Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis)

Most human granulocytic ehrlichiosis (HGE) studies carried out in horses use needle inoculation of infected leucocytes or cell cultures. This route of inoculation does not accurately reflect natural infection of the tick-borne agent. To investigate whether tick transmission influences the course of granulocytic ehrlichiosis in the horse model, experimental transmission through infected laboratory-reared Ixodes scapularis ticks was attempted into two healthy horses. One additional horse served as negative control and was exposed to uninfected ticks. Eleven days after exposure to nymphal or adult ticks infected with Anaplasma phagocytophila (HGE agent) the two horses developed severe clinical and laboratory signs consistent with granulocytic ehrlichiosis. Bacteraemia was determined at various time points in the two horses by observation of morulae within neutrophils and by detection of A. phagocytophila genomic DNA by PCR of peripheral blood leucocytes. Further, both horses seroconverted. In contrast the control horse stayed uninfected. The results demonstrate that A. phagocytophila can be experimentally transmitted by infected nymphal and adult ticks and that the agent is able to produce a severe disease, similar to naturally occurring cases. Therefore, tick transmission is highly reproducible and can be successfully used in the equine animal model in order to study HGE.     

Evidence of host adaptation in Lawsonia intracellularis infections

Background

Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate.

Materials and methods

Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (10⁹L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response.

Results

Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate.

Conclusions

Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.     

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated intravenously with blood containing an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila, and identical to the human granulocytic ehrlichiosis agent with respect to its 16S rRNA gene sequence. The clinical signs were evaluated, and blood samples were collected for haematology, serum biochemistry and serology. Ehrlichial inclusions in the blood were monitored by microscopy, and ehrlichial DNA was detected by the polymerase chain reaction (PCR). Two of the dogs were injected with prednisolone on days 54 to 56 and days 152 to 154 after infection, and the other was injected with prednisolone on days 95 to 97 after infection. The dogs were euthanased and examined postmortem. Ehrlichial inclusions were demonstrated in the neutrophils and seroconversion occurred shortly after inoculation. Two of the dogs developed acute disease with rectal temperatures above 39.0 degrees C, after which no further clinical signs were observed. The administration of corticosteroids seemed to facilitate the detection of ehrlichial inclusions. Ehrlichial DNA was detected intermittently by PCR in blood samples from two of the dogs throughout the study. Persistent infection was demonstrated up to five-and-a-half months after inoculation.     

Death of a horse infected experimentally with Anaplasma phagocytophilum

A 19-year-old horse that was one of a group of six horses infected experimentally with Anaplasma phagocytophilum for a study of the pathogenesis of equine granulocytic ehrlichiosis died suddenly two days after first showing clinical signs of disease. The clinical signs and laboratory findings observed before its death were similar to all those of the other infected horses, and to previous reports of this disease. A postmortem examination revealed widespread haemorrhaging in its internal organs, and vasculitis and thrombosis in the kidneys. These changes are consistent with disseminated intravascular coagulation, which has previously been reported in human beings infected with the presumably identical agent of human granulocytic ehrlichiosis.     

Experimental transmission of granulocytic ehrlichial organisms in dogs

Canine granulocytic ehrlichial organisms were transmitted from an infected dog from Missouri to two male, 10-month-old dogs by an intravenous injection of whole blood. Physical or behavioral abnormalities were not detected during the 98 days of evaluation other than a mild pyrexia from Day 18 to 20. Ehrlichial morulae were found in blood granulocytes of Dog 1 from Day 13 to 44 and of Dog 2 from Day 14 to 34 with the peak rickettsemia occurring on Day 16 for both dogs. By Day 21 after inoculaiuon, both dogs had positive titers to Ehrlichia canis. The highest titers for both dogs were found 63 days after inoculation, after which the titers decreased. Most of the hematologic abnormalities (i.e., neutropenia, lymphocytosis, thrombocytopenia) and fever occurred between 18 and 24 days after inoculation. The pathologic bases of these abnormalities were not investigated but their concurrent presence suggested an association with the dogs' immunologic responses to the granulocytic ehrlichial agent. Results from the study indicated that the canine granulocytic ehrlichial agent of Missouri may produce subclinical infections and suggested that dogs may be able to clear the organism without antimicrobial therapy.     

Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum

Six horses were experimentally infected by administration of horse blood containing a Swedish strain of Anaplasma phagocytophilum. The polymerase chain reaction (PCR) signal was consistently detected 2-3 days before appearance of clinical signs and persisted 4-9 days beyond abatement of clinical signs, whereas diagnostic inclusion bodies were 1st noted on average 2.6 +/- 1.5 (SD) days after onset of fever. Clinical signs and hematologic changes were largely indistinguishable from those previously reported for diseases caused by A phagocytophilum (formerly Ehrlichia equi--"Californian agent") and the human-derived human granulocytic ehrlichiosis agent. Horses 1st demonstrated antibody response 12-16 days after inoculation, 2 cases of which were still febrile, and serotiters rapidly peaked within 3-7 days of clinical illness. One horse died during the acute stage of disease, but initial clinical signs and hematologic changes were similar to those of other infected horses. This report shows that, despite minor genetic differences, a European equine-derived strain of A. phagocytophilum may be similar in pathogenicity to the Californian agent. The PCR used holds promise to widen the diagnostic window and would also be diagnostic during the initial days of clinical disease when inclusions in neutrophils in blood smears are not yet apparent.     

Anaplasma phagocytophilum infection in a dog: identifying the causative agent using PCR

A diagnosis of Anaplasma phagocytophilum infection was confirmed in a two-year-old male golden retriever displaying few clinical and haematological abnormalities. This was achieved by demonstrating ehrlichial organisms in circulating neutrophils, by indirect immunofluorescence assay using A phagocytophilum as an antigen, and by detecting DNA specific for the 16S rRNA gene of granulocytic Anaplasma by PCR. After treatment with doxycycline for 10 days the dog showed improvement and the laboratory values returned to normal.     

Experimental infection of calves with bovine respiratory syncytial virus (Quebec strain).

An experiment was designed to evaluate the clinical, haematological, viral and serological aspects of bovine respiratory syncytial virus infection in calves. Eleven calves were inoculated intranasally with bovine respiratory syncytial virus (Quebec strain) in aerosol. Clinical, haemotological and serological responses of the calves and virus shedding were monitored. The experimentally infected animals manifested moderate to severe signs of respiratory disease. The parameters used to evaluate the severity of the disease included ocular discharge, conjunctivitis, lung sounds, nasal discharge, pyrexia and leukopenia. The animals were scored accordingly (scale infected 70.8-148.5, control 22-29.3). Highest disease scores were observed between day 6-9 after infection. Virological and serological assessment demonstrated that the observed clinical picture was due to bovine respiratory syncytial virus infection.     

Larval viability and serological response in horses with long-term Trichinella spiralis infection

The horse is considered an aberrant host for the nematode parasite Trichinella spiralis, and many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood. It has been reported that experimentally-infected horses produce a transient serological response to infection and that muscle larvae are cleared more rapidly than in parasite-adapted hosts such as the pig and humans. However, limited numbers of animals have been studied, and both the longevity of larvae in horse musculature and the immune response to Trichinella larvae remain unclear. In this study, we infected 35 horses with 1000, 5000, or 10,000 T. spiralis muscle larvae and followed the course of infection for 1 year, assessing larval burdens in selected muscles, the condition and infectivity of recovered larvae, and the serological response of infected horses. The results demonstrated that T. spiralis establishes infection in horses in a dose dependent manner. Anti-Trichinella IgG antibodies peaked between weeks 6-10 post-inoculation. Viable, infective larvae persisted in horse musculature for the duration of the study (12 months), and exhibited no apparent reduction in muscle burdens over this period. Encapsulated larvae showed no obvious signs of degeneration in histological sections. Larval capsules were surrounded by infiltrates consisting of mature plasma cells and eosinophils. Macrophages were notably absent. Given the lack of a detectable serological response by 26 weeks p.i. and the persistence of infective muscle larvae for at least 1 year, parasite recovery methods are currently the only suitable detection assays for both meat inspection and epidemiological studies of Trichinella infection in the horse.     

Application of microbiological cancer test to cattle infected with bovine leucosis virus

A microbiological cancer test, previously verified in men and dogs using a clostridium strain (Clostridium butyricum CNRZ 528), was applied to cattle infected with bovine leucosis virus (BLV). An extended period of time was allowed to pass after infection with BLV, which had been checked up through specific serological and virological examinations. The cattle belonged to different age groups and stages of infection (with and without haematological alterations [preleukosis], with incipient tumour development [swelling of externally visible and palpable lymph nodes]). Controls included BLV-infected cows as well as test animals to which isotonic saline had been applied or healthy BLV-free cattle in which the clostridium strain had been used. The serological investigation was carried out in a blind test. 3 of 6 BLV-infected spore-treated heads of cattle responded positively to the cancer test, while the other 3 were negative. The 3 cows with positive cancer test were haematologically and serologically leucosis-positive animals with clinically detectable enlargement of lymph nodes. The 3 negative ones of this group, also serologically and haematologically leucosis-positive, were younger animals without signs of tumorous process. 3 spore-treated BLV-free cows and 2 BLV-infected animals, treated with isotonic saline, were cancer test-negative, as well. Finally, 4 BLV-infected and 2 BLV-free cattle, all of them without spore injection, were completely cancer test-negative. 1 cow of the BLV-infected group did not produce spore antibodies after spore treatment, while 1 cow of the BLV-free untreated control group developed spore antibodies.     

Reactivity of serum samples of dogs and horses tested by use of class-specific recombinant-based enzyme-linked immunosorbent assays for detection of granulocytic ehrlichiosis

OBJECTIVE: To test serum samples of dogs and horses by use of class-specific recombinant-based ELISA for establishing a diagnosis of granulocytic ehrlichiosis attributable to infection with organisms from the Ehrlichia phagocytophila genogroup. SAMPLE POPULATION: Serum samples from 43 client-owned dogs and 131 horses (81 with signs of acute illness and 50 without signs of disease). PROCEDURE: Serum samples were analyzed, using ELISA with a recombinant 44-kd protein antigen for IgM and IgG antibodies to the human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain). Western blot analyses, using infected human promyelocytic leukemia cells, were conducted on 38 serum samples of horses and 11 serum samples of dogs to verify reactivity to the 44-kd peptide. RESULTS: IgM or IgG antibodies to the HGE agent were detected in 5 to 28% of dog serum samples and 5 to 37% of horse serum samples. Thirty-five of 38 (92%) horse serum samples had corresponding results on both tests (2 positive results for 26 samples and 2 negative results for 9 samples), using an ELISA for IgG antibodies or immunoblotting for total immunoglobulins. All 11 serum samples of dogs had positive results for both methods. CONCLUSION AND CLINICAL RELEVANCE: These ELISA with recombinant 44-kd antigen are suitable for detecting IgM or IgG antibodies to the HGE agent in serum samples of dogs and horses. Positive results for serum samples of horses from Connecticut, New York, Virginia, and Georgia indicate that the HGE agent is widely distributed in tick-infested areas of the eastern United States.     

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay.cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healhty horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16–35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.     

Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis.

OBJECTIVE: To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. ANIMALS: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. PROCEDURE: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horses were tested by PCR to detect ehrlichial DNA. RESULTS: Antibodies to Erlichial equi were detected in serum samples obtained during all seasons; seropositivity rates ranged from 50 to 93%. In IFA assays using the BDS or NCH-1 strain, seropositivity rates were 70 and 79%, respectively, whereas in immunoblot analyses using the NCH-1 strain, a seropositivity value of 79% was recorded. By immunoblot analysis, all serum samples of all seropositive horses were reactive to a protein having molecular mass of about 44 kd. Blood samples from 29 of 69 (42%) acutely ill horses contained ehrlichial DNA. CONCLUSION AND CLINICAL RELEVANCE: Results of the various serologic testing procedures were in close agreement with each other. All serologic testing methods are suitable for laboratory diagnosis of equine granulocytic ehrlichiosis.     

Clinical signs and humoral immune response in horses following equine herpesvirus type-1 infection and their susceptibility to equine herpesvirus type-4 challenge.

A group of three horses was experimentally infected with equine herpesvirus type 1 (EHV-1) and showed clinical signs characterised by a biphasic febrile response, leucopenia and cell associated viraemia accompanied by virus shedding from the nasopharynx. A second exposure to the virus 18 days later resulted in the isolation of virus from the nasopharynx of one horse. This and a further group of three EHV-1 seropositive horses were subsequently infected with equine herpesvirus type 4 (EHV-4) 147 days after the initial EHV-1 infection and virus was shed from the nasopharynx in the absence of clinical disease. Following the first EHV-1 infection, virus specific immunoglobulin M (IgM) was present by day 5 and remained high until the second exposure at day 18 at which point levels decreased. In contrast, EHV-1 specific IgG, detected at day 6 peaked at day 18, after which time levels remained high. Virus neutralising antibodies and antibodies able to mediate antibody-dependent cellular cytotoxicity were present by day 10. The immune response to EHV-1 is discussed with reference to the disease.     

Studies on experimental enteric salmonellosis in ponies.

Clinical, bacteriological, serological and haematological observations were made on 13 adult ponies orally inoculated with Salmonella typhimurium. The results were compared to two control ponies and four others infected by accidental transmission. The clinical responses in inoculated ponies included pyrexia lasting four days and neutropaenia during the first five days after inoculation followed by a neutrophilia. Pyrexia and neutropaenia was associated with maximal shedding of organisms in the rectal faeces. Changes in the character of the faeces occurred between one and two days after inoculation and appeared to be associated with the serological response. Serological responses occurred in all the infected ponies except one. At necropsy, of the 14 ponies with positive cultures in the colon, seven had negative cultures in the rectal faeces. Serological studies performed on 43 clinically normal horses indicated a correlation between age and salmonella agglutination titre.     

Effects of the dose of Ehrlichia phagocytophila on the severity of experimental infections in lambs

Twenty-one lambs were used to investigate whether their response to an infection with Ehrlichia phagocytophila was dose-dependent Four groups of four lambs were infected intravenously with a dilution in physiological saline of E phagocytophila-infected sheep blood containing either 1.3 x 10(5) infected cells, or approximately 43 infected cells, 4.3 infected cells, or 1.3 infected cells (mean values) and four lambs were left uninfected. The incubation period was significantly shorter in the lambs infected with the highest dose of E phagocytophila. However, the clinical and haematological changes observed, and the weekly weight gains of the lambs were independent of the dose of E phagocytophila. As little as one Ephagocytophila infected cell may be enough to transmit the infection.     

Susceptibility of cattle to infection with Ehrlichia equi and the agent of human granulocytic ehrlichiosis

OBJECTIVE: To determine susceptibility of cattle to infection with Ehrlichia equi and the agent of human granulocytic ehrlichiosis (HGE). DESIGN: Experimental disease and prevalence survey. ANIMALS: 6 cattle, 2 horses, and 2,725 serum samples from healthy cattle. PROCEDURE: 2 cattle and 1 horse were inoculated with E equi, 2 cattle and 1 horse were inoculated with the HGE agent, and 2 cattle served as sham-inoculated controls; inoculated animals were evaluated via clinical, hematologic, serologic, and real-time polymerase chain reaction tests. Prevalence of antibodies against E equi in 2,725 healthy cattle was determined by use of an indirect immunofluorescent technique. RESULTS: No abnormal clinical or hematologic findings or inclusion bodies within granulocytes were observed in the cattle after inoculation, and results of all polymerase chain reaction tests were negative. Seroconversion in inoculated cattle developed 10 to 12 days after inoculation (reciprocal titers, 160). Both horses developed clinical signs of ehrlichiosis. Five of 2,725 (0.18%) cattle were seropositive for E equi, with titers ranging from 20 to 80. All seropositive cattle originated from the same tick-rich region in the Sierra Nevada foothills. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle are not susceptible to infection with E equi or the agent of HGE and that prevalence of exposure to E equi in healthy cattle is low. Therefore, E equi and the agent of HGE are likely of negligible importance for cattle in North America.     

Inoculation of lactating ewes by the intramammary route with Mycoplasma agalactiae: comparative pathogenicity of six field strains

Contagious agalactia affects goats and sheep. In most infected sheep, the causal agent, Mycoplasma agalactiae, induces mastitis and/or agalactia, keratoconjunctivitis and arthritis. However, a few strains of M. agalactiae were isolated from tank milk from flocks without any clinical signs. The present study was undertaken to compare these apparently "asymptomatic" strains to classical virulent strains in order to assess the pathogenicity of four "asymptomatic" strains. Six groups of lactating ewes were inoculated by the intramammary route with 10(8) viable mycoplasmas of each strain. The clinical signs were regularly evaluated; the excretion of bacteria in milk and the serological response were measured. Ewes were necropsied 7 weeks after inoculation and the level of infection in retromammary lymph nodes was determined. Among the 4 apparently "asymptomatic" strains, 2 were fully virulent as were the strains isolated from discased animals, and the other 2 induced somewhat less severe clinical symptoms. The other parameters, in particular the level of excretion in milk and the level of infection of regional lymph nodes following necropsy were similar for all strains. Mean antibody response was also comparable between the apparently "asymptomatic" and virulent strains, in spite of great individual variability. This observation shows that flocks without any clinical sign from which M. agalactiae is isolated in bulk milk, must be kept under strict control since mycoplasmas may induce severe outbreaks later with changing conditions of breeding.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).