Acute Anaplasmosis Research Articles

Study to Compare the Efficacy of Three Oxytetracycline Regimens for the Treatment of Anaplasma Marginale Carrier Status in Beef Cattle

Anaplasmosis is a blood-borne disease of cattle and other ruminants caused by Anaplasma marginale. Animals recovering from clinical anaplasmosis and those treated with therapeutic doses of oxytetracycline remain persistently infected with microscopically undetectable levels of the organism. These animals serve as reservoirs of the disease which can be spread mechanically or through arthropod vectors. Tetracyclines are the only compounds approved for treatment of acute anaplasmosis infections in the United States; however no compounds are approved for eliminating the carrier state. This has contributed to the restricted movement of cattle from endemic areas of the US to anaplasmosis-free territories such as Canada. Anaplasmosis is estimated to cost the US cattle industry $100 million per year, with some estimating the cost of resticted trade to be near $300 million annually. The objective of this study was to compare the effects of three oxytetracycline treatment regimens on eliminating Anaplasma marginale infection from carrier beef steers.

Characterization of Anaplasma marginale isolated from North American bison.

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present study was undertaken to confirm A. marginale infection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of this A. marginale bison isolate for cattle. The results of this study showed that these A. marginale isolates obtained from bison were similar to ones from naturally infected cattle.

Selective in vivo depletion of CD4(+) T lymphocytes with anti-CD4 monoclonal antibody during acute infection of calves with Anaplasma marginale.

To investigate the in vivo role of CD4(+) T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4(+) T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain of Anaplasma marginale in two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3(+) CD4(+) and CD4(+) CD45R(+) (naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection with A. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginale antibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginale major surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4(+)-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4(+) T lymphocytes would abrogate resistance to acute infection.

An in vivo model to investigate lymphocyte-mediated immunity during acute hemoparasitic infections. Use of a monoclonal antibody to selectively deplete CD4+ T lymphocytes from thymectomized calves.

Thymectomized calves were selectively depleted of CD4+ T lymphocytes with a monoclonal antibody (mAb) specific for the bovine CD4 monomer (ILA-11). Calves were treated with high loading doses of ILA-11 during the first week of the study then treated with subsequent lower maintenance doses. Depletion of CD4+ T lymphocytes was assessed weekly by flow cytometric analysis of PBMC and mononuclear cells from lymph node and spleen biopsies. Treatment with high doses of ILA-11 resulted in rapid and marked depletion of CD4+ T lymphocytes from the peripheral blood, peripheral lymph nodes, and spleen. Although CD4+ T lymphocytes slowly returned to the peripheral blood, peripheral lymph nodes, and spleen by day 21 post-treatment, the numbers of CD4+ T lymphocytes in depleted calves remained below pre-depletion levels for the duration of the study. CD4+ T lymphocytes failed to be effectively depleted from a non-thymectomized calf treated with the mAb ILA-11. Development of a T lymphocyte depletion model in thymectomized calves will permit testing of the hypothesis that CD4+ T lymphocytes and IFN-gamma are required in cattle for control of acute anaplasmosis. In subsequent planned studies, thymectomized calves depleted of CD4+ T lymphocytes will be experimentally infected with A. marginale and parameters of disease compared between depleted and non-depleted calves.

Anaplasma marginale inactivated vaccine: dose titration against a homologous challenge

The present study was performed to dose-titrate an Anaplasma marginale experimental immunogen derived from partially purified initial bodies from three geographically different Mexican strains. Three five-bovine groups were inoculated twice on days zero and 21 with A. marginale initial bodies equivalent to 1.5×10 10 (group I), 3×10 10 (group II) or 6×10 10 (group III) infected erythrocytes mixed with STDCM® adjuvant. A similar group served as non-vaccinated controls. All four groups were challenged with 1×10 8 infected erythrocytes from a donor cow with an increasing rickettsemia of strain MEX-15 on day 87 post-vaccination. The prepatent period was very similar for all four groups. All five non-vaccinated controls presented typical acute anaplasmosis syndrome reaching a mean of 30.9% rickettsemia and a loss of 73.4% in the packed cell volume (PCV). Two of five controls died of acute anaplasmosis. Within the vaccinated groups only one animal (group II) suffered acute disease and died. Although all the other vaccinated animals were free of clinical signs, they developed very low rickettsemias (3.2, 3.8 and 4.3%) and PCV losses of 49.9, 47.8, and 49.3% for groups I, II and III. The starting mean weight was very similar for all four groups. All animals lost weight following challenge but losses for groups I and II were lower and significantly different from group IV losses ( P⩽0.1). Although there were no significant differences among vaccinated groups, group III was more severely affected. Taken altogether, these results show a 93.3% protection against both illness and death for all groups; and 100% protection for groups I and III, and 80% for group II.

Effect on intraerythrocytic Anaplasma marginale of soluble factors from infected calf blood mononuclear cells.

Blood mononuclear cells (lymphocytes and monocytes) were isolated from infected calves during in vivo control of acute anaplasmosis and cultured with Anaplasma marginale organisms. Supernatants from the cultures reduced the proportion of erythrocytes containing viable A. marginale in vitro, indicating that an antibody-independent mechanism of rickettsemia control might occur during acute anaplasmosis.

EFFICACY OF LO],;G • ACTING OXYTETRACYCLlNE ON BOVINE ANAPLASMOSIS

: Anaplasmosis ıvas diagnosed in 4 COM'S and l3 young bulls belongiııg to the Farm of the Faculty of Veterinary Medicine of Ankara University, while 8 of them revealed typical symptoms of acute anaplasmosis (Group i), the others were identified as A. nıarginale can'iers (Group Il). Three injections of long-acting oxytetracycline were administered intramus('ulary (20 mg! kg) at 3-day intervals to Group i. Two iııjectioııs of the same preparatioıı were administered in the same manner to Group ll. lt was concluded that long-acting oxytetracycline could be used successJully in both preventing acute anaplasmosis and eliminating carrier state of anaplasmosis.

Vaccination with Anaplasma centrale: response after an experimental challenge with Anaplasma marginale.

The haematological and clinical responses to vaccination with Anaplasma centrale and to subsequent challenge with Anaplasma marginale were evaluated. Twenty Holstein steers 14 to 16 months of age were divided into two groups of 12 and eight animals respectively (groups I and II). Group I was inoculated on day zero with 10(7) A. centrale-infected erythrocytes and group II was kept as a control. On day 125 both groups were challenged with 5 X 10(7) A. marginale-infected erythrocytes. A. centrale inoculation produced low parasitaemias (maximum mean 2.7%), moderate packed cell volume (PCV) falls (minimum mean 20.5%) and no clinical symptoms. After the challenge group I had significantly lower parasitaemia (maximum mean 2.3%) and higher PCV (minimum mean 20.1%) than group II (7.5% and 14.5% respectively). Four steers from group II developed acute anaplasmosis and required treatment.

Observations on histamine, serotonin, cortisol and cortisol binding globulins in bovine anaplasmosis

Changes in histamine, serotonin, cortisol and cortisol binding globulin (CBG) levels were observed in Anaplasma marginale-infected bovine calves. In addition, packed cell volume (PCV), platelet number and percentage parasitaemia were also recorded. The whole blood histamine and serotonin values rose significantly ( P<0.01 and P<0.025, respectively) during the acute stage of Anaplasma infection. Higher serum cortisol and CBG levels ( P<0.05) were observed in acute and carrier infections, respectively. A sharp drop in thrombocyte count (59%) and PCV (33%) was also noticed in clinical anaplasmosis. The results suggest that the higher levels of biogenic amines which are known to produce increased vasodilation, capillary permeability and tissue anoxia and hypercortisolaemia to protect animals from stress and cell damage may play a similar role in the pathogenesis of acute anaplasmosis.

Anemia associated with anestrus in beef heifers inoculated with [formula omitted][formula omitted]: Endocrinology and ovarian changes

Eleven cyclic Hereford heifers were synchronized to estrus with a commercial luteolytic agent. Eight heifers had been inoculated with Anaplasma marginale . Six of the eight inoculated heifers became anemic, i.e. packed cell volume, red blood cell counts and hemoglobin decreased. White blood cell counts were elevated during severe anemia. Anemic heifers did not show behavioral estrus or ovulate, and during this stage, serum concentrations of progesterone and estradiol-17β were significantly depressed. Mature beef heifers that have acute anaplasmosis suffer from inhibited ovarian function and anestrus.

Anestrus in cycling beef heifers experimentally inoculated with [formula omitted][formula omitted

Normal estrous cycles were established for twenty beef heifers. Ten heifers were inoculated with blood from a known Anaplasma marginale carrier. The inoculated heifers experienced anemia, anorexia, and positive Anaplasma marginale complement-fixing antibody titers, and A . marginale was observed on the erythrocytes while uninoculated control heifers remained normal. Further observation following inoculation revealed that five of ten inoculated heifers experienced anestrus while controls continued to cycle normally. Anestrus coincided with clinical signs of acute anaplasmosis. Normal estrus patterns returned following treatment and recovery. This study provides evidence that acute anaplasmosis in beef heifers may cause anestrus and therefore lead to reproductive inefficiency.

Effect of Anaplasma marginale Infection upon Blood Gases and Electrolytes in Splenectomized Calves

The principal pathologic manifestation of Anaplasma marginale infection in bovines is anemia, which in acute cases may cause packed cell volumes (PCV) to fall below 10% within 4 to 5 days after the onset of parasitemia. Mortality seen in acute cases appears directly related to the anemic process. However, the underlying mechanisms connecting anemia and mortality have not been well documented. Furthermore, the death or survival of an infected animal is difficult to predict from its PCV, parasite count, and outward appearance. The rapid destruction of 02and CO2-carrying red blood cells (RBC's) and the concomitant rapid release of RBC contents within the host in acute anaplasmosis could conceivably lead to imbalances in blood pH and salts. Thus we examined blood gases, pH, and electrolytes to determine whether any of these parameters change during A. marginale infection in a manner incompatible with life. The 6to 10-month-old splenectomized calves studied were animals involved in other continuing experiments at this station. The use of splenectomized calves was justified by the findings of Roby et al. (1961, Am. J. Vet. Res. 22: 982-985) who found that splenectomized, mature cows were similar to nonsplenectomized animals in susceptibility to inoculation with anaplasma-infected blood. Ten calves were infected with A. marginale (1.4 x 108 infected particles), four calves received no inoculum. In addition, observations were made on three calves that spontaneously revealed Eperythrozoon wenyoni infections after splenectomy and survived. Blood samples for gas and pH analyses were collected anaerobically in heparinized syringes by jugular venipuncture, iced immediately, and analyzed at 37 C within an hour on a Corning blood gas analyzer. Blood samples for other analyses were collected in evacuated tubes with lithium-heparin as an anticoagulant. Plasma K+ and Na+ were assayed by flame photometry; chloride, by titration (Schales, 1953. In Standard methods of clinical chemistry, Vol. 1, M. Reiner, [ed.], Academic Press, New York, New York, pp. 37-42); and blood urea nitrogen (BUN), colorimetrically (Mattenheimer, 1970. Micromethods for the Clinical and Biochemical Laboratory, Ann Arbor Science Pub., Ann Arbor, Michigan, pp. 126-127). Hemoglobin (Hb) was assayed as methemoglobin (Mattenheimer, 1970, loc. cit. p. 88). Percent reticulocytosis was estimated from counts of at least 3,000 Giemsa-stained RBC's. Until the time of anemic crisis, the only noticeable changes in all infected calves were decreases in Hb and in oxygen content (OC, sum of free, dissolved 02 and 02 bound to Hb), which roughly paralleled decreases in Hb. However, at anemic crisis, dramatic reductions in pH and HCO3concentration were observed in the calves that died. Thus, we compared all measured or calculated values of all the infected calves at anemic crisis (the time of lowest Hb concentration, usually the last observations on the calves in the AMF group), and contrasted them with values from the control group. The infected calves were divided into three groups: A. marginale fatalities (AMF), A. marginale survivors (AMS), and E. wenyoni survivors (EWS); data were subjected to analyses of variance, and the significance of differences among infected groups and between infected and control groups was determined by Duncan's multiple range test at the 5% level. No significant difference in the mean plasma levels of Na+ or Clwas found among the four groups (data not shown). The mean OC's of all infected groups were lower than the mean of the control, but they did not differ significantly from one another (Table I). The mean BUN's of all infected groups were higher than the controls but were not high enough to indicate glomerular dysfunction

Blood Levels of Oxytetracycline After the Use of a Long-Acting Formulation to Eliminate the Carrier State of Bovine Anaplasmosis

The purpose of this paper is to present data on the blood plasma levels of oxytetracycline which resulted when cattle were injected intramuscularly (IM) once a week with a strengthened and improved formulation of oxytetracycline containing 200 mg/ml of base drug. One injection of this formulation has been reported to have an inhibitory effect on the parasitemia in acute anaplasmosis equivalent to three daily injections of oxytetracycline containing 50 mg/ml (2). Also, the former formulation has been recently reported to effect the elimination of the carrier state of bovine anaplasmosis when administered IM at seven-day intervals (3).&#x0D; The use of tetracycline drugs at relatively high dosage levels for varying periods of time has been an accepted procedure for eliminating A. marginale infection. Either prolonged feeding of chlortetracycline or daily injection of oxytetracycline have been used for this purpose in a number of earlier studies. The present study suggests that the use of a long-acting oxytetracycline will effectively eliminate the carrier state of sensitive strains of A. marginale. Also, the present study shows that in a limited ·number of cattle, detectable blood levels were present in 10 of 12 cattle seven days after one treatment with the long-acting formulation of oxytetracycline. It will be necessary to further evaluate the use of this treatment regimen to determine if similar results can be achieved under field conditions.

Scanning Electron Microscopic Studies of Paranaplasma sp. in Erythrocytes of a Cow

Blood from a California cow naturally infected with Paranaplasma organisms was examined utilizing a scanning electron microscope to study the 3-dimensional morphology of the parasite. Although blood films stained with Wright-Leishman stain revealed that 63% of the red cells were parasitized, the morphology of nonhemolyzed erythrocytes fixed directly in glutaraldehyde gave little indication of the organism's presence. A rapid, simple sample preparation technique which entailed simultaneous hemolysis and fixation of the parasitized red cells in blood films was found highly satisfactory in that it allowed easy viewing of the entire parasite. Dumbbell, comet, ring, and bipolar discoid forms were observed. The tails of the organisms appeared as distinct cylindrical structures intimately associated with the marginal bodies or heads. Viewing in 3 dimensions made apparent the structural complexity of the organism. Boynton (1932), in a study of anaplasmosis in California cattle, observed ring and tailed forms of Anaplasma. The ring and taillike structures associated with the typical marginal bodies of Anaplasma were considered by him to be either erythrocytic substance or a portion of the parasite itself. With phase contrast and electron microscopy, Espana et al. (1959) demonstrated ring, match, comet, and dumbbell-like forms in unstained lysed erythrocytes from both naturally and experimentally infected cattle. Signet ring and comet forms in California calves with acute anaplasmosis were also demonstrated by staining blood films with new methylene blue (Schalm et al., 1962). Tailed organisms were described (Pilcher et al., 1961; Madden, 1962) for the Oregon strain of Anaplasma. Madden (1962), by fluorescent antibody technique, showed that the taillike structures were an integral part of the organism. These observations revealed the existence of two morphologically different forms of Anaplasma: the classical punctate structures located marginally in the erythrocytes, as described by Theiler (1909) and observed in blood smears stained with Romanowsky stains, and the more complex tailed forms. Kreier and Ristic (1963a, b, c) made an extensive comparison of Oregon and Florida isolates of Anaplasma. The Florida isolate was found to consist of only one morphological class appearing as typical round marginal bodies. Three morphological classes were identified for the Oregon isolate; the predomReceived for publication 5 September 1972. inant form was the round marginal body class while th other two classes were those having a marginally situated head with a body and a tail and those of bipolar disc shape. The Florida isolate and the round marginal body class of the Oregon strain were found to be morphologically and antigenically distinct (Kreier and Ristic, 1963b). Kreier and Ristic (1963c), on the basis of sheep and deer passage studies, proposed that the tailed and bipolar disc forms of the Oregon isolate were d stinct organisms and should be designated Paranaplasma caudata and Paranaplasma discoides, respectively. The Anaplasma body found at the margin of the erythrocyte has been the focus of umerous electron micrographic studies (De Rob rtis and Epstein, 1951; Foote et al., 1958; Ristic, 1960a, 1967; Ristic and Watrach, 1961, 1963; Scott et al., 1961; Gates et al., 1967; Simpson et al., 1967; Summers and Padgett, 1970). In comparison, the structural features of the filamentous portion of the tailed organisms have received less attention. In hemolyzed, parasitized erythrocytes, Espana et al. (1959) observed various tailed and ring forms including parasites seemingly joined tail to head and tail to tail. Ritchie (1962), using a dialysis technique to remove the hemoglobin, noted the filamentous portion of the organism to be a saclike structure which had apparently lysed in a manner similar to the erythrocytes. Simpson et al. (1965) carried out both surface and thin section studies of the tails or filamentous portions of Anaplasma which they referred to as bands. The bands appeared as

The Inhibitory Effect of Imidocarb on Experimental Anaplasmosis in Splenectomized Calves

Summary The development of acute infections of Anaplasma marginale in splenectomized calves was inhibited by subcutaneous injection of imidocarb [3,3′-bis-(2-imidazolin-z-yl)-carbanilide dihydrochloride] in single doses of 2·5, 5·0, or 10·0 mg./kg. A single dose of 25·0 mg./kg. administered orally was not effective. Six untreated calves died from acute anaplasmosis, whereas 6 calves treated by the subcutaneous route survived. There was a rapid decrease in numbers of Anaplasma bodies in the blood following the injection of imidocarb.

The Inhibitory Effect of a Dithiosemicarbazone on Acute Infections of Anaplasma marginale

Acute infections of Anaplasma marginale in splenectomized calves and adult cattle were inhibited by administering a micronized suspension of α-ethoxy-ethylglyoxal dithiosemicarbazone. Intravenous administration produced the best results. A comparison of treated principals with untreated infected controls showed that a single dose of 5 mg./kg. body-weight suppressed parasite development, lessened anaemia and increased the survival rate. Acute anaplasmosis did not develop in a yearling steer given a subcutaneous dose of 10 mg./kg. suspended in Freund’s incomplete adjuvant