Observations on erythrocytic acetylcholinesterase activity in experimentally induced anaplasmosis in calves

Erythrocytic acetylcholinesterase (AChE) activity was assessed in splenectomized bovine calves inoculated with Anaplasma marginale. Other parameters, i.e., haematological values (haemoglobin, packed-cell volume, and total erythrocytic count), percentage parasitaemia and the ensuing host reactions during Anaplasma infection were also studied in relation to AChE activity. There was significant inhibition of red blood cell-AChE activity commencing soon after inoculation of infected blood; this preceded a substantial decrease in haematological values along with high parasitaemia. The changes in AChE activity may possibly be associated with increased cell membrane permeability, thus triggering the entry of A. marginale organisms into red blood cells.     

Attempted transmission to cattle of Anaplasma marginale from overwintered Dermacentor andersoni ticks.

Since the 1983 summer outbreak of anaplasmosis in southern Saskatchewan, the role of the tick, Dermacentor andersoni as an overwintering reservoir for Anaplasma marginale has been questioned. The purpose of this study was to determine if spring-collected ticks carried virulent A. marginale. Sixteen splenectomized calves were assigned randomly to two groups of 14 principals and two controls. Adult D. andersoni, collected in April from areas having high transmission rates of A. marginale, were confined to the ears of the principals by special bags and allowed to feed for eight days. The two control calves were subsequently challenged intravenously with blood from a calf infected with the Virginia strain of A. marginale. Principals and controls were monitored for 60 and 50 days postexposure respectively for signs of infection by clinical, hematological and serological procedures. None of the principals developed anaplasmosis but both control calves developed signs of disease.     

Epidemiologic aspects of bovine anaplasmosis in semiarid range conditions of south central Idaho

The prevalance of Anaplasma marginale-infected cows, as determined by use of the modified rapid card agglutination (MRCA) test, was measured during a 4-year period (1980-1983). The prevalence of A marginale-infected cows, defined as positive reactors on the MRCA test, remained constant (31%-37%). The apparent incidence of A marginale transmission to susceptible cows was approximately 7% from 1980 to 1981, 8% from 1981 to 1982, and no transmission from 1982 to 1983. The occasional MRCA-positive cow became negative on the MRCA test, and 1 cow was determined to be free of A marginale infection by subinoculation of 100 ml of the cow's blood into a susceptible, splenectomized calf. Dermacentor andersoni, a known vector of A marginale, was often found on the cattle and in their environment. However, A marginale was not transmitted to susceptible, splenectomized calves, using collected ticks. Of 56 calves born to MRCA-positive cows, 82% were MRCA-positive within the first 3 months of life. These calves converted to MRCA-negative status and were determined to be free of A marginale infection by subinoculation of their blood into susceptible, splenectomized calves, indicating the passive transfer of colostral antibodies.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Pathogenicity of Eperythrozoon suis alone and when mixed with Babesia trautmanni in experimentally-infected pigs

The pathogenicity of a single infection with Eperythrozoon suis and of a mixed infection of E. suis and Babesia trautmanni in experimentally-infected pigs, was described. In the pigs with a single infection, parasitaemia of E. suis was observed 4 days after blood inoculation. Although parasitaemia with this parasite was also observed on the 4th day in a mixed infection, the parasitaemia of B. trautmanni was not noted until 14 days after inoculation at a period when that of E. suis had started to fall. No parasitaemia was observed in any of the pigs in the splenectomized and unsplenectomized uninoculated control animals. The pigs carrying either single or mixed infections had pyrexia and their temperatures were consistently higher than those of the control animals. Infected pigs also showed anaemia with significant decline in Pcv, Hb and RBC values. The total WBC count rose in the infected pigs and the rise was more pronounced in pigs carrying E. suis alone. In addition, it was only in pigs infected by E. suis alone that a decline in the percentage of neutrophils and a rise in percentage lymphocytes was observed.     

Establishment and characterization of an Oklahoma isolate of Anaplasma marginale in cultured Ixodes scapularis cells

Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The Virginia isolate of A. marginale was propagated previously in a cell line derived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma (VA-tc) was passaged continuously for over 4 years and retained its infectivity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a naturally infected cow in Oklahoma (OK-tc). Blood from the infected cow was subinoculated into a splenectomized calf and blood collected at peak parasitemia was frozen, thawed and used as inoculum on confluent tick cell monolayers. Colonies of Anaplasma were apparent in low numbers at 9 days post exposure (PE) and infection in monolayers reached 100% by 4-5 weeks PE. Cultures were passaged by placing supernatant onto fresh tick cell monolayers at a dilution of 1:5 or 1:10. By the third passage development of the OK-tc was similar to that of the VA-tc and a 1:5 dilution resulted in 100% infection in 10-12 days. Inoculation of OK-tc into a splenectomized calf caused clinical anaplasmosis and Dermacentor ticks that fed on this calf transmitted the organism to a second susceptible calf. Major surface proteins (MSPs) 1-5 of the OK-tc were compared with homologous proteins present on VA-tc and the erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were conserved on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 were the major proteins recognized by serum from infected cattle. Two-dimensional gels also identified positional differences between VA-tc and OK-tc in MSP2 and MSP3. The OK-tc may have potential to be used as antigen for development of an improved vaccine for anaplasmosis in the South Central United States.     

High-yield preparation of purified Anaplasma marginale from infected bovine red blood cells

Purification of Anaplasma marginale from infected bovine RBC was achieved through enzyme treatment and density-gradient centrifugation. A relative yield of 41.6% was obtained by dividing the number of organisms in the final purified preparation by the number of A marginale-infected RBC. Purified parasites were verified as A marginale by light microscopy, electron microscopy, and immunologic tests. The purified parasites reacted positively with calf and rabbit anti-A marginale sera in interfacial and slide agglutination tests. Anti-bovine RBC serum did not agglutinate purified A marginale, indicating absence of any contaminating RBC stroma. Anaplasma marginale was antigenic, but did not cause infection when the preparation was inoculated into a susceptible calf. The density of A marginale was determined to be 1.19 g/ml and cell diameters ranged from 0.25 to 0.63 micron. This method provided procedures for obtaining A marginale free of bovine RBC antigens for accurate biochemical assays and vaccine production.     

Evaluation of jackrabbits as nonruminant hosts for Anaplasma marginale

Two black-tailed jackrabbits (Lepus californicus), 1 splenectomized and 1 intact, were inoculated with 0.2 ml of a 1:5 dilution of a Florida Anaplasma marginale stabilate. Five months later, both hares were inoculated with 1 ml of whole blood from a calf with acute anaplasmosis. Neither hare developed any signs of clinical anaplasmosis. Pooled blood (7 ml) from these jackrabbits which was inoculated into 2 Anaplasma-susceptible, splenectomized calves failed to induce hematologic or serologic signs of anaplasmosis for at least 90 days. Two susceptible, splenectomized calves were inoculated with 35 ml of pooled whole blood from 9 wild-collected black-tailed jackrabbits from a known anaplasmosis enzootic area. Both steers remained free of anaplasmosis signs for 90 days.     

In vitro and in vivo glucose consumption in swine eperythrozoonosis

One complication of swine eperythrozoonosis is the hypoglycemia that occurs during parasitemia. To determine the cause of the hypoglycemia, we studied glucose consumption in splenectomized pigs infected with Eperythrozoon suis. With the rapid rise of erythroparasites, the in vitro glucose consumption of parasited whole blood increased dramatically, and hypoglycemia developed. Because mature porcine erythrocytes are impermeable to glucose, the increased glucose consumption is most logically the result of E. suis metabolism. Iodoacetamide and sodium fluoride (which inhibit glycolysis), but not sodium cyanide (which prevents cellular respiration), and tetracycline (which is used to treat eperythrozoonosis) inhibited glucose consumption. In vivo glucose turnover studies before infection and during peak parasitemia indicated an increased glucose production by infected pigs during parasitemia. The results suggest that hypoglycemia occurs during swine eperythrozoonosis because the parasite uses glucose faster than the gluconeogenic pathways can provide it.     

Longevity of colonies of Anaplasma marginale in midgut epithelial cells of Dermacentor andersoni

Colonies of Anaplasma marginale in midgut epithelial cells of experimentally infected Dermacentor andersoni were studied in adult ticks 1, 3, and 6 months old. Longevity of the parasite in ticks was assessed by evaluating its infectivity for splenectomized calves; calves were exposed by feeding ticks and by inoculation of tick gut homogenates. Longevity was also evaluated by determining size, type, and density of colonies in male and female ticks. The effect of incubation (2.5 days at 37 C) on colony density was also examined for ticks at each age period. All methods used to assess longevity of A marginale in ticks (tick transmission, calf inoculation, and histologic studies) indicated a decrease of the numbers of organisms in 6-month-old ticks. Furthermore, when tick gut homogenates from 6-month-old nonincubated ticks were not infectious for susceptible calves, incubation of ticks before dissection restored infectivity of homogenates. Colonies of A marginale were detected in gut tissues of 6-month-old ticks that were not infective; therefore, infectivity of ticks could not be confirmed merely by presence of A marginale colonies.     

Isolation of Theileria mutans from Kenyan buffalo,and transmission by Amblyomma gemma

Blood from 2 buffalo harbouring Theileria organisms was inoculated into a splenectomized Ayrshire calf. The calf developed an infection which extended over a long period. The infection was transmitted to two cattle with Amblyomma gemma by transstadial transmission between the larvae and nymphs. Severe anaemia developed in these cattle and correlated with the parasitaemia. Schizonts morphologically characteristic of T. mutans were detected for short periods in the lymphoid cells of cattle infected by the ticks. The antigens and sera prepared from the cattle reacted with T. mutans sera and antigens in the indirect fluorescent antibody test. After recovery from the primary parasitaemia, the cattle had detectable organisms and antibodies to T. mutans for more than two years.     

Clinical and hematologic effects of experimental infection of dogs with recently identified Babesia gibsoni-like isolates from Oklahoma

OBJECTIVE: To characterize clinical and hematologic responses in dogs following experimental inoculation with Babesia gibsoni-like isolates from infected dogs in Oklahoma. DESIGN: Prospective study. ANIMALS: 6 mixed-breed dogs. PROCEDURE: 2 dogs were inoculated with organisms from a naturally infected dog, and 3 were inoculated with organisms from a second naturally infected dog (1 of these 3 dogs was splenectomized 1 week prior to inoculation). One dog was not inoculated. Complete blood counts were performed weekly. RESULTS: In the 5 dogs inoculated with organisms, parasites were initially detected 1 to 5 weeks after inoculation, and severity of parasitemia peaked with 1.9 to 6.0% of RBC infected by 4 to 6 weeks after inoculation. Parasitemia was easily detectable (> 0.1% of RBC infected) for 3 to 4 weeks. Clinical abnormalities included lethargy, fever, and pale mucous membranes but were mild to nearly inapparent in 2 dogs. All dogs developed regenerative anemia and marked thrombocytopenia. Thrombocytopenia developed before and lasted longer than the parasitemia. Profound but transient neutropenia was detected in some dogs. The splenectomized dog developed more severe parasitemia and anemia and more pronounced clinical abnormalities. Three dogs with intact spleens recovered without treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 2 or more genotypically distinct, but morphologically identical, small Babesia parasites can infect dogs in the United States. Compared with infection with small Babesia parasites from California, infection with these isolates resulted in less severe parasitemia and clinical abnormalities. Parasitemia was transient, indicating that identification of organisms in blood smears may be difficult in some dogs.     

Efficacy of enrofloxacin against severe experimental Anaplasma marginale infections in splenectomized calves

Four Anaplasma marginale-infected splenectomized calves with greater than 25% parasitized erythrocytes received enrofloxacin at 12.5 mg/kg SC twice, 48 hours apart. Two infected splenectomized calves were designated as untreated controls. A precipitous decline in percent parasitized erythrocytes from 39.13% to less than 1% was observed over 12 days following treatment. However, a self-limiting recrudescence of A. marginale parasites was observed within 30 days after treatment. Untreated control calves became moribund and were euthanized. These data indicate that the regimen of enrofloxacin tested herein ameliorates, but does not eliminate, A. marginale infections in splenectomized calves.     

The effect of Eperythrozoon suis infection on the osmotic fragility of erythrocytes]

Osmotic fragility of erythrocytes was tested in weaned pigs experimentally infected with Eperythrozoon (E.) suis. Acute eperythrozoonosis of splenectomized pigs led to an increase of osmotic fragility. It is supposed that E. suis infection causes a structural change in erythrocyte membrane. Possible mechanisms of this cell membrane injury are discussed.     

Comparison of three oxytetracycline regimes for the treatment of persistent Anaplasma marginale infections in beef cattle

Anaplasmosis, caused by the tick-borne rickettsia, Anaplasma marginale, is an economically important disease of cattle in the United States and worldwide. Cattle that recover from acute infection become carriers in which low or microscopically undetectable A. marginale rickettsemia persists. Tetracycline antimicrobials are currently the only drug used in the US for treatment of acute anaplasmosis. There are currently no drugs specifically licensed for elimination of persistent infections. This study tested the efficacy of three oxytetracycline treatment regimens to clear A. marginale from cattle that were persistently infected. Forty Angus x Simmental steers, aged 6-12 months were experimentally infected with A. marginale. After the steers recovered from acute infection, seroconverted, and were confirmed infected using nested PCR followed by DNA hybridization, the carrier status of each animal was ascertained by sub-inoculation of blood into a separate, splenectomized Holstein calf. The steers were then blocked by bodyweight and randomly assigned as follows to four treatment groups: Treatment A, 300 mg/ml solution of oxytetracycline (Tetradure LA-300, Merial Canada Inc.) administered at 30 mg/kg, by intramuscular (i.m.) injection on day 0; Treatment B, the same 300 mg/ml solution of oxytetracycline administered at 30 mg/kg, i.m. on day 0 and again on day 5; Treatment C, a 200 mg/ml solution of oxytetracycline (Liquamycin LA-200, Pfizer Animal Health) administered at 22 mg/kg, intravenously (i.v.), q 24 h for 5 days (a treatment dose that corresponds with current Office International des Epizooties (OIE) recommendations for treatment prior to export). The fourth group consisted of untreated infected control cattle. All steers were still nested PCR and cELISA positive at 60 days after treatment. Infection was confirmed by subinoculation of blood into a splenectomized Holstein calf. These results demonstrated that the treatment regimens tested failed to clear A. marginale infections in carrier cattle.     

The transovarial transmission of Babesia trautmanni by Rhipicephalus simus to domestic pigs.

Rhipicephalus simus was, for the first time, experimentally proven to be a transovarial vector of Babesia trautmanni of domestic pigs. The nymphal and adult progeny of experimentally infected female ticks transmitted the infection to 2 susceptible splenectomized pigs. Features of the infection included a prepatent period of 6-8 days post-tick infestation, a febrile reaction for 3 days and a maximum parasitaemia score of 15 (more than 6 parasites per 300 red blood cells). Other clinical signs in both pigs were mild inappetence and listlessness. Both pigs recovered without any antibabesial therapy.     

Comparison of the efficacy of enrofloxacin, imidocarb, and oxytetracycline for clearance of persistent Anaplasma marginale infections in cattle

This study compared enrofloxacin and imidocarb dipropionate treatments with an oxytetracycline regimen proposed by the World Organization for Animal Health for elimination of persistent Anaplasma marginale infections in cattle. The effect of therapy on competitive ELISA and polymerase chain reaction (PCR) reactivity was also assessed. Twelve A. marginale-infected carrier calves were randomly assigned to groups receiving either enrofloxacin (5 mg/kg IV q24h for 5 days), imidocarb (5 mg/kg IM twice, 7 days apart), or oxytetracycline (22 mg/kg IV q24h for 5 days). One calf infected with an Oklahoma isolate in the imidocarb group and one infected with a Virginia isolate in the oxytetracycline group failed to infect a splenectomized calf following blood subinoculation. Both became competitive ELISA negative by 44 days after treatment, but the imidocarb-treated calf remained PCR positive. None of the tested treatments reliably eliminated persistent A. marginale infections in all cattle. Furthermore, PCR was not a reliable means of determining the success of chemosterilization in calves.     

Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Effect of five daily intravenous treatments with oxytetracycline hydrochloride on the carrier status of bovine anaplasmosis.

Intravenous administration of oxytetracycline hydrochloride at the rate of 22 mg/kg daily for 5 days was effective in rendering parasite-free 11 adult cattle that were naturally infected Anaplasma marginale carriers. The treatment did not cause any noticeable distress or side effect. Through 12 posttreatment months, the efficacy of the treatment procedure was evaluated by serologic tests and subinoculation of blood into susceptible splenectomized calves. Results of the rapid card agglutination test were positive for 5 cattle at 2 months after treatment, but negative for all cattle at 4 through 12 months. Complement-fixation titers were variable and transient in posttreatment serologic studies. After subinoculation of blood into splenectomized calves (at 4 and 12 months after chemotherapy), serologic, hematologic, or clinical evidence of infection with A marginale was not seen during a 60-day observation period.     

Effect of age on the immune response of cattle experimentally infected with Babesia bigemina

Two different age groups of Holstein Friesian cattle were experimentally infected with Babesiabigemina. Calves of group A (6 months old) did not show noticeable symptoms of babesiosis and had relatively low (0.6%) numbers of parasites in their red blood cells (RBCs). Group B calves (1 year old) had typical signs of the disease; parasites were found in 6.6% of their RBCs. Blood from both groups inoculated into splenectomized calves at 3, 6, 12 and 18 months following initial inoculation demonstrated the presence of B. bigemina, while after 22 months no parasites could be demonstrated.The indirect fluorescent antibody (IFA) test detected babesial antibodies at 4–5 days post inoculation (PI) and reached a maximum titre of 1 : 640 at 2 weeks PI. Following challenge at 2–3 months after initial inoculation, the antibody titre rose sharply to 1 : 2560, then decreased gradually but was still detectable 22 months PI. No correlation was found between antibody titre and the presence of the parasite hin the peripheral blood.