retrieved 21 Anaplasmataceae species, six Rickettsiaceae species and Coxiella burnetii. Meta-analysis was performed for a total of 11 target pathogens. Anaplasma marginale, Ehrlichia ruminantium and Anaplasma centrale were the most prevalent in African bovids (13.9 %, CI: 0-52.4 %; 20.9 %, CI: 4.1-46.2 %; 13.9 %, CI: 0-68.7 %, respectively). Estimated TBPs prevalences were further stratified per animal order, family, species and sampling country. DISCUSSION: We discussed the presence of a sylvatic cycle for A. marginale and E. ruminantium in wild African bovids, the need to investigate A. phagocytophilum in African rodents and non-human primates as well as E. canis in the tissues of wild carnivores, and a lack of data and characterization of Rickettsia species and C. burnetii. CONCLUSION: Given the lack of epidemiological data on wildlife diseases, the current work can serve as a starting point for future epidemiological and/or experimental studies.

A surveillance system for diseases of companion animals in the Veneto region (Italy).

Experts and international public health organisations stress the lack of surveillance systems for companion animal diseases and the need to implement such surveillance as a priority of the 'One Health' perspective. This paper presents the features of a system for the collection, analysis, interpretation and dissemination of data regarding the health status of pets in the Veneto region (Italy). The system involved the construction of a Web-based database containing the diagnoses of transmissible and non-transmissible diseases of dogs and cats made by veterinarians in their practices, hospitals, kennels and catteries. Each diagnosis constitutes a single record, also containing data on the identification of the individual animal and on several characteristics of epidemiological relevance. The World Health Organization (WHO) 10th revision of the International Classification of Diseases (ICD-10) for human diseases has been adapted to canine and feline diseases to standardise the diagnostic nomenclature. Software has been specifically created for online data entry and data management. The first results show that the main disorders were digestive (21%), dermatological (18%) and cardiovascular (11%) among 1,087 diagnostic records in dogs, and digestive (23%), dermatological (15%) and urinary (14%) among 289 diagnostic records in cats. The main causes of death are represented by cardiovascular (21%) and gastrointestinal (21%) diseases in dogs and by urinary (31%) disorders in cats. At present, no institutional surveillance system for companion animal health exists in Italy, and veterinarians joining this project and sharing the outcomes of their clinical activity are acting on a voluntary basis.

Aussi bien les experts que les organisations internationales oeuvrant dans le domaine de la santé publique soulignent l'absence de systèmes de surveillance dédiés aux maladies des animaux de compagnie, alors que la mise en place de cette surveillance constitue une priorité dans une perspective « Une seule santé ¼. Les auteurs décrivent les caractéristiques d'un système introduit en Vénétie (Italie) pour collecter, analyser, interpréter et diffuser des données sur la situation sanitaire des animaux de compagnie. Le système repose sur une base de données en ligne alimentée par les rapports de diagnostic sur les maladies transmissibles et non transmissibles des chiens et des chats établis par les vétérinaires dans leur cabinet, à l'hôpital ou dans les élevages ou pensions pour chiens et chats. Chaque diagnostic fait l'objet d'une notification spécifique où sont également consignées les données d'identification individuelle de l'animal et les caractéristiques pertinentes au plan épidémiologique. La classification internationale statistique des maladies (ICD­10) de l'Organisation mondiale de la santé (OMS), qui concerne les maladies humaines, a été adaptée aux maladies des chiens et des chats afin d'utiliser une nomenclature standardisée des diagnostics. Un logiciel spécifique a été créé pour la saisie en ligne des données et leur gestion. D'après les premiers résultats, les principales affections diagnostiquées étaient, chez le chien (sur 1 087 rapports de diagnostic), des maladies digestives (21 %), dermatologiques (18 %) et cardio-vasculaires (11 %) et, chez le chat (sur 289 rapports de diagnostic), des maladies digestives (23 %), dermatologiques (15 %) et urinaires (14 %). Les principales causes de mortalité étaient respectivement les maladies cardio-vasculaires (21 %) et gastro-intestinales (21 %) chez le chien et les maladies du système urinaire (31 %) chez le chat. À l'heure actuelle, aucun système institutionnel de surveillance n'est en place en Italie pour les animaux de compagnie, de sorte que les vétérinaires qui participent à ce projet et partagent leurs résultats cliniques le font sur une base volontaire.

Tanto especialistas como organizaciones internacionales dedicadas a temas de salud pública hacen hincapié en la ausencia de sistemas de vigilancia de las enfermedades de los animales de compañía y en la necesidad de instaurar tal vigilancia como elemento prioritario de los planteamientos de «Una sola salud¼. Los autores presentan las características de un sistema destinado a reunir, analizar, interpretar y difundir datos sobre el estado de salud de los animales de compañía en la región italiana del Veneto. Para instituir ese sistema se creó una base de datos en línea que centraliza información sobre los diagnósticos de enfermedades transmisibles y no transmisibles de perros y gatos realizados por veterinarios en el ejercicio de su labor en consultorios, hospitales y residencias caninas y felinas. Cada diagnóstico constituye un registro único, que también contiene datos sobre la identidad del animal en cuestión y sobre una serie de aspectos de importancia epidemiológica. Con objeto de normalizar la nomenclatura de diagnóstico se adaptó a las enfermedades caninas y felinas la Clasificación Internacional de Enfermedades, décima revisión (CIE­10), de la Organización Mundial de la Salud (OMS), que se aplica a las enfermedades humanas. También se crearon programas informáticos destinados específicamente a la introducción de datos en línea y a su gestión. Los primeros resultados muestran que los principales trastornos en los perros, de los 1.087 diagnósticos registrados, fueron los digestivos (21%), seguidos de los dermatológicos (18%) y los cardiovasculares (11%). En el caso de los gatos, con 289 diagnósticos registrados, las dolencias más importantes fueron las digestivas (23%), las dermatológicas (15%) y las urinarias (14%). En el perro, las principales causas de mortalidad fueron las enfermedades cardiovasculares (21%) y gastrointestinales (21%), y en el gato las patologías urinarias (31%). Actualmente no existe en Italia ningún sistema institucional de vigilancia de la salud de los animales de compañía, y los veterinarios que participan en este proyecto y comparten los resultados de su praxis clínica lo hacen con carácter voluntario.

A novel stop codon mutation within the hepatitis B surface gene is detected in the liver but not in the peripheral blood mononuclear cells of HIV-infected individuals with occult HBV infection.

To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV-) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV-DNA was measured by a quantitative real-time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV- patients, HBV-DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV-DNA was found in 6/7 (86%) PBMC of occult-positive and none of occult-negative LT. Significantly lower HBV-DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult-positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper-mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV-DNA detection in PBMC may offer a useful tool to identify OHB in serum-negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.

Comparative field study to evaluate the performance of three different traps for collecting sand flies in northeastern Italy.

Three standard methods for collecting sand flies (sticky trap, CDC light trap, and CO2 trap) were compared in a field study conducted from June to October, 2012, at a site located in the center of a newly established autochthonous focus of canine leishmaniasis in northeastern Italy. Six traps (two sticky traps, two CDC light traps, and two CO2 traps) were activated at the same time for a single night every two weeks during the season of sand fly activity. A total of 5,667 sand flies were collected and 2,213 identified, of which 82.1% were Phlebotomus perniciosus, 17.4% P. neglectus, 0.3% Sergentomya minuta, and 0.2% P. mascitti. The performances of all traps were influenced by their position inside the site, increasing with proximity to the animal shelters. CO2 traps were more attractive for females of P. perniciosus and P. neglectus. CDC light traps showed an intermediate efficiency and were more attractive for P. neglectus, compared to other two traps. Results suggest that in northern Italy the CO2 trap is a suitable sampling method for sand fly monitoring programs that include transmitted pathogen surveillance.

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle.

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.

New insights into the epidemiology of bovine piroplasmoses in Italy.

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques. Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively. Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.

Canine piroplasmosis in Italy: epidemiological aspects in vertebrate and invertebrate hosts.

In Italy, canine piroplasmosis is believed to be widespread, but few data are available on its presence in most areas. In 2005 and 2006, vertebrate and invertebrate hosts were investigated in Central and Northern Regions of the Country. Microscopy on blood smears, molecular tools and serological tests were applied to 420 blood samples collected from dogs, in order to evaluate the presence of these protozoa and to identify possible risk factors. Moreover, ticks were analyzed by molecular techniques. Microscopy identified as positive 2.8% of the animals, all from Central Italy, and PCR detected 'piroplasm' DNA in 6.0%. Serology evidenced a mean prevalence of 34.0% with a decreasing trend from Central to Northern areas. The 507 collected ticks were identified as belonging to 8 species, mostly represented by Rhipicephalus sanguineus (n=376) and Ixodes ricinus (n=58). Molecular analyses evidenced the presence of babesial parasites (Babesia canis canis, B. canis vogeli, B. microti-like) in 25 (4.9%) of them; in Rh. sanguineus there was also demonstration of the vertical transmission of B. canis canis. Statistical analysis identified 'kennel' as risk factor for Babesia infection. Our findings evidenced that different species of piroplasms potentially infectious for dogs are circulating in Italy, and that epidemiological aspects of these infections are more complex than expected. Vector importance of both Rh. sanguineus and I. ricinus is hypothesized, but further investigation is needed.

Low replication and variability of HBV pre-core in concomitant infection with hepatitis B and hepatitis C viruses.

In an attempt to define the virological profile of HBV in HCV co-infection, we analysed the viral load, the infecting genotype, and the mutational pattern of the HBV pre-core region (pre-C), which is involved in viral encapsidation and DNA replication. Eighty-six patients were studied: 32 with serological HBV/HCV-1b co-infection (group BC), 32 infected by HBV alone (group B), and 22 by HCV-1b alone (group C). Sequence analysis of the HBV pre-S and pre-C regions identified genotypes and mutational patterns. The HBV viral load was significantly lower in group BC than in group B (p < 0.001), and the distribution of HBV pre-C mutations showed a higher prevalence of wild type in concomitant infection than in the control group (p < 0.006). The predominant HBV infecting strain was genotype D in both the BC (96%) and B (87%) groups. No difference was observed in HCV viremia levels between the two groups, whereas in HBV/HCV infection, the low levels of circulating HBV were closely associated with the low degree of variability of pre-C domain (p = 0.005). In conclusion, in HBV/HCV infection, the virological pattern was characterised by the dominance of HCV associated with lower HBV replication capacity and decreased emergence of HBV pre-C variants.

Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma.

We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre-C/C) domain in hepatocellular carcinoma (HCC). Thirty-eight consecutive patients with HCC were included in the study - 18 of them with HBV/HCV co-infection and 20 with HBV single infection. Twenty-three additional patients with co-infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre-C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co-infection in comparison with single-infected HCC, whereas two different HBV viraemia profiles were detected in co-infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre-C/C region was found in co-infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co-infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre-C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre-C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co-infection could be linked with accelerated HBV/HCV-related disease progression.

Mutations in the E2-PePHD region of hepatitis C virus type 1b in patients with hepatocellular carcinoma.

An interaction between the protein kinase (PKR)-eIF2-alpha phosphorylation homology domain (PePHD) within the E2 protein of hepatitis C virus (HCV) and cell protein kinase (PKR) may affect the control of protein synthesis and cell growth. In an attempt to investigate the genetic variability of the E2-PePHD domain in hepatocellular carcinoma (HCC), we studied sera and liver tissues from HCC patients. The partial E2-PePHD region was analysed by direct sequencing of the sera of 47 HCCs in cirrhotic livers and 31 cases of chronic active hepatitis (CAH), and tumoral and non-tumoral liver tissues from 13 HCC patients. A similar number of mutations was detected within the E2 domain in the HCC and CAH cases, but nine of the 47 HCCs (19%) showed an amino acid (aa) mutation at position 660, eight of which involved a change in the same aa (alanine instead of serine; A/S). No such mutation was detected in any of the PePHD sequences from the CAH patients: this difference was statistically significant (P = 0.008). The aa change at position 660 was also found in two sequences from tumoral but not non-tumoral tissue from the same liver. The analysis of 461 sequences obtained from GenBank supports the conclusion that the observed aa change is an infrequent event in HCV-infected patients, thus suggesting that it could be associated with HCC.

Genetic variability of hepatitis C virus in HBV/HCV co-infection and HCV single-infection.

To describe the virological profile of HCV in HBV/HCV co-infection, we investigated the variability of HVR-1 and NS5A domains, which may be involved in viral persistence and replication efficiency. We studied 95 patients: 37 with serological markers of HBV/HCV co-infection, 33 with single HBV and 25 with single HCV infection. HVR-1 complexity and NS5A gene variability were respectively explored by means of PCR-SSCP and direct sequencing. Serum HBV genomes were detected in all coinfected patients: 19 also had circulating HCV particles (group BC-I), whereas HCV were undetectable in the other 18 (group BC-II). Group BC-I was characterised by a significantly lower HBV replication capacity, that reflects the replicative dominance of HCV, although the dominant virus had the same degree of variability as the HCV in single infection. HBV viral load was higher in group BC-II, but not significantly different from that observed in the single infection. Our data indicate an alternation in replicative dominance in co-infection: HBV can suppress HCV replication to undetectable levels, whereas HCV may reduce but does not abrogate the replication capacity of HBV. Furthermore, in the cases of HCV dominance, circulating HBV genomes did not have a significant effect on the viral heterogeneity of HCV.

New infection with heterotypic hepatitis C virus in a patient with long-term hepatitis C virus eradication.

BACKGROUND: Experimental hepatitis C virus infection in chimpanzees has shown that natural hepatitis C virus infection does not induce protective immunity and reinfection can occur in seroconverted animals. AIM: To study the clinical, virological and histological outcome of a new infection sustained by a different hepatitis C virus strain after a primary infection with eradication of the original virus. PATIENTS AND METHODS: A young Italian man with chronic hepatitis C virus type 4 hepatitis was treated with Interferon therapy and achieved a sustained biochemical and virological response. After long follow-up, an asymptomatic flare-up of alanine transaminase occurred. This alanine transaminase increase was associated with serum hepatitis C virus RNA positivity and a low viral load, and the infecting hepatitis C virus genotype was type 3. The clinical and virological course of this new infection is described. RESULTS AND CONCLUSIONS: This report shows that there is no protective immunity against hepatitis C virus type 3 after infection by hepatitis C virus type 4 strain.