Prevalence and diversity of Hepatozoon canis in naturally infected dogs in Japanese islands and peninsulas

Canine hepatozoonosis is a worldwide protozoal disease caused by Hepatozoon canis and Hepatozoon americanum and is transmitted by ixodid ticks, Rhipicephalus and Amblyomma spp., respectively. H. canis infection is widespread in Africa, Europe, South America, and Asia, including Japan. The objective of this study was to study the distribution pattern and diversity of H. canis in naturally infected dogs in nine Japanese islands and peninsulas. Therefore, 196 hunting dogs were randomly sampled during the period from March to September 2011 and the ages and sexes were identified. Direct microscopy using Giemsa-stained blood smears revealed H. canis gametocytes in the peripheral blood of 45 (23.6 %) dogs. Polymerase chain reaction (PCR) was performed on EDTA-anticoagulated blood, initially with the common primer set (B18S-F and B18S-R) amplifying the 1,665-bp portion of the 18S rRNA gene, and then with the specific primer set (HepF and HepR) amplifying about 660 bp fragments of the same gene. Based on PCR, 84 (42.9 %) dogs were positive using the common primer and 81 (41.3 %) were positive using the specific primer. The current investigation indicated that all screened areas, except for Sado Island and Atsumi Peninsula, were infected. Yaku Island had the highest infection rate (84.6 % in males and 100.0 % in females), while Ishigaki Island showed the lowest infection rates (8.3 % in males and 17.7 % in females). Both sexes were infected with no significant difference. However, diversity of infection among the surveyed islands and peninsulas was significantly different (P?<?0.05). Although H. canis has previously been reported in dogs in Japan, the higher infection rate described in the current study and the diversity of infection in a wide range of islands strongly encourage prospective studies dealing with the prevention and treatment of the infection in dogs, as well as control of ticks.     

Molecular characterization of Hepatozoon canis in dogs from Colombia

Hepatozoonosis is a tick-borne disease whose transmission to dogs occurs by ingestion of oocysts infected ticks or feeding on preys infested by infected ticks. Until now, there is no previous report of molecular characterization of Hepatozoon sp. in dogs from Colombia. EDTA blood samples were collected from 91 dogs from central-western region of Colombia (Bogotá, Bucaramanga, and Villavicencio cities) and submitted to 18S rRNA Hepatozoon sp. PCR and blood smears confection. Phylogenetic analysis was used to access the identity of Hepatozoon species found in sampled dogs. From 91 sampled dogs, 29 (31.8%) were positive to Hepatozoon sp. (25 dogs were only positive in PCR, 1 was positive only in blood smears, and 3 were positive in both blood smears and PCR). After sequencing, the found Hepatozoon sp. DNA showed 100% of identity with Hepatozoon canis DNA isolates. The phylogenetic tree supported the identity of the found Hepatozoon sp. DNA, showing that the isolates from Colombia were placed in the same clade than other H. canis isolates from Venezuela, Spain, and Taiwan. This is the first molecular detection of H. canis in dogs from Colombia.     

C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis.

To elucidate whether acute-phase protein responses occur in dogs infected with Ehrlichia canis, C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) levels were serially measured in the plasma of five dogs experimentally inoculated with E. canis and 10 sham-inoculated or noninoculated control dogs. The CRP concentration was measured by a canine-specific capture enzyme-linked immunosorbent assay, and the AAG concentration was measured by a canine-specific radial immunodiffusion method. In all E. canis-inoculated dogs, a 3.3- to 6.5-fold increase in the plasma CRP concentration and a 1.9- to 8.6-fold increase in the plasma AAG concentration over the preinoculation level occurred at days 4 to 6 postexposure. Despite the persistence of E. canis and high antibody titers, both CRP and AAG concentrations gradually declined to preexposure levels by day 34 postexposure. E. canis-infected dogs had mild and transient clinical signs which resolved without treatment by day 14 postexposure. The CRP and AAG concentrations in control inoculated or nontreated dogs remained within the normal range throughout the experimental period. Of 12 dogs naturally infected with E. canis, 75% had greater than 50 micrograms of CRP per ml and 83% had greater than 500 micrograms of AAG per ml. All of these 12 dogs had chronic and severe clinical signs of canine ehrlichiosis. Thus, elevations in the levels of acute-phase proteins occur in both acute and chronic canine ehrlichiosis. Determination of CRP and AAG concentrations may help in assessing the severity of inflammatory damage in dogs with E. canis infections.     

Changes in serum adenosine deaminase activity during normal pregnancy

Adenosine deaminase (ADA), an enzyme essential for the differentiation of lymphoid cells, has been used for monitoring diseases with altered immunity. The purpose of this study was to investigate the changes in serum ADA activity throughout normal pregnancy. We measured the catalytic values of serum ADA from 202 normal pregnant women using a commercial kit. Subjects were divided into four groups according to the gestational age in weeks (Gwks) (Group I: 5-9 Gwks [n=58]; Group II: 15-20 Gwks [n= 63]; Group III: 24-30 Gwks [n=34]; Group IV: 30-39 Gwks [n=47]). The serum ADA levels for the Groups I, II, III, and IV were as follows: 20.1 +/- 6.9 IU/L, 20.0 +/- 7.6 IU/L, 37.9 +/- 19.9 IU/L, and 24.5 +/- 8.6 IU/L, respectively. The serum ADA activity of group III was significantly higher than the other groups (p<0.05). However, there was no significant correlation between the Gwks and the serum ADA activity. Furthermore, other parameters, such as maternal age (p=0.29), gestational age at delivery (p=0.07), delivery mode (p=0.39), and birth weight (p=0.59) had no correlation with ADA activity. Reference values of serum ADA in normal pregnancy may provide important database for making clinical decisions in pregnancies complicated by conditions where cellular immunity has been altered.     

Report of a clinical case of dog infected by Hepatozoon canis in Southern Brazil

This article aims to describe a clinical case of a dog naturally infected with Hepatozoon canis. Clinical findings, hematological parameters, parasitological, and molecular diagnosis and treatments were evaluated. On physical examination, this animal showed apathy, anorexia, pale mucosal, nasal and eye secretion, coughing, fever, and progressive weight loss. Gamonts of H. canis were observed within neutrophils in thin blood smears. Important hematological findings observed in this case consisted of severe anemia, thrombocytopenia, and leukocytosis. A partial sequence of the 18S rRNA gene amplified and sequenced from the dog blood was 100% identical to H. canis. The animal was treated with imidocarb dipropionate, which had great curative efficacy to clinical hepatozoonosis.     

Increased serum adenosine deaminase activity in schizophrenic receiving antipsychotic treatment

Adenosine is an important modulator of the nervous system that has been implicated in the pathophysiology of schizophrenia. We studied peripheral adenosine metabolism by determining the activity of serum adenosine deaminase, which converts adenosine into inosine, and 5'-nucleotidase, which converts AMP into adenosine, in 26 DSM-IV male schizophrenic patients under antipsychotic monotherapy and 26 healthy volunteers balanced for age and race. Schizophrenic patients treated either with typical antipsychotics or clozapine showed increased serum adenosine deaminase activity compared to controls (controls=18.96+/-4.61 U/l; typical=25.09+/-10.98 U/l; clozapine=30.32+/-10.83 U/l; p<0.05, ANOVA) and 5'-nucleotidase activity was also increased in patients on clozapine. After adjusting for confounding factors, adenosine deaminase, but not 5'-nucleotidase, alterations remained significant particularly in the clozapine group. This result suggests that either altered adenosine metabolism is present in schizophrenic patients or is influenced by treatment with antipsychotics, particularly clozapine.     

Diagnostic value of serum and pleural fluid adenosine deaminase activity in tuberculous pleurisy

The biological diagnosis of tuberculous pleurisy poses the problem of the time required to obtain results and of the sensitivity of the usual diagnostic methods. The determination of adenosine deaminase (ADA) activity has been proposed for the diagnosis of tuberculous pleural effusion and for the follow-up. In the present study, ADA in pleural effusion (p-ADA) and in serum (s-ADA) has been measured in 2 groups of patients: tuberculosis (27) and non-tuberculosis (53) patients. The upper limit of the normal values was fixed at 37 U/L. Comparing these 2 groups, we observed a specificity of 81.2% and a sensitivity of 66.6%. The PPV and the NPV were respectively 64.3% and 82.7%. We used p-ADA/s-ADA ratio for diagnosis of tuberculous pleural effusion, a threshold value of 1.8 gave a sensitivity of 82.6% and a specificity of 84.8%. Within the tuberculosis patient group, the activity of ADA decreased after the appropriate treatment initiation. Thus, the determination of ADA activity and/ or the p-ADA/s-ADA ratio, can help to recognize the tuberculosis origin of pleural effusions.     

Allosteric modification of adenylate deaminase activity initiation of adenosine deaminase activity by potassium ions

Activation of purified adenylate deaminase from the duck myocardium by K⁺ ions is accompanied by a change in the substrate specificity of the enzyme and appearance of ability to deaminate adenosine and adenine. Adenosine deaminase activity appears with K⁺ in a concentration exhibiting maximal stimulating effect (0.15 M) and it increases with an increase in the K⁺ concentration, parallel with a decrease in Hill's coefficient. It can be concluded from the pH dependence, the character of inhibition by phosphate, and the effect of cations of the alkali metals that deamination of adenosine takes place at natural combining sites of adenylate deaminase, the conformation of which is modified by the activator.Laboratory of Biochemistry of Amines and Other Nitrogenous Bases, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 535–537, November, 1978.     

Comparison of serological detection methods for diagnosis of Ehrlichia canis infections in dogs

We determined the value of four serological assays for the diagnosis of canine monocytic ehrlichiosis by comparing them to the indirect fluorescent-antibody assay "gold standard." The specificity of Dip-S-Ticks was significantly lower than that of all of the other tests evaluated. The sensitivity of Dip-S-Ticks was significantly higher than that of Snap3Dx or the Snap Canine Combo. The sensitivity of the rMAP2 enzyme-linked immunosorbent assay (ELISA) was significantly higher than that of the Snap Canine Combo. The accuracy levels of the rMAP2 ELISA, Snap3Dx, Dip-S-Ticks, and Snap Canine Combo were 97.0, 89.8, 85.1, and 82.9%, respectively.     

Urinary creatinine to serum creatinine ratio and renal failure index in dogs infected with Babesia canis

Urinary creatinine to serum creatinine (UCr/SCr) ratio and renal failure index (RFI) are useful indices of renal damage. Both UCr/SCr ratio and RFI are used in differentiation between prerenal azotaemia and acute tubular necrosis. In this work the authors calculated the UCr/SCr ratio and RFI in dogs infected with Babesia canis and the values of these indices in azotaemic dogs infected with the parasite. The results of this study showed significantly lower UCr/SCr ratio in dogs infected with B. canis than in healthy dogs. Moreover, in azotaemic dogs infected with B. canis the UCr/SCr ratio was significantly lower and the RFI was significantly higher than in non-azotaemic dogs infected with B. canis. The calculated correlation between RFI and duration of the disease before diagnosis and treatment was high, positive and statistically significant (r = 0.89, p < 0.001). The results of this study showed that during the course of canine babesiosis caused by B. canis in Poland acute tubular necrosis may develop.     

Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies

Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis.     

Reisolation of Ehrlichia canis from blood and tissues of dogs after doxycycline treatment.

We present evidence that supports the carrier status of dogs experimentally infected with Ehrlichia canis after treatment with doxycycline. Canine ehrlichiosis was induced in five dogs by intravenous inoculation with E. canis-infected DH82 cells. All animals developed mild clinical signs of transient fever, body weight loss, thrombocytopenia, and increased gamma globulin levels in plasma. An indirect fluorescent-antibody test (IFA) revealed that all dogs had seroconverted (titer, 5,120) by day 10 postinoculation (p.i.). E. canis was reisolated from blood samples collected at intervals throughout the 2-month period p.i. Doxycycline was administered orally once daily at 10 mg/kg of body weight per day for 1 week starting at 2 months p.i. Following treatment, gamma globulin levels in plasma were decreased. At necropsy on days 54 to 59 after the start of treatment, spleen, liver, kidney, and lymph nodes were collected for E. canis culture and histopathologic examination. Although the dogs did not show significant clinical signs during or after treatment with the antibiotic, E. canis was reisolated from the blood and tissue samples of three of five dogs. A 16-fold reduction in IFA titer was noted in two dogs which were negative for E. canis reisolation at day 49 after the start of treatment, whereas a zero- to fourfold reduction in IFA titer was seen in the remaining three dogs. Western immunoblot reactions to higher-molecular-size E. canis antigens in the sera of two dogs which were negative for E. canis on blood culture decreased, whereas they remained continuously high or only transiently decreased for the duration of the study for antigens in the sera of three dogs from which E. canis was reisolated. Histopathologically, prominent plasmacytosis in the kidney cortex was present in three dogs from which E. canis was reisolated, whereas the kidney cortices of two dogs had moderate to minor plasmacytosis. These findings pose questions regarding the efficacy, dosage and duration of doxycycline treatment in dogs with E. canis infection. In addition, it was shown that IFA and Western immunoblotting may aid in assessing the efficacy of antibiotic therapy when definitive reisolation procedures are not readily available.     

Enteric disease in dogs naturally infected by a novel canine astrovirus

Infection by a novel canine astrovirus was associated with gastroenteritis in two dogs. The virus displayed 70.3 to 73.9% amino acid identity to other canine astroviruses in the full-length capsid. Specific antibodies were detected in the convalescent-phase sera of the dogs, indicating seroconversion. Also, the virus appeared weakly related antigenically to the prototype canine astrovirus isolate ITA/2008/Bari.     

Genetic and antigenic evidence supports the separation of Hepatozoon canis and Hepatozoon americanum at the species level

Recognition of Hepatozoon canis and Hepatozoon americanum as distinct species was supported by the results of Western immunoblotting of canine anti-H. canis and anti-H. americanum sera against H. canis gamonts. Sequence analysis of 368 bases near the 3' end of the 18S rRNA gene from each species revealed a pairwise difference of 13.59%.     

Relation between calcium levels and adenosine deaminase activity in serum in pre- and postpartum of dairy cow

The transition period is marked by intense metabolic changes that defy calcium homeostasis, and compromise the immune capacity of dairy cows, what denotes interaction between metabolic status and immune activity. The adenosine deaminase (ADA) regulates adenosine levels; a molecule with anti-inflammatory properties that can act in reducing the production of pro-inflammatory cytokines and reactive oxygen species (ROS), which in exacerbated production is harmful to health. Therefore, the aim of this study was to evaluate the relationship between seric levels of calcium levels and ADA in prepartum and postpartum of dairy cows. To evaluate these variables, Holstein cows were subjected to blood collection in the transition period (days 20, 10, and 5 prepartum; and days 3, 7, 12, and 20 postpartum) and where we evaluated the serum levels of calcium and the ADA activity. An increase in the ADA activity was observed mean day 20 to day 10 postpartum (P < 0.001). Numerically, in other periods evaluated, ADA activity was higher when compared to day 20 prepartum. The serum calcium significantly reduced from 5 days prepartum, keeping smaller the day 20 prepartum until day 12 postpartum (P < 0.02). There was a negative correlation between serum calcium levels and ADA activity. The increase of ADA can be related to modulation of the inflammatory response that occurs during the transition period for cows.     

Seroprevalence of Ehrlichia canis and of Canine Granulocytic Ehrlichia Infection in Dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

Regulation of thymocyte proliferation by endogenous adenosine and adenosine deaminase

Spontaneous proliferation of thymocytes after 20-25 h of culture was significantly increased by the presence of adenosine deaminase (ADA) or theophylline. The effect of ADA was counteracted by the ADA inhibitor EHNA. When given alone, EHNA inhibited proliferation. This effect was not blocked by inhibition of adenosine uptake with dipyridamol. These results suggest that proliferation in culture is regulated by a balance between endogenous adenosine and ADA, controlling the influence of adenosine on the intracellular cyclic AMP level via an adenosine receptor on the surface of thymocytes. According to the hypothesis, ADA would stimulate proliferation by decreasing extracellular adenosine levels and theophylline by blocking adenosine receptors on thymocytes. EHNA would inhibit proliferation by increasing extracellular adenosine levels. In accordance with this interpretation, the adenosine analogue phenylisopropyl adenosine (PIA) inhibited proliferation and the effect could be inhibited by theophylline. The postulated effect of endogenous adenosine could not be mimicked by a single administration of exogenous adenosine. Whereas most doses of adenosine were without effect, a high dose of adenosine (0.1 mM) in combination with EHNA unexpectedly stimulated proliferation. Since the effect was blocked by dipyridamol, an intracellular site of action for adenosine is suggested in this case.