Targeted genotyping by sequencing: a new way to genome profile the cat.

Targeted GBS is a recent approach for obtaining an effective characterization for hundreds to thousands of markers. The high throughput of next-generation sequencing technologies, moreover, allows sample multiplexing. The aims of this study were to (i) define a panel of single nucleotide polymorphisms (SNPs) in the cat, (ii) use GBS for profiling 16 cats, and (iii) evaluate the performance with respect to the inference using standard approaches at different coverage thresholds, thereby providing useful information for designing similar experiments. Probes for sequencing 230 variants were designed based on the Felis_catus_8.0. 8.0 genome. The regions comprised anonymous and non-anonymous SNPs. Sixteen cat samples were analysed, some of which had already been genotyped in a large group of loci and one having been whole-genome sequenced in the 99_Lives Cat Genome Sequencing Project. The accuracy of the method was assessed by comparing the GBS results with the genotypes already available. Overall, GBS achieved good performance, with 92-96% correct assignments, depending on the coverage threshold used to define the set of trustable genotypes. Analyses confirmed that (i) the reliability of the inference of each genotype depends on the coverage at that locus and (ii) the fraction of target loci whose genotype can be inferred correctly is a function of the total coverage. GBS proves to be a valid alternative to other methods. Data suggested a depth of less than 11× is required for greater than 95% accuracy. However, sequencing depth must be adapted to the total size of the targets to ensure proper genotype inference.

Doxycycline levels and anti-Wolbachia antibodies in sera from dogs experimentally infected with Dirofilaria immitis and treated with a combination of ivermectin/doxycycline.

Sera from Dirofilaria immitis-experimentally infected dogs treated with a combination of ivermectin/doxycycline were analysed for doxycycline levels by HPLC and anti-Wolbachia Surface Protein (rWSP) antibodies by ELISA and compared with sera from dogs treated with doxycycline alone. Results show that doxycycline levels were not statistically different between the two groups. Circulating anti-WSP antibody titres were markedly lower in both treatment groups when compared to control D. immitis infected dogs, indicating that doxycycline is able to reduce Wolbachia and prevent the immune response against the bacteria. The combination treatment protocol has been shown to be highly adulticidal and further studies are needed to better understand the interaction between doxycycline and ivermectin in D. immitis infected dogs.

The adulticide effect of a combination of doxycycline and ivermectin in Dirofilaria immitis-experimentally infected dogs is associated with reduction in local T regulatory cell populations.

In a previous study, dogs experimentally infected with Dirofilaria immitis were treated with either ivermectin or doxycycline or a combination of both. The adulticide effect was significantly higher in the dogs treated with both drugs and was similar to that observed in dogs treated with melarsomine hydrochloride. In the present study, lung tissue samples from these dogs were evaluated for the presence of T regulatory (Foxp3+) cells by immunohistochemistry. Cells were enumerated for each dog in the four groups and compared with untreated controls. There was a significantly lower number of Treg cells in those dogs treated with a combination of both drugs when compared either to the control group or to the other groups treated with either drug alone or with melarsomine. These results suggest that successful adulticide effects of doxycycline and ivermectin are associated with a decrease in immune regulation towards the parasite.

Molecular characterization of Echinococcus granulosus in south-eastern Romania: evidence of G1-G3 and G6-G10 complexes in humans.

Clin Microbiol Infect ABSTRACT: Echinococcus granulosus is the aetiological agent of cystic echinococcosis (CE), which is a public health problem in many eastern European countries, particularly in Romania, where the infection causes a high number of human and animal cases. To shed light on the transmission patterns of the parasite, we performed a genotyping analysis on 60 cyst samples obtained from patients who live in south-eastern Romania and who underwent surgery for liver or lung CE. DNA was extracted from the endocysts or the cyst fluids, and fragments of cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 1 mitochondrial genes (cox1 and nd1, respectively) were amplified by PCR and sequenced. We found that most of the samples analysed (59/60) belonged to the G1-G3 complex (E. granulosus sensu stricto), which contains the most widespread and infective strains of the parasite. We also identified the first human patient infected by a non-G1-G3 genotype of E. granulosus in this country. As the DNA sequence of this cyst sample showed maximum homology with the G6-G10 complex (Echinococcus canadensis), this is, in all likelihood, a G7 genotype, which is often found in pigs and dogs in most countries of eastern and south-eastern Europe.

Genetic variability of Haemonchus contortus (Nematoda: Trichostrongyloidea) in alpine ruminant host species.

Genetic variability of the ovine parasite Haemonchus contortus from the Alpine area was investigated using mitochondrial DNA (nd4 gene), internal transcribed spacers 1 and 2 and microsatellites, in order to assess whether cross-transmission between domestic and wild ruminants occurs. The dataset was composed of 78 individual adult male H. contortus collected from chamois (Rupicapra r. rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domestic goat (Capra hircus) and sheep (Ovis aries) from different alpine areas. The data obtained show low host specificity and high genetic variation within H. contortus populations. The analyses indicate the presence of two mitochondrial haplotype clusters among host species and the absence of cryptic parasite species, confirming H. contortus as a generalist nematode and suggesting that parasite transmission between populations of domestic and wild ruminants normally occurs.

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.

Prevalence of Setaria equina microfilaraemia in horses in Hungary.

Peripheral blood samples were collected randomly from 195 horses in various parts of Hungary, and the presence of microfilariae was evaluated by the Knott technique. On the basis of morphological identification 18 of the horses (9.2 per cent) were infected with Setaria equina, and the infection was confirmed in 10 animals by pcr and sequencing. The level of microfilaraemia was between 1 and 1138 larvae in 2 ml of blood. There was no correlation between the time of sampling or the sex of the animals (stallions versus mares) and the prevalence of infection, but the prevalence decreased with age. There was a significant association between the prevalence of microfilaraemia and the presence of still waters; positive samples were collected either in the region of Lake Balaton, the largest lake in the country, or at places with nearby ponds.

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.

iNOs expression is stimulated by the major surface protein (rWSP) from Wolbachia bacterial endosymbiont of Dirofilaria immitis following subcutaneous injection in mice.

The bacterial endosymbiont Wolbachia of several species of filarial nematodes plays an important role in the inflammatory pathology of filariasis. Nitric oxide (NO) production has also been implicated in the immune response during filarial infections. Here we present data indicating that a recombinant Wolbachia surface protein (rWSP) induces iNOs mRNA expression and NO production, as well as IFN-gamma and a Th1-type antibody response, in inoculated BALB/c mice. This effect is not observed when mice are inoculated with a recombinant heat shock protein from Wolbachia (GroEL).

Dogs with patent Dirofilaria immitis infection have higher expression of circulating IL-4, IL-10 and iNOS mRNA than those with occult infection.

Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival.

Is Wolbachia complicating the pathological effects of Dirofilaria immitis infections?

Human and animal parasitic filarial nematodes, which often are the cause of severe disease, harbor intracellular bacteria of the genus Wolbachia (Rickettsiaceae). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. In order to determine the possible role of Wolbachia in heartworm disease, dogs naturally infected with Dirofilaria immitis were studied for specific antibody response to Wolbachia surface protein (WSP). Antibody subclasses were analyzed to determine immune response polarization. Dogs that died from heartworm disease were necropsied, and various organs were studied by immunohistochemistry to determine whether Wolbachia-derived molecules were present in tissue from infected dogs. Humoral response to the WSP was present in all infected dogs and appeared to be predominantly of the Th1-type. Several organs, including lung, liver, and kidney, contained positive-staining cells for WSP, confirming that the canine host does come into contact with Wolbachia-derived molecules.

[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.

Molecular characterisation of a field strain of bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological reactivation.

Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.

Recent advances in heartworm disease.

This compilation of articles consists of four papers presented at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (held in New Orleans, LA, USA, on 10­14 August 2003) in a symposium session titled " Recent Advances in Heartworm Disease," organized and chaired by JohnW. McCall and Jorge Guerrero. The first paper(Guerrero) covered the American Heartworm Society's most recent revision of their guidelines for the diagnosis, prevention, and management of heartworm infection in dogs, based on new research and clinical experience, particularly in the areas of heartworm chemoprophylaxis, adulticide therapy,and serologic testing and retesting. The entire updated 2003 "Guidelines" are presented herein.One paper (McCall) reviewed the "soft-kill" adulticidal and "safety-net" (reach-back, retroactive,clinical prophylactic) activity of prolonged dosing of prophylactic doses of macrocyclic lactones,concluding that ivermectin is the most effective in this way, milbemycin oxime is the least effective,and the activity of injectable moxidectin and selamectin lies between that of ivermectin and milbemycin oxime. The two remaining papers provided an overview of the discovery, rediscovery,phylogeny, and biological association between Wolbachia endosymbionts and filarial nematodes(Genchi and co-authors) and compelling evidence that Wolbachia may play a major role in the immunopathogenesis of filarial diseases of man and animals (Kramer and co-authors).

Sequencing of the complete gene coding for the GroEL of the Wolbachia of Dirofilaria immitis and expression and purification of the recombinant protein.

Wolbachia are intracellular bacteria that infect arthropods and filarial nematodes. These bacteria play an important role in the immunology and pathogenesis of filarial diseases through their proteins and, possibly, other molecules. GroEL is a constitutively expressed bacterial protein; it is highly conserved among bacteria and is involved in the correct folding of newly synthesized proteins. Here we report the production of recombinant GroEL from the Wolbachia of Dirofilaria immitis. Our goal is to test the hypothesis that GroEL is involved in the immunopathology of filariases. The complete groel gene was PCR-amplified, sequenced and cloned into an expression vector. The recombinant GroEL was purified by affinity chromatography by using high-performance liquid chromatography (HPLC).

Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8.

Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.