Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement.

Essentials A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development. Mutation type-inhibitor risk association was explored in 231 patients with severe hemophilia A. A 2-fold increase in INH development for in silico null vs. non-null mutations was found. A 3.5-fold increase in INH risk for antigen negative vs. antigen positive mutations was found. SUMMARY: Background The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non-null. Objectives To explore the mutation type-inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) randomized trial. Methods The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma. Results A two-fold increase in inhibitor development for in silico null mutations as compared with in silico non-null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84-5.17) and a 3.5-fold increase in inhibitor development for antigen-negative mutations as compared with antigen-positive mutations (HR 3.61, 95% CI 0.89-14.74] were found. Conclusions Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance.

Loss of CD45 cell surface expression in canine T-zone lymphoma results from reduced gene expression.

Canine T-zone lymphoma (TZL) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant CD45-negative phenotype, commonly recognized by flow cytometry (FC). Recent studies have described clinical presentation and behavior, but to date the mechanisms behind the loss of CD45 protein expression have never been investigated. The aims of this study were: 1) to confirm the absence of CD45 in canine TZL via the concomitant use of FC and immunohistochemistry with two different sources of antibody; and 2) to investigate the amount of CD45 transcript and the presence of CD45 gene in the neoplastic cells of dogs affected by TZL. 57 lymph node aspirates were included in the present study: 40 (70.2%) TZLs, 7 (12.3%) high grade T-cell lymphomas and 10 (17.5%) reactive lymph nodes. Neoplastic cells and normal T-cells were isolated from TZL and reactive lymph nodes, respectively, via cell sorting. Immunohistochemistry was performed on 2 TZL, 2 reactive lymph nodes and 2 Peripheral T-cell Lymphomas. Total RNA and genomic DNA were extracted from lymph-nodes aspirates. Two different quantitative real-time PCR experiments were designed, to determine the amount of the CD45 transcript and of the corresponding gene fragment. All TZL cases were negative for CD45 at immunohistochemistry. CD45 transcript amount was significantly lower in TZL compared to controls (p<0.001). This difference was not significant (p=0.584) for CD45 DNA load, that was similar between TZL and controls. Moreover, CD45 transcript amount was inversely correlated with the percentage of neoplastic cells in each TZL sample (p=0.010). These results confirm that CD45 protein is lacking on cell surface irrespective of the technique and antibody source adopted. This phenotypic aberrancy is apparently due to the absence of gene transcription, as CD45 DNA was present, whereas CD45 transcript was virtually absent in the neoplastic cells. The data here reported support further studies investigating possible factors impairing CD45 gene transcription.

VEGF and MMP-9: biomarkers for canine lymphoma.

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF-ß) stimulates VEGF and MMPs production. VEGF and TGF-ß plasma levels were tested by ELISA, MMP-2 and MMP-9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act-MMP-9 (P < 0.01) and VEGF (P < 0.05), and lower TGF-ß than controls, and a positive correlation between act-MMP-9 and VEGF (P < 0.001). Act-MMP-9 and VEGF were significantly higher in T-cell lymphomas, and in stage V compared with stages III-IV disease, regardless of immunophenotype. VEGF was higher in high-grade compared with low-grade T-cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act-MMP-9 and VEGF decreased in B-cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers.

Immunophenotype-related microRNA expression in canine chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are posttranscriptional regulatory noncoding RNAs used to profile human hematopoietic tumors. In this study, some mature miRNAs was quantitated in peripheral blood from dogs with chronic lymphocytic leukemia (CLL). Relative expression data were normalised against four endogenous controls (let-7a, miR-17-5p, miR-26b, and miR-223) selected by geNorm analysis. The results revealed distinct miRNA patterns in CLL depending on the immunophenotype. Also in dogs, the different miRNAs expression could reflect developmental lineage and tumor differentiation. The similar genetics, physiology and exposure to environment in dogs and humans make the miRNA expression study in canine CLL attractive for comparative oncology.

Prenatal diagnosis and preimplantation genetic diagnosis: novel technologies and state of the art of PGD in different regions of the world.

Prenatal diagnosis (PND) aims to provide accurate, rapid results as early in pregnancy as possible. Conventional PND involves sampling cells of foetal origin by chorionic villus sampling at 11-14th weeks of pregnancy or amniocentesis after 15th week. These are invasive procedures and have a small but significant rate of 0.5% to 1% for loss of pregnancy. An alternative to existing methods for conventional PND for couples at risk of transmitting a genetic disease to their child is preimplantation genetic diagnosis (PGD). PGD is a newly emerging form of a very early prenatal diagnosis. The technique combines assisted reproductive technology with molecular genetics and cytogenetics to allow the identification of abnormality in embryos prior to implantation. The diagnosis of genetic disease in human preimplantation embryos was pioneered in the late 1980s for testing of aneuploidy, single gene and X-linked disease, such as cystic fibrosis, haemophilia and chromosomal abnormalities. The PGD-related legal and ethical issues have been debated at many levels both nationally and internationally. The attitude towards PGD varies substantially not only in different parts of the world but also within the Europe, owing to scientific, cultural and religious differences. PGD has become widely practised throughout the world for various indications and can substantially decrease the eventual risks of passing a genetic undesired condition of the offspring. Nevertheless, its extension to some new and non-medical indications has raised ethical concerns, in particular its potential eugenic dimension.

Non-invasive tool for foetal sex determination in early gestational age.

Free foetal DNA in maternal blood during early pregnancy is an ideal source of foetal genetic material for non-invasive prenatal diagnosis. The aim of this study was to evaluate the use of free foetal DNA analysis at early gestational age as pretest for the detection of specific Y-chromosome sequences in maternal plasma of women who are carriers of X-linked disorders, such as haemophilia. Real-time quantitative PCR analysis of maternal plasma was performed for the detection of the SRY or DYS14 sequence. A group of 208 pregnant women, at different gestational periods from 4 to 12 weeks, were tested to identify the optimal period to obtain an adequate amount of foetal DNA for prenatal diagnosis. Foetal gender was determined in 181 pregnant women sampled throughout pregnancy. Pregnancy outcome and foetal gender were confirmed using karyotyping, ultrasonography or after birth. The sensitivity, which was low between 4th and 7th week (mean 73%), increased significantly after 7+1th weeks of gestation (mean 94%). The latter sensitivity after 7+1th week of gestation is associated to a high specificity (100%), with an overall accuracy of 96% for foetal gender determination. This analysis demonstrates that foetal gender determination in maternal plasma is reliable after the 9th week of gestation and it can be used, in association with ultrasonography, for screening to determine the need for chorionic villus sampling for prenatal diagnosis of X-linked disorders, such as haemophilia.

Immunophenotype predicts survival time in dogs with chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a hematologic disorder in dogs, but studies on prognostic factors and clinical outcome are lacking. In people, several prognostic factors have been identified and currently are used to manage patients and determine therapy. OBJECTIVES: The aim of the study was to determine if the immunophenotype of neoplastic cells predicts survival in canine CLL. DESIGN: Retrospective study. ANIMALS: Forty-three dogs with CLL. PROCEDURES: Records of dogs with a final diagnosis of CLL were reviewed. For each included dog, a CBC, blood smear for microscopic reevaluation, and immunophenotyping data had to be available. Data on signalment, history, clinical findings, therapy, follow-up, as well as date and cause of death were retrieved. RESULTS: Seventeen dogs had B-CLL (CD21+), 19 had T-CLL (CD3+ CD8+), and 7 had atypical CLL (3 CD3- CD8+, 2 CD3+ CD4- CD8-, 1 CD3+ CD4+ CD8+, and 1 CD3+ CD21+). Among the variables considered, only immunophenotype was associated with survival. Dogs with T-CLL had approximately 3-fold and 19-fold higher probability of surviving than dogs with B-CLL and atypical CLL, respectively. Old dogs with B-CLL survived significantly longer than did young dogs, and anemic dogs with T-CLL survived a significantly shorter time than dogs without anemia. CONCLUSIONS: Although preliminary, results suggested that immunophenotype is useful to predict survival in dogs with CLL. Young age and anemia are associated with shorter survival in dogs with B-CLL and T-CLL, respectively.

Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood.

Dirofilaria immitis and Dirofilaria repens are the most common species of filarial nematodes described in the dogs with increasing spread into new geographical areas. The diagnosis of canine dirofilariosis is usually based upon the microscopical detection and identification of circulating microfilariae together with ELISA detection of serum circulating heartworm antigens or antibodies. The identification of the parasite species using the traditional approaches sometimes can be difficult and can lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. In this paper we report a new molecular method based on single-step multiplex PCR to detect and differentiate simultaneously and unequivocally D. immitis and D. repens on DNA extracted from canine peripheral blood. The amplification was performed using a set of primers designed on a portion of the small subunit ribosomal RNA gene of the mitochondrion (12S rDNA). The single-step multiplex PCR here described ensured high (4 mf/ml) sensitivity and specificity with reduced cost and time saving. The multiplex PCR assay represents an additional tool for epidemiological studies and routine disease assessment in areas co-endemic for the two Dirofilaria species.

Identification of suitable endogenous controls and differentially expressed microRNAs in canine fresh-frozen and FFPE lymphoma samples.

The elucidation of microRNA (miRNA) expression pattern in canine lymphoma is attractive for veterinary and comparative oncology due to similar genetics, physiology and exposure to environment in dogs and humans. In this work, the expression of a panel of mature miRNAs was quantitated in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) lymph nodes from canine lymphoma. The major findings were: the detection of a panel of miRNAs expressed in canine lymph node; the identification of three suitable endogenous controls (let-7a, miR-16, and miR-26b) by NormFinder and geNorm analysis; the concordance between results obtained from fresh-frozen and FFPE samples; the detection of upregulation of miR-17-5p and miR-181a in B- and T-cell lymphomas respectively. This is the first study aimed to the application of miRNAs analysis in canine lymphoma.

ZAP-70 and Syk expression in canine lymphoid cells and preliminary results on leukaemia cases.

Zeta-chain-associated protein (ZAP-70) and spleen tyrosine kinase (Syk) are structurally and functionally homologous tyrosine kinases playing a role in the T- and B-cell signal transduction. Their activation can lead to lymphokine production, cytolitic activity, antibody secretion, cell proliferation, differentiation, survival and phagocytosis. Anomalous ZAP-70 and Syk expression is reported to be related to tumor formation and progression, and ZAP-70 immunoreactivity is a good prognostic marker of disease progression in human chronic lymphocytic leukaemia (CLL). Until now, to our knowledge, there are no reports about canine ZAP-70 and Syk expression profiles. In the present study, a RT-PCR procedure for the quali-quantitative evaluation of canine ZAP-70 and Syk transcripts was designed. The expression patterns of canine ZAP-70 and Syk mRNAs were evaluated in canine leukocyte subpopulations and in peripheral whole blood samples from healthy dogs and from dogs with different types of leukaemia. Similarly to humans, normal canine CD4+ and CD8+ T cells showed high expression of ZAP-70, whereas Syk was abundantly expressed in normal CD21+ B cells. The expression profile of ZAP-70 and Syk was markedly different in canine normal and leukaemic blood. Decreased Syk expression was detected in dogs with T-cell CLL, whereas decreased ZAP-70 expression was detected in dogs with B-cell CLL and B-cell acute lymphocytic leukaemia (ALL). The comparison of ZAP-70 and Syk mRNA levels between normal and leukaemic peripheral whole blood showed that the expression ratio ZAP-70/Syk is subjected to modification depending on the leukaemia status of patients. The results of the present work open an interesting topic for leukaemogenesis investigation and are the basis for further studies for a proper evaluation of the potential utility of these parameters for the diagnosis and prognosis of canine T- and B-cell leukaemia.

Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection.

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0-6, 10-12, 16-18, 22-26, and 28-34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.

Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs.

There is still a pressing need for effective adulticide treatment for human and animal filarial infections. Like many filarial nematodes, Dirofilaria immitis, the causative agent of canine heartworm disease, harbours the bacterial endosymbiont Wolbachia, which has been shown to be essential for worm development, fecundity and survival. Here the authors report the effect of different treatment regimens in dogs experimentally infected with adult D. immitis on microfilariemia, antigenemia, worm recovery and Wolbachia content. Treatment with ivermectin (IVM; 6 microg/kg per os weekly) combined with doxycycline (DOXY; 10 mg/kg/day orally from Weeks 0-6, 10-12, 16-18, 22-26 and 28-34) resulted in a significantly faster decrease of circulating microfilariae and higher adulticidal activity compared with either IVM or DOXY alone. Quantitative PCR analysis of ftsZ (Wolbachia DNA) and 18S rDNA (nematode DNA) absolute copy numbers showed significant decreases in Wolbachia content compared with controls in worms recovered from DOXY-treated dogs that were not, however, associated with worm death. Worms from IVM/DOXY-treated dogs, on the other hand, had Wolbachia/nematode DNA ratios similar to those of control worms, suggesting a loss of both Wolbachia and nematode DNA as indicated by absolute copy number values. Histology and transmission electron microscopy of worms recovered from the IVM/DOXY combination group showed complete loss of uterine content in females and immunohistochemistry for Wolbachia was negative. Results indicate that the combination of these two drugs causes adult worm death. This could have important implications for control of human and animal filarial infections.

Wolbachia surface protein (WSP) inhibits apoptosis in human neutrophils.

Polymorphonuclear cells (PMNs) are essential for the innate immune response against invading bacteria. At the same time, modulation of PMNs' apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Wolbachia bacteria are Gram-negative endosymbionts of filarial nematodes and arthropods, phylogenetically related to the genera Anaplasma, Ehrlichia and Neorickettsia (family Anaplasmataceae). Although several pathogens are known to interfere with apoptosis, there is only limited information on specific proteins that modulate this phenomenon. This is the first evidence for the anti-apoptotic activity of a surface protein of Wolbachia from filarial nematode parasites (the Wolbachia surface protein, WSP). The inhibition of apoptosis was demonstrated on purified human PMNs in vitro by different methods. TUNEL assay showed that the percentage of dead cells was reduced after stimulation with WSP; Annexin V-FITC binding assay confirmed that cell death was due mainly to apoptosis and not to necrosis. Reduced caspase-3 activity in stimulated cells also confirmed an inhibition of the apoptotic process.

Is heartworm disease really spreading in Europe?

Based on recently published surveys and newly acquired data, a study was conducted to verify the distribution of filarial worm (Filarioidea) infections in Europe, with particular emphasis on canine heartworm infection (Dirofilaria immitis). A Geographic Information System based on thermal regimen was constructed as a means to identify areas potentially suitable for heartworm transmission, taking into account that the development of D. immitis larvae in the mosquito does not occur below the threshold temperature of approximately 14 degrees C. Furthermore, a bionomic model of D. immitis in its mosquito vectors, which calculates the moving cumulative heartworm development unit parameter, was applied using the available temperature data to assess the theoretic transmission timing of heartworm in Europe. The results show that the earliest infection risk occurs in Spain on March 21 and the latest risk occurs in Spain on September 11. The longest risk period occurs in Spain (Murcia station: March 21-November 11), and the shortest risk period occurs in northeastern Europe. The study also provides the first risk assessment maps for Europe and suggests that if the actual climatic trend continues, filarial infection should spread into previously infection-free areas.

Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections.

Sera from three groups of cats under different experimental conditions were studied by ELISA to assess the host's immune response against synthetic peptides derived from Dirofilaria immitis (Dipp) and against the surface protein of its endosymbiont, Wolbachia (WSPr). In experimentally infected cats (Group 1), an increase of IgG antibody against both Dipp and WSPr was observed from 2 months post-infection until the end of the study, 6 months post-infection. In experimentally infected cats, treated against infective larvae (Group 2), anti-Dipp IgG decreased dramatically from 4 months post-infection (3 months post treatment), showing very low values till the end of the study (6.5 months from infection, 5.5 months from treatment), while anti-WSP IgG increased constantly till the end of the study. Of 49 outdoor, asymptomatic cats exposed to a high risk of natural infection (Group 3), 9 were positive for anti-Dipp IgG and for a validated, in-clinic commercial antibody diagnostic kit for cats. Two cats were also found positive for circulating antigens of adult female worm. Anti-WSPr IgG were found in five of nine anti-Dipp IgG-positive sera and from eight ELISADipp-negative sera. Our results confirm the strong IgG response in heartworm infected cats and demonstrate the involvement of the Wolbachia endosymbiont in the immune reaction to the parasite both in experimentally infected cats and in cats exposed to a high risk of natural infection.

[Quantitative PCR in the diagnosis of Leishmania]. / PCR quantitativa nella diagnosi di Leishmania.

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

[Monitoring of canine leishmaniasis in northern Italy: an update from a scientific network]. / Monitoraggio della leishmaniosi canina in nord Italia: aggiornamenti da un network scientifico.

Canine leishmaniasis (CanL) due to Leishmania infantum is a disease of great veterinary importance and a serious public health problem. In humans, L. infantum causes visceral (VL) and cutaneous leishmaniasis (CL) and the distribution of VL overlaps that of CanL. Currently, VL is considered by WHO as an emerging zoonosis in southern Europe. The dog is the only domestic reservoir of the infection and phlebotomine sandflies are the only proven vectors of leishmaniasis for dogs and humans. CanL is endemic in Italy, particularly in central and southern regions, including islands. Until 1983, all regions of northern Italy but Liguria and some territories of Emilia Romagna were considered free from CanL. From early '90s new stable foci of CanL have appeared, most of them located within classical endemic areas including territories of Emilia Romagna, Tuscany, Umbria, Marche, and Abruzzi regions. But the most relevant aspect, from an epidemiological point of view, has been the appearance of stable CanL foci in northern Italy, namely in Veneto and Piedmont regions. In these two foci, entomological surveys showed the presence of P. perniciosus and of a second phlebotomine vector, P. neglectus, which may have played a role in the CanL diffusion in some parts of northern Italy. Furthermore, in these areas, autochthonous human VL cases have occurred. There is therefore a realistic risk that CanL infection could rapidly spread through northern latitudes and a surveillance activity is strongly needed. For this reason, in October 2002, thanks to the collaboration and support of Intervet Italia, the network "LeishMap" was created, with the main purpose of monitoring the spread of CanL and vectors in northern Italy. LeishMap consists of scientific and sanitary institutions with proven experience both in field surveys and diagnostic methodologies on CanL and phlebotomine vector. It is organised in 4 Operational Units (OU), represented by researchers of the Veterinary Faculties of the University of Bologna, Padua, Milan and Turin, under the scientific coordination of the MIPI Department, ISS of Rome and with the collaboration of private and public veterinarians operating in the regions under study. During the first year of activity, each OU was involved in the serological and entomological surveillance of several territories in the respective regions, where recent autochthonous CanL cases were registered. The studies have involved five regions, namely Valle D'Aosta, Piedmont, Lombardia, Veneto, Trentino-Alto Adige and Emilia Romagna. In the Symposium 6 of this Congress we report detailed results of a retrospective analysis of data concerning CanL and vectors in northern Italy till 2002 and the preliminary results of 2003 on the seroprevalence rates observed in foci studied and on the entomological surveys carried out. In summary, the results outlined that already known foci of CanL are expanding from the original sites. Several new foci have been identified and many others are at high risk of evolving toward a stable endemicity. P. perniciosus has been found in all but one the suspected new foci. In Emilia Romagna region P. perfiliewi was identified in 2 areas and in one was the only species present. The occurrence of P. neglectus was confirmed in three regions, Veneto, Lombardia and Piedmont. In conclusion, from the 2002-2003 LeishMap activities it appears that further monitoring activities are necessary to identify new endemic foci of CanL, this representing the prerequisite for the implementation of programs for leishmaniasis control in northern Italy.

Molecular systematics and diagnosis.

This collection of articles provides an account of six presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology(WAAVP) (held in New Orleans, Louisiana, USA, from 10 to 14 August 2003) in a symposium session on Molecular Systematics and Diagnosis, organised and chaired by R.B. Gasser and D.S. Zarlenga. The focus was on recent advances in molecular tools for specific and genotypic identification,diagnosis, systematics and population genetics, with special emphasis on investigations of parasitic nematodes and protists.