Detection of vector-borne pathogens in owned dogs with cranial cruciate ligament rupture living in the Mediterranean area.

BACKGROUND: Cranial cruciate ligament rupture (CCLR) results from a multifactorial degenerative process that leads to rupture of the ligament. Vector-borne pathogens (VBP) in dogs can induce joint disease but their role in CCLR has not been previously investigated. The aim of the present work is to evaluate the prevalence of VBP in dogs with CCLR. METHODS: This was a prospective study that included 46 dogs presented for CCLR surgical treatment and 16 control dogs euthanized for diseases unrelated to the joints. Specimens collected included blood, synovial fluid, and synovial membrane biopsy. Pathogen testing consisted of serology for Leishmania infantum (quantitative ELISA), Ehrlichia canis/ewingii, Borrelia burgdorferi, Anaplasma phagocytophilum/platys, and Dirofilaria immitis (4DX IDEXX test), and PCR for L. infantum, Ehrlichia/Anaplasma spp., Bartonella spp., piroplasms (Babesia spp. and Theileria spp.), and filariae (D. immitis, Dirofilaria repens, Acanthocheilonema dracunculoides, Acanthocheilonema reconditum, and Cercopithifilaria spp.) on both EDTA-whole blood (EB) and synovial fluid (SF) samples. SF cytology and histopathological evaluation of synovial membrane were also performed. RESULTS: The prevalence of VBP was 19.6% in the CCLR group and 18.8% in the control group, with no statistical difference among them. The presence of synovitis was not more frequent in CCLR dogs (45.6%) than in control dogs (43.7%). Lymphoplasmacytic infiltration was the most common inflammatory pattern detected in the joints of both groups of dogs. CONCLUSIONS: This study failed to demonstrate a role of canine VBP in CCLR or the presence or different pattern of joint inflammation in pathogen-positive dogs.

In Situ Plant Virus Nucleic Acid Isothermal Amplification Detection on Gold Nanoparticle-Modified Electrodes.

Solid-phase isothermal recombinase polymerase amplification (RPA) offers many benefits over the standard RPA in homogeneous phase in terms of sensitivity, portability, and versatility. However, RPA devices reported to date are limited by the need for heating sources to reach sensitive detection. With the aim of overcoming such limitation, we propose here a label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle (AuNP)-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Plant disease ( Citrus tristeza virus (CTV)) diagnostics was selected as a relevant target for demonstration of the proof-of-concept. RPA assay for amplification of the P20 gene (387-bp) characteristic of CTV was first designed/optimized and tested by standard gel electrophoresis analysis. The optimized RPA conditions were then transferred to the AuNP-modified electrode surface, previously modified with a thiolated forward primer. The in situ-amplified CTV target was investigated by EIS in a Fe(CN6)4-/Fe(CN6)3- red-ox system, being able to quantitatively detect 1000 fg µL-1 of nucleic acid. High selectivity against nonspecific gene sequences characteristic of potential interfering species such as Citrus psorosis virus (CPsV) and Citrus caxicia viroid (CCaV) was demonstrated. Good reproducibility (RSD of 8%) and long-term stability (up to 3 weeks) of the system were also obtained. Overall, with regard to sensitivity, cost, and portability, our approach exhibits better performance than RPA in homogeneous phase, also without the need of heating sources required in other solid-phase approaches.

Skin mites in mice (Mus musculus): high prevalence of Myobia sp. (Acari, Arachnida) in Robertsonian mice.

Myobia sp. and Demodex sp. are two skin mites that infest mice, particularly immunodeficient or transgenic lab mice. In the present study, wild house mice from five localities from the Barcelona Roberstonian system were analysed in order to detect skin mites and compare their prevalence between standard (2n = 40) and Robertsonian mice (2n > 40). We found and identified skin mites through real-time qPCR by comparing sequences from the mitochondrial 16S rRNA and the nuclear 18S rRNA genes since no sequences are available so far using the mitochondrial gene. Fourteen positive samples were identified as Myobia musculi except for a deletion of 296 bp out to 465 bp sequenced, and one sample was identified as Demodex canis. Sampling one body site, the mite prevalence in standard and Robertsonian mice was 0 and 26%, respectively. The malfunction of the immune system elicits an overgrowth of skin mites and consequently leads to diseases such as canine demodicosis in dogs or rosacea in humans. In immunosuppressed mice, the probability of developing demodicosis is higher than in healthy mice. Since six murine toll-like receptors (TLRs) are located in four chromosomes affected by Robertsonian fusions, we cannot dismiss that differences in mite prevalence could be the consequence of the interruption of TLR function. Although ecological and/or morphological factors cannot be disregarded to explain differences in mite prevalence, the detection of translocation breakpoints in TLR genes or the analysis of TLR gene expression are needed to elucidate how Robertsonian fusions affect the immune system in mice.

Corrigendum: Individual Signatures Define Canine Skin Microbiota Composition and Variability.

[This corrects the article on p. 6 in vol. 4, PMID: 28220148.].

Drivers of Bartonella infection in micromammals and their fleas in a Mediterranean peri-urban area.

People living at the human/wildlife interface are at risk of becoming infected with Bartonella for which micromammals act as reservoir. We aimed to determine the factors related to the prevalence of Bartonella and its haplotype diversity in micromammals and in their fleas in a Mediterranean peri-urban environment. We analyzed 511 micromammals, chiefly 407 wood mice (Apodemus sylvaticus), captured into Barcelona metropolitan area (Spain) in spring and autumn from 2011 to 2013 in two natural and two adjacent residential areas, their fleas (grouped in 218 monospecific pools) and 29 fetuses from six Bartonella-positive female wood mice. Amplification of a fragment of ITS was carried out by real time PCR. Prevalence was 49% (57% in the dominant species, the wood mouse), and 12 haplotypes were detected. In general, prevalence was higher in those hosts more heavily infested by fleas, coincident with higher rates of capture, in autumn than in spring, and in adults than in juveniles. Prevalence did not differ between natural and residential areas except for one prevalent haplotype, which was more frequent in natural areas. Prevalence in flea pools (58%) was only explained by Bartonella occurrence in the pool host. In 56.4% of the flea pools with identified Bartonella haplotypes, we found the same haplotype in the host and in its flea pool. Prevalence in wood mouse fetuses was 69%, with at least one infected fetus in all litters, and two litters with all the fetuses infected. indicating that vertical transmission might be important in Bartonella epidemiology in the wood mouse. There is a hazard of Bartonella infection for people living in residential areas and those visiting peri-urban natural areas in Barcelona.

First description of Bartonella koehlerae infection in a Spanish dog with infective endocarditis.

BACKGROUND: Bartonella koehlerae has been recently described as a new cat- and cat fleas-associated agent of culture-negative human endocarditis. It has been also encountered in one dog from Israel and six dogs from the USA, but other clinically relevant reports involving this bacterium are lacking. RESULTS: A 7-year-old intact male mixed dog presented with clinico-pathological signs consistent with mitral endocarditis and cutaneous hemangiosarcoma. Molecular studies revealed the presence of Bartonella koehlerae DNA in samples from blood and mitral valve tissue. CONCLUSIONS: This is the first description of B. koehlerae in Spain, corroborating that it can also be detected in dogs. Bartonella koehlerae infection should also be considered in Spain in humans and dogs presenting with clinical disease suggestive of it, such as culture-negative endocarditis.

Afoxolaner and fluralaner treatment do not impact on cutaneous Demodex populations of healthy dogs.

BACKGROUND: Fluralaner and afoxolaner are isoxazolines licensed for the treatment of flea and tick infestations. Isoxazolines have also shown efficacy for treatment of demodicosis. Nothing is known about the impact of these compounds on the populations of Demodex in healthy dogs. HYPOTHESIS/OBJECTIVES: The objective of this study was to measure the prevalence of Demodex in the skin of healthy dogs prior to and following the use of either afoxolaner or fluralaner, using real-time PCR (RT-PCR) for Demodex DNA. Our hypothesis was that the use of an isoxazoline at the labelled dose would eliminate Demodex populations from the skin of healthy dogs. ANIMALS AND METHODS: Twenty healthy dogs with no history of skin disease were recruited. Dogs were divided into two groups of ten, with each group receiving afoxolaner or fluralaner for the 90 day study period. Hairs were plucked from three body sites on Day 0 prior to medication administration, then again on days 30 and 90. RT-PCR amplifying Demodex DNA was performed on all samples. RESULTS: At Day 0 (prior to treatment), five of the 20 dogs were positive for Demodex DNA at least in one skin site (25%). At Day 60, three of 18 dogs were positive (16.7%) and on Day 90, six of 20 dogs were positive (30%). No significant difference in numbers of positive dogs was found between groups or timepoints. CONCLUSION: Treatment with afoxolaner or fluralaner does not impact on cutaneous Demodex populations of normal dogs over a 90 day period.

Molecular detection of vector-borne pathogens in blood and splenic samples from dogs with splenic disease.

BACKGROUND: The spleen is a highly perfused organ involved in the immunological control and elimination of vector-borne pathogens (VBP), which could have a fundamental role in the pathogenesis of splenic disease. This study aimed to evaluate certain VBP in samples from dogs with splenic lesions. METHODS: Seventy-seven EDTA-blood and 64 splenic tissue samples were collected from 78 dogs with splenic disease in a Mediterranean area. Babesia spp., Bartonella spp., Ehrlichia/Anaplasma spp., Hepatozoon canis, Leishmania infantum, hemotropic Mycoplasma spp. and Rickettsia spp. were targeted using PCR assays. Sixty EDTA-blood samples from dogs without evidence of splenic lesions were included as a control group. RESULTS: More than half (51.56%) of the biopsies (33/64) were consistent with benign lesions and 48.43% (31/64) with malignancy, mostly hemangiosarcoma (25/31). PCR yielded positive results in 13 dogs with spleen alterations (16.67%), for Babesia canis (n = 3), Babesia gibsoni (n = 2), hemotropic Mycoplasma spp. (n = 2), Rickettsia massiliae (n = 1) and "Babesia vulpes" (n = 1), in blood; and for B. canis, B. gibsoni, Ehrlichia canis and L. infantum (n = 1 each), in spleen. Two control dogs (3.3%) were positive for B. gibsoni and H. canis (n = 1 each). Benign lesions were detected in the 61.54% of infected dogs (8/13); the remaining 38.46% were diagnosed with malignancies (5/13). Infection was significantly associated to the presence of splenic disease (P = 0.013). There was no difference in the prevalence of infection between dogs with benign and malignant splenic lesions (P = 0.69); however B. canis was more prevalent in dogs with hemangiosarcoma (P = 0.006). CONCLUSIONS: VBP infection could be involved in the pathogenesis of splenic disease. The immunological role of the spleen could predispose to alterations of this organ in infected dogs. Interestingly, all dogs with B. canis infection were diagnosed with hemangiosarcoma in the present survey. As previously reported, results support that VBP diagnosis could be improved by analysis of samples from different tissues. The sample size included here warrants further investigation.

Individual Signatures Define Canine Skin Microbiota Composition and Variability.

Dogs present almost all their skin sites covered by hair, but canine skin disorders are more common in certain skin sites and breeds. The goal of our study is to characterize the composition and variability of the skin microbiota in healthy dogs and to evaluate the effect of the breed, the skin site, and the individual. We have analyzed eight skin sites of nine healthy dogs from three different breeds by massive sequencing of 16S rRNA gene V1-V2 hypervariable regions. The main phyla inhabiting the skin microbiota in healthy dogs are Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, and Bacteroidetes. Our results suggest that skin microbiota composition pattern is individual specific, with some dogs presenting an even representation of the main phyla and other dogs with only a major phylum. The individual is the main force driving skin microbiota composition and diversity rather than the skin site or the breed. The individual is explaining 45% of the distances among samples, whereas skin site explains 19% and breed 9%. Moreover, analysis of similarities suggests a strong dissimilarity among individuals (R = 0.79, P = 0.001) that is mainly explained by low-abundant species in each dog. Skin site also plays a role: inner pinna presents the highest diversity value, whereas perianal region presents the lowest one and the most differentiated microbiota composition.

Early reduction of Leishmania infantum-specific antibodies and blood parasitemia during treatment in dogs with moderate or severe disease.

BACKGROUND: Leishmania infantum-specific antibodies are used extensively for the diagnosis and monitoring of treatment in canine leishmaniosis. Different views have been described for the measurement of L. infantum antibody levels for the monitoring of anti-leishmanial treatment. In addition, molecular techniques using blood are frequently employed in the clinical setting. However, there are not enough studies to prove the usefulness of PCR in diagnosis, treatment monitoring and in assessing the prognosis of the disease. The objectives of this study were to evaluate L. infantum-specific antibodies and blood parasitemia at the time of diagnosis and during treatment and to correlate these with the dog's clinical status. METHODS: Thirty-seven dogs were diagnosed and followed-up during treatment (days 30, 180 and 365). The treatment protocol consisted of a combination of meglumine antimoniate for one month and allopurinol for at least one year. Leishmania infantum-specific antibodies and blood parasitemia were assessed by an end point sera dilution ELISA and by real-time PCR, respectively. RESULTS: The majority of dogs were classified as LeishVet stage II (moderate disease) at the time of diagnosis (86 %) and the rest as stage III. Results showed variable levels of specific antibodies at the time of diagnosis [median ± interquartile range (IQR): 1372 ± 8803 ELISA units (EU)]. Twenty-three seropositive dogs (64 %) were detected as PCR-positive at the time of diagnosis. Interestingly, a rapid significant antibody level reduction was observed by day 30 of treatment (median ± IQR: 604 ± 2168 EU). A continuing significant decrease of specific antibodies was also found at days 180 (median ± IQR: 201 ± 676 EU) and 365 (median ± IQR: 133 ± 329 EU) in association with clinical improvement. A significant blood parasitemia reduction was also observed at all time points studied. Mean parasites/ml ± SD were 19.4 ± 79.1 on day 0, 2.2 ± 11.7 on day 30, 0.9 ± 2.9 on day 180, and 0.3 ± 0.7 on day 365. CONCLUSIONS: This study reports a significant reduction of L. infantum antibodies measured by an end point sera dilution ELISA method after 30 days of treatment associated with clinical improvement. A low proportion of sick dogs with moderate disease were negative by blood real-time PCR at the time of diagnosis.

Molecular detection of Leishmania infantum, filariae and Wolbachia spp. in dogs from southern Portugal.

BACKGROUND: Leishmaniosis caused by the protozoan Leishmania infantum and dirofilariosis caused by the nematodes Dirofilaria immitis or Dirofilaria repens are vector-borne zoonoses widely present in the Mediterranean basin. In addition, some studies reported that the endosymbiont Wolbachia spp. play a role in the biology and pathogenesis of filarial parasites. The aim of this work was to evaluate the frequency of mono- and co-infections by L. infantum, filariae and Wolbachia spp. and their association with clinical signs in dogs from the south of Portugal. Leishmanial, filarial and Wolbachia spp. DNA were evaluated by specific real-time polymerase chain reaction (qPCR) assays in blood samples from 230 dogs. FINDINGS: One hundred and thirty-nine (60.4 %) dogs were qPCR-positive for L. infantum and 26 (11.3 %) for filariae (24 for D. immitis only, one D. immitis and for Acanthocheilonema dracunculoides and another one for Acanthocheilonema reconditum only). Wolbachia spp. DNA was amplified from 16 (64.0 %) out of the 25 D. immitis-positive dogs. Nineteen (8.3 %) dogs were co-infected with L. infantum and D. immitis, including the one (0.4 %) A. drancunculoides-positive animal. In dogs without clinical signs consistent with leishmaniosis and/or dirofilariosis, L. infantum prevalence was 69 %, whereas in those dogs with at least one clinical manifestation compatible with any of the two parasitoses prevalence was 42.7 %. Leishmania prevalence was significantly higher in apparently healthy mongrels (77.2 %) and pets (76.9 %) than in defined-breed dogs (including crosses; 58.8 %) and in dogs with an aptitude other than pet (i.e. farm, guard, hunting, shepherd or stray), respectively, whereas in those dogs with at least one clinical sign, the detection of L. infantum DNA was higher in males (53.3 %) and in those dogs not receiving insect repellents (52.8 %). CONCLUSIONS: The molecular detection of canine vector-borne disease (CVBD) agents, some of which are zoonotic, reinforces the need to implement efficient prophylactic measures, such as insect repellents and macrocyclic lactones (including compliance to administration), in the geographical areas where these agents are distributed, with the view to prevent infection and disease among mammalian hosts including humans.

Detection of vector-borne pathogens in cats and their ectoparasites in southern Italy.

BACKGROUND: Vector-borne pathogens are the subject of several investigations due to the zoonotic concern of some of them. However, limited data are available about the simultaneous presence of these pathogens in cats and their ectoparasites. The aim of the present study was to define the species of ectoparasites found on cats as well as to investigate vector-borne pathogens in cats and their ectoparasites in southern Italy. METHODS: Blood from 42 cats and fleas or flea pools (n = 28) and ticks (n = 73) collected from them were investigated by quantitative PCR for the detection of vector-borne pathogens. Feline serum samples were tested by IFAT to detect IgG antibodies against Leishmania infantum, Bartonella henselae, Rickettsia conorii, Rickettsia felis, Rickettsia typhi, Babesia microti, Ehrlichia canis and Anaplasma phagocytophilum antigens. RESULTS: Only one flea species (Ctenocephalides felis) and four tick species belonging to the genera Rhipicephalus and Ixodes were identified on cats from southern Italy. Molecular evidence of Bartonella spp., Rickettsia spp., hemoplasmas, Babesia vogeli and L. infantum was found in ectoparasites (fleas and/or ticks) while DNA from Hepatozoon felis and Ehrlichia/Anaplasma spp. was not detected. Likewise, DNAs from Bartonella, hemoplasma and Leishmania were the only pathogens amplified from feline blood samples. Cats had also antibodies against all the investigated pathogens with the exception of Rickettsia typhi. Agreement between serological and molecular results in individual cats and their ectoparasites was not found. The only exception was for Bartonella with a fair to moderate agreement between individual cats and their ectoparasites. Bartonella clarridgeiae was the species most frequently found in cats and their fleas followed by B. henselae. CONCLUSIONS: In conclusion, cats harboring ticks and fleas are frequently exposed to vector-borne pathogens. Furthermore, ticks and fleas harbored by cats frequently carry pathogens of zoonotic concern therefore appropriate feline ectoparasiticide preventative treatments should be used in cats.

Magnetic Bead/Gold Nanoparticle Double-Labeled Primers for Electrochemical Detection of Isothermal Amplified Leishmania DNA.

A novel methodology for the isothermal amplification of Leishmania DNA using labeled primers combined with the advantages of magnetic purification/preconcentration and the use of gold nanoparticle (AuNP) tags for the sensitive electrochemical detection of such amplified DNA is developed. Primers labeled with AuNPs and magnetic beads (MBs) are used for the first time for the isothermal amplification reaction, being the amplified product ready for the electrochemical detection. The electrocatalytic activity of the AuNP tags toward the hydrogen evolution reaction allows the rapid quantification of the DNA on screen-printed carbon electrodes. Amplified products from the blood of dogs with Leishmania (positive samples) are discriminated from those of healthy dogs (blank samples). Quantitative studies demonstrate that the optimized method allows us to detect less than one parasite per microliter of blood (8 × 10(-3) parasites in the isothermal amplification reaction). This pioneering approach is much more sensitive than traditional methods based on real-time polymerase chain reaction (PCR), and is also more rapid, cheap, and user-friendly.

Molecular detection of vector-borne pathogens in wild and domestic carnivores and their ticks at the human-wildlife interface.

Urbanization of natural areas is considered one of the causes of the current apparent emergence of infectious diseases. Carnivores are among the species that adapt well to urban and periurban environments, facilitating cross-species disease transmission with domestic dogs and cats, and potentially with their owners. The prevalence of vector-borne pathogens (VBP) of zoonotic and veterinary interest was studied in sympatric wild and domestic carnivores into Barcelona Metropolitan Area (NE Spain). Blood or spleen samples from 130 animals, including 34 common genets (Genetta genetta), 12 red foxes (Vulpes vulpes), 10 stone martens (Martes foina), three Eurasian badgers (Meles meles), 34 free-roaming domestic cats and 37 dogs with outdoor access, were collected either in protected or adjacent residential areas. A total of 309 ticks (chiefly Rhipicephalus turanicus) were collected on these animals. The samples were analyzed with a battery of PCR assays targeting the DNA of Rickettsia spp., Anaplasmataceae, Coxiella burnetii, Bartonella spp., and Piroplasmida, and the amplicons were sequenced. The fox showed the highest prevalence (58%) and diversity of VBP (four pathogens), whereas none of the dogs were infected. Bartonella spp. (including B. clarridgeiae, B. henselae, and B. rochalimae) was the most prevalent pathogen. Infection of wild carnivores with Ehrlichia canis, C. burnetii, Theileria annae and Babesia vogeli was also confirmed, with some cases of coinfection observed. The presence of DNA of T. annae and B. vogeli was also confirmed in tick pools from four species of wild carnivores, supporting their role in piroplasmid life-cycle. By the sequencing of several target genes, DNA of Rickettsia massiliae was confirmed in 17 pools of Rh. turanicus, Rh. sanguineous, and Rh. pusillus from five different species, and Rickettsia conorii in one pool of Rh. sanguineous from a dog. None of the hosts from which these ticks were collected was infected by Rickettsia. Although carnivores may not be reservoir hosts for zoonotic Rickettsia, they can have an important role as mechanical dispersers of infected ticks.

Ticks and associated pathogens collected from cats in Sicily and Calabria (Italy).

BACKGROUND: Limited information is available about the species of ticks infesting the cat and the pathogens that they harbor. The aims of the present study were to identify the species of ticks removed from cats living in Sicily and Calabria (Italy) and to detect DNA of vector-borne pathogens in the same ticks. FINDINGS: Morphological identification of 132 adult ticks collected throughout the year from cats was carried out. Real-time PCRs for Hepatozoon felis, Piroplasmid, Ehrlichia/Anaplasma spp., Rickettsia spp., Bartonella spp., Mycoplasma spp. and Leishmania infantum were performed from each individual tick. Ticks belonging to Rhipicephalus (R. sanguineus sensu lato, R. pusillus) and Ixodes (I. ricinus, I. ventalloi) genera were identified. Ixodes ventalloi was the most frequently found tick species (47 %). The positivity rate to at least one pathogen was 14.4 % (19/132 ticks). Leishmania infantum, Rickettsia spp. (R. monacensis and R. helvetica), Bartonella spp. (B. clarridgeiae), Piroplasmid (Babesia vogeli), and Ehrlichia/Anaplasma spp. (E. canis) DNAs were amplified in 8.3, 5.3, 1.5, 0.75 and 0.75 % of ticks, respectively. Hepatozoon felis, Anaplasma spp. and hemotropic Mycoplasma spp. DNAs were not detected. Four (21.1 %) out of nineteen positive ticks were co-infected. CONCLUSIONS: This study provides novel data about ticks infesting cats and the DNA of pathogens that they harbor. In Southern Italy, anti-tick prophylaxis should be implemented throughout the year in cats without neglecting winter time.

Identification of a third feline Demodex species through partial sequencing of the 16S rDNA and frequency of Demodex species in 74 cats using a PCR assay.

BACKGROUND: Demodex cati and Demodex gatoi are considered the two Demodex species of cats. However, several reports have identified Demodex mites morphologically different from these two species. The differentiation of Demodex mites is usually based on morphology, but within the same species different morphologies can occur. DNA amplification/sequencing has been used effectively to identify and differentiate Demodex mites in humans, dogs and cats. HYPOTHESIS/OBJECTIVES: The aim was to develop a PCR technique to identify feline Demodex mites and use this technique to investigate the frequency of Demodex in cats. METHODS: Demodex cati, D. gatoi and Demodex mites classified morphologically as the third unnamed feline species were obtained. Hair samples were taken from 74 cats. DNA was extracted; a 330 bp fragment of the 16S rDNA was amplified and sequenced. RESULTS: The sequences of D. cati and D. gatoi shared >98% identity with those published on GenBank. The sequence of the third unnamed species showed 98% identity with a recently published feline Demodex sequence and only 75.2 and 70.9% identity with D. gatoi and D. cati sequences, respectively. Demodex DNA was detected in 19 of 74 cats tested; 11 DNA sequences corresponded to Demodex canis, five to Demodex folliculorum, three to D. cati and two to Demodex brevis. CONCLUSIONS AND CLINICAL IMPORTANCE: Three Demodex species can be found in cats, because the third unnamed Demodex species is likely to be a distinct species. Apart from D. cati and D. gatoi, DNA from D. canis, D. folliculorum and D. brevis was found on feline skin.

Infection and exposure to vector-borne pathogens in rural dogs and their ticks, Uganda.

BACKGROUND: In rural parts of Africa, dogs live in close association with humans and livestock, roam freely, and usually do not receive prophylactic measures. Thus, they are a source of infectious disease for humans and for wildlife such as protected carnivores. In 2011, an epidemiological study was carried out around three conservation areas in Uganda to detect the presence and determine the prevalence of vector-borne pathogens in rural dogs and associated ticks to evaluate the risk that these pathogens pose to humans and wildlife. METHODS: Serum samples (n = 105), blood smears (n = 43) and blood preserved on FTA cards (n = 38) and ticks (58 monospecific pools of Haemaphysalis leachi and Rhipicephalus praetextatus including 312 ticks from 52 dogs) were collected from dogs. Dog sera were tested by indirect immunofluorescence to detect the presence of antibodies against Rickettsia conorii and Ehrlichia canis. Antibodies against R. conorii were also examined by indirect enzyme immunoassay. Real time PCR for the detection of Rickettsia spp., Anaplasmataceae, Bartonella spp. and Babesia spp. was performed in DNA extracted from FTA cards and ticks. RESULTS: 99% of the dogs were seropositive to Rickettsia spp. and 29.5% to Ehrlichia spp. Molecular analyses revealed that 7.8% of the blood samples were infected with Babesia rossi, and all were negative for Rickettsia spp. and Ehrlichia spp. Ticks were infected with Rickettsia sp. (18.9%), including R. conorii and R. massiliae; Ehrlichia sp. (18.9%), including E. chaffeensis and Anaplasma platys; and B. rossi (1.7%). Bartonella spp. was not detected in any of the blood or tick samples. CONCLUSIONS: This study confirms the presence of previously undetected vector-borne pathogens of humans and animals in East Africa. We recommend that dog owners in rural Uganda be advised to protect their animals against ectoparasites to prevent the transmission of pathogens to humans and wildlife.

An iridium oxide nanoparticle and polythionine thin film based platform for sensitive Leishmania DNA detection.

An impedimetric label-free genosensor for high sensitive DNA detection is developed. This system is based on a screen-printed carbon electrode modified with the thionine layer and iridium oxide nanoparticles (IrO2 NP). An aminated oligonucleotide probe is immobilized on the IrO2 NP/polythionine modified electrode and ethanolamine was used as a blocking agent. Different diluted PCR amplified DNA samples have been detected. The selectivity and reproducibility of this system are studied and the system was highly reproducible with RSD ≈ 15% and sensitive enough while using 2% of ethanolamine during the blocking step employed for genosensor preparation.

Serum chemistry reference values for the common genet (Genetta genetta): variations associated with Leishmania infantum infection.

BACKGROUND: The role of wildlife in the epidemiology of leishmaniosis in under debate, and determining whether infection with Leishmania infantum causes illness in wild carnivores is important to determine its potential role as a reservoir. OBJECTIVES: To provide for the first time serum biochemistry reference values for the common genet (Genetta genetta), and to determine variations associated with L. infantum infection. METHODS: Twenty-five serum biochemistry parameters were determined in 22 wild-caught genets. Blood samples were analyzed for L. infantum DNA by means of real-time polymerase chain reaction (PCR). RESULTS: Two female genets were positive for L. infantum DNA but did not show any external clinical sign upon physical examination. Among other variations in the biochemistry values of these genets, one presented a higher concentration of gamma-globulins and cholesterol, whereas the other genet presented increased creatinine, bilirubin, and chloride levels when compared to uninfected females. Sex-related differences in some parameters were also reported. CONCLUSION: Infection with L. infantum may sometimes be accompanied by abnormal serum biochemistry in wild carnivores. CLINICAL IMPORTANCE: Clinical disease may occur in L. infantum-infected wild carnivores. This has implications in the epidemiology of leishmaniosis. In addition, the data provided here would also be useful as reference values for researchers or rehabilitators working with the common genet.

Non-synonymous genetic variation in exonic regions of canine Toll-like receptors.

BACKGROUND: Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) considered to be the primary sensors of pathogens in innate immunity. Genetic variants could be associated to differences in breed innate immune response to pathogens and thus to susceptibility to infections or autoimmune diseases. There is therefore great interest in the characterization of canine TLRs. RESULTS: Polymorphisms in canine TLRs have been characterized by massive sequencing after enrichment of their exonic regions. DNAs from 335 dogs (seven different breeds) and 100 wolves (two different populations) were used in pools. The ratio of SNP discovery was 76.5% (in relation to CanFam 3.1); 155 out of 204 variants identified were new. Functional annotation identified 64 non-synonymous variants (43 new), 73 synonymous variants (56 new) and 67 modifier variants (57 new). 12 out of 64 non-synonymous variants are breed or wolf specific. TLR5 has been found to be the most polymorphic among canine TLRs. Finally, a TaqMan OpenArray® plate containing 64 SNPs with a possible functional effect in the protein (4 frameshifts and 60 non-synonymous codons) has been designed and validated. CONCLUSIONS: Non-synonymous genetic variation has been characterized in exonic regions of canine Toll-like Receptors. The TaqMan OpenArray® plate developed to capture the individual variability that affects protein function will allow high-throughput genotyping either to study association to infection susceptibility or even TLR evolution in the canine genome.