Detection of Metalloproteases and Cysteine Proteases RNA Transcripts of Leishmania (Leishmania) infantum in Ear Edge Skin of Naturally Infected Dogs.

Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive success these parasites to the mammalian hosts. Since these enzymes are poorly studied in naturally infected dogs, this work aims to show the differences in metalloprotease and cysteine proteases gene expression in ear edge skin of dogs naturally infected by Leishmania (Leishmania) infantum. A cohort of dogs (n = 20) naturally infected by L. (L.) infantum was clinically classified as asymptomatic, oligosymptomatic, and polysymptomatic and the parasite load range estimated. The analysis of proteases expression by RT-PCR in the ear edge skin was also assessed, suggesting more transcripts of proteases in cDNA samples from polysymptomatic dogs than oligosymptomatic and asymptomatic ones. Metalloprotease RT-PCR assays yielded products (202 bp) in all assessed cDNA dog samples. In contrast, cysteine proteases transcripts (227 bp) had shown to be better detected in cDNA samples of polysymptomatic dogs, compared with cDNA samples from asymptomatic and oligosymptomatic dogs. Predictive in silico assays suggested that secondary structures of metalloproteasee mRNAs can be more stable than cysteine proteases at the skin temperature of dogs. Evidence is presented that during natural infection of dogs by L. (L.) infantum, this parasite produces transcripts of metalloprotease and cysteine protease RNA in the skin from asymptomatic, oligosymptomatic, and polysymptomatic dogs.

Obesity impairs resistance to Leishmania major infection in C57BL/6 mice.

An association between increased susceptibility to infectious diseases and obesity has been described as a result of impaired immunity in obese individuals. It is not clear whether a similar linkage can be drawn between obesity and parasitic diseases. To evaluate the effect of obesity in the immune response to cutaneous Leishmania major infection, we studied the ability of C57BL/6 mice fed a hypercaloric diet (HSB) to control leishmaniasis. Mice with diet-induced obesity presented thicker lesions with higher parasite burden and a more intense inflammatory infiltrate in the infected ear after infection with L. major. There was no difference between control and obese mice in IFN-gamma or IL-4 production by auricular draining lymph node cells, but obese mice produced higher levels of IgG1 and IL-17. Peritoneal macrophages from obese mice were less efficient to kill L. major when infected in vitro than macrophages from control mice. In vitro stimulation of macrophages with IL-17 decreased their capacity to kill the parasite. Moreover, macrophages from obese mice presented higher arginase activity. To confirm the role of IL-17 in the context of obesity and infection, we studied lesion development in obese IL-17R-/- mice infected with L. major and found no difference in skin lesions and the leukocyte accumulation in the draining lymph node is redcuced in knockout mice compared between obese and lean animals. Our results indicate that diet-induced obesity impairs resistance to L. major in C57BL/6 mice and that IL-17 is involved in lesion development.

Comparison of three methods for the diagnosis of otoacariasis due to Otodectes cynotis in dogs and cats.

BACKGROUND: The diagnosis of otoacariasis due to Otodectes cynotis is based on the visualization of the parasite, either directly by otoscopy or indirectly after microscopic examination of cerumen collected by several methods. OBJECTIVES: To compare the sensitivity of three techniques: conventional handheld otoscopy, Volkmann's curette sampling and cotton-tipped swabbing. ANIMALS: Five dogs and 12 cats (30 naturally infested ears). METHODS AND MATERIALS: For each case, following otoscopy, the order of examinations (swab or curette) was chosen randomly and the samples were observed on a slide mixed with lactophenol and covered with a coverslip. Parasite detection was noted as positive or negative and parasitic stages were counted separately. RESULTS: The diagnostic sensitivity of otoscopy alone was 67% (positive in 20 of 30 cases), using the curette sampling it was 93% (28/30) and 57% (17/30) for the swabbing. The curette technique had a significantly higher sensitivity than classic ear-swabbing (P = 0.001) or otoscopy alone (P = 0.02). Combining otoscopy and the curette, we obtained a sensitivity of 100% compared to 86% when otoscopy and swabbing were combined. Moreover, the parasite count in the curette samples (average 25 ± 30 SD) was significantly higher than the swab samples (4.5 ± 11) (P < 0.001). CONCLUSIONS AND CLINICAL IMPORTANCE: When suspecting O. cynotis infestation, otoscopic examination should be performed. To confirm the nature of the parasites observed or whenever this examination result is negative, doubtful or cannot be performed, the curettage sampling method for microscopic cerumen examination is recommended.

Transcutaneous immunization using SLA or rLACK skews the immune response towards a Th1 profile but fails to protect BALB/c mice against a Leishmania major challenge.

Leishmaniasis is an expanding health threat worldwide complicated by the absence of an effective vaccine. We investigated transcutaneous immunization (TCI) as a needle-free immunization route which exploits the abundance of antigen presenting cells in the skin to induce both mucosal and systemic immunity. Leishmania (L.) major soluble antigens (SLA) or recombinant Leishmania homolog of receptors for activated C-kinase (rLACK) antigens were delivered transcutaneously together with cholera toxin (CT), to BALB/c mice. Mice were immunized at weeks 1, 4, and 7 with PBS, CT, SLA/CT or rLACK/CT. Two weeks after the final boost, antigen-specific IgG titers, IFN-γ ELISpot, and cytokine levels were assessed in half of the mice and the remainder were challenged with an intradermal (ear) injection of 5 × 104L. major metacyclic parasites. Mice were monitored weekly and sacrificed after 7 weeks to assess the parasite burden and to study the ear lesion immunohistopathology. Our results show that TCI with SLA or rLACK yielded high levels of anti-SLA, anti-rLACK and anti-CT IgG antibodies. A Th1-type of immune response was demonstrated with a high frequency of IFN-γ secreting cells, high levels of IFN-γ production, and lower levels of IL-10 resulting in a high IFN-γ/IL-10 ratio in mice immunized with SLA/CT or rLACK/CT. After parasite challenge, rLACK immunization was not associated with protection. In addition, SLA/CT immunized mice had larger ear lesions and an increased parasite load in the ear. Immunohistochemistry of ear biopsies stained for nitric oxide synthase revealed that staining intensity was diminished in the SLA/CT group compared to the control group. This finding suggested that less parasite killing occurred at the site of the infection. In conclusion, despite a strong Th1 type profile induced by TCI, exacerbation of infection occurred after challenge with L. major. This also correlated with low induction of nitric oxide.

A single subcutaneous administration of a sustained-release ivermectin suspension eliminates Psoroptes cuniculi infection in a rabbit farm.

BACKGROUND: Psoroptes cuniculi mites are the most common ear parasites infesting breeding female rabbits. The suffering rabbits show cutaneous signs of the infestation in the ears and are prone to secondary infections. OBJECTIVES: This trial was conducted to eliminate P. cuniculi in farm rabbits with a sustained-release ivermectin-loaded solid dispersion suspension (IVM-SD) suspension, and studied the stability of the formulation. ANIMALS: There were 986 breeding female Hyplus rabbits naturally infected with P. cuniculi. METHODS: All rabbits infected with P. cuniculi were subcutaneously administered with a single dose of IVM-SD suspension at 2 mg/kg body weight. Twenty-seven rabbits with severe infections were observed daily and examined on days 0 and 14 to score the lesions and count mites in crusts. RESULTS: Fourteen days after the treatment no live mites were detected, demonstrating 100% therapeutic efficacy. The mean lesion scores decreased from 4.33 to 0.11 in the left ears and from 4.22 to 0.22 in the right ears. No reinfection occurred within 60 days of treatment. CONCLUSIONS: A single subcutaneous administration of the IVM-SD suspension at 2 mg/kg was effective in eliminating P. cuniculi infection in the rabbit farm.

Expression of matrix metalloproteinase-2 and metalloproteinase-9 in the skin of dogs with visceral leishmaniasis.

The skin is the first organ to be infected by the parasite in canine visceral leishmaniasis. The enzyme matrix metalloproteinase (MMP) acts towards degradation of the extracellular matrix (ECM) and modulation of the inflammatory response against many kinds of injuries. The aims of this study were to evaluate the expression of MMP-2 and MMP-9 through immunohistochemistry and zymography on the skin (muzzle, ears, and abdomen) of dogs that were naturally infected by Leishmania spp. and to compare these results with immunodetection of the parasite and with alterations to the dermal ECM. Picrosirius red staining was used to differentiate collagen types I and III in three regions of the skin. The parasite load, intensity of inflammation, and production of MMP-2 (latent) and MMP-9 (active and latent) were higher in the ear and muzzle regions. MMP-9 (active) predominated in the infected group of dogs and its production was significantly different to that of the control group. Macrophages, lymphocytes, and plasma cells predominated in the dermal inflammation and formed granulomas in association with degradation of mature collagen (type I) and with discrete deposition of young collagen (type III). This dermal change was more pronounced in dogs with high parasite load in the skin. Therefore, it was concluded that the greater parasite load and intensity of inflammation in the skin led consequently to increased degradation of mature collagen, caused by increased production of MMPs, particularly active MMP-9, in dogs with visceral leishmaniasis. This host response profile possibly favors systemic dissemination of the parasite.

A Case of Cutaneous Leishmaniasis guyanensis Mimicking Otitis Externa.

Cutaneous leishmaniasis typically presents as a painless papule progressing to an ulcer or plaque. In this case study of the ear, the disease manifested as a small painful bump progressing into redness and swelling about the ear with purulent drainage. After multiple oral/intravenous antipseudomonal, antistaphylococcal, and antifungal treatments, there was no improvement. The skin progressed to an erythematous plaque and hemorrhagic ulcer; punch biopsy and speciation revealed Leishmaniasis guyanensis. The patient was switched to a seven-dose course of intravenous L-amphotericin B (visceral leishmaniasis protocol). Within 21 days, pain and edema resolved and the ulcers healed. Three-month follow-up demonstrated no recurrence. Further studies are needed to evaluate the use of L-amphotericin B in Leishmaniasis guyanensis.

First report of spinose ear tick, Otobius megnini (Acari, Argasidae), in Sweden.

A dog that had travelled to Sweden was inspected by a veterinarian. In one ear canal a tick was found. It was later identified as a spinose ear tick, Otobius megnini. In this case report we also review the previously known reports of O. megnini in Europe and the recent introduction and spread in Turkey.

Parasitism and inflammation in ear skin and in genital tissues of symptomatic and asymptomatic male dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is transmitted through vector, although venereal transmission has been suggested. This study aimed to compare the parasitic loads and inflammatory processes in genital tissues with ear skin from seropositive male dogs. Forty-five seropositive dogs were separated into groups containing symptomatic (n = 23) and asymptomatic (n = 22) animals. The control group (n = 2) healthy animals with seronegative and negative results in direct parasitological test. Samples of ear tip skin, prepuce, glans penis, testis, epididymis, and prostate were collected for evaluation of parasitic load and inflammatory infiltrate. Although ear tip skin was the most intensely parasitized, prepuce and epididymis revealed no difference in parasitism when compared with ear tip skin (P > 0.05). Parasitic loads in testis and prostate were lower than other tissues (P < 0.05). Parasitism in glans penis was high, similar to prepuce and epididymis, but lower than ear tip skin. High parasitism was more frequent in symptomatic dogs than asymptomatic animals. Severe inflammatory processes were more frequent within the symptomatic animals compared with asymptomatic and more predominant in prepuce and epididymis. Ear tip skin and genital tissues presented signs of chronic inflammation. There were weak and moderate positive correlations between parasitic loads and inflammatory processes. Our results demonstrate that, likewise with the ear tip skin, the genital of seropositive dogs can carry a large number of Leishmania infantum amastigotes and this process are more intense in symptomatic animals. These data have important implications for understanding the possibility of venereal transmission of CVL.

Comparison of parasitic mite retrieval methods in a population of community cats.

Objectives This study compared methods of mite retrieval from community cats in the Ohio River Valley region of the USA and determined incidence of parasitic mites in this region. Methods In total, 493 community cats were humanely trapped and anesthetized for a trap-neuter-return program. Cats received a dermatologic examination, ear swabs, superficial skin scraping, flea combing, acetate tape preparation and feces collection. All samples were examined microscopically. Large volumes of hair and scale from flea combing were dissolved in 10% potassium hydroxide and centrifuged with Sheather's solution. Fecal samples were mixed with Sheather's solution, filtered and centrifuged. Results Ear swabs were significantly ( P <0.05) better than other methods for finding chigger mites and Otodectes cynotis, and skin scraping was significantly better than ear swabs for finding Cheyletiella species. Only cats with O cynotis had clinical lesions. Mites remained identifiable for 6 months at room temperature. Mite incidence rates were as follows: Notoedres cati (1/493 cats), 0.002 (95% confidence interval [CI] 0-0.006); Lynxacarus radovskyi (2/493 cats), 0.004 (95% CI 0-0.01); Demodex gatoi (5/493 cats), 0.01 (95% CI 0.001-0.019); chigger mites (10/493 cats), 0.02 (95% CI 0.008-0.033); Cheyletiella species (12/493 cats), 0.024 (95% CI 0.011-0.038); and O cynotis (124/493 cats), 0.252 (95% CI 0.213-0.29). Conclusions and relevance Ear swabs are recommended when O cynotis or chigger mites are suspected. Skin scraping is more likely to yield positive results than ear swabs, but was not significantly better than acetate tape preparations, flea combing or fecal flotation for finding Cheyletiella species. Mites can remain identifiable for prolonged periods at room temperature. With the exception of O cynotis, the incidence of feline parasitic mites in the Ohio River Valley region of the USA is low; however, D gatoi and L radovskyi were present in the area and should be considered in cats with dermatologic disease attributable to them. In this population of community cats, asymptomatic carriage of mites was common.

Pilot Study: Occurrence of Ear Mites and the Otic Flora in Domestic Ruminants on St. Kitts.

Psoroptes, a globally occurring mite, and Raillietia, a primarily tropical mite, have been attributed to otitis in ruminants. Within the Caribbean, little is known about the prevalence of these mites and their relation to the microbial flora of the ears. From May 2015 to May 2016, ears of cattle, sheep and goats brought to the St. Kitts abattoir were examined post-slaughter for mite infestations. No Raillietia spp. were seen and none of the sheep had ear mites. Psoroptes spp. were found in 2.6% of the cattle and 22.6% of the goats. Tick control programs, focused on cattle and sheep, might influence the occurrence seen in this study.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

HISTOPATHOLOGY AND RISK FACTORS ASSOCIATED WITH NEOTROMBICULA MICROTI INFESTATION IN THE ENDANGERED AMARGOSA VOLE (MICROTUS CALIFORNICUS SCIRPENSIS).

The Amargosa vole (Microtus californicus scirpensis) is a profoundly endangered rodent found only in the Central Mojave Desert, Inyo County, California, US. In 2010, severe cases of trombiculiasis, caused by larval Neotrombicula microti mites, were discovered among voles and sympatric small mammals. We evaluated Amargosa voles and sympatric rodents for infestation with N. microti December 2011-November 2012 and evaluated histopathology of ear tissue from 13 actively N. microti-infested Amargosa voles and 10 Amargosa voles with no gross evidence of current or past infestation. Rodents with current infestation had mites visible on tissue, typically ear pinnae, whereas mites were not seen on rodents with presumptive past infestation, but some of these animals had gross tissue scarring and loss consistent with healing from infestation. Ears from infested voles had severe granulocytic and necrotizing dermatitis, most associated with stylostome fragments, whereas few lesions were present in grossly uninfested voles. There was no association between body condition and infestation or severity of lesions. Significantly more voles were infested (37%) with N. microti than sympatric rodents (3%), suggesting that sympatric rodents do not serve as an important source of N. microti exposure to voles. Although this chigger infestation was common and induced severe localized pathology, we did not detect a fitness cost to infestation and recommend further evaluation of the disease to discern its significance in this conservation context.

Rodent species as natural reservoirs of Borrelia burgdorferi sensu lato in different habitats of Ixodes ricinus in The Netherlands.

Rodents are natural reservoirs for human pathogenic spirochaetes of the Borrelia burgdorferi complex [B. burgdorferi sensu lato (s.l.)], and the pathogens are transmitted by Ixodes ricinus ticks to humans in The Netherlands. B. burgdorferi s.l. infection prevalence in questing ticks, rodents, and ticks feeding on these rodents, all sampled within the same short time span of five days in three different areas in The Netherlands, were compared in order to establish the relationship between ticks, reservoir hosts, and B. burgdorferi s.l. Questing nymphs were found in all 3 areas and numbers differed per area and even per site within areas. Infection prevalence in questing nymphs ranged between 0 and 20%. Apodemus sylvaticus and Myodes glareolus were the dominant rodents captured, and their numbers differed per area. Infection prevalence, determined by ear biopsies, ranged between 0 and 33.3% for both rodent species. Larvae were most frequently found feeding on these rodents, and their Borrelia infection prevalence ranged between 0 and 6.3% (A. sylvaticus) and between 0 and 29.4% (M. glareolus). The burden of nymphs feeding on rodents was low and varied per area with only 2 of 42 nymphs infected. Comparisons made on the basis of infection prevalence indicated that there was no clear relationship between rodents and questing nymphs when sampled within the same short time span. However, a possible relationship was present when questing ticks were sampled over longer periods in time (months) within or near the same areas (range of infection prevalence between 3.7 and 39.4). Confounding factors thus play a role in the interaction between rodents, ticks, and B. burgdorferi s.l., and it is very likely that other reservoir host species are responsible for the observed fluctuations. It is concluded that the local variations in rodent-Borrelia-tick interactions only partially explain the Lyme borreliosis risk in the sites studied and that other ecological determinants, notably vertebrate hosts and vegetation structure, should be incorporated in future studies of Lyme borreliosis risk.

Antibody responses to Sarcoptes scabiei apolipoprotein in a porcine model: relevance to immunodiagnosis of recent infection.

No commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive. The recombinant Sarcoptes scabiei apolipoprotein antigen Sar s 14.3 is a promising immunodiagnostic, eliciting high levels of IgE and IgG in infected people. Limited data are available regarding the temporal development of antibodies to Sar s 14.3, an issue of relevance in terms of immunodiagnosis. We utilised a porcine model to prospectively compare specific antibody responses to a primary infestation by ELISA, to Sar s 14.3 and to S. scabiei whole mite antigen extract (WMA). Differences in the antibody profile between antigens were apparent, with Sar s 14.3 responses detected earlier, and declining significantly after peak infestation compared to WMA. Both antigens resulted in >90% diagnostic sensitivity from weeks 8-16 post infestation. These data provide important information on the temporal development of humoral immune responses in scabies and further supports the development of recombinant antigen based immunodiagnostic tests for recent scabies infestations.

Dual effect of Lutzomyia longipalpis saliva on Leishmania braziliensis infection is mediated by distinct saliva-induced cellular recruitment into BALB/c mice ear.

BACKGROUND: Leishmania parasites are transmitted to their vertebrate hosts by infected Phlebotomine sand flies during the blood meal of the flies. Sand fly saliva is known to enhance Leishmania spp. infection, while pre-exposure to saliva protects mice against parasitic infections. In this study, we investigated the initial inflammatory leucocyte composition induced by one or three inocula of salivary gland extract (SGE) from Lutzomyia longipalpis in the presence or absence of Leishmania braziliensis. RESULTS: We demonstrated that inoculating SGE once (SGE-1X) or three times (SGE-3X), which represented a co-inoculation or a pre-exposure to saliva, respectively, resulted in different cellular infiltrate profiles. Whereas SGE-1X led to the recruitment of all leucocytes subtypes including CD4(+) T cells, CD4(+)CD25(+) T cells, dendritic cells, macrophages and neutrophils, the immune cell profile in the SGE-3X group differed dramatically, as CD4(+) T cells, CD4(+)CD25(+) T cells, dendritic cells, macrophages and neutrophils were decreased and CD8(+) T cells were increased. The SGE-1X group did not show differences in the ear lesion size; however, the SGE-1X group harbored a higher number of parasites. On the other hand, the SGE-3X group demonstrated a protective effect against parasitic disease, as the parasite burden was lower even in the earlier stages of the infection, a period in which the SGE-1X group presented with larger and more severe lesions. These effects were also reflected in the cytokine profiles of both groups. Whereas the SGE-1X group presented with a substantial increase in IL-10 production, the SGE-3X group showed an increase in IFN-γ production in the draining lymph nodes. Analysis of the inflammatory cell populations present within the ear lesions, the SGE-1X group showed an increase in CD4(+)FOXP3(+) cells, whereas the CD4(+)FOXP3(+) population was reduced in the SGE-3X group. Moreover, CD4(+) T cells and CD8(+) T cells producing IFN-γ were highly detected in the ears of the SGE-3X mice prior to infection. In addition, upon treatment of SGE-3X mice with anti-IFN-γ monoclonal antibody, we observed a decrease in the protective effect of SGE-3X against L. braziliensis infection. CONCLUSIONS: These results indicate that different inocula of Lutzomyia longipalpis salivary gland extract can markedly modify the cellular immune response, which is reflected in the pattern of susceptibility or resistance to Leishmania braziliensis infection.

Nasal, oral and ear swabs for canine visceral leishmaniasis diagnosis: new practical approaches for detection of Leishmania infantum DNA.

BACKGROUND: The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P=0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05). CONCLUSIONS: Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.

Ixodes ricinus infestation in free-ranging cervids in Norway--a study based upon ear examinations of hunted animals.

Prevalence, abundance and instar composition of Ixodes ricinus as found on one ear collected from 1019 moose (Alces alces), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), shot during hunting (August-December) 2001-2003, are reported. The animals originated from 15 coastal municipalities (CM), seven municipalities bordering to coastal municipalities (BCM) and four inland municipalities (IM), in Norway, between latitudes 58-66° N. I. ricinus occurred endemically in all CM and BCM up to 63°30' N, whereas it was non-endemic further north and in the IM. This geographical distribution of the tick along the coast of southern Norway was largely in accordance with that reported as far back as the 1940s. Our results therefore did not indicate any large scale northwards expansion of I. ricinus in Norway during the 60 year-period between the two studies. However, the prevalence of infestation and tick abundance were significantly higher in CM as compared to BCM. The prevalence and abundance by month were highest during August and September, gradually decreasing towards December. The considerable prevalence of ticks in November, as well as findings in December, would seem to indicate a prolonged tick season as compared with the studies carried out 60 years ago. A total of 8920 ticks were isolated from 439 of the 603 animals examined in endemic municipalities, and the maximum number of ticks found on one single ear was 204. Attached adult ticks were primarily found among the long hairs at base of the ear, whereas nymphs and larvae were seen all over the outer surface of the pinna, for larvae especially at the edge and tip of the ear. Nymphs were the dominant instar, constituting 74% of the total tick count. The proportion of larvae and adult ticks was 13% and 12%. A significantly higher proportion of adult ticks and lower proportion of immature stages were found in moose, as compared to red deer and roe deer. The same apparently size-associated preference of adult ticks was also found for adult animals (all species) as compared to calves. Other grossly detected ectoparasites included the lice Solenopotes burmeisteri in red deer and Damalinia meyeri in roe deer, and the deer ked fly, Lipoptena cervi, in moose and roe deer. This is believed to be the first systematic study on the instar composition by I. ricinus infestation in free-ranging cervids. The examination of ears from hunted cervids should be recognized as a rational way of obtaining data on the geographical distribution and abundance of this tick in nature.

Clinical efficacy of botanical extracts from Eupatorium adenophorum against the Sarcoptes scabiei (Sarcoptidae: Sarcoptes) in rabbits.

The aims of present study were to evaluate the therapeutic efficacy of extracts from Eupatorium adenophorum against Sarcoptes scabiei. A 30-day experiment was performed using New Zealand rabbits that were naturally infested with S. scabiei in the toes (n=30) or artificially infected in the external ear margin with S. scabiei (n=30). Rabbits were randomly divided into five groups (6 animals per group, A-E groups for rabbits of naturally infested and F-J groups for artificially infected rabbits), respectively. All 60 rabbits were treated twice on days 0 and 7 successively. Animals in groups A/F, B/G, and C/H were treated on each toe/external ear margin with topical E. adenophorum ethanol extract at 1.0, 0.5 and 0.25 g/ml (w/v), respectively. Animals in groups D/I and E/J were treated with ivermectin by injections (positive controls) or by glycerol with water only rubbed onto the affected area (negative controls). After two treatments with extracts of E. adenophorum with relatively high concentrations of 0.5 and 1g/ml, the S. scabiei was completely eliminated in rabbits between days 14 and 30. Our results showed that rabbits treated with ivermectin (positive controls) and those treated with the extracts of concentrations of 1.0 or 0.5 g/ml achieved remarkable therapeutic efficacy; no mites were present in toes of rabbits in these groups on day 14, which confirmed a 100% therapeutic efficacy rate up to day 30 of the end of the trial. The clinical effects of treatment with 1.0 and 0.5 g/ml E. adenophorum extracts (groups A and B) were similar to ivermectin treatment. However, the therapeutic efficacy in group C and E rabbits only reached 43.25% and 7.13% by day 14. Furthermore, the therapeutic efficacy improved slightly by the end of the experiment on day 30, and rabbits in groups F, G and I also achieved good efficacy according to the recovery scoring criteria. These results indicate that E. adenophorum contains potent compounds for the effective control of sarcoptidosis.

PCR amplification and DNA sequencing of Demodex injai from otic secretions of a dog.

BACKGROUND: The identification of Demodex mites from dogs is usually based on morphology and location. Mites with uncharacteristic features or from unusual locations, hosts or disease manifestations could represent new species not previously described; however, this is difficult to determine based on morphology alone. HYPOTHESIS/OBJECTIVES: The goal of this study was to identify and confirm Demodex injai in association with otitis externa in a dog using PCR amplification and DNA sequencing. METHODS: Otic samples were obtained from a beagle in which a long-bodied Demodex mite was identified. For comparison, Demodex mite samples were collected from a swab and scraping of the dorsal skin of a wire-haired fox terrier and an otic sample from a dog with generalized and otic demodicosis. To identify the Demodex mite, DNA was extracted, and 16S rRNA was amplified by PCR, sequenced and compared with Demodex sequences available in public databases and from separate samples morphologically diagnosed as D. injai and Demodex canis. RESULTS: PCR amplification of the long-bodied mite rRNA DNA obtained from otic samples was approximately 330 bp and was identical to that from the mite morphologically identified as D. injai obtained from the dorsal skin of a dog. Furthermore, the examined mite did not have any significant homology to any of the reported genes from Demodex spp. CONCLUSIONS: These results confirmed that the demodex mites in this case were D. injai.