Molecular and histopathological detection of Hepatozoon canis in red foxes (Vulpes vulpes) from Portugal.

BACKGROUND: Hepatozoon canis is a protozoan tick-borne pathogen of dogs and wild canids. Hepatozoon spp. have been reported to infect foxes in different continents and recent studies have mostly used the polymerase chain reaction (PCR) for the detection and characterization of the infecting species. Surveying red foxes (Vulpes vulpes) may contribute to better understanding the epidemiology of canine vector-borne diseases, including hepatozoonosis caused by H. canis in domestic dogs. The present study investigated the prevalence of Hepatozoon spp. by means of histopathology and molecular analysis of different tissues in red foxes from different parts of Portugal. METHODS: Blood and tissues including bone marrow, heart, hind leg muscle, jejunum, kidney, liver, lung, popliteal or axillary lymph nodes, spleen and/or tongue were collected from 91 red foxes from eight districts in northern, central and southern Portugal. Tissues were formalin-fixed, paraffin-embedded, cut and stained with hematoxylin and eosin. Polymerase chain reaction (PCR) amplified a ~650 bp fragment of the 18S rRNA gene of Hepatozoon spp. and the DNA products were sequenced. RESULTS: Hepatozoon canis was detected in 68 out of 90 foxes (75.6%) from all the sampled areas by PCR and sequencing. Histopathology revealed H. canis meronts similar in shape to those found in dogs in the bone marrow of 11 (23.4%) and in the spleen of two (4.3%) out of 47 foxes (p = 0.007). All the 11 foxes found positive by histopathology were also positive by PCR of bone marrow and/or blood. Positivity by PCR (83.0%) was significantly higher (p < 0.001) than by histopathological examination (23.4%) in paired bone marrow samples from the same 47 foxes. Sequences of the 18S rRNA gene of H. canis were 98-99% identical to those in GenBank. CONCLUSIONS: Hepatozoon canis was found to be highly prevalent in red fox populations from northern, central and southern Portugal. Detection of the parasite by histopathology was significantly less sensitive than by PCR. Red foxes are a presumptive reservoir of H. canis infection for domestic dogs.

Redescription of Hepatozoon felis (Apicomplexa: Hepatozoidae) based on phylogenetic analysis, tissue and blood form morphology, and possible transplacental transmission.

BACKGROUND: A Hepatozoon parasite was initially reported from a cat in India in 1908 and named Leucocytozoon felis domestici. Although domestic feline hepatozoonosis has since been recorded from Europe, Africa, Asia and America, its description, classification and pathogenesis have remained vague and the distinction between different species of Hepatozoon infecting domestic and wild carnivores has been unclear. The aim of this study was to carry out a survey on domestic feline hepatozoonosis and characterize it morphologically and genetically. METHODS: Hepatozoon sp. DNA was amplified by PCR from the blood of 55 of 152 (36%) surveyed cats in Israel and from all blood samples of an additional 19 cats detected as parasitemic by microscopy during routine hematologic examinations. Hepatozoon sp. forms were also characterized from tissues of naturally infected cats. RESULTS: DNA sequencing determined that all cats were infected with Hepatozoon felis except for two infected by Hepatozoon canis. A significant association (p = 0.00001) was found between outdoor access and H. felis infection. H. felis meronts containing merozoites were characterized morphologically from skeletal muscles, myocardium and lungs of H. felis PCR-positive cat tissues and development from early to mature meront was described. Distinctly-shaped gamonts were observed and measured from the blood of these H. felis infected cats. Two fetuses from H. felis PCR-positive queens were positive by PCR from fetal tissue including the lung and amniotic fluid, suggesting possible transplacental transmission. Genetic analysis indicated that H. felis DNA sequences from Israeli cats clustered together with the H. felis Spain 1 and Spain 2 sequences. These cat H. felis sequences clustered separately from the feline H. canis sequences, which grouped with Israeli and foreign dog H. canis sequences. H. felis clustered distinctly from Hepatozoon spp. of other mammals. Feline hepatozoonosis caused by H. felis is mostly sub-clinical as a high proportion of the population is infected with no apparent overt clinical manifestations. CONCLUSIONS: This study aimed to integrate new histopathologic, hematologic, clinical, epidemiological and genetic findings on feline hepatozoonosis and promote the understanding of this infection. The results indicate that feline infection is primarily caused by a morphologically and genetically distinct species, H. felis, which has predilection to infecting muscular tissues, and is highly prevalent in the cat population studied. The lack of previous comprehensively integrated data merits the redescription of this parasite elucidating its parasitological characteristics.

Efficacy of rifampicin in the treatment of experimental acute canine monocytic ehrlichiosis.

OBJECTIVES: To assess the efficacy of rifampicin in achieving clinical and haematological recovery and clearing infection in dogs with experimentally induced acute monocytic ehrlichiosis. METHODS: Five Ehrlichia canis-infected Beagle dogs were treated with rifampicin (10 mg/kg/24 h orally for 3 weeks), nine E. canis-infected dogs received no treatment (infected untreated dogs) and two dogs served as uninfected controls. Clinical score, platelet counts, immunofluorescent antibody titres and PCR detection of E. canis-specific DNA in blood, bone marrow and spleen aspirates were evaluated on post-inoculation days 21 (start of rifampicin), 42 (end of rifampicin) and 98 (end of the study). RESULTS: By day 21 post-inoculation, all infected dogs became clinically ill and thrombocytopenic, seroconverted and were PCR positive in at least one tissue. Clinical scores and antibody titres did not differ between the treated and infected untreated dogs throughout the study. The rifampicin-treated dogs experienced an earlier resolution of their thrombocytopenia (Kaplan-Meier survival plot, P=0.048), and the median platelet counts were significantly higher in the treated compared with the infected untreated dogs on post-inoculation days 42 (P=0.0233) and 98 (P=0.0195). At the end of the study, three treated and six untreated infected dogs remained PCR positive in one tissue each. CONCLUSIONS: The rifampicin treatment regimen applied in this study hastened haematological recovery, but was inconsistent in eliminating the acute E. canis infection.

Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria.

BACKGROUND: Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep. CONCLUSIONS/SIGNIFICANCE: The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents.

First description of natural Ehrlichia canis and Anaplasma platys infections in dogs from Argentina.

Bacteria belonging to the Anaplasmataceae family are vector transmitted agents that affect a variety of vertebrate hosts including the tick-borne pathogens Ehrlichia canis and Anaplasma platys, which cause canine monocytic ehrlichiosis and cyclic thrombocytopenia, respectively. These two infections, typically reported from tropical and sub-tropical regions, have not been previously reported in dogs from Argentina. A total of 86 blood samples from dogs with suspected rickettsial disease and 28 non-suspected dogs were studied. Analysis included evaluation of hematological findings, PCR for Ehrlichia and Anaplasma species and sequencing of the positive PCR products. E. canis was detected in the blood of six dogs and A. platys in eighteen. All the dogs categorized as non-suspected were negative by PCR. Co-infection with Hepatozoon canis and Babesia vogeli was documented. This first report of E. canis and A. platys infections in dogs from Argentina indicates that these tick-borne infections have a considerably broader range than previously recognized in South America.

Evaluation of an attenuated strain of Ehrlichia canis as a vaccine for canine monocytic ehrlichiosis.

Canine monocytic ehrlichiosis is an important tick-borne disease worldwide. No commercial vaccine for the disease is currently available and tick control is the main preventive measure against the disease. The aim of this study was to evaluate the potential of a multi-passaged attenuated strain of Ehrlichia canis to serve as a vaccine for canine monocytic ehrlichiosis, and to assess the use of azithromycin in the treatment of acute ehrlichiosis. Twelve beagle dogs were divided into 3 groups of 4 dogs. Groups 1 and 2 were inoculated (vaccinated) with an attenuated strain of E. canis (#611A) twice or once, respectively. The third group consisted of naïve dogs which served as controls. All 3 groups were challenged with a wild virulent strain of E. canis by administering infected dog-blood intravenously. Transient thrombocytopenia was the only hematological abnormality observed following inoculation of dogs with the attenuated strain. Challenge with the virulent strain resulted in severe disease in all 4 control dogs while only 3 of 8 vaccinated dogs presented mild transient fever. Furthermore, the mean blood rickettsial load was significantly higher in the control group (27-92-folds higher during days 14-19 post challenge with the wild the strain) as compared to the vaccinated dogs. The use of azithromycin was assessed as a therapeutic agent for the acute disease. Four days treatment resulted in further deterioration of the clinical condition of the dogs. Molecular comparison of 4 genes known to express immunoreactive proteins and virulence factors (p30, gp19, VirB4 and VirB9) between the attenuated strain and the challenge wild strain revealed no genetic differences between the strains. The results of this study indicate that the attenuated E. canis strain may serve as an effective and secure future vaccine for canine ehrlichiosis.

Prevalence of Trypanosoma evansi in horses in Israel evaluated by serology and reverse dot blot.

Trypanosoma evansi is the cause of surra in horses, camels and other domestic animals. Following the first outbreak of surra in horses and camels in Israel in 2006, a survey of the prevalence of the parasite in the Israeli horse population was conducted using serology, PCR followed by the reverse dot blot (RDB) technique and blood smear microscopy. In total, 614 horses from 7 regions were sampled. The CATT/T. evansi kit was used for serology for all the horses. Horses from the Arava and Dead Sea region, where the first outbreak occurred, were sampled again one year later and both samples were subjected to serology and the RDB technique. The country wide seroprevalence was 4.6% (28/614). The seroprevalence in the Arava and Dead Sea region was 6.5% (9/139) in the first sampling compared with 4.1% (5/122) in the second, whereas the prevalence of RDB-positivity was 18.7% (26/139) in the first sampling and only 0.8% (1/122) in the second. All horses were asymptomatic except for one horse from the Arava and Dead Sea region that demonstrated clinical signs of surra combined with positive serology and RDB. The results of this study indicated that surra is prevalent in most regions of the country and thus should be considered an important differential diagnosis in horses and other domestic animals in Israel with chronic weight loss, edema or neurological signs.

Oocysts of Hepatozoon canis in Rhipicephalus (Boophilus) microplus collected from a naturally infected dog.

Canine hepatozoonosis is a tick-borne disease caused by protozoans of the genus Hepatozoon. Several tick species have been implicated as potential vectors. Therefore, extensive studies are needed to determine the 'natural' endemic cycle of this parasite. This paper presents the first report of the presence of Hepatozoon canis oocysts in Rhipicephalus (Boophilus) microplus collected from an infected dog.

Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli.

Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/µl in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs.

Failure of imidocarb dipropionate to eliminate Hepatozoon canis in naturally infected dogs based on parasitological and molecular evaluation methods.

The efficacy of imidocarb dipropionate for the treatment of Hepatozoon canis infection was studied in three naturally infected asymptomatic dogs followed longitudinally over 8 months. Response to treatment was followed by monitoring blood counts, parasitemia levels in blood, parasite in concentrated buffy-coat smears and by PCR. The dogs were initially treated with a low dose of 3 mg/kg imidocarb dipropionate twice a month and when parasitemia persisted after five treatments, with the regular dose of 6 mg/kg. In one dog, H. canis gamonts were no longer detectable by blood and buffy-coat microscopy after 2 months of therapy with 6 mg/kg while in the two other dogs gamonts were intermittently found in blood but persistently detectable in buffy-coat smears during the whole study period. Furthermore, combined therapy with doxycycline monohydrate administered at 10 mg/kg/day PO for 4 weeks also failed to eliminate H. canis. PCR revealed that parasite DNA was present in the blood of all dogs at all sampling dates regardless of treatment refuting the effectiveness of treatment suggested by negative blood microscopy. Detection of H. canis in buffy coat was found to be twice as sensitive than by blood smear and detection by PCR was even more sensitive revealing infection in eight samples (16% of total samples) negative by blood and buffy-coat microscopy. In conclusion, imidocarb dipropionate was not effective in eliminating H. canis from dogs treated repeatedly over 8 months. Microscopical detection is not sufficient for the evaluation of treatment response in H. canis infection and follow up by molecular techniques is recommended.

Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR.

The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S-23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.

Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.

Babesia canis canis and Babesia canis vogeli infections in dogs from northern Portugal.

Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408bp fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis and B. canis vogeli in dogs from Portugal.

Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera.

Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC-EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC-EIA were highly correlated (R2=0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera.

Role of M3 protein in the adherence and internalization of an invasive Streptococcus pyogenes strain by epithelial cells.

Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process.

Role of CsrR, hyaluronic acid, and SpeB in the internalization of Streptococcus pyogenes M type 3 strain by epithelial cells.

Internalization of group A streptococcus by human epithelial cells has been extensively studied during the past 6 years. It is now clear that multiple mechanisms are involved in this process. We have previously demonstrated that the CsrR global regulator controls the internalization of an invasive M type 3 strain through regulation of the has (hyaluronic acid synthesis) operon, as well as another, unknown gene(s). Recently, it was reported that the CsrR-regulated cysteine protease (SpeB) is also involved in bacterial uptake. In this study we have examined the roles of CsrR, hyaluronic acid capsule, and SpeB in streptococcal internalization. We have constructed isogenic mutants of the M3 serotype deficient in the csrR, hasA, and speB genes and tested their ability to be internalized by HEp-2 epithelial cells. Inactivation of csrR abolished internalization, while inactivation of either hasA or speB increased the internalization efficiency. Mutation in csrR derepressed hasA transcription and lowered the activity of SpeB, while no effect on speB transcription was observed. The speB mutant expressed smaller amounts of capsule, while the hasA mutant transcribed more csrR and speB mRNAs. Thus, it seems that complex interactions between CsrR, SpeB, and capsule are involved in modulation of group A streptococcus internalization.