Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): An overview

Canine monocytotropic ehrlichiosis (CME), caused by the rickettsia Ehrlichia canis, an important canine disease with a worldwide distribution. Diagnosis of the disease can be challenging due to its different phases and multiple clinical manifestations. CME should be suspected when a compatible history (living in or traveling to an endemic region, previous tick exposure), typical clinical signs and characteristic hematological and biochemical abnormalities are present. Traditional diagnostic techniques including hematology, cytology, serology and isolation are valuable diagnostic tools for CME, however a definitive diagnosis of E. canis infection requires molecular techniques. This article reviews the current literature covering the diagnosis of infection caused by E. canis.     

Detection and identification of Toxocara canis DNA in bronchoalveolar lavage of infected mice using a novel real-time PCR

Toxocarosis is a zoonosis with worldwide distribution caused by Toxocara spp. of dogs and cats. In humans, diagnosis relies mainly on detection of parasite-specific antibodies. Although serological assays in current use have defined sensitivity and specificity, the problem of cross-reactivity still remains, particularly in areas of endemic polyparasitism. Microscopic detection of the parasite in tissue biopsies is not recommended for diagnosis because larvae can be difficult to locate, and finding the parasite eggs in faeces is not applicable since the larvae do not develop to the adult stage in the human host. In this study we describe a novel real-time PCR (‘Nemo-PCR’) that, in combination with DNA sequencing, allows the detection and identification of Toxocara canis and other nematodes in the Superfamily Ascaridoidea. Results indicate that this approach can detect Toxocara spp. DNA in bronchoalveolar lavage (BAL) of experimentally-infected mice. For diagnostic purposes further studies are necessary to evaluate this assay including testing human BAL fluid. The availability of such a direct assay would improve diagnosis of toxocarosis particularly for patients with pulmonary signs and symptoms.     

A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10³ colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.     

Prevalence and molecular characterization of Hepatozoon canis in dogs from urban and rural areas in Southeast Brazil

The objective of this survey was to investigate the prevalence of Hepatozoon infection in dogs in the rural and urban areas of Uberlândia, Brazil by PCR and molecular characterization. DNA was obtained from blood samples collected from 346 local dogs from both genders and various ages. Seventeen PCR products from positive blood samples of urban dogs and 13 from the rural dogs were sequenced. Partial sequences of the 18S rRNA gene indicated that all 30 dogs were infected with Hepatozoon canis similar in sequence to H. canis from southern Europe. Four local dog sequences were submitted to GenBank (accessions JN835188; KF692038; KF692039; KF692040). This study indicates that H. canis is the cause of canine hepatozoonosis in Uberlândia and that infection is similarly widespread in rural and urban dogs.     

Recovery threshold of Toxocara canis eggs from soil

The purpose of this study was to evaluate the threshold of Toxocara canis eggs form soil samples through utilisation of a centrifuge-flotation technique (CFT). Aliquots of soil (1 g each) were artificially contaminated with known numbers of T. canis eggs (1, 10, 25, 50, 100, and 200 eggs). The threshold was evaluated based on a CFT using zinc sulphate (Zn₂SO₄) and sodium nitrate (Na₂NO₃) solutions at a specific gravity of 1.20. The number of eggs recovered was directly proportional to the number of eggs employed to seed the soil. Both solutions enabled full recovery of samples containing merely three eggs; only Zn₂SO₄ demonstrated efficiency in soil contaminated with a single egg. A recovery rate of 100% was obtained for all tests with samples containing 10 and 25 eggs for Zn₂SO₄ and Na₂NO₃, respectively_. There was no difference in the mean number of recovered eggs regarding either the efficacy of the solutions or the repetition of evaluations in the same trial (p > 0.05). Therefore, the CFT is efficient for the detection of Toxocara eggs, even in samples containing low egg numbers.     

Molecular characterization of norovirus and sapovirus detected in animals and humans in Costa Rica: Zoo-anthropozoonotic potential of human norovirus GII.4

Background:Noroviruses (NoV) and sapoviruses (SaV) are major causes of acute viral gastroenteritis in humans worldwide, as well as gastrointestinal infections in animals. However, it has not been determined whether these viruses are zoonotic pathogens.Aim:In this study, we investigated the presence of NoV and SaV in stool samples from dogs, pigs, cows, and humans to determine some aspects of the molecular epidemiology and the genetic relationship of several strains present in these species.Methods:Polymerase chain reaction and sequencing of NoV and SaV strains present in stool samples from humans and dogs with diarrhea, pigs, and cattle with and without diarrhea were carried out during fragmented periods from 2002 to 2012.Results:Of all samples analyzed, 11.6% (123/1,061) of the samples were positive for NoV and 0.88% (9/1,023) were positive for SaV. The phylogenetic analysis confirmed 16 human strains of NoV (HuNoV) belonging to HuNoV G?/GII.P2 (1), GII.4/GII.P4 (5), G?/GII.P4 (9), and GII.6/GII.P6 (1) and allowed us to verify and assign three strains of human SaV to genotypes GI.2 (1) and GII.5 (2). In dogs, eight strains of NoV [HuNoV G?/GII.P4 (4) and canine G?/GVI.P1 (4)] and two strains of canine SaV were determined. In pigs, six strains were assigned to HuNoV G?/GII.P4 and four strains to porcine SaV were assigned to genogroup GIII (2), GVIII (1), and GXI (1). In bovines, five strains were characterized as HuNoV G?/GII.P4.Conclusions:This study showed that NoV and SaV prototype strains have been present in humans and dogs in Costa Rica. Additionally, it revealed that the zoonotic potential of SaV is very limited, while the zoonotic implications for HuNoV GII.4 are stronger due to the simultaneous circulation of strains related to HuNoV GII.4 in four species, which suggests a zoo-anthropozoonosis.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

Experimental inoculation of beagle dogs with Ehrlichia species detected from Ixodes ovatus

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.     

A parasitological, molecular and serological survey of Hepatozoon canis infection in dogs around the Aegean coast of Turkey

Canine hepatozoonosis is caused by the tick-borne protozoon Hepatozoon spp. The prevalence of the infection in the Aegean coast of Turkey was investigated by examination of blood smear parasitology and polymerase chain reaction (PCR) using blood samples from 349 dogs collected from Central Aydin, Kusadasi, Selcuk, Central Manisa, Bodrum and Marmaris within the Aegean coast of Turkey. The indirect fluorescent antibody test (IFAT) for the detection of Hepatozoon canis antibodies was also used to detect the exposure rate to H. canis. PCR amplifying a 666bp fragment of 18S rRNA gene of Hepatozoon spp. was used in the epidemiological survey. The prevalence of Hepatozoon spp. infection was 10.6% by blood smear parasitology and 25.8% by PCR. IFAT revealed that 36.8% of serum samples were positive for antibodies reactive with Hepatozoon spp. The PCR products of 18S rRNA gene of Hepatozoon spp. isolated from six infected dogs, one isolate originating from each of the six different locations, were sequenced. The results of sequence analysis indicate that they are closely related to Indian and Japanese isolates of H. canis. This is the first epidemiological study on the prevalence of H. canis infection in the dog, in Turkey.     

Detection and molecular characterization of Theileria sp. in fallow deer (Dama dama) and ticks from an Italian natural preserve

The prevalence of piroplasms in a closed population of fallow deer (Dama dama L.) living in the Italian preserve of “Bosco della Mesola” - Ferrara (Mesola wood) was investigated. Blood samples and ticks were collected from 62 fallow deer. On microscopic observation, 28 (45.0%) blood samples were positive for piroplasms while PCR provided evidence for piroplasms infection in 47 (75.8%) fallow deer. The 67 ticks, collected from positive and negative animals, were identified as Ixodesricinus L., 1758 (89.6%) and Haemaphysalisconcinna Koch, 1844 (10.4%). At the PCR, four samples of I. ricinus were positive for piroplasms. The sequences of the 18S rRNA gene from both blood and ticks were identical and showed high identity (99.6%) with Theileria sp. 3185/02 (DQ866842) and Theileria capreoli (AY726011) from roe deer. Interestingly, the phylogenetical analyses evidenced differences between the Theileria strain from Mesola wood and the ones isolated in fallow deer from other Italian areas.     

The molecular epidemiology of Cryptosporidium and Giardia infections in coyotes from Alberta,Canada, and observations on some cohabiting parasites

Coyotes from southern Alberta and Saskatchewan, Canada, were examined for the presence of Giardia and Cryptosporidium and cohabiting helminths. Toxascaris was present in over 90% of the 70 animals examined, and Taenia sp. in 6.5–25% of the two groups of animals studied. Giardia (12.5–21.7%) and Cryptosporidium (0–17.4%) were also common and molecular characterisation revealed both zoonotic and host-adapted genotypes of Giardia, whereas the Cryptosporidium proved to be a variant of the canine species C. canis. The seasonal variation observed in the occurrence of Cryptosporidium may be related to stress-induced shedding of the parasite.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

The isolation and characterization of Campylobacter jejuni bacteriophages from free range and indoor poultry

Six hundred and sixty one samples – primarily fresh chicken faeces – were processed to isolate wild type Campylobacter jejuni bacteriophages, via overlay agar methods using C. jejuni NCTC 12662. The aims of this study were to isolate and purify bacteriophages and then test for their ability to lyse field strains of C. jejuni in vitro. Of all samples processed, 130 were positive for bacteriophages. A distinct difference was observed between samples from different poultry enterprises. No bacteriophages could be isolated from indoor broilers. The majority of bacteriophages were isolated from free range poultry – both broilers and egg layers. Bacteriophages were purified and then selected for characterization based on their ability to produce clear lysis on plaque assay, as opposed to turbid plaques. Two hundred and forty one C. jejuni field isolates were tested for sensitivity to the bacteriophages. Lysis was graded subjectively and any minimal lysis was excluded. Using this system, 59.0% of the C. jejuni isolates showed significant sensitivity to at least one bacteriophage. The sensitivity to individual bacteriophages ranged from 10.0% to 32.5% of the C. jejuni isolates. Five bacteriophages were examined by electron microscopy and determined to belong to the Myoviridae family. The physical size, predicted genetic composition and genome size of the bacteriophages correlated well with other reported Campylobacter bacteriophages. The reasons for the observed difference between indoor broilers and free range poultry is unknown, but are postulated to be due to differences in the Campylobacter population in birds under different rearing conditions.     

First isolation of Neospora caninum from the faeces of a dog from Portugal

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of γ-interferon knockout mice with 10² sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.     

Prevalence and antibiotic resistance profiles of Enterococcus species in chicken at slaughter level; absence of vanA and vanB genes in E. faecalis and E. faecium

The prevalence of enterococci in neck skin samples of poultry from Ankara region in Turkey was investigated and their antibiotic resistance patterns were determined. In the study, 83 of 106 analyzed neck skin samples were positive for Enterococcus, with E. faecium as the most prevalent species (48%) followed by E. durans (23%) and E. faecalis (19%). Lower numbers were detected for E. gallinarum, E. hirae, E. mundtii and E. casseliflavus. Using the disc diffusion method, it was established that over 90% of E. faecium and E. faecalis isolates were high-level resistant against erythromycin and tetracycline. Four E. faecium isolates were additionally resistant to chloramphenicol, gentamicin and streptomycin, though they were susceptible to penicillin G. The most frequently observed multiple resistance in E. faecium (25%) was against erythromycin, tetracycline, chloramphenicol and streptomycin. Of the E. faecalis isolates, 44% were multiple resistant to erythromycin, tetracycline and streptomycin. Vancomycin resistance could not be demonstrated phenotypically and vanA or vanB genes were not detected by multiplex PCR in any of the isolates. Nevertheless, the observed resistance patterns are of concern for public health.     

Hepatozoon canis infecting dogs in the State of Espírito Santo, southeastern Brazil

From May 2007 to March 2008, blood samples were collected from 92 healthy dogs living in 21 households (17 farms in rural area, and 4 homes in urban area) in 6 counties of the State of Espírito Santo, southeastern Brazil. In addition, ticks were collected from these dogs. A mean of 4.4 ± 3.0 dogs (range: 1–12) were sampled per household; 78 and 14 dogs were from rural and urban areas, respectively. Polymerase chain reaction (PCR) designed to amplify fragments of the 18S rDNA gene of Babesia spp or Hepatozoon spp revealed amplicons of the expected size in 20 (21.7%) dogs for Babesia, and 54 (58.7%) dogs for Hepatozoon. All Babesia-positive dogs were also Hepatozoon-positive. Among the 21 households, 15 (71.4%) from 3 counties had at least one PCR-positive dog, including 13 farms (rural area) and 2 homes (urban area). A total of 40 PCR products from the Hepatozoon-PCR, and 19 products from the Babesia-PCR were submitted to DNA sequencing. All generated sequences from Hepatozoon-PCR were identical to each other, and to corresponding 18S rDNA sequences of H. canis in GenBank. Surprisingly, all generated sequences from the Babesia PCR were also identical to corresponding 18S rDNA sequences of H. canis in GenBank. Dogs from 10 rural and 2 urban households were found infested by Rhipicephalus sanguineus ticks. Immature of Amblyomma cajennense ticks were found in dogs from only 4 rural households (also infested by R. sanguineus). All but one household with R. sanguineus-infested dogs had at least one Hepatozoon-infected dog. Statistical analysis showed that the presence of ticks (i.e. R. sanguineus) infesting dogs in the households was significantly (P < 0.05) associated with at least one PCR-positive dog. There was no significant association (P > 0.05) between PCR-positive dogs and urban or rural households. Canine hepatozoonosis caused by H. canis is a high frequent infection in Espírito Santo, Brazil, where it is possibly vectored by R. sanguineus. Since all infected dogs were found apparently healthy, the pathogenicity of H. canis for dogs in Espírito Santo is yet to be elucidated.     

A perfect time to harness advanced molecular technologies to explore the fundamental biology of Toxocara species

Toxocarosis is of major canine health and socioeconomic importance worldwide. Although many studies have given insights into toxocarosis, to date, there has been limited exploration of the molecular biology, biochemistry, genetics, epidemiology and ecology of Toxocara species as well as parasite–host interactions using ‘-omic’ technologies. The present article gives a background on Toxocara species and toxocarosis, describes molecular tools for specific identification and genetic analysis, and provides a prospective view of the benefits that advanced molecular technologies will have towards better understanding the parasites and disease. Tackling key biological questions employing a ‘systems biology’ approach should lead to new and improved strategies for the treatment, diagnosis and control of toxocarosis.     

Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.