Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.     

Theileria parva: Kinetics of infection in the lymphoid system of cattle

The kinetics of infection with Theileria parva in cattle were studied by examining the total cellularity and numbers of parasites in a range of lymphoid organs from animals killed at intervals during the course of the infection. With the dose of T. parva stabilate used, macroschizonts were initially detected in the drainage lymph node about 7 days after inoculation and death of the host resulted on Day 18–19. Associated with the initial detection of parasites, there was a marked increase in cellularity of the drainage lymph node and a more gradual and less pronounced increase in cellularity of the other lymphoid organs. From about Day 12 onward, there was a gradual decrease in the cellularity in all of the lymphoid organs, so that in animals examined in the terminal stages of the infection there was often cellular depletion. The pattern of these cellular changes was similar in groups of Boran and Friesian cattle, although both the increase in cellularity and the terminal depletion were more marked in the Friesians. Blood leukocyte counts in infected Boran started to drop as early as Day 7 of infection and by Day 14 had reached values less than 25% of normal. Quantitation of parasitic schizonts indicated that the numbers of parasites in the lymphoid organs do not increase in a simple exponential manner. Rather, there appears to be an early rapid increase in parasite numbers followed by a phase of less rapid multiplication. Because of the marked changes which occured in total cellularity of the lymphoid organs during the course of the infection, a significant discrepancy was found between the replication rate of the parasite as calculated using total numbers of parasites and that obtained using schizont index (SI). These results indicated that the use of SI, as described in previous studies, is not a reliable method of determining the replication rate of the parasite.     

The antitheilerial effects of Theileria parva parva reaction and recovery sera in vitro

Peripheral blood leucocytes (PBL) of cattle were infected in vitro with the sporozoites of Theileria parva spp. The transformed cell lines were adapted to grow in sera from the PBL donors. The cattle were then infected with T. p. parva stabilate and either treated with parvaquone or the disease allowed to run its course. Sera harvested during severe disease reaction or early recovery were substituted for pre infection sera and caused the intracellular degeneration of the Theileria macroschizonts. Cell lines passaged in these sera died out as the parasites were eliminated. The antiparasitic effects of sera were short lived and were neither host nor parasite isolate restricted.     

Risk factors associated with exposure to Crimean-Congo haemorrhagic fever virus in animal workers and cattle,and molecular detection in ticks,South Africa

Crimean-Congo haemorrhagic fever (CCHF) is a severe tick-borne viral zoonosis endemic to parts of Africa, Europe, the Middle East and Central Asia. Human cases are reported annually in South Africa, with a 25% case fatality rate since the first case was recognized in 1981. We investigated CCHF virus (CCHFV) seroprevalence and risk factors associated with infection in cattle and humans, and the presence of CCHFV in Hyalomma spp. ticks in central South Africa in 2017–18. CCHFV IgG seroprevalence was 74.2% (95%CI: 64.2–82.1%) in 700 cattle and 3.9% (95%CI: 2.6–5.8%) in 541 farm and wildlife workers. No veterinary personnel (117) or abattoir workers (382) were seropositive. The prevalence of CCHFV RNA was significantly higher in Hyalomma truncatum (1.6%) than in H. rufipes (0.2%) (P = 0.002). Seroprevalence in cattle increased with age and was greater in animals on which ticks were found. Seroprevalence in cattle also showed significant geographic variation. Seroprevalence in humans increased with age and was greater in workers who handled livestock for injection and collection of samples. Our findings support previous evidence of widespread high CCHFV seroprevalence in cattle and show significant occupational exposure amongst farm and wildlife workers. Our seroprevalence estimate suggests that CCHFV infections are five times more frequent than the 215 confirmed CCHF cases diagnosed in South Africa in the last four decades (1981–2019). With many cases undiagnosed, the potential seriousness of CCHF in people, and the lack of an effective vaccine or treatment, there is a need to improve public health awareness, prevention and disease control.     

Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test

A schizont antigen for the indirect fluorescent antibody test was prepared from an in vitro culture suspension of lymphoid cells infected with Theileria annulata macroschizonts. Two cattle recovered from T. annulata infection showed marked rises in antibody titer to this schizont antigen, with peak titers of 1:40,960 and 1:2560. Using sera from these recovered cattle, T. annulata cell culture schizont antigen was shown to cross-react markedly with Theileria parva and Theileria lawrencei cell culture schizonts and with Theileria mutans piroplasms in the indirect fluorescent antibody test. In contrast, using high-titer antisera to T. parva, T. lawrencei, and T. mutans, serological cross-reactions with T. annulata schizonts were only detected with T. parva and T. lawrencei antisera, and in both instances these were minimal. The value of the indirect fluorescent antibody test in the differential diagnosis of Theileria species pathogenic for cattle is discussed.     

Theileria parva: cell culture analysis of clones of macroschizont-infected bovine lymphoblastoid cells

Three clones (H7, D7, and C5) were established from single cells of a bovine lymphoblastoid cell line (IR.TPM.1) infected with macroschizonts of the protozoan parasite Theileria parva. The cloning efficiency using feeder layers was 0.3–0.4. The mean parasite size (the number of parasite nuclei per cell) was different in each clone and was correlated to the growth rate. The fast growing clone, C5 (population doubling time 24 hr), contained smaller (mean parasite nuclear number, 12) parasites than a slow growing clone, D7 (population doubling time, 73 hr; mean number of parasite nuclei per cell, 35.3). The third clone, H7, had an intermediate growth rate (population doubling time, 49 hr) and parasite size (mean nuclei number, 18.1). There was variation in the incidence of microschizonts among the clones but microschizont-free clones were not isolated. When the clones were subjected to 4.3 × 10^?7M aminopterin, 20–25% of the cell population of clones H7 and C5 and the uncloned parent line lost their parasites in 4 days, while it took 7 days to reach a similar result (31% parasite-free cells) in clone D7. We were unable to isolate parasite-free clones from cells treated with aminopterin. Hydroxyurea (4 × 10^?4M) inhibited the growth of clone C5, but the macroschizonts continued to proliferate, and the incidence of cells with microschizonts increased. The size profile analysis showed that most of the aminopterin-treated cells were 9.0 μm, the hydroxyurea-treated cells 14.7 μm, and the untreated cells 10.8 μm in diameter.     

Cryopreservation of infective particles of Theileria parva

Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology3: 583–587. Infective particles of Theileria parva, the causative organism of East Coast fever of cattle, were obtained from infected Rhipicephalus appendiculatus ticks, either by using an in vitro feeding technique or by grinding the ticks in a suitable medium. If foetal calf serum containing 15% glycerol (v/v) was added to the infective material and it was then distributed either to glass capillary tubes (in vitro tick feed) or glass tubes (ground tick supernate) it could be slowly frozen to either ?80°C or ?196°C without loss of viability. Stabilates, tested by rapid thawing and inoculation into ECF-susceptible cattle, remained viable for up to a year at these temperatures.     

Theileria parva: isolation of macroschizonts from in vitro propagated lymphoblastoid cells of cattle

A method for purifying macroschizonts of Theileria parva from bovine lymphoblastoid cells, propagated in vitro, was developed. This method involved three steps. First, the macroschizonts were liberated by disrupting host cells suspended in growth medium at 4 × 10⁶ cells/ml at 300–400 psi, using the Stansted cell disrupter. This yielded 80–90% disrupted cells while causing minimum damage to the macroschizonts. Second, the host cell nuclei were separated by (a) centrifuging the lysate at 300g for 60 min, (b) resuspending the pellet in 0.02 times the volume of initial host cell suspension in Leibovitz's L15 growth medium, and (c) lysing the host cell nuclei by adding nucleus-lysing buffer (NLB, containing 0.14 M Tris, 0.1 M HCl, 0.12 M glucose, and 0.5 M NaCl adjusted with NaOH to pH 7) to 0.2 times the volume of initial host cell suspension. The resulting chromatin precipitate was removed by adding DE-52 cellulose equilibrated with NLB and allowing the precipitate to sediment. Lastly, the final suspension obtained in the second step was applied on a DE-52 cellulose column which was equilibrated with the elution buffer (NLB with 10% fetal, or newborn, bovine serum, pH 7). Macroschizonts free of intact host cells and naked host cell nuclei were collected in the eluate. The protein yield was 2.7 mg per 10⁹ starting undisrupted host cells, which was 1.7% of the total starting protein.     

Quantifying the Association between Bovine and Human Trypanosomiasis in Newly Affected Sleeping Sickness Areas of Uganda

Background

Uganda has active foci of both chronic and acute HAT with the acute zoonotic form of disease classically considered to be restricted to southeast Uganda, while the focus of the chronic form of HAT was confined to the northwest of the country. Acute HAT has however been migrating from its traditional disease focus, spreading rapidly to new districts, a spread linked to movement of infected cattle following restocking. Cattle act as long-term reservoirs of human infective T. b. rhodesiense showing few signs of morbidity, yet posing a significant risk to human health. It is important to understand the relationship between infected cattle and infected individuals so that an appropriate response can be made to the risk posed to the community from animals infected with human pathogens in a village setting.

Methodology/Principal Findings

This paper examines the relationship between human T. b. rhodesiense infection and human infective and non-human T. brucei s.l. circulating in cattle at village level in Kaberamaido and Dokolo Districts, Uganda. The study was undertaken in villages that had reported a case of sleeping sickness in the six months prior to sample collection and those villages that had never reported a case of sleeping sickness.

Conclusions and Significance

The sleeping sickness status of the villages had a significant effect with higher odds of infection in cattle from case than from non-case villages for T. brucei s.l. (OR: 2.94, 95%CI: 1.38–6.24). Cattle age had a significant effect (p<0.001) on the likelihood of T. brucei s.l. infection within cattle: cattle between 18–36 months (OR: 3.51, 95%CI: 1.63–7.51) and cattle over 36 months (OR: 4.20, 95%CI: 2.08–8.67) had significantly higher odds of T. brucei s. l. infection than cattle under 18 months of age. Furthermore, village human sleeping sickness status had a significant effect (p<0.05) on the detection of T. b. rhodesiense in the village cattle herd, with significantly higher likelihood of T. b. rhodesiense in the village cattle of case villages (OR: 25, 95%CI: 1.2–520.71). Overall a higher than average T. brucei s.l. prevalence (>16.3%) in a village herd over was associated with significantly higher likelihood of T. b. rhodesiense being detected in a herd (OR: 25, 95%CI: 1.2–520.71).     

Leishmaniadonovani s.l.: Evaluation of the proliferation potential of promastigotes using CFSE staining and flow cytometry

Leishmania infantum causes visceral leishmaniasis in all countries in the Mediterranean basin. It uses Phlebotomine sandflies as vectors where the promastigote stage develops, reproduces and becomes infective. Therefore the reproductive power of the promastigotes determines the inoculum size of the isolate. Ten Leishmania strains from Cyprus: two Leishmania donovani and eight L. infantum were used to study the proliferation capacity of the promastigotes. Population increase during a 6-day culture period was assessed quantitatively, by haematocytometer enumeration, and qualitatively by following the division history of each population during the same period by CFSE staining and flow cytometry. The strains exhibited different proliferation rates with L. infantum showing higher multiplication rates than L. donovani. These differences may represent their fitness capabilities and their ability to synchronize the multiplication activity of individual members in the population for the production of a sizeable inoculum in time for the vector’s blood meal.     

Survival of the parasitic protozoan, Babesia bigemina, in blood cooled at widely different rates to -196 degrees C

Survival of the parasitic protozoan, Babesia bigemina, in blood cooled at widely different rates to ? 196°C. International Journal for Parasitology4: 169–172. The infectivity of Babesia bigemina in blood containing 2 m DMSO was tested in 99 cattle after the blood had been cooled to ? 196°C at eight rates ranging from 0·73–3070°C/min. Blood cooled at each rate was infective; 95 of the recipients became infected, the exceptions being four of the seven cattle inoculated with blood cooled at 3070° C/min. The infectivity of blood cooled at 39, 82 and 212°C/min was higher than that of blood cooled at slower or faster rates. Least depression of infectivity occurred at 82°C/min.     

Factors associated with acquisition of human infective and animal infective trypanosome infections in domestic livestock in Western Kenya

Background

Trypanosomiasis is regarded as a constraint on livestock production in Western Kenya where the responsibility for tsetse and trypanosomiasis control has increasingly shifted from the state to the individual livestock owner. To assess the sustainability of these localised control efforts, this study investigates biological and management risk factors associated with trypanosome infections detected by polymerase chain reaction (PCR), in a range of domestic livestock at the local scale in Busia, Kenya. Busia District also remains endemic for human sleeping sickness with sporadic cases of sleeping sickness reported.

Results

In total, trypanosome infections were detected in 11.9% (329) out of the 2773 livestock sampled in Busia District. Multivariable logistic regression revealed that host species and cattle age affected overall trypanosome infection, with significantly increased odds of infection for cattle older than 18 months, and significantly lower odds of infection in pigs and small ruminants. Different grazing and watering management practices did not affect the odds of trypanosome infection, adjusted by host species. Neither anaemia nor condition score significantly affected the odds of trypanosome infection in cattle. Human infective Trypanosoma brucei rhodesiense were detected in 21.5% of animals infected with T. brucei s.l. (29/135) amounting to 1% (29/2773) of all sampled livestock, with significantly higher odds of T. brucei rhodesiense infections in T. brucei s.l. infected pigs (OR = 4.3, 95%CI 1.5-12.0) than in T. brucei s.l. infected cattle or small ruminants.

Conclusions

Although cattle are the dominant reservoir of trypanosome infection it is unlikely that targeted treatment of only visibly diseased cattle will achieve sustainable interruption of transmission for either animal infective or zoonotic human infective trypanosomiasis, since most infections were detected in cattle that did not exhibit classical clinical signs of trypanosomiasis. Pigs were also found to be reservoirs of infection for T. b. rhodesiense and present a risk to local communities.     

Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures

Dalgliesh R. J. and Stewart N. P. 1979. Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures. International Journal for Parasitology9: 115–120. The temperature of incubation of unfed Boophilus microplus larvae infected with Babesia bovis influenced the morphology and infectivity of the Babesia within the tick. Incubation at 37°C for 1–3 days stimulated the development of parasites morphologically similar to those usually observed in fed larvae harvested from cattle; similar forms appeared more slowly in larvae incubated at 31°C or 25°C. Extracts prepared from larvae after incubation at 37°C for 3–5 days or 30°C for 8 days were consistently infective for cattle. Prior storage of larvae at 14°C for up to 28 days enhanced the development of infectivity at 37°C; infectivity could still be produced after 65 days storage at 14°C but not after 76 days. Larvae released on a host transmitted B. bovis sooner if they had been incubated at 37°C for 4 days. It was concluded that the development of B. bovis to an infective stage in B. microplus is temperature dependent and does not require the stimulus of feeding by the host.     

Influence of Alfalfa Plant Growth on the Multiplication Rates and Ceiling Population Density of Meloidogyne hapla

The rates of reproduction and multiplication of Meloidogyne hapla decreased as a result of self-regulatory, density-dependent processes with time and nematode population increase in the soil and roots of Medicago sativa cv. Cuf 101. Juvenile, egg, and mature female population densities increased at a maximum rate until damage to the host resulted in alfalfa yield reductions. Temporal differences in multiplication and reproduction rates of M. hapla were observed to be a function of initial population density (Pi), host damage, and root biomass, indicating increased levels of competition for a constant but limited number of feeding sites. Over time, a log linear relationship emerged between multiplication rate of M. hapla and Pi. Slopes of -0.90953 for combined eggs and juveniles and -0.71349 for mature females indicated a gradual approach to ceiling densities. Reproductive rates decreased exponentially from an initial maximal value of 200 to a relatively constant rate of 53 eggs per female.     

In vitro migration of Dictyocaulus viviparus larvae: migration of the infective stage inagar gel.

The infective stage of the cattle lungworm, Dictyocaulus viviparus, was able to migrate in agar gel when activated by bile. The number of larvae which penetrated the upper surface of a 2 mm thick agar layer was counted and found to be independent from the agar concentration. Larvae which had migrated out of the agar remained on the surface and did not re-enter. The agar migration process was temperature dependent. Influence of time as well as dependency of agar thickness was investigated. The significance of these findings is discussed.     

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: Interspecific and intraspecific differences in infectivity for insects

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.     

Tracking gastrointestinal nematode risk on cattle farms through pasture contamination mapping

Gastrointestinal nematode (GIN) parasites in grazing cattle are a major cause of production loss and their control is increasingly difficult due to anthelmintic resistance and climate change. Rotational grazing can support control and decrease reliance on chemical intervention, but is often complex due to the need to track grazing periods and infection levels, and the effect of weather on larval availability. In this paper, a simulation model was developed to predict the availability of infective larvae of the bovine GIN, Ostertagia ostertagi, at the level of individual pastures. The model was applied within a complex rotational grazing system and successfully reproduced observed variation in larval density between fields and over time. Four groups of cattle in their second grazing season (n = 44) were followed throughout the temperate grazing season with regular assessment of GIN faecal egg counts, which were dominated by O. ostertagi, animal weight and recording of field rotations. Each group of cattle was rotationally grazed on six group-specific fields throughout the 2019 grazing season. Maps and calendars were produced to illustrate the change in pasture infectivity (density of L3 on herbage) across the 24 separate grazing fields. Simulations predicted differences in pasture contamination levels in relation to the timing of grazing and the return period. A proportion of L3 was predicted to persist on herbage over winter, declining to similar intensities across fields before the start of the following grazing season, irrespective of contamination levels in the previous year. Model predictions showed good agreement with pasture larval counts. The model also simulated differences in seasonal pasture infectivity under rotational grazing in systems that differed in temperature and rainfall profiles. Further application could support individual farm decisions on evasive grazing and refugia management, and improved regional evaluation of optimal grazing strategies for parasite control. The integration of weather and livestock movement is inherent to the model, and facilitates consideration of climate change adaptation through improved disease control.     

Studies on cell fusion between Babesia rodhaini-infected mouse erythrocytes and baby hamster kidney cells.

Heterokaryons were formed by fusion of B. rodhaini-infected mouse erythrocytes and baby hamster kidney (BHK) cells, using Sendai virus. The erythrocyte membrane rapidly lysed inside the BHK cell cytoplasm releasing free parasites. There was no evidence that parasite multiplication occurred inside the BHK cells, nor that parasitized BHK cells were infective for mice.Transient erythrocyte homokaryons were observed in some preparations.The approach indicates a possible method for the in vitro cultivation of Babesia.     

Theileria lawrencei: infection in the Indian water buffalo, Bubalus bubalis

Two Indian water buffalo (Bubalus bubalis), experimentally infected with Theileria lawrencei (Serengeti) by inoculation of stabilate material, died from acute theileriosis. Macroschizonts were first detected in both buffalo in the peripheral lymph node regional to the site of inoculation 8 days later. No microschizonts were seen in either animal and intraerythrocytic theilerial piroplasms were only detected in one in very low numbers. Both buffalo had marked febrile responses and died on Days 15 and 16 after exposure, when only low numbers of lymphoid cells were parasitized with macroschizonts, the vast majority of which contained few nuclei. Both buffalo showed evidence of terminal leucopenia and thrombocytopenia. On post mortem both animals showed generalised hyperplasia and edema of all lymphoid tissues, gross edema of the lungs, and hemorrhages in the heart and kidneys. T. lawrencei (Serengeti) infections of water buffalo and of cattle are compared and shown to be very similar.     

Longer uncommon polyamines have a stronger defense gene-induction activity and a higher suppressing activity of Cucumber mosaic virus multiplication compared to that of spermine in Arabidopsis thaliana

Key message

Our work suggests that long chain polyamines and their derivatives are potential chemicals to control viral pathogens for crop production.

Abstract

Previously we showed that two tetraamines, spermine (Spm) and thermospermine (T-Spm), induce the expression of a subset of defense-related genes and repress proliferation of Cucumber mosaic virus (CMV) in Arabidopsis. Here we tested whether the longer uncommon polyamines (LUPAs) such as caldopentamine, caldohexamine, homocaldopentamine and homocaldohexamine have such the activity. LUPAs had higher gene induction activity than Spm and T-Spm. Interestingly the genes induced by LUPAs could be classified into two groups: the one group was most responsive to caldohexamine while the other one was most responsive to homocaldopentamine. In both the cases, the inducing activity was dose-dependent. LUPAs caused local cell death and repressed CMV multiplication more efficiently as compared to Spm. LUPAs inhibited the viral multiplication of not only avirulent CMV but also of virulent CMV in a dose-dependent manner. Furthermore, LUPAs can activate the systemic acquired resistance against CMV more efficiently as compared to Spm. When Arabidopsis leaves were incubated with LUPAs, the putative polyamine oxidase (PAO)-mediated catabolites were detected even though the conversion rate was very low. In addition, we found that LUPAs induced the expression of three NADPH oxidase genes (rbohC, rbohE and rbohH) among ten isoforms. Taken together, we propose that LUPAs activate two alternative reactive oxygen species evoked pathways, a PAO-mediated one and an NADPH-oxidase-mediated one, which lead to induce defense-related genes and restrict CMV multiplication.