Serologic and molecular evidence of Ehrlichia spp. in coyotes in California

In order to determine the role of coyotes in the epidemiology of granulocytic and monocytic ehrlichial agents in California (USA), we tested 149 serum samples for antibodies against Ehrlichia equi, E. risticii, and E. canis, using an indirect immunofluorescent antibody test. Polymerase chain reaction (PCR) assay was used to survey for the presence of members of the E. phagocytophila genogroup, E. risticii and E. canis in blood samples of 95 coyotes. Sixty-eight (46%) samples were seropositive for E. equi, two (1%) for E. risticii and none of the samples had antibodies reactive to E. canis. Two and one coyote were positive for E. risticii and members of the E. phagocytophila genogroup by PCR assay, respectively. In contrast, the 95 samples were negative for E. canis by PCR. Ninety-five percent of the 68 E. equi seropositive coyotes and the one coyote PCR positive for members of the E. phagocytophila genogroup originated from a coastal area. However, the two E. risticii seropositive coyotes and the two coyotes PCR positive for E. risticii were from northern California. Sequence analysis of the three amplified PCR products revealed the agent to be similar in two coyotes to the sequences of E. risticii from horses originating from northern California and identical in one coyote to the agent of human granulocytic ehrlichiosis and E. equi from California. Thus, coyotes are exposed to granulocytic ehrlichiae and E. risticii and may play a role in the epidemiology of these ehrlichial agents in California.     

Antigenic and genomic relatedness among Ehrlichia risticii, Ehrlichia sennetsu, and Ehrlichia canis.

Antigenic and genomic relatedness among Ehrlichia risticii, E. sennetsu, and E. canis was analyzed by enzyme-linked immunosorbent assay, Western blotting (immunoblotting) and DNA-DNA hybridization. E. risticii and E. sennetsu were serologically related, and their Western blot antigen profiles were nearly identical. Two antigens of E. sennetsu corresponding to the 28- and 51-kDa antigens of E. risticii were apparently larger than the E. risticii antigens, and the 55-kDa antigen of E. risticii appeared to be unique to this species. The 110-, 70-, and 44-kDa antigens of these two species were identical, as determined by the use of monospecific antibodies. DNA homology between these two species was high. On the other hand, E. canis was antigenically least reactive with the antisera to E. risticii and E. sennetsu. However, a dog convalescent-stage E. canis antiserum recognized antigens in the other two species which were different from those recognized by their homologous antisera. Similarly, homology between the DNA of E. canis and the DNAs of the other two species was very minimal. These results indicate that E. risticii and E. sennetsu are closely related both at the genomic and antigenic levels and that the relationship of these two species with E. canis is minimal.     

Experimental infection of dusky-footed wood rats (Neotoma fuscipes) with Ehrlichia phagocytophila sensu lato

Dusky-footed wood rats, Neotoma fuscipes, have been implicated in the natural maintenance of Ehrlichia phagocytophila sensu lato, the agent of human granulocytic ehrlichiosis (HGE), in northern California based on high seroprevalence and amplification of E. phagocytophila s.l. DNA from wood rat blood. In order to further assess granulocytic ehrlichiosis in wood rats, we examined wild-caught wood rats for infection and then performed experimental intra-peritoneal infections with E. phagocytophila s.l. in horse or wood rat blood, and tested animals for 120 days by polymerase chain reaction (PCR) and serology. Of 15 wood rats collected from northern California, three were antibody and PCR-positive for E. phagocytophila s.l. at the time of capture. The naturally infected wood rats remained PCR-positive for a mean of 52 days (+/- 7 SD). Experimental i.p. passage of E. phagocytophila s.l. in wood rat blood was successful in three of four wood rats and the mean duration of PCR-positivity was 75 days (+/- 21.2 SD). Experimental infection with E. phagocytophila s.l. in horse blood succeeded in all four of the recipients and the mean duration of PCR-positivity of 81 days (+/- 17.5 SD). No infected individual appeared to be ill based on feeding behavior, activity, and hydration status. These data confirm that wood rats are susceptible to E. phagocytophila s.l., may develop prolonged infection without clinical ehrlichiosis, and may play a role in maintaining E. phagocytophila s.l. in nature.     

Retention and attempted mechanical transmission of Ehrlichia risticii by Stomoxys calcitrans

Abstract. The ability of adult Stomoxys calcitrans (L.) (Diptera: Muscidae) to retain viable Ehrlichia risticii (Rickettsiaceae), the aetiologic agent of Potomac horse fever (PHF), and mechanically transmit the pathogen from citrated bovine blood artificially infected with E.risticii to susceptible mice was studied. Viable E. risticii were found in the digestive tract of S.calcitrans 3h after the flies had engorged to repletion on infected blood; however, no E. risticii were detected in flies ≥2 days after feeding. Subsequently, groups of S.calcitrans were fed for 20 s on infected blood, then fed to repletion on mice 30, 60,130, 180 or 220 min after having feeding interrupted. Mice displayed no clinical signs of PHF and did not produce anti- E. risticii antibodies when assayed 30 days after S. calcitrans had fed. Although S.calcitrans were able to harbour viable E.risticii for at least 3h, transmission of the disease agent to susceptible mice during interrupted feeding was not demonstrated under these experimental conditions.     

Tick-Borne Fever in Lambs of Different Ages

Four groups of lambs aged 1 week, 4 weeks, 1/2 year and 1 year old respectively were inoculated with Ehrlichia phagocytophila infected blood. Clinical signs, temperature reactions, haematological changes and parasitaemia were more moderate in lambs inoculated with E. phagocytophila at the age of 1 week than those recorded in the older animals. The clinical response to tick-borne fever (TBF) appears to be more severe with increasing age of the lambs. The lymphocyte reactivity to mitogens was reduced in the TBF infected lambs, and was most pronounced in lambs in the 3 older age groups.     

Ehrlichiosis in a moose calf in Norway

A case of granulocytic ehrlichiosis in a moose calf (Alces alces) in Norway is described. The animal was heavily infested with ticks (Ixodes ricinus), and died from a Klebsiella pneumoniae septicemia. Examination of blood smears from the calf revealed cytoplasmic inclusions (morulae) typical of infection with Ehrlichia phagocytophila in the granulocytes. Ehrlichia sp. was detected by polymerase chain reaction (PCR) in blood from the calf, and in the ticks. Sequence determination identified it as E. phagocytophila. This is the first report of ehrlichiosis in moose.     

Recrudescence of Entopolypoides macaci Mayer, 1933 (Babesiidae) infection secondary to stress in long-tailed macaques (Macaca fascicularis)

Parasites were found in red blood cells of two long-tailed macaques (Macaca fascicularis) imported from Indonesia and housed in the Washington Regional Primate Research Center breeding colony for 7 years or longer. Both macaques developed parasitemias secondary to stress (type D retrovirus in one case and severe trauma in the other). Entopolypoides macaci (Babesiidae) was diagnosed on the basis of morphology from peripheral blood smears stained with Wright's stain. Antibodies against Babesia sp. were detected by immunofluorescence assay (IFA) from one infected macaque, which showed antibody cross-reactions (high titer) to B. bigemina, B. bovis, B. canis, and (low titers) to Plasmodium falciparum. Five feral long-tailed macaques that had been imported recently from the same country had no detectable antibodies. This is the first report of IFA as an aid to diagnose E. macaci in nonhuman primates.     

Molecular cloning and characterization of a cDNA encoding cellobiose dehydrogenase from the wood-rotting fungus Grifola frondosa

A total of 305 Ixodes ricinus ticks (243 nymphs and 62 adults) were collected from three different regions of Thuringia in Middle Germany which are known to be endemic for Borrelia burgdorferi. Our aim was to investigate the carrier rate of ticks for granulocytic Ehrlichia species. The presence of ehrlichial 16S ribosomal DNA was investigated by polymerase chain reaction. Using primers specific for the Ehrlichia phagocytophila group PCR fragments of 151 bp and 943 bp, respectively, were produced in positive samples. Adult ticks showed a significantly higher infection rate (4/62; 6.5%) compared to nymphs (3/243; 1.2%). Prevalence rates varied between 0 and 3.8% regarding the different areas under investigation. The nucleotide sequences showed high similarity (between 97.5% and 99% identity) to the known sequences of the three E. phagocytophila group members HGE agent, E. phagocytophila and Ehrlichia equi. The sequence data did not allow a final classification to a particular member of this group.     

Profile of the Caprine arthritis-encephalitis virus (CAEV) in blood, semen from bucks naturally and experimentally infected in the semi-arid region of Brazil

The aim of this study was to report the chronology of Caprine arthritis-encephalitis virus elimination and compare the blood and semen viral profiles of animals naturally and experimentally infected by SRLV raised in the semi-arid region of Brazil. The experiment was carried out at the Brazilian Center for Goat Research (Embrapa). Nine bucks were selected, four naturally infected by CAEV and five animals proven negative that were inoculated with the goat lentivirus (CAEV-Cork strain). Every week the animals were submitted to semen collection using an artificial vagina. The blood was collected by puncturing the jugular vein with tubes containing EDTA, 7 days after inoculation (experimentally infected group) or at the start of the experiment (naturally infected group) and then at every 30 days. The genomic viral DNA was extracted from semen and blood and then Nested-PCR was applied. An agar gel microimmunodiffusion was performed to detect anti-CAEV antibodies. The results were described in percentage and analyzed by the Chi square test (P < 0.05). The presence of anti-CAEV antibodies was detected in the 16th week after inoculation that characterized the seroconversion from four of the five naturally infected goat bucks (80%). The fifth reproducer presented late seroconversion, totaling 32 weeks post-inoculation. A quantity was observed in the total of samples collected of 12.50 and 17.14% positive results in the blood and 10.98 and 11.25% positive results in the semen of the naturally and experimentally infected animals, respectively, and there was no statistical difference. No statistically significant differences were observed regarding the presence of proviral DNA in the blood and semen of the naturally and experimentally infected animals. A viral elimination pattern was not identified during the assessment period, but the presence of proviral DNA was shown at shorter intervals after the 18th week and the 22nd week, for the experimentally and naturally infected bucks, respectively. Therefore, recently infected goats in the period prior to seroconversion eliminated small ruminant lentivirus proviral DNA in the semen and are important sources of infection that should be considered in a control program of this lentivirus, and the Nested-PCR technique is a relevant tool to select virus-free ejaculates.     

Epizootiologic investigations of selected infectious disease agents in free-ranging Eurasian lynx from Sweden

Serum samples from 106 Eurasian lynx (Lynx lynx) from across Sweden, found dead or shot by hunters in 1993-99, were investigated for presence of antibodies to feline parvovirus (FPV), feline coronavirus, feline calicivirus, feline herpesvirus, feline immunodeficiency virus, Francisella tularensis, and Anaplasma phagocytophila, and for feline leukemia virus antigen. In addition, tissue samples from 22 lynx submitted in 1999 were analyzed by real-time polymerase chain reaction (PCR) to detect nucleic acids specific for viral agents and A. phagocytophila. Except for FPV antibodies in one lynx and A. phagocytophila in four lynx, all serology was negative. All PCR results also were negative. It was concluded that free-ranging Swedish lynx do not have frequent contact with the infectious agents considered in this study.     

Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients,animals and ticks in the Czech Republic

We report moderately severe cases of human ehrlichiosis and a lethal one caused by human granulocytic Ehrlichia, the HGE agent, closely related to Ehrlichia phagocytophila and Ehrlichia equi. Their vector is the Ixodes ricinus tick, which also transmits Borrelia burgorferi sensu lato in central, west and east regions of the Czech Republic. The diagnosis was established by PCR with sequence analysis of the genes encoding 16S rRNA of Ehrlichia and with reverse hybridization by using enzyme linked immunosorbent assay with different covalently coupled probes to the activated plate.Ten out of 47 patients and 10 huntsmen were PCR positive and 7 of them seroconverted to the HGE. Coinfection of Ehrlichia phagocytophila with Borrelia burgdorferi sensu lato was detected in 3 patients. Ehrlichia spp., the HGE agent, was isolated and propagated only from one patient in the HL-60 promyelocytic cell line. The maintenance of Ehrlichia in culture and in patients was assayed also by immunocytological staining and electron microscopy. Sequence or hybridization analysis of PCR results in different wild mammals and birds showed significant sources of Ehrlichia fagocytophila in nature. Three variants of E. phagocytophila in wild roe deer and boars, as well as for the first time in birds, have been described. Cultures from the blood of horses, and from the spleen and kidney specimens of roes and boars, PCR positive for Ehrlichia spp., displayed a disappearing level of the pathogen or contamination with other bacteria.     

Persistence of Granulocytic Ehrlichia Infection During Wintertime in Two Sheep Flocks in Norway

Granulocytic Ehrlichia infection in sheep is common in Norway in areas with Ixodes ricinus. In this study, 2 sheep flocks that had been grazing on I. ricinus infested pastures the previous season, were blood sampled after being housed indoors for nearly 6 months during wintertime. Thirty animals from each flock were examined for granulocytic Ehrlichia infection in the peripheral blood by blood inoculation studies, stained blood smear evaluation, polymerase chain reaction (PCR) analysis and serology (IFAantibodies). The animals were sampled twice within a three-week period, the first time before and the second time after lambing. Two sheep in one flock were found Ehrlichia positive by both blood smear evaluation and PCR before lambing, and 3 sheep were found positive after lambing; 2 by blood smear examination and 3 by PCR. In the other flock, no sheep was found infected before lambing, but 2 ewes were found positive after lambing by both blood smear evaluation and PCR. In the first flock, 87% of the animals were found seropositive before lambing, and the mean antibody titre (log 10 ± SD) to E. equi was 2.45 ± 0.401. In the second flock, 40% were found seropositive before lambing, and the mean antibody titre was 1.93 ± 0.260. Seroprevalence and mean antibody titre in these 2 flocks were significantly different (p < 0.001). The present study indicates that sheep may be a reservoir host for granulocytic Ehrlichia infection from one grazing season to the next under natural conditions in Norway.     

Experimental Infection of Lambs with an Equine Granulocytic Ehrlichia Species Resembling the Agent that Causes Human Granulocytic Ehrlichiosis (HGE)

Five lambs were inoculated with a granulocytic Ehrlichia species originally isolated from a Swedish horse with granulocytic ehrlichiosis (EGE). The 16S rRNA gene sequence of the Swedish Ehrlichia sp. causing EGE was identical to the sequence of the agent causing human granulocytic ehrlichiosis (HGE). After the inoculation, infected neutrophils and a low serologic response were seen in all lambs, but no clinical symptoms were observed. In one lamb 17% of the neutrophils were infected without a corresponding fever. Six weeks later the lambs were inoculated with an ovine isolate of E. phagocytophila. After challenge with E. phagocytophila the lambs reacted with fever and infected granulocytes. The results presented herein show that the equine Ehrlichia isolate was infective for lambs but generated weak immune response and no distinctive protection from subsequent challenge with E. phagocytophila.     

Neorickettsia risticii is vertically transmitted in the trematode Acanthatrium oregonense and horizontally transmitted to bats

Potomac horse fever is known to be transmitted through the ingestion of caddisflies parasitized with Neorickettsia (formerly Ehrlichia) risticii-infected metacercaria. However, the species of trematode involved and how N. risticii is maintained in nature are unknown. In this study, gravid trematodes were recovered from the intestines of 12 out of 15 Eptesicus fuscus big brown bats and eight out of nine Myotis lucifugus little brown bats from various sites in Pennsylvania, USA. Trematode specimens isolated from six E. fuscus bats contained N. risticii DNA. The trematode was identified as Acanthatrium oregonense. N. risticii was detected within individual trematode eggs by polymerase chain reaction as well as by immunofluorescence labelling with an anti-N. risticii antibody, indicating that N. risticii is vertically transmitted (from adult to egg) in A. oregonense. Furthermore, N. risticii DNA was detected in the blood, liver or spleen of 23 out of 53 E. fuscus and M. lucifugus bats, suggesting that N. risticii can also be transmitted horizontally from trematode to bat. These results indicate that A. oregonense is a natural reservoir and probably a vector of N. risticii.     

Studies on humoral immunity against Taenia taeniaeformis infection in rats

Rats were infected with doses of 100, 1000, 5000 and 10 000 eggs of Taenia taeniaeformis. Haemagglutinating antibody to cysticerus antigen was detected at the 4th week of infection. The appearance and levels of antibody titre did not vary greatly with the infective dose. An IgM peak appeared at the 6th week, with IgG appearing slightly later and continuing to rise. Transfer of serum from the 1st week onwards from infections with 1000 eggs however could confer significant protection. Dilutions of hyperimmune serum (1 ml volumes) of up to 1/32 conferred significant protection on normal recipients. Hyperimmune serum transferred up to 4 days before challenge could confer 80% protection whereas serum transferred 4 days after challenge was totally non-protective. The significance of this finding is discussed in the light of current knowledge of metacestode immunity.     

感染流行性出血热病毒家兔病毒血症的研究

本文采用流行性出血热病毒114株实验感染家兔,用免疫荧光法及病毒培养技术研究了家兔病毒血症动态,发现感染后第6天,病毒抗原首先在淋巴细胞及单核细胞中出现;次日,亦可见于粒细胞中,第10—12天的抗原反应较强,第15天则明显减弱至消失。而在红细胞及血小板中始终未见明显的抗原反应。从感染后第3—13天的血浆中分离出病毒;感染后第6—15天,外周血单核细胞病毒分离阳性。结果表明,流行性出血热病毒在接种局部增殖后,侵入血液,并在白细胞中复制增殖,可能使病毒随血循环播散至全身其它组织脏器,造成因血传播引起的靶器官感染。     

Viremia and serum antibodies in Macacus rhesus monkey after inapparent infection with European tick-borne encephalitis virus.

1. Inapparent infection was called forth in M. rhesus monkeys by means of subcutaneous inoculation of ETBE virus. 2. Viremia was found in 18 (86 per cent) of 21 monkeys. In all the 18 monkeys, specific virus-neutralizing antibodies were found; in 17 of them complement-fixing antibodies developed in addition. 3. Neutralizing antibodies (N greater than or equal to 1.7 log10) were first recorded on 10th day with a peak on 32nd day, retaining, after a small decrease, a relatively high level in all reacting animals. 4. Complement-fixing antibodies were first found on 18th day, showing a peak on 22nd day whereafter they dropped considerably or even disappeared so that on 150th day they were present in a low titre in only 35 per cent of the originally reacting monkeys. 5. The findings were compared with the situation in naturally infected humans and in hamsters infected inapparently (experimentally) with ETBE virus.     

Larval recovery of Toxocara canis in organs and tissues of experimentally infected Rattus norvegicus

The aim of this note was to record for the first time the recovery of Toxocara canis larvae from tissues and organs of Rattus norvegicus (Berkenhout, 1769), Wistar strain, until the 60th day after experimental infection. Rats were orally infected with embryonated T. canis eggs, killed on days 3, 5, 8, 10, 15, 30, and 60 after inoculation and larvae were recovered from liver, lungs, kidneys, brain, and carcass after acid digestion, showing a pattern of migration similar of that previously observed in mice.     

Cross-reactions of sera from Toxocara canis-infected mice with Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval excretory-secretory (E/S) products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worm extract were used to determine possible cross-reactions in BALB/c and C57BL/10 mice, inoculated with embryonated eggs or adult worm extract of T. canis in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. canis against different heterologous antigens, we observed no cross-reactions in BALB/c mice against A. suum E/S and adult worm extract antigens with a single dose. In multiple doses this was absent too against T. leonina adult worm extract in BALB/c mice, and in both strains against A. suum E/S and adult worm extract. In BALB/c mice inoculated with adult worm extract of T. canis we did not observe cross-reactions with A. suum E/S antigen with both inoculation doses. In the remainder of the experiments, we observed cross-reactions of different intensities.     

Trypanosoma evansi: effects of zinc and copper in experimentally infected rats

The aim of this study was to evaluate the effects of a treatment using injectable zinc and copper in rats infected with Trypanosoma evansi. 48 rats were divided into eight groups of six animals each. Group A was composed of uninfected animals. Animals from groups B-H were inoculated at the 5th day of experiment with 1.2×10(6) trypanosomes. Group B was used as a positive control. The infected groups received prophylactic (C, D and E) and therapeutic (F, G and H) treatments with the zinc and copper, both at a dose of 5 mg kg(-1). The effectiveness of treatment was confirmed by negative blood smears and Polymerase Chain Reaction (PCR) at the end of study. All treated animals had their prepatent period and survival prolonged when compared with control group (group B). Treatment efficacy was 17% (C: zinc), 33% (D: copper), 50% (E: zinc+copper), 0% (F: zinc), 50% (G: copper) and 50% (H: zinc+copper). Thus, we can conclude that treatment with zinc and copper are capable of controlling and/or curing T. evansi infection in rats, delaying the parasitemia and prolonging their survival.