Evaluation of anaemia in Nigerian goats using FAMACHA© eye colour chart: a preliminary study

A preliminary study was carried out on the possibility of using the FAMACHA eye colour chart to predict the level of anaemia in 280 Nigerian goats of varied ages slaughtered at the Nsukka abattoir, in Enugu State, Nigeria. Three indices of anaemia, namely packed cell volume (PCV), red blood cell (RBC) counts and haemoglobin (Hb) concentrations were compared with the colour of the ocular membranes of the goats. The colours of the ocular conjunctiva of all animals were scored on a 1–5 scale using the FAMACHA© card, and blood samples were collected from each animal for determination of PCV, RBC counts and Hb concentration. Correlations between eye colour scores and the duo of PCV and Hb concentrations were highly significant and negative. Haemonchus contortus was the most predominant gastrointestinal nematode parasite observed in the study with average larval recovery of 70.18 %. Age has no effect on the predictability of anaemia by the FAMACHA© technique. It was concluded that the FAMACHA method can be used by farmers in Nigeria to identify anaemic goats particularly in conditions of haemonchosis which is one of the main causes of anaemia in goats and the most predominant gastrointestinal nematode in small ruminants in the study area. It is, therefore, believed that these preliminary findings will form a basis for further work on validating the use of FAMACHA© in Nigerian goats.     

Hypophagia-induced hypometabolism during experimental anaemia in rats

Several investigators have reported a drop in oxygen (O₂) consumption (VO₂) and body temperature in laboratory animals during normobaric or hypobaric hypoxia. Hypophagia, with normal efficiency of protein utilisation for growth, was also observed. It has recently also been observed that hypometabolism is present during anaemic hypoxia. The present study was designed to test the experimental hypothesis that anaemic hypoxia induces hypometabolism secondary to hypophagia. Episodes of anaemia were created in adult male rats by either blood withdrawal through cardiac puncture (haemorrhagic anaemia) or phenylhydrazine administration (haemolytic anaemia). Haematrocrit, VO₂, and food consumption, as indirect estimations of the level of anaemia, energy production, and appetite, respectively, were serially measured in all animals during 7 days (acute experiments) or 17 days (chronic experiments). Positive correlations were found between the three parameters during development of and recovery from anaemia during each anaemic episode. When the amount of food offered to non-anaemic rats was equalised to that freely eaten by anaemic rats, VO₂ dropped in the former to almost the level found in the latter. Body composition changed during chronic anaemia because of a decrease in the lipid fraction of the body. The results confirmed the working hypothesis that hypometabolism, which has been considered as an immediate, emergency-type response to both hypoxic and anaemic hypoxia, can be considered as a response secondary to hypophagia because of depressed appetite. How appetite is adapted to the mechanisms which control O₂ convection and O₂ availability is not known at present.     

Seroprevalence of tick-borne diseases among cattle in the Sudan

This study was carried out to determine the prevalence of Theileria annulata, Theileria mutans, Babesia bigemina, and Anaplasma marginale antibodies among cattle in the Sudan. A total of 600 serum samples were collected from indigenous (zebu) and crossbred cattle (zebu × Friesian) of both sex and different age groups. Indirect enzyme-linked immunosorbent assay was used to assess antibodies against tick-borne diseases in apparently healthy cattle. The overall prevalence rates of T. annulata, T. mutans, B. bigemina, and A. marginale antibodies were found to be 30.8%, 6.1%, 10.7%, and 38.9%, respectively. The highest seroprevalence of T. annulata was reported in Atbara and El Damer, Northern Sudan. There were no significant associations for the seroprevalence of all tick-borne diseases reported among different age groups. Although there were no significant differences between the two breeds of cattle examined for T. annulata, T. mutans, and B. bigemina antibodies, there was a significant difference for prevalence of antibodies against A. marginale, with highest percentages of antibodies in indigenous cattle. Six different combinations of mixed infection were detected. This is the first report in which antibodies against A. marginale among cattle in Northern Sudan is reported. The findings imply that antibodies to tick-borne infections are widely distributed in the region. The need for further investigations using more advanced techniques is recommended.     

Haematological changes in sheep experimentally infected with Trypanosoma evansi

 Sheep were infected with 2×10⁶ Trypanosoma evansi TREU 2143 through the external jugular vein. The parasite kinetics as well as the effects on body temperature, packed cell volume (PCV), erythrocyte counts and total and differential white blood cell counts were monitored twice weekly for 3 months. The results showed that T. evansi produced a chronic form of the disease in sheep characterised by low-level and often cryptic parasitaemia, with self-cure occurring in two cases; mild anaemia as evidenced by decreases in PCV and erythrocyte counts; and significant (P<0.02) leucocytosis by day 22 post infection (p.i.). The leucocytosis was a result of marked lymphocytosis whose significant rises (P<0.02) parallelled the rises in total white blood cell (TWBC) counts. These changes were less obvious in the animals that underwent self-cure. We conclude that T. evansi produces pathological changes in the peripheral blood of sheep similar to those produced by its tsetse-transmitted counterparts. It would thus appear that the sheep/T. evansi model is suitable for long-term study of the immunopathology of pathogenic trypanosomes since the sheep is easily available, easy to handle and a natural host to all pathogenic trypanosomes. Received: 17 July 1995 / Accepted: 11 January 1996     

Efficacy of purifiedanaplasma marginale initial bodies as a vaccine against anaplasmosis

Anaplasma marginale initial bodies of the Florida strain were purified from infected erythrocytes using a combination of ultrasonic disruption, nonionic detergent and differential centrifugation. Immunochemical analysis revealed at least 12A. marginale proteins in the molecular mass (m) range 81–15 kDa with a prominent band at 38 kDa. Several of these proteins remained insoluble in the presence of nonionic detergent. Preparations of purifiedAnaplasma initial bodies contained negligible erythrocytic contamination, as confirmed by the minimal induction of isoantibodies against bovine blood group antigens and the absence of delayed-type hypersensitivity to erythrocytic antigens in immunized animals. A total of 33 crossbred and purebred Holstein cattle were vaccinated with either 1.5, 1.0, or 0.1 mg protein of intact initial bodies, or with 1.0 mg of solubilizedAnaplasma protein. The immunogens were supplemented with 3.0 mg Quil-A saponin adjuvant and administered in 2 subcutaneous injections given at a 4-week interval. A similar number of nonvaccinated cattle served as controls. Three months after vaccination, all cattle were challenged by inoculation of 10⁹ virulenta. marginale of either the homologous (Florida) or heterologous (Venezuelan) strains. Vaccinated cattle showed solid protection after homologous and heterologous challenge, characterized by parasite clearance and minimal hematocrit reductions. Initial data from four field vaccine trials revealed a reduced incidence of clinical anaplasmosis among immunized animals. Use of immunogens consisting of purifiedA. marginale initial bodies offers a potential immunoprophylactic approach to control of bovine anaplasmosis.     

Measurement of Mouse Plasma Erythropoietin by an Improved ELISA

We have examined whether mouse plasma erythropoietin (EPO) can be measured by an improved enzyme-linked immunosorbent assay (ELISA) using milk proteins (Block Ace) both as a blocking reagent and as a diluent for standard recombinant human EPO (rHuEPO) and for plasma samples. Block Ace brought about high slope sensitivity of the standard curve, with a low background. The dose–response curves of normal or anaemic mouse plasma and of rHuEPO were linear and parallel to each other. The anaemic plasma had an additive effect with rHuEPO by increasing the absorbance at 405 nm. The coefficients of variation in the intra- and interassays ranged from 4.2% to 15.3%. The plasma EPO levels in 22 normal mice were 18.3 ± 10.3 mU/ml. An inverse relationship between the logarithm of plasma EPO concentrations and blood haemoglobin concentrations, red blood cell counts or packed cell volumes was found in normal mice and in mice with iron deficiency anaemia (IDA). These results show the validity for the use of the new improved ELISA method for measuring circulating murine EPO.     

Anaplasma marginale: failure of sera from immune cattle to confer protection in passive-transfer experiments

High levels of immunity toAnaplasma marginale were induced in cattle either by vaccination using sonically disruptedA. marginale-infected erythrocytes or by repeated infection with different strains of the rickettsia. In both instances, high levels of anti-A. marginale antibody were detected in the sera of the immune cattle by immunoblotting. Serum from one animal that had been made immune by repeated infection was transferred intravenously toA. marginale-susceptible calves (three non-splenectomised and two splenectomised) undergoing initialA. marginale infection at serum doses of 2–10 ml/kg. Neither the course nor the outcome of infection as indicated by the parasite levels attained or the level of anaemia induced was altered in the calves that received the immune serum relative to the course or outcome of infection in control calves (two non-splenectomised and two splenectomised) that received serum from anA. marginale-naive donor animal. In a similar experiment, a pool of sera from four steers that had been vaccinated with sonically disruptedA. marginale initial bodies was transfused into two intactA. marginale-susceptible calves during the early stage ofA. marginale infection at a dose of 10 ml/kg. No difference was observed in the course or outcome of infection in these calves relative to the course or outcome of infection in the two non-splenectomised calves that were transfused with non-immune serum. The failure of serum obtained from animals that had been made immune toA. marginale either by infection or by vaccination with disrupted initial bodies to confer any protection against infection following its transfer into splenectomised or non-splenectomised naive calves indicates that antibody per se is not a significant factor in immunity to this parasite.This work was supported by the Australian Meat Research Corporation     

Measurement of mouse plasma erythropoietin by an improved ELISA

We have examined whether mouse plasma erythropoietin (EPO) can be measured by an improved enzyme-linked immunosorbent assay (ELISA) using milk proteins (Block Ace) both as a blocking reagent and as a diluent for standard recombinant human EPO (rHuEPO) and for plasma samples. Block Ace brought about high slope sensitivity of the standard curve, with a low background. The dose–response curves of normal or anaemic mouse plasma and of rHuEPO were linear and parallel to each other. The anaemic plasma had an additive effect with rHuEPO by increasing the absorbance at 405 nm. The coefficients of variation in the intra- and interassays ranged from 4.2% to 15.3%. The plasma EPO levels in 22 normal mice were 18.3 ± 10.3 mU/ml. An inverse relationship between the logarithm of plasma EPO concentrations and blood haemoglobin concentrations, red blood cell counts or packed cell volumes was found in normal mice and in mice with iron deficiency anaemia (IDA). These results show the validity for the use of the new improved ELISA method for measuring circulating murine EPO.     

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.     

The haematology ofTrypanosoma congolense infection in cattle I. Sequential cytomorphological changes in the blood and bone marrow of Boran cattle

Five adult Boran cattle (Bos indicus), infected with a clone ofTrypanosoma congolense IL13-E3 three years earlier and treated, were re-challenged with the same clone. Changes in the peripheral blood were monitored twice weekly, and events in the bone marrow (BM) were assessed by weekly biopsies of the sternal BM, until day 98 postinfection (dpi) when the three surviving animals were treated with diminazene aceturate. One animal died on 57 dpi whereas another was treated on 63 dpi when the packed cell volume was 15%. The infected animals developed anaemia, leucopenia and thrombocytopenia during the first peak of parasitaemia which persisted until the experiment was terminated. Three phases of BM response were demonstrated on light microscopic examination of BM smears. The first, the preparasitaemic phase represented by samples taken on 15 dpi, was an immunological response with slight but significant increases in lymphoblasts, lymphocytes, plasma cells and macrophages (Mø) whereas erythroid and granulocytic cells were unchanged. The second, the early parasitaemic or acute phase (21–57 dpi) associated with the development of anaemia, leucopenia and thrombocytopenia, was characterised by intensification of the immunological response, and an early but transient granulocytic hyperplasia. The third, the late parasitaemic or chronic phase (63–98 dpi) associated with persisting pancytopenia, was characterised by erythroid, megakaryocytic and Mø hyperplasia, dyserythropoiesis, granulocyte hypoplasia and return of lymphoid cell counts to preinfection numbers. Transmission electron microscopy confirmed these findings and showed that intact trypanosomes were not observed in the sinusoids and haemopoiesic compartment of the BM.This study demonstrates thatT. congolense infection the various blood cell lineages depending on the stage of infection. This suggests a fine control mechanism, presumably cytokine-mediated. Erythropoiesis, thrombopoiesis and monocytopoiesis were generally upregulated, whereas granulopoiesis was downregulated. However, haemopoiesis was generally ineffective as numbers of circulating blood cells remained below preinfection levels throughout the period of the study.     

The role of the bone marrow in bovine trypanotolerance I. Changes in blood and bone marrow inTrypanosoma congolense-infected cattle

This study compared the changes in the bone marrow (BM) of five trypanotolerant N'Dama cattle with those of four trypanosusceptible Boran cattle during trypanosome infection. In the early parasitaemic phase, from 12 to 21 days postinfection (DPI), tsetsetransmitted primaryTrypanosoma congolense IL 1180 infection induced parasitaemia, slight depression in packed cell volume (PCV), marked leucopenia due to lymphocytopenia and eosinopenia, and thrombocytopenia which were of similar intensity in Boran and N'Dama cattle. However, from 28 DPI until the end of the experiment on 112 DPI, the parasitaemia was higher in the Boran than in the N'Dama. Severe anaemia and leucopenia characterised by lymphopenia, neutropenia, eosinopenia and monocytopenia persisted in Boran cattle. In contrast, the PCV values dropped gradually in N'Dama cattle and from 77 DPI recovered slowly to values just below preinfection levels by 112 DPI. The total and differential leucocyte counts of the N'Dama cattle stabilised at approximately two-thirds of preinfection values between 28 and 112 DPI, and were double those of the Boran. Marked thrombocytopenia occurred in both breeds. The anaemia was initially macrocytic hypochromic but terminally became microcytic hypochromic in both breeds. Light and electron microscopic studies of sequential biopsies of the BM of these animals showed that the BM response was the key to these differences between the N'Dama and Boran. The biopsies of the BM of the N'Dama cattle were hypercellular (scored 4.5±1.0 compared to 4.0 for controls) with mild hyperplasia of erythroid cells and mild hypoplasia of myeloid cells from 28 to 112 DPI, endowing the animals with higher haemopoietic potential that enabled them to replace most lost cells. In contrast, the Boran cattle had hypocellular (scored 2.4±1.1) BM biopsies with relative erythroid hyperplasia and myeloid hypoplasia, resulting in low capacity of cell replacement manifested as severe unremitting anaemia and leucopenia. The BM of both breeds showed moderate hyperplasia of cells of the mononuclear phagocyte system. Therefore, this study showed, for the first time, that BM response is a key determinant factor of trypanotolerance as it determines the animal's capability for blood cell regeneration.     

Infection of Tick Cells and Bovine Erythrocytes with One Genotype of the Intracellular Ehrlichia Anaplasma marginale Excludes Infection with Other Genotypes

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, is endemic in several areas of the United States. Many geographic isolates of A. marginale that occur in the United States are characterized by the major surface protein 1a, which varies in sequence and molecular weight due to different numbers of tandem repeats of 28 or 29 amino acids. Recent studies (G. H. Palmer, F. R. Rurangirwa, and T. F. McElwain, J. Clin. Microbiol. 39:631-635, 2001) of an A. marginale-infected herd of cattle in an area of endemicity demonstrated that multiple msp1α genotypes were present but that only one genotype was found per individual bovine. These findings suggested that infection of cattle with other genotypes was excluded. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in infected bovine erythrocytes and cultured tick cells. Two tick-transmissible isolates of A. marginale, one from Virginia and one from Oklahoma, were used for these studies. In two separate trials, cattle inoculated with equal doses of the two isolates developed infection with only one genotype. Tick cell cultures inoculated with equal doses of the two isolates became infected with only the Virginia isolate of A. marginale. When cultures were inoculated with different ratios of the Oklahoma and Virginia isolates of A. marginale, the isolate inoculated in the higher ratio became established and excluded infection with the other. When cultures with established infections of one isolate were subsequently infected with the other, only the established isolate was detected. We documented infection exclusion during initial infection in cell culture by labeling each isolate with a different fluorescent dye. After 2 days in culture, only a single isolate was detected per cell by fluorescence microscopy. Finally, when Anaplasma ovis infections were established in cultures that were subsequently inoculated with the Virginia or Oklahoma isolate of A. marginale, A. marginale infection was excluded. These studies confirm that infection exclusion occurs with A. marginale in bovine erythrocytes and tick cells, resulting in the establishment of only one genotype, and appears to be the first report of infection exclusion for Anaplasma and Ehrlichia species.     

Peripheral blood lymphocyte proliferative responses in cattle infected with or vaccinated against Anaplasma marginale

 An assay was developed for measurement of the peripheral blood lymphocyte proliferative response (PBLPR) in cattle infected with or immunised against Anaplasma marginale. PBLPR was not evident in all cattle that had recovered from A. marginale infection. However, A. marginale-sensitised lymphocytes were detected in the spleens of all immune cattle tested in the absence of detectable PBLPR. During the course of initial infection, cattle exhibited detectable PBLPR for a period corresponding with and up to 2 weeks after patent parasitaemia, followed by a second, usually larger peak in PBLPR corresponding to the time of sub-clinical relapse of cattle. Analysis of the PBLPR of A. marginale chronically infected cattle demonstrated highly variable PBLPR between individuals and over time. A positive PBLPR was induced in cattle by vaccination using a crude A. marginale antigen preparation. The PBLPR of vaccinated cattle subsequently infected with A. marginale was markedly different from that of naive cattle, with reduced PBLPR being associated with the onset of parasitaemia. The antigen used in the PBLPR assay was inactivated by proteolysis. Proteolysis also abolished immunity that had been induced in cattle vaccinated using the antigen preparation. A. marginale-sensitised PBL did not proliferate in response to antigen from the heterologous species A. centrale. A. centrale-sensitised PBL, however, responded to A. marginale antigen. Interferon-γ (IFN-γ) was detected in PBLPR-assay supernatants and was associated with a strong PBLPR. Received: 7 December 1995 / Accepted: 12 December 1995     

Babesia shortti infection in a common kestrel (Falco tinnunculus) in Catalonia (northeastern Spain)

We report the presence of the avian piroplasm Babesia shortti in a common kestrel (Falco tinnunculus) admitted in a wildlife recovery centre in Catalonia, Spain. The bird, which was in a very poor condition and had respiratory distress, was anaemic (packed cell volume of 22%). The animal died within days. No postmortem examination was performed, but the extremely high parasitaemia of almost 45% and the anaemia might have caused death.     

Kinetics of haemopoietic progenitor cells during a Trypanosoma congolense rechallenge infection in Boran cattle

In a preliminary study, using clonogenic assays, the in vitro kinetics of committed haemopoietic progenitors were monitored during a Trypanosoma congolense rechallenge infection in five trypanosusceptible Boran cattle. Early in the infection (week 2), in the absence of any detectable parasitaemia, a drop in the number of nucleated marrow cells was recorded. This was accompanied by a marked but transient decrease in the levels of the colony-forming units-erythroid (CFU-E) followed by a partial recovery by weeks 3–4 after infection. The burst-forming units-erythroid (BFU-E) and the colony-forming units-granulocyte macrophage (CFU-GM) also significantly decreased between weeks 2 and 4. After a transient rise at weeks 3–5 postinfection, the CFU-GM steadily declined and remained below preinfection levels throughout the infection. The BFU-E remained below preinfection levels until the end of the experiment. The drop in nucleated marrow cells associated with the decreased numbers of CFU-E, BFU-E and CFU-GM was suggestive of a defect at the pluripotential stem cell level early in the infection (week 2). The erythrocyte indices, i.e. mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), were unchanged until week 10 postinfection. Two animals became severely anaemic; one was euthanised at week 8 and one treated at week 9. The three remaining animals developed chronic anaemia with mean packed cell volume (PCV) fluctuating around 18%–19% between weeks 11 and 14. Low parasitaemia levels were recorded during that period. A CFU-E peak above preinfection levels was noted at week 12 and BFU-E appeared in the peripheral blood culture of two animals between weeks 11 and 14. A progressive rise in MCV associated with a gradual decrease in MCHC also characterised that period. A return to near preinfection levels was recorded for the numbers of all three progenitors three weeks after trypanocidal treatment followed by a full recovery five months after treatment. Although ineffective haemopoiesis has been suggested to contribute to the anaemia of bovine trypanosomiasis, this is the first demonstration of a negative effect on erythroid development in cultures of bone marrow of trypanosome-infected cattle.     

Identification,Molecular Characterization,and Occurrence of Two Bovine Hemoplasma Species in Swiss Cattle and Development of Real-Time TaqMan Quantitative PCR Assays for Diagnosis of Bovine Hemoplasma Infections

Concomitantly with an outbreak of fatal anaplasmosis in a cattle herd in Switzerland in 2002, we detected two bovine hemoplasma species in diseased animals: Mycoplasma wenyonii (formerly Eperythrozoon wenyonii) and a second, novel bovine hemoplasma species later designated “Candidatus Mycoplasma haemobos” (synonym, “Candidatus Mycoplasma haemobovis”). The second species was characterized by a shorter 16S rRNA gene. The aims of the present study were to provide a detailed molecular characterization of this species, to develop specific quantitative real-time PCR assays for the two bovine hemoplasma species, and to apply these assays in order to evaluate the prevalence and clinical significance of the hemoplasmas. Sequencing of the near-complete 16S rRNA gene of the second hemoplasma revealed that it was 94% identical to that of Mycoplasma haemofelis, an anemia-inducing feline hemoplasma species, but less than 85% identical to that of the bovine hemoplasma M. wenyonii. Using the newly developed assays, a total of 159 animals from the anaplasmosis outbreak were reexamined. In addition, we tested 57 clinically ill and 61 healthy Swiss cattle, as well as 47 calves. Both hemoplasmas were highly prevalent in adult cattle but occurred rarely in calves. Animals from the herd with the fatal anemia outbreak were more frequently infected with M. wenyonii and exhibited higher M. wenyonii blood loads than animals with unrelated diseases and healthy animals. Coinfections may increase the pathogenicity and clinical significance of bovine hemoplasmosis.In connection with an outbreak of anaplasmosis in a cattle herd in eastern Switzerland in 2002, more than 300 animals were culled. Most of these cattle exhibited pronounced anemia. The anemia was statistically associated with the detection of Anaplasma marginale, Babesia spp., Theileria spp., and Mycoplasma wenyonii in the blood of diseased animals (5).M. wenyonii, first described in 1934, was formerly known as Eperythrozoon wenyonii (1, 13). The species was recently reclassified within the group of hemotropic mycoplasmal species based on the 16S rRNA gene sequence (11-13). M. wenyonii is a cell wall-free bacterium that parasitizes bovine red blood cells (11). In our study of the above-mentioned outbreak, we reported two distinct hemotropic mycoplasma species: M. wenyonii and a second, unknown, agent (5). The 16S rRNA gene of the second agent was shorter than that in M. wenyonii and was 95% identical to the 16S rRNA gene found in Mycoplasma haemofelis, the causative agent of feline infectious anemia (24, 25). A similar bovine hemoplasma species has since been detected in China and Japan using molecular assays, and the name “Candidatus Mycoplasma haemobos” has been proposed (20). Other bovine hemoplasmas that were found to be distinct from M. wenyonii using other methods are described elsewhere (3, 30-32). Characterization included morphological and immunogenic differences, as well as different localization of the agents within the host (28, 29, 31, 32, 38).The clinical relevance of M. wenyonii is controversial (16, 18); in the United States, infection with M. wenyonii is considered to be of low pathogenicity. However, a study with splenectomized calves showed that a preexisting M. wenyonii infection followed by an A. marginale superinfection led to severe anemia, with packed-cell volumes (PCV) dropping from 30% to 9.5% when M. wenyonii was found in the blood and about 2 weeks before A. marginale appeared (13). The clinical relevance of “Candidatus Mycoplasma haemobos” remains unclear (14, 20).The aims of the present study were to characterize the two bovine hemotropic mycoplasma species identified in 2002 using molecular techniques, to develop specific real-time TaqMan PCR assays for the detection and quantification of these species, and to determine the occurrence of the two bovine hemoplasmas in sick and healthy cattle in order to evaluate their clinical importance.     

Effects of Duration of Storage and Storage Temperature on Cell Counts of Bovine Blood Samples as Determined by an Automated Haematology Analyser

Analysis of blood cells is an important part of many scientific investigations in the field of cattle herd health. Over the last 30 years, automated blood analysis has all but replaced manual counting of blood cells using counting chambers. The present study investigated the effects of prolonged storage and storage temperatures on cell counts as determined by a haematology analyser. Blood samples from 20 clinically healthy cows were repeatedly analysed with a Cell-Dyn 3500 (Abbott Diagnostika, Delkenheim), within 24 hours after collection and after storage at either 4° C or 20° C. The counts of most blood cells were more stable in samples stored at 20° C than those stored at 4° C. For at least 8 h, the counts of all analysed cell types, with the exception of lymphocytes, remained within ±3 standard deviations that were calculated for fresh samples, provided that the blood was stored at 20° C. Correspondence and offprint requests to: Dr Ulrich Bleul, Klinik für Fortpflanzungskunde, Universit?t Zürich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.     

The effect of <Emphasis Type="Italic">Iresine herbstii</Emphasis> Hook on some haematological parameters of experimentally induced anaemic rats

Iresine herbstii is fed to livestock in South-eastern Nigeria in the belief that it boosts their blood supply. The aim of this work is to study the effect of the methanolic extract of I. herbstii Hook on some blood parameters of experimentally induced anaemic albino rats. For acute toxicity study, graded doses of the methanolic leaf extract of I. herbstii at 10, 100, 1000, 1600, 2900 and 5000 mg/kg were administered orally to rats randomly allotted to six groups of three animals each and signs of toxicity were observed for 24 h. To evaluate its haematological effects, 100, 200, and 400 mg/kg of extract was administered orally to three out of five groups of six rats each for 14 days after the induction of acute blood loss anaemia. Parameters monitored were packed cell volume (PCV), haemoglobin concentration (Hb), weight gain and red blood cell counts (RBC). Phytochemical, proximate and nutritional analysis of the plant was done. I. herbstii had an LD₅₀?>?5000 mg/kg. Mean group weight gain and RBC were significantly (p?<?0.05) higher in treated than untreated groups. PCV and Hb did not differ significantly between the treated and untreated groups. Plant contained flavonoids; phenols; alkaloids; 22.85 % crude protein; 18.58 % ash; 9.62 % crude fibre; 1.5 % ether extract; 12.05 % moisture; vitamins A, C, E, B1, B3, B5, B6 and B12; zinc; iron; phosphorus; calcium; and magnesium. Methanolic extract of I. herbstii significantly improved red blood cell count of anaemic rats at the dose of 400 mg/kg bw and the body weights of anaemic rats at all treatment doses (100, 200 and 400 mg/kg bw).     

Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin

Subolesin was recently shown by both gene silencing and immunization with the recombinant protein to protect against tick infestations, and to cause reduced tick survival and degeneration of gut and salivary gland tissues. In this research, we extended these studies by testing whether targeting subolesin by RNAi or vaccination interfered with the ability of ticks to become infected with two tick-borne pathogens, Anaplasma marginale which causes bovine anaplasmosis and Anaplasma phagocytophilum, the causative agent of human granulocytin anaplasmosis. For the A. marginale studies, Dermacentor variabilis males were injected with subolesin dsRNA or saline and then were allowed to feed on cattle with ascending rickettsemias, while for the A. phagocytophilum studies, mice were immunized with the recombinant subolesin protein, infected with the pathogen and then infested with larval Ixodes scapularis. Tick infections were determined by quantitative polymerase chain reaction of gut and salivary gland tissues. In both experimental approaches, tick infections were significantly reduced. These results suggest that subolesin appears to be a candidate vaccine antigen that may contribute to control of multiple tick species and the reduction of tick-borne pathogens.     

Livestock trypanosomosis in Uganda: parasite heterogeneity and anaemia status of naturally infected cattle, goats and pigs

The prevalence and pathogenic effects of trypanosomosis were determined in cattle, goats and pigs reared in Kasese, Jinja and Rakai districts, Uganda; presence of trypanosomes was detected by buffy coat technique (BCT). The overall prevalence of trypanosomosis in cattle was 7.6 % (144/1,891), 0.7 % in goats (4/573) and 2.3 % in pigs (9/386). Internal transcribed spacer 1 (ITS1) of ribosomal DNA polymerase chain reaction was utilised to identify trypanosomes to species level and revealed infections in 108 of the 144 trypanosome-positive cattle while all infected goats and pigs gave amplicons. Trypanosoma vivax was the most prevalent trypanosome species in cattle in single and mixed infections compared to infections involving Trypanosoma congolense and Trypanosoma brucei; in pigs, eight were mixed infections with one single T. vivax infection. No predominant trypanosome species was detected in goats. Anaemia, the main trypanosomosis pathological feature, was investigated by determining packed cell volume (PCV). Mean PCV values by t test in infected individuals were significantly lower than non-infected individuals (P?<?0.05) for all animal species. However, the proportion of anaemic animals was not significantly different in infected and non-infected individuals. In addition, the percent of infected animals by Fisher’s exact test depended on district of origin and species but not sex. These findings show that trypanosomosis is a major cause of anaemia in livestock in endemic areas. Cattle were the major animal species affected by trypanosomosis; similar genotypes of trypanosomes were detected in the three animal species. BCT was more effective than ITS1 rDNA detecting trypanosomes in naturally infected cattle.