Prevalence of Trypanosoma theileri in cows and fetuses at slaughter

Blood from 171 pregnant and 65 nonpregnant cows was taken at slaughter for culture and examined during the culturing period for Trypanosoma theileri once a week for 5 weeks. Of the 171 fetuses from the pregnant cows, 119 (69.6%) were greater than or equal to 4 months gestation; blood samples from these fetuses were also taken for culture. Of 236 cows (81 of 171 [47.4%] pregnant and 20 of 65 [30.8%] nonpregnant cows), 101 (42.8%) were culture positive. More of the pregnant than nonpregnant cows were culture positive (P less than 0.05). More beef cows (48.0%) than dairy cows (34.1%) were culture positive (P less than 0.025). Two of the 119 (1.7%) fetus samples were found culture positive. The percentages of positive cultures from Brucella-reactor cows, 18 of 40 (45.0%), and from non-Brucella-reactor cows, 83 of 196 (42.3%), were similar. However, both of the culture-positive fetuses were from Brucella-reactor cows.     

Observations on Trypanosoma theileri Infection in Cattle

Naturally occurring Trypanosoma theileri infection was studied in two cattle herds. Herd A was a dairy herd of approximately 250. Herd B was an isolated herd of 32 and contained both dairy and beef breeds. Blood samples were collected from all animals in Herd A during July and August on two successive years. Samples were collected from Herd B at monthly intervals. Total leukocyte and differential counts packed cell volume determinations, and trypanosome cultures were made on each sample.

Infection was detected in all age groups between seven months and fifteen years but it was rare in calves. Infected animals were not consistently positive for trypanosomes on consecutive blood cultures and there was considerable variation between infected individuals. Positive cultures were usually obtained from some animals while others were positive intermittently. No correlation was found between trypanosome isolations and the season of the year.

A correlation was found between trypanosome isolation and lymphocytosis. Of the 920 blood samples examined, approximately one in every five trypanosome positive samples had lymphocyte levels in the Bendixen positive range. Approximately one in every twenty trypanosome negative samples had lymphocyte numbers in the Bendixen positive range. Evidence indicated that trypanosome isolation from animals with lymphocytosis was not caused by increased numbers of infected buffy coat cells in the inoculum cultured.

Eight calves were inoculated intravenously with trypanosome-infected blood. Lymphocyte numbers increased an average of 3549 per cumm above pre-inoculation levels in seven and remained essentially unchanged in one. Prior to inoculation with infective blood, two of the calves were intravenously inoculated with trypanosome-infected blood that had been frozen and thawed to kill the trypanosomes contained in it. Neither developed lymphocytosis following this inoculation.

No clinical disease problems which could be attributed to trypanosome infection were found.

     

First Isolation of Trypanosoma theileri in Sicilian cattle

Veterinary Research Communications -     

Comparative in vitro isolation of Trypanosoma theileri from cattle in Belgium

Ten blood samples randomly collected from cows on a farm nearby Antwerp, Belgium, were inoculated into KIVI culture medium (Kit for In Vitro Isolation of trypanosomes) and RPMI 10%+feeder medium. Within 3 weeks of incubation all KIVI cultures and four RPMI 10%+feeder revealed presence of Trypanosoma theileri. Some practical implications regarding the use of KIVI for isolation of pathogenic African trypanosomes from cattle and other Bovidae are discussed.     

Genetic diversity in Trypanosoma theileri from Sri Lankan cattle and water buffaloes

Trypanosoma theileri is a hemoprotozoan parasite that infects various ruminant species. We investigated the epidemiology of this parasite among cattle and water buffalo populations bred in Sri Lanka, using a diagnostic PCR assay based on the cathepsin L-like protein (CATL) gene. Blood DNA samples sourced from cattle (n = 316) and water buffaloes (n = 320) bred in different geographical areas of Sri Lanka were PCR screened for T. theileri. Parasite DNA was detected in cattle and water buffaloes alike in all the sampling locations. The overall T. theileri-positive rate was higher in water buffaloes (15.9%) than in cattle (7.6%). Subsequently, PCR amplicons were sequenced and the partial CATL sequences were phylogenetically analyzed. The identity values for the CATL gene were 89.6–99.7% among the cattle-derived sequences, compared with values of 90.7–100% for the buffalo-derived sequences. However, the cattle-derived sequences shared 88.2–100% identity values with those from buffaloes. In the phylogenetic tree, the Sri Lankan CATL gene sequences fell into two major clades (TthI and TthII), both of which contain CATL sequences from several other countries. Although most of the CATL sequences from Sri Lankan cattle and buffaloes clustered independently, two buffalo-derived sequences were observed to be closely related to those of the Sri Lankan cattle. Furthermore, a Sri Lankan buffalo sequence clustered with CATL gene sequences from Brazilian buffalo and Thai cattle. In addition to reporting the first PCR-based survey of T. theileri among Sri Lankan-bred cattle and water buffaloes, the present study found that some of the CATL gene fragments sourced from water buffaloes shared similarity with those determined from cattle in this country.     

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca²⁺ transients and TGF-β-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.     

High infection rates of the tick Hyalomma anatolicum anatolicum with Trypanosoma theileri

A crossbred calf (3 months old) obtained from a farm where regular control of ticks was practised and found to be free of blood parasites was inoculated with 20 ml pooled blood collected from four field cattle which had very low Trypanosoma theileri parasitaemias (one parasite per 70 microl blood as determined by the haematocrit centrifugation technique). Trypanosoma theileri was present in the blood 6 days after injection and a peak parasitaemia of 42 parasites per 70 microl blood was recorded by day 12. Hyalomma anatolicum anatolicum nymphs were applied on the ears of the calf on day 8 and they dropped engorged by days 13 and 14. The resulting adult ticks were examined for the presence of T. theileri by severing a leg and making a smear of the clear haemolymph which exuded from the wound. The smear was fixed in methanol and stained with Giemsa stain. The infection rate with T. theileri in the ticks was 43.3% (26 out of 60). The intensity of infection was very high and various developmental stages of the flagellates were observed (epimastigotes, sphaeromastigotes, trypomastigotes and other intermediate stages). The haemolymph from 12 ticks was also collected in tissue culture medium and the trypanosomes survived for 25 weeks before eventually dying. The results demonstrated unequivocally the high vectorial capacity of the tick H. a. anatolicum for T. theileri.     

Trypanosoma theileri: a literature review and report of incidence in New York cattle

The incidence of infection in adult dairy cattle in New York State with Trypanosoma (Megatrypanum) theileri (Laveran 1902) was determined by culturing buffy coats of peripheral blood samples in tissue culture growth media. Three sample groups of cattle were studied and revealed an overall rate of trypanosome infection of 44.4% (67 of 151) as determined by a single culture. Fifty-seven cows, representing four herds from three different geographic locations in the Southern-tier region of the state, comprised the first group. The infection rates of these herds ranged from 56.5 to 100%. The second group consisted of 81 cows randomly selected from animals admitted to the Large Animal Clinic of the New York State College of Veterinary Medicine. Trypanosoma theileri was cultured from 25.9% (21 of 81) of these animals. Repeated blood cultures from a third group of thirteen yearling heifers during the spring and summer revealed an increase in the infection rate from 0% in May, to 66.7% (8 of 12) in September, with 76.9% (10 of 13) positive at least once during this period. These findings are compared with the reported incidence of bovine T. theileri infections in other areas of the United States and in other countries.     

泰氏锥虫抗原制备及实验室诊断研究

应用体外培养的泰氏锥虫制备可溶性抗原Ⅰ、抗原Ⅱ和代谢抗原.经测定,其蛋白含量每毫升分别为6.5mg、7.4mg和7.1mg.薄层等电聚焦电泳测定结果表明,抗原Ⅰ出现22条区带,抗原Ⅱ21条区带,代谢抗原28条区带,对照的伊氏锥虫琼脂免疫扩散抗原14条区带.经分析,泰氏锥虫抗原和伊氏锥虫抗原有4条区带在同一迁移率上.琼脂免疫扩散反应中,3种泰氏锥虫抗原均与相应免疫兔血清发生沉淀反应,抗原Ⅰ出现1条致密沉淀线,抗原Ⅱ和代谢抗原出现2～3条沉淀线,抗原效价为1:4～16.免疫电泳显示了类似的结果,抗原Ⅰ与免疫兔血清出现1条弧形沉淀线,抗原Ⅱ和代谢抗原与免疫兔血清出现了3条弧形沉淀线.间接血凝试验结果表明,泰氏锥虫自然感染牛血清效价为1:20～40,免疫兔血清为1:1280～5120.所制泰氏锥虫抗原对伊氏锥虫和媾疫锥虫血清也能很好地发生交叉反应,3份伊氏锥虫病马血清和3份伊氏锥虫人工感染兔血清血凝效价分别为1:10～40和1:8～1024;5份媾疫马血清有4份血凝效价为1:20～320.4份环形泰勒虫病牛血清均为阴性.     

The density of equine tissue at 37 degrees C.

The density of the following equine tissues was measured: white and grey brain matter, myocardium, lung parenchyma, liver, spleen, gastrointestinal tract and contents, renal medulla and cortex, muscle, fat, tendon and bone. Statistical analysis indicated that there were highly significant differences in the values for density between horses and between tissues within horses. Values for density of eight different muscles were obtained for 18 horses and these were shown to differ highly significantly between horses and between muscles within horses. The values for splenic density differed highly significantly between those for knackery killed horses and for the barbiturate killed post morten room horses.