Haemoparasites of free-roaming dogs associated with several remote Aboriginal communities in Australia.

BACKGROUND: Tick-borne haemoparasites Babesia vogeli and Anaplasma platys are common among the free-roaming canine populations associated with Aboriginal communities in Australia, whilst the prevalence of haemoplasmas, which are also suspected to be tick-borne, remained unexplored. The aim of this study was to determine the prevalence of haemoplasma infection in these populations, and to identify any correlation with other haemoparasites. Blood was collected from 39 dogs associated with four Aboriginal communities and screened for infection using PCR and serology. DNA was purified and PCR analyses for piroplasms, Anaplasmataceae family bacteria and haemoplasmas performed. Serum was analysed using a commercial haemoparasite ELISA. Prevalence of infection was compared between communities. RESULTS: Seventeen dogs (44%) were infected (PCR positive) with Mycoplasma haemocanis, eight (21%) with 'Candidatus Mycoplasma haematoparvum', 20 (51%) with A. platys, and 17 (44%) with B. vogeli. Two dogs were infected with a novel haemoplasma as determined by DNA amplification and sequencing. Two dogs (5%) were serologically positive for Dirofilaria immitis antigens, one (3%) was positive for Ehrlichia canis antibodies and nine (24nbsp;%) were positive for A. platys antibodies. Co-infections were frequent. Haemoplasma prevalence was highest (73%, 16/22) in Central Australia and lowest (22%, 2/9) in Western Australia (p = 0.017). In contrast, B. vogeli prevalence was low in Central Australia (18%, 4/22) but higher (78%, 7/9) in Western Australia (p = 0.003). CONCLUSIONS: This is the first time haemoplasma infections, including a novel species, have been molecularly documented in Australian dogs. The wide regional variation in prevalence of some of the haemoparasite infections detected in this study warrants further investigation.

Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA.

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.

Prevalence of hemotropic mycoplasmas in healthy and unhealthy cats and dogs in Spain.

The aims of the present study were to determine the prevalence of hemoplasmas in cats and dogs from the Barcelona area of Spain with the use of species-specific quantitative polymerase chain reaction (qPCR) assays and to evaluate any associations between hemoplasma infection, clinical presentation, and vector-borne infections. Blood samples from cats (191) and dogs (182) were included and were classified as healthy (149) or unhealthy (224). Ethylenediamine tetra-acetic acid blood samples underwent DNA extraction and qPCR analysis. Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis' were detected in cats, whereas Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' were detected in dogs, with prevalences of 3.7%, 9.9%, 0.5%, 14.3%, and 0.6%, respectively. In cats, no association between hemoplasma infection and health status, age, breed, presence of anemia, Feline leukemia virus status, and other vector-borne infections was found, but outdoor access (P = 0.009), male sex (P = 0.01), and Feline immunodeficiency virus status (P < 0.001) were significantly associated with hemoplasma infection. In dogs, sex, age, health status, presence of anemia, and breed were not significantly associated with hemoplasma infection, but a significant association was found between hemoplasma infection and vector-borne infections (P < 0.001). The present report documents the occurrence of feline 'Candidatus M. turicensis' and canine 'Candidatus M. haematoparvum' infections in Spain.

Canine pulmonary angiostrongylosis: the influence of climate on parasite distribution.

The geographic range of Angiostrongylus vasorum is expanding, leading to increased disease. Although observed cases of canine pulmonary angiostrongylosis have been dutifully reported in the literature, the state of biological knowledge remains too poor to predict future patterns of spread with any confidence. Nevertheless, there is an urgent need to identify areas that are likely to be suitable for parasite establishment. Preliminary attempts to do this using a climatic envelope approach suggest that several new areas are open to colonisation, even without invoking climate change. The risk of parasite importation into these areas should be mitigated, e.g. by restricting movement of dogs unless tested or treated for A. vasorum, and monitored by focused surveillance of definitive and intermediate hosts. These efforts will benefit from newly developed diagnostic tests.

Canine leishmaniosis in the United Kingdom: a zoonotic disease waiting for a vector?

Leishmaniosis is an important sand fly transmitted protozoan disease of dogs and humans. In northern Europe, infection is mainly restricted to dogs that have travelled to and/or from endemic areas of the Mediterranean region during periods when there is high sand fly exposure, mostly between March and November. Infected dogs in these areas in northern latitudes are a potential reservoir should incursion of a competent vector occur. However, information on the scale of the potential reservoir in the UK is lacking. Confirmed cases of canine leishmaniosis entering the United Kingdom between 2005 and 2007 were identified using diagnostic samples submitted to the Department of Clinical Veterinary Science, University of Bristol and from collaborating laboratories (n=257). All study dogs had clinico-pathological signs compatible with leishmaniosis, as typically reported in endemic countries and were leishmania positive in real time or conventional PCR tests, IFA serology and/or tissue microscopic examination for amastigote identification. Information obtained from each case included travel history, habitat, clinico-pathological findings and geographical location once located in the UK. The majority of dogs with complete travel history (n=183) had spent at least 6 months in Spain (105/183), 28/183 were rescued from re-homing centres in the country of origin and 26/183 entered the UK with confirmed leishmaniosis. Once located in the UK, the majority of positive cases were resident in south and central England. The spectrum of clinico-pathological signs for this group of dogs is similar to that reported in endemic countries. These data confirm that a potentially significant reservoir of infected dogs is resident in areas where future climatic conditions may support introduction of competent vectors.

Association of calcium intake and adiposity in postmenopausal women.

OBJECTIVE: The purpose of this study was to examine the relationship between calcium intake and 1) body composition and 2) body fat distribution in postmenopausal women. METHODS: Archived data from a previous study including forty-nine Caucasian women, aged 51-73 and at least three years postmenopausal, were used for analysis. Data included medical and diet history from a questionnaire, average nutrient intake from computerized analysis of a 3-day diet record, anthropometric measures including height, weight, body mass index (BMI), waist circumference, and waist-to-hip ratio (WHR). Calcium intake represented both food and supplement sources of calcium. Lean and fat mass were determined from dual energy x-ray absorptiometry (DXA) total body scans, and abdominal fat mass was defined as fat mass between the top of the iliac crest and L1 on the DXA scan. RESULTS: Subjects were 60.5 +/- 0.9 y/o with an average BMI of 26.9 +/- 0.7 and percent body fat of 42.8 +/- 1.2. Average calcium intake was 1151 +/- 83 g/d. There was a significant inverse relationship between calcium intake and percent body fat (r = -0.36, p < 0.01) and abdominal fat mass (r = -0.25, p < 0.05), but there was no significant correlation between calcium intake and body mass index, fat mass, lean mass, waist circumference, or WHR. When kcalories were controlled, the inverse correlation between dietary calcium intake and percent body fat remained (r = -0.24, p < 0.05). Total fat was significantly greater (p < 0.05) in the low dairy intake (1-2 servings/d) vs. high dairy intake group (3-4 servings/d), but there were no significant differences between the groups in other body composition variables. CONCLUSIONS: Increased calcium intake was associated with lower percent body fat and higher dairy intake was associated with lower fat mass in postmenopausal women, but there was no association between calcium intake and body fat distribution measures in this population.

High-resolution genetic fingerprinting of European strains of Anaplasma phagocytophilum by use of multilocus variable-number tandem-repeat analysis.

Anaplasma phagocytophilum is a widely distributed tick-borne pathogen of humans, livestock, and companion animals. We used in silico methods to identify 10 variable-number tandem-repeat (VNTR) loci within the genome sequence of the A. phagocytophilum HZ strain and used these data to develop a multilocus VNTR-based typing scheme for the species. Having confirmed the stability of four of the loci in replicates of the A. phagocytophilum strain that had been subjected to different numbers of passages through cell cocultures in vitro, we then used this typing scheme to discriminate between 20 A. phagocytophilum strains of diverse geographical and host provenances. Extensive diversity was found at each of the four loci studied, with total allele numbers ranging from 13 to 18 and Hunter-Gaston discriminatory index values ranging from 0.93 to 0.99. Only 2 of the 20 strains examined shared alleles at all four loci. The discriminatory power of VNTR analysis was found to be greater than that of either partial msp4 or 16S rRNA gene sequence comparison. The extremely high sensitivity of this novel approach to the genetic fingerprinting of A. phagocytophilum strains should serve well in molecular epidemiological studies of infection transmission, particularly when fine-scale strain delineation is required.

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs.

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.

Prevalence of Rickettsia felis DNA in the blood of cats and their fleas in the United States.

Rickettsia felis is associated with fever, headache, myalgia, and macular rash in some infected humans and has been detected in the cat flea (Ctenocephalides felis) in many countries around the world. While some naturally exposed cats have been assessed for antibodies against R felis, to our knowledge, no one has reported use of polymerase chain reaction (PCR) to attempt to amplify R felis DNA from client-owned cats and the fleas collected from them. In this study, we assayed 92 pairs of cat blood and flea extracts from Alabama, Maryland and Texas, using PCR assays that amplify a region of the citrate synthase gene (gltA) and the outer membrane protein B gene (ompB). Of the 92 pairs, 62 of 92 (67.4%) flea extracts and none of the cat blood samples were positive for R felis DNA.

A review of household drinking water intervention trials and an approach to the estimation of endemic waterborne gastroenteritis in the United States.

The incidence of acute gastrointestinal illness (AGI) attributable to public drinking water systems in the United States cannot be directly measured but must be estimated based on epidemiologic studies and other information. The randomized trial is one study design used to evaluate risks attributable to drinking water. In this paper, we review all published randomized trials of drinking water interventions in industrialized countries conducted among general immunocompetent populations. We then present an approach to estimating the incidence (number of cases) of AGI attributable annually to drinking water. To develop a national estimate, we integrate trial results with the estimated incidence of AGI using necessary assumptions about the estimated number of residents consuming different sources of drinking water and the relative quality of the water sources under different scenarios. Using this approach we estimate there to be 4.26-11.69 million cases of AGI annually attributable to public drinking water systems in the United States. We believe this preliminary estimate should be updated as new data become available.

Polygenic detection of Rickettsia felis in cat fleas (Ctenocephalides felis) from Israel.

The presence of Rickettsia felis, an emerging bacterial pathogen, was investigated in 79 cat flea (Cteno-cephalides felis) pools from Israel (5 to 20 fleas each) by polymerase chain reaction (PCR) and sequencing of 5 different genes. Amplified targets included both metabolic (gltA and fusA) and surface antigen (ompA, ompB, and the 17-kDa antigen) genes. R. felis DNA was detected in 7.6% of the flea pools. Two genotypes similar in their housekeeping gene sequences but markedly different in their surface antigenic genetic milieus were characterized. This is the first detection of this flea-transmitted rickettsia within its vector in Israel and the Middle East. Although no clinical case has been reported in human beings in Israel to date, these findings suggest that this infection is prevalent in Israel.

Prevalence of house dust mites and dermatophagoides group 1 antigens collected from bedding, skin and hair coat of dogs in south-west England.

The house dust mites Dermatophagoides farinae (Df) and D. pteronyssinus (Dpt) are commonly implicated as allergens causing canine atopic dermatitis in the UK. However, there are few studies that characterize the exposure of UK pet dogs to these mites. The objectives of this study were to determine the prevalence of the mite species on the skin, hair coat and bedding of a population of pet dogs. Dust samples (n = 68) were collected from both dogs and their beds using a standardized vacuuming technique and stored at -20 degrees C. Mites were identified using accepted morphological criteria. House dust mite allergen concentrations were assayed using standardized ELISA for Dpt and Df group 1 allergens (Der p 1 and Der f 1). Mites were identified in 15/68 samples (22%) and Dpt was the most common. Df mites were not present. Der p 1 allergens were detected in 60% of samples, and Der f 1 in 6% of samples. There were no significant differences between the number of Der p 1 positive samples from dogs and the number of those from their bedding, or between the average Der p 1 concentrations from dogs and the number of those from their bedding. Contrary to studies elsewhere in Europe and the USA, these findings support studies of human asthma patients in the UK, where exposure to Df is rare, but to Dpt is common. As the prevalence of positive intradermal and serological reactions to Df in atopic dogs is high, further investigations are warranted to clarify true Df hypersensitivity or potential immunological cross-reactivity between mite allergens.

Angiostrongylus vasorum: a real heartbreaker.

Recent reports suggest that the canine heartworm Angiostrongylus vasorum is expanding from traditional endemic foci in several parts of the world. We are ill placed to judge the causes and potential consequences of this expansion because of a lack of knowledge about fundamental aspects of the biology of the parasite. We call for a renewed focus on this important but neglected nematode.

Demonstration of two distinct hemotropic mycoplasmas in French dogs.

In North America it has been shown that distinct hemotropic mycoplasmas exist in dogs. Blood samples from 460 French dogs were analyzed by PCR to evaluate hemoplasma infection status. Seventy-one dogs (15.4%) were positive; of these, 44 (9.6%) were infected with an organism closely related to "Candidatus Mycoplasma haemoparvum" only, 15 (3.3%) were infected with Mycoplasma haemocanis only, and 12 dogs (2.6%) were dually infected with both organisms.

Infection with a proposed new subspecies of Babesia canis, Babesia canis subsp. presentii, in domestic cats.

Parasitemia with a large Babesia species was identified in two domestic cats from Israel. One cat, also coinfected with feline immunodeficiency virus and "Candidatus Mycoplasma haemominutum," had profound icterus and anemia which resolved after therapy, whereas a second cat was an asymptomatic carrier. Amplification and sequencing of the 18S rRNA gene, followed by phylogenetic analyses, indicated that infection was caused by Babesia canis. However, the sequences of the internal transcribed and 5.8S rRNA regions of the ribosomal operon used for subspeciation of B. canis were markedly different from the recognized subspecies of B. canis, which include B. canis vogeli, B. canis canis, and B. canis rossi. Based on phylogenetic comparisons of the 18S rRNA gene, 5.8S, and internal transcribed spacer sequences of the isolates from the cats and on the smaller sizes of the merozoite and trophozoite stages of this parasite, which distinguish it from the subspecies of B. canis present in dogs, we propose to identify the novel feline genotype of B. canis described in the present study as a new subspecies, B. canis subsp. presentii.

Chemotherapy of canine leishmaniosis.

Visceral leishmaniosis is a widespread and potentially fatal disease of dogs and humans common in the Mediterranean region, the Middle East, and South America. Canine leishmaniosis is most frequently treated with the drugs meglumine antimoniate, allopurinol, amphotericin B, or a combination of meglumine antimoniate and allopurinol. Therapy with the currently used drugs often achieves temporary clinical improvement and changes in immunologic parameters with restoration of the ability to mount parasite-specific cell mediated responses and decrease in anti-leishmanial antibody titers. However, treatment usually does not prevent relapse of disease or eliminate parasite carriage. Due to the current lack of an ultimate and effective therapy for canine leishmaniosis, new drugs, delivery systems and treatment strategies are necessary to achieve a consistent parasitological cure in infected dogs.