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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.

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.

The tick-transmitted Anaplasma phagocytophilum has been recorded in a range of mammal species and causes granulocytic ehrlichiosis in humans, horses, and companion animals as well as tick-borne fever in ruminants. Although deer and other ruminant species are known to be natural hosts, the distribution among sympatric deer populations is unexplored. Blood from 80 deer of four species were screened using an A. phagocytophilum-specific real-time polymerase chain reaction. Overall, 29% (19–40) of deer tested positive. Fallow deer (Dama dama), the most numerous species, had significantly lower prevalence (21%) than roe (Capreolus capreolus), red (Cervus elaphus), or sika (Cervus nippon) deer (average 50%). It is suggested that patterns of habitat use influence infection levels in different deer species. The role of deer as reservoirs of anaplasmosis remains unknown; however, prevalence in deer could be a useful index of local infection pressure and the risk of disease in domestic animals and humans.

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.

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.

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.

The nematode parasite Angiostrongylus vasorum is an increasingly important cause of respiratory and other diseases in dogs. Geographical spread from previously limited endemic foci has occurred rapidly. This paper investigates parasite epidemiology around the location of the first reported case in Scotland in 2009: by detection of A vasorum-specific DNA in gastropod intermediate hosts, and in dogs circulating DNA and specific antibodies, and first stage larvae in faeces. Overall prevalence in gastropods was 6.7 per cent (16/240), with parasite DNA found in slugs in the Arion ater and Arion hortensis species aggregates and the snail Helix aspersa (syn. Cornu aspersum). Of 60 dogs presenting with clinical signs compatible with angiostrongylosis, none tested positive using PCR on peripheral blood or Baermann test on faeces, and none of 35 tested for circulating anti-A vasorum antibodies were positive. PCR prevalence in gastropods was highest (11 per cent) in the park frequented by the canine angiostrongylosis index case. Molecular survey for infection in gastropods is a potentially informative and efficient method for characterising the distribution of A vasorum and therefore local risk of canine infection. However, there appears to be a complex relationship between prevalence in gastropods and emergence of canine clinical disease, which requires further work to advance understanding of parasite transmission and geographical disease spread.

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.

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 .

Tick-transmitted infections are an emerging problem in dogs. In addition to causing serious disease in traditional tropical and semi-tropical regions, they are now increasingly recognized as a cause of disease in dogs in temperate climates and urban environments. Furthermore, subclinically infected companion animals could provide a reservoir for human tick-transmitted infectious agents, such as Ehrlichia chaffeensis, Ehrlichia ewingii, the Ehrlichia phagocytophila group and Rickettsia conorii. Here, we discuss the emergence of new canine tick-transmitted diseases, which results from several factors, including the expansion of the tick range into urban and semi-urban areas worldwide, the movement of infected dogs into previously non-endemic areas, and the advent of novel molecular techniques for diagnosis and pathogen identification.

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.

Bartonella henselae (Rhizobiales: Bartonellacae), the agent of cat-scratch disease, is an emerging bacterial pathogen which can be transmitted via infective faecal material of Ctenocephalides felis Bouche (Siphonaptera: Pulicidae). Worldwide, B. henselae has been identified in 1-53% of felines and 2.9-17.4% of fleas. Although culture is the routine method for detection, the procedure is time-consuming and is rarely used for isolation directly from flea vectors. The current study reports the development of a quantitative real-time polymerase chain reaction (qPCR) to detect and quantify B. henselae organisms from vector samples. The qPCR is specific and detects as few as 2.5 genome copies. To enable direct quantification of Bartonella organisms in different vector samples, we developed a qPCR to detect C. felis DNA that also acts as an extraction control. Combining both PCRs into a multiplex format validates B. henselae results when sampling flea populations, although there is a reduction in sensitivity. This reduction might be counteracted by a different combination of probe fluorophores.

Polymerase chain reaction (PCR) analysis is regularly used to detect pathogens within arthropod vectors, but has also been applied to investigate vector DNA. This study details a novel highly sensitive quantitative PCR (qPCR) which detects and quantifies DNA from Ixodes ricinus , the European vector of Anaplasma phagocytophilum . By pairing this with a qPCR to detect A. phagocytophilum , valid comparisons of pathogen load can be made between different sized tick-tissue samples. These qPCRs were validated in I. ricinus that were fed A. phagocytophilum -infected blood using an artificial membrane feeder. Pathogens were detected in the tick haemolymph within 36 h, indicating that successful infection had taken place. This study illustrates the application of vector-targeted qPCRs to confirm and validate pathogen load in samples as part of investigations of vector–pathogen interactions.

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.

To the Editor: Rickettsia felis is a bacterium transmitted by the cat flea (Ctenocephalides felis), which also acts as a reservoir by means of transovarial transmission (1–3). The distribution of R. felis is potentially as wide as that of its insect host, and to date, its presence has been confirmed in cat flea populations in North and South America and southern Europe (4,5). R. felis was first identified as a human pathogen in 1994 (6), and cases of “flea-borne spotted fever,” which have signs and symptoms of febrile exanthema, have now been reported in the United States, Mexico, Brazil, France, and Germany (7,8). To our knowledge, reports on the presence of R. felis, or indeed any other spotted fever group rickettsia, in the United Kingdom have not been published. To determine whether R. felis is present in the United Kingdom, we surveyed cat fleas collected from dogs and cats seen at veterinary practices in southern England and Northern Ireland. A total of 31 dogs and 79 cats from veterinary practices in Bristol, Dorset, London, Devon, Gloucestershire, Hampshire, and Antrim were included in our study. Fleas were collected by combing these animals for 10 minutes. All fleas from each animal were pooled in 70% ethanol. A total of 316 Ct. felis (Bouche, 1835), identified by using accepted morphologic criteria, were obtained, with each animal yielding one to five fleas. DNA was extracted from each of the 110 flea pools by using a standard silica cartridge method (QiaAmp DNA mini kit, QIAGEN Ltd., Crawley, West Sussex, U.K.) using the manufacturer’s instructions for tissue DNA extraction. The presence of rickettsial DNA was determined by using the polymerase chain reaction (PCR) with oligonucleotide primers that target rickettesial ompB (5) or gltA (2) genes. Positive control material was cultured R. felis. Rigorous controls to limit contamination were carried out, including the use of separate, dedicated rooms for DNA extraction, PCR setup, and gel analysis. Amplification products obtained from ompB and gltA PCRs were analyzed by using DNA sequencing. Sequences obtained were edited by using BioEdit (available from: URL: http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Similarity to published sequences was determined with the BLAST program (available from: URL: http://www.ncbi.nlm.nih.gov) hosted by the National Centre for Biotechnology Information. Eighteen flea DNA pools were positive for spotted fever group rickettsia. All 18 yielded PCR products with both ompB and gltA-targeting PCRs. The ompB and gltA DNA sequences of all PCR products were 100% identical to those published for R. felis, thereby providing evidence for the presence of R. felis in fleas collected from >16% of the animals surveyed. PCR-positive fleas were collected from 4 dogs and 14 cats from Bristol, Hampshire, Dorset, and Northern Ireland. Taking into account the number of fleas in each pool, we estimate that 6% to 12% of the fleas collected were infected with R. felis. This study represents the first description of a spotted fever group rickettsia endemic to the United Kingdom. The species detected, R. felis, has clear public health implications. The bacterium appears to be widely distributed within the country, infecting a geographically dispersed population of Ct. felis. Up to 12% of Ct. felis may be infected with R. felis, a flea that is by far the most common species of ectoparasite encountered on cats and dogs in the U.K. mainland. Furthermore, Ct. felis often feeds on humans. Clinicians encountering patients with fever or rash (or both) and a history of cat contact or flea bites should consider a diagnosis of R. felis. Laboratory confirmation of infection is not easy, but in vitro culture of R. felis, and hence material for a serologic assay for the diagnosis of human R. felis infections, has recently been described, and serology appears to be an accurate indicator of exposure (9). As with other spotted fever group rickettsial infections, molecular diagnostics may provide a useful alternative approach to detecting and identifying R. felis in infected tissues. In culture, R. felis has been shown to be resistant to erythromycin (unlike other rickettsia), gentamicin, amoxicillin, and trimethoprim-sulfamethoxazole. Thus, infection with this bacterium should be considered in cases of antibiotic-insensitive fever with a rash, especially in young, old, and immunosuppressed persons. The organism is sensitive to doxycyclin, rifampicin, thiamphenicol, and fluoroquinolones (10)

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.

Serum C4 concentrations and C4 allotypes were determined wherever possible on eight dogs with an autoimmune disorder characterized by antinuclear antibody (ANA) positivity, pyrexia, and seronegative arthritis. Three of the six dogs tested serially showed hypocomplementemia at disease onset with return to normal concentrations with clinical remission following steroid therapy. All five dogs C4 allotyped were phenotypically identical. The phenotype (C4-4) was significantly increased in the diseased dogs. Thus C4 allotype may provide a marker for susceptibility to development of an autoimmune disorder in the dog. Serum C4 concentrations may help in disease diagnosis and monitoring the effectiveness of therapy.