Search

Background: Identification of populations to be targeted for individual treatment and broad-spectrum therapy in schistosomiasis-endemic areas, assessment of therapy efficacy, morbidity, and evaluation of control strategies need to be based on reliable diagnostic tools. Kato-Katz is routinely used and remains the standard diagnostic technique for schistosomiasis, despite its many challenges. This study was conducted in Nyamanga village, Mbita, western Kenya, and evaluated the diagnostic performance of Kato-Katz, Mini-Parasep and modified Mini-FLOTAC techniques in detection of Schistosoma mansoni and soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and hookworm) ova., Methods: Stool samples from 132 individuals were screened for eggs of S. mansoni by the 3 techniques. Mini-Parasep faecal parasite concentrator (Apacor Ltd, England), a single-use diagnostic device with a built-in filter for faecal concentration of helminth eggs by sedimentation was employed on stool samples fixed in 10% formalin. A modified Mini-FLOTAC (University of Naples, Italy) was based on floatation of helminths eggs with two different solutions (FS2 and FS7) using a closed system (Fill-FLOTAC) with 5% formalin. Kato-Katz was performed following WHO recommendation. Prevalence of S. mansoni and STH, sensitivity and degree of agreement among the 3 techniques were determined., Results: Prevalence of S. mansoni was 47.0%, 34.1% and 20.5% by Mini-Parasep, Kato-Katz and modified Mini-FLOTAC FS7 techniques, respectively. Prevalence of any STH infection was 6.1%, 3.0%, 6.1% and 6.8% by Mini-Parasep, Kato-Katz, modified Mini-FLOTAC FS2 and modified Mini-FLOTAC FS7 techniques, respectively. Considering the pooled results of the three methods (Mini-Parasep, Kato-Katz and modified Mini-FLOTAC FS7) as diagnostic 'gold' standard, the sensitivity of Mini-Parasep, Kato-Katz and modified Mini-FLOTAC FS7 for S. mansoni was 77.5%, 56.1%, and 33.8%, respectively. Mini-Parasep and modified Mini-FLOTAC FS7 techniques had moderate (κ = 0.46) and fairly good (κ = 0.25) agreements with Kato-Katz for S. mansoni, respectively. Mini-Parasep detected a higher proportion of light intensity S. mansoni infections compared to Kato-Katz, which detected high proportions of heavy infections. Mini-Parasep detected a similar mean number of S. mansoni eggs per gram (EPG) of stool compared to the standard Kato-Katz (62.9 vs 97.3; t (131) = -0.49, P = 0.6265) and significantly higher EPG compared to the modified Mini-FLOTAC FS7 (62.9 vs 34.6; t (131) = 5.39, P < 0.0001)., Conclusions: The high sensitivity of Mini-Parasep suggests its promising potential as an alternative tool in enhancing diagnosis and in monitoring schistosomiasis transmission and determining endpoint of intervention programs, especially in low endemicity areas. Mini-Parasep is also easy to operate, safe and also permits work with fresh stool.

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC50 of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50 of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Human Immunodeficiency Virus (HIV) and Schistosomiasis co-infection is common among residents at the shores of Lake Victoria in Kenya. About 36% of this population initiating antiretroviral therapy (ART) experience Immune Reconstitution Inflammatory Syndrome (IRIS) that complicates recovery. Several IL-23R alleles have been associated with susceptibility to both autoimmune and inflammatory diseases through T-helper type 17 (TH17) cells. However, whether or not variants within the IL-23R increase susceptibility to IRIS in western Kenya is unknown. The objective of the current study was to determine the association between IL-23R gene polymorphisms, CD4+ cell counts and HIV RNA levels and IRIS in HIV and Schistosoma mansoni co-infected patients undergoing highly active anti-retroviral therapy (HAART)., Methods: A three-month case-control study was conducted on antiretroviral naïve schistosomiasis/HIV co-infected fishermen starting HAART in Uyoma Rarieda, Siaya County, Kenya. Seventy one patients were sampled at baseline and followed up for three months, to establish if they developed Schistosoma-related IRIS. In addition, the CD4+ cell counts and HIV RNA levels were determined in pre- and post-administration of HAART. Variations at five polymorphic sites of IL-23R (rs1884444, rs11465754, rs6682925, rs7530511 and rs7539625) based on >10% minor allele frequency in Yoruban reference population was determined using Allelic Discrimination Assay. The association between the five variants and susceptibility to IRIS was determined using logistic regression while controlling for potential confounders. In addition, the functional differences between the baseline CD4 + Cell counts and viral loads were determined using medians while across IL-23R genotypes were determined using Kruskal-Wallis tests., Results: Overall, 26 (36.6%) patients developed schistosomiasis-associated IRIS at a median age of 35.5 years. Carriage of the TT genotype at the non-synonymous rs1884444 T > G relative to GG, was associated with a decreased risk of schistosomiasis-associated IRIS (OR, 0.25, 95% CI, 0.07-0.96, P = 0.043) while both baseline CD4+ cell counts and viral loads had no association with IRIS., Conclusion: These findings indicate that the non-synonymous variant rs1884444 T > G of IL-23R is associated with a decreased risk to schistosomiasis-associated IRIS. However, additional studies in a larger cohort and with an all inclusive polymorphic variants in the synonymous and non-synonymous regions need to be evaluated.

Endemic Burkitt lymphoma (eBL) is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum coinfections. Malaria appears to dysregulate immunity that would otherwise control EBV, thereby contributing to eBL etiology. Juxtaposed to human genetic variants associated with protection from malaria, it has been hypothesized that such variants could decrease eBL susceptibility, historically referred to as "the protective hypothesis." Past studies attempting to link sickle cell trait (HbAS), which is known to be protective against malaria, with protection from eBL were contradictory and underpowered. Therefore, using a case-control study design, we examined HbAS frequency in 306 Kenyan children diagnosed with eBL compared to 537 geographically defined and ethnically matched controls. We found 23.8% HbAS for eBL patients, which was not significantly different compared to 27.0% HbAS for controls [odds ratio (OR) = 0.85; 95% confidence interval (CI) 0.61-1.17; p-value = 0.33]. Even though cellular EBV titers, indicative of the number of latently infected B cells, were significantly higher (p-value < 0.0003) in children residing in malaria holoendemic compared to hypoendemic areas, levels were not associated with HbAS genotype. Combined, this suggests that although HbAS protects against severe malaria and hyperparasitemia, it is not associated with viral control or eBL protection. However, based on receiver operating characteristic curves factors that enable the establishment of EBV persistence, in contrast to those involved in EBV lytic reactivation, may have utility as an eBL precursor biomarker. This has implications for future human genetic association studies to consider variants influencing control over EBV in addition to malaria as risk factors for eBL., (© 2013 UICC.)

Clinical immunity to malaria declines in the absence of repeated parasite exposure. However, little is known about how B cell populations and antigen-specific memory B cells change in the absence of P. falciparum infection. A successful indoor residual insecticide spraying campaign in a highland area of western Kenya, led to an absence of blood-stage P. falciparum infection between March 2007 and April 2008. We assessed memory B cell responses in 45 adults at the beginning (April 2008) and end (April 2009) of a subsequent 12-month period during which none of the adults had evidence of asymptomatic parasitemia or clinical disease. Antibodies and memory B cells to the 42-kDa portion of the merozoite surface protein-1 (MSP-142) were measured using ELISA and ELISPOT assays, respectively. B cell populations were characterized by flow cytometry. From 2008 to 2009, the prevalence of MSP-142-specific memory B cells (45% vs. 55%, respectively, P = 0.32) or antibodies (91% vs. 82%, respectively, P = 0.32) did not differ significantly, although specific individuals did change from positive to negative and vice versa, particularly for memory B cells, suggesting possible low-level undetected parasitemia may have occurred in some individuals. The magnitude of MSP-142-specific memory B cells and levels of antibodies to MSP-142 also did not differ from 2008 to 2009 (P>0.10 for both). However, from 2008 to 2009 the proportions of both class-switched atypical (CD19+IgD-CD27-CD21-IgM-) and class-switched activated (CD19+IgD-CD27+CD21-IgM-) memory B cells decreased (both P<0.001). In contrast, class-switched resting classical memory B cells (CD19+IgD-CD27+CD21+IgM-) increased (P<0.001). In this area of seasonal malaria transmission, a one- year absence of detectable P. falciparum infection was not associated with changes in the prevalence or level of MSP-142 specific memory B cells, but was associated with major changes in overall memory B cell subsets.

High levels of environmental contamination, often associated with improper waste and excreta management, are widespread among informal settlements within urban areas in developing countries. We determined the level of faecal contamination in domestic water sources and evaluated the potential contribution of these water sources to intestinal helminthiases in seven informal settlements of Kisumu City, western Kenya. Membrane filtration technique was used for enumeration of total and faecal (Escherichia coli) coliform bacteria in water samples collected from dams, rivers, springs and wells. Out of the 80 water sources sampled, 76 (95%) were highly contaminated with E coli. All water samples from unprotected wells (26) and 92.6% of samples from protected wells (25) were positive for E. coli. The highest and lowest E. coli densities were observed in samples from dams (3,800 ± 1,807 coliforms per 100 ml) and boreholes (419 ± 223 coliforms per 100 ml), respectively (p = 0.0321). Distance from pit latrines was negatively associated with E. coli coliform density for wells (r = -0.34, n = 53, p = 0.0142). Untreated well-water may not be suitable for human consumption, and its continued use constitutes a major health risk for the inhabitants of these informal settlements.

Background: Plasmodium falciparum infection leads to alterations in B cell subset distribution. During infancy, development of peripheral B cell subsets is also occurring. However, it is unknown if infants living a malaria endemic region have alterations in B cell subsets that is independent of an age effect., Methods: To evaluate the impact of exposure to P. falciparum on B cell development in infants, flow cytometry was used to analyse the distribution and phenotypic characteristic of B cell subsets in infant cohorts prospectively followed at 12, 18 and 24 months from two geographically proximate regions in western Kenya with divergent malaria exposure i.e. Kisumu (malaria-endemic, n = 24) and Nandi (unstable malaria transmission, n = 21)., Results: There was significantly higher frequency and absolute cell numbers of CD19+ B cells in Kisumu relative to Nandi at 12(p = 0.0440), 18(p = 0.0210) and 24 months (p = 0.0493). No differences were observed between the infants from the two sites in frequencies of naïve B cells (IgD+CD27-) or classical memory B cells (IgD-CD27+). However, immature transitional B cells (CD19+CD10+CD34-) were higher in Kisumu relative to Nandi at all three ages. In contrast, the levels of non-class switched memory B cells (CD19+IgD+CD27+) were significantly lower overall in Kisumu relative to Nandi at significantly at 12 (p = 0.0144), 18 (p = 0.0013) and 24 months (p = 0.0129)., Conclusions: These data suggest that infants living in malaria endemic regions have altered B cell subset distribution. Further studies are needed to understand the functional significance of these changes and long-term impact on ability of these infants to develop antibody responses to P. falciparum and heterologous infections.

Background: Although schistosomiasis is generally considered a rural phenomenon, infections have been reported within urban settings. Based on observations of high prevalence of Schistosoma mansoni infection in schools within the informal settlements of Kisumu City, a follow-up malacological survey incorporating 81 sites within 6 informal settlements of the City was conducted to determine the presence of intermediate host snails and ascertain whether active transmission was occurring within these areas., Methods: Surveyed sites were mapped using a geographical information system. Cercaria shedding was determined from snails and species of snails identified based on shell morphology. Vegetation cover and presence of algal mass at the sites was recorded, and the physico-chemical characteristics of the water including pH and temperature were determined using a pH meter with a glass electrode and a temperature probe., Results: Out of 1,059 snails collected, 407 (38.4%) were putatively identified as Biomphalaria sudanica, 425 (40.1%) as Biomphalaria pfeifferi and 227 (21.5%) as Bulinus globosus. The spatial distribution of snails was clustered, with few sites accounting for most of the snails. The highest snail abundance was recorded in Nyamasaria (543 snails) followed by Nyalenda B (313 snails). As expected, the mean snail abundance was higher along the lakeshore (18 ± 12 snails) compared to inland sites (dams, rivers and springs) (11 ± 32 snails) (F(1, 79) = 38.8, P < 0.0001). Overall, 19 (1.8%) of the snails collected shed schistosome cercariae. Interestingly, the proportion of infected Biomphalaria snails was higher in the inland (2.7%) compared to the lakeshore sites (0.3%) (P = 0.0109). B. sudanica was more abundant in sites along the lakeshore whereas B. pfeifferi and B. globosus were more abundant in the inland sites. Biomphalaria and Bulinus snails were found at 16 and 11 out of the 56 inland sites, respectively., Conclusions: The high abundance of Biomphalaria and Bulinus spp. as well as observation of field-caught snails shedding cercariae confirmed that besides Lake Victoria, the local risk for schistosomiasis transmission exists within the informal settlements of Kisumu City. Prospective control interventions in these areas need to incorporate focal snail control to complement chemotherapy in reducing transmission.

This cross-sectional study determined the prevalence and distribution of schistosome and soil-transmitted helminth (STH) infections among 1,308 children aged 10-18 years in 34 primary schools in 8 informal urban settlements in Kisumu City, western Kenya. Stool samples were collected and examined for eggs of Schistosoma mansoni and STH (Hookworms, Ascaris lumbricoides and Trichuris trichiura) using the Kato-Katz technique. Haematuria was used as a proxy indicator of urinary schistosomiasis. Schools and water bodies were mapped using a geographical information system. Overall, 34% of children were infected with one or more helminth species whereas 16·2% of children were infected with one or more STH species. Schools in closest proximity to Lake Victoria and River Nyamasaria had the highest S. mansoni prevalence while schools with STH were more homogenously distributed. Mean school prevalence of S. mansoni infection was 21% (range=0-69·7%), S. haematobium 3·6% (range=0-12%), hookworms 6·1% (range=0-20%), A. lumbricoides 4·9% (range=0-18·4%), and T. trichiura 7·7% (range=0-18·6%). Helminth-related morbidities were not associated with infection. Our study demonstrates that schistosomiasis and STH are important health priorities among schools in informal settlements of Kisumu City, and highlights the need for routine deworming in similar settings.

Background: The appropriate use of anti-malarial drugs determines therapeutic efficacy and the emergence and spread of drug-resistant malaria. Strategies for improving drug compliance require accurate information about current practices at the consumer level. This is to ascertain that the currently applied new combination therapy to malaria treatment will achieve sustained cure rates and protection against parasite resistance. Therefore, this cross-sectional study was designed to determine knowledge and behaviour of the consumers in households (n = 397) in peri-urban location in a malaria holoendemic region of western Kenya., Methods: The knowledge and behaviour associated with anti-malarial use were evaluated. Using clusters, a questionnaire was administered to a particular household member who had the most recent malaria episode (within <2 weeks) and used an anti-malarial for cure. Mothers/caretakers provided information for children aged <13 years., Results: Consumers' knowledge on dosage and duration/frequency demonstrated that only 29.4% used the correct artemisinin-based combination therapy (ACT) dosage. Most respondents who used quinine identified the correct duration of use (96.4%) since its administration was entirely at health facilities. To assess behaviours during use of anti-malarial drugs, respondents were stratified into those who took drugs with prescription (39.4%) and without prescription (61.6%). For those without prescription, the reasons given were; procedure of acquisition less costly (39.0%), took same drug for similar symptoms (23.0%), not satisfied with health services (15.5%), neighbour/friend/relative previously taken the same drug (12.5%) and health institution was far from their location (10%)., Conclusion: Majority of consumers in the study area were knowledgeable on the symptoms of malaria. In addition, majority acquired ineffective anti-malarial drugs for treatment and reported sub-optimal treatment regimens with the currently recommended drugs. Furthermore, behaviours which constrain the successful up-scaling of ACT were common, creating a challenge in the desire to turn efficacy to effectiveness of the combination therapy programme. It will be important to direct and focus interventions in creating awareness on the importance of using recommended drugs to lessen the use of less efficacious anti-malarials. In addition, the consumers need to be educated on the importance of drug adherence in such areas to reduce the emergence and spread of drug-resistant malaria.

Background: Interventions to reverse trends in malaria-related morbidity and mortality in Kenya focus on preventive strategies and drug efficacy. However, the pattern of use of anti-malarials in malaria-endemic populations, such as in western Kenya, is still poorly understood. It is critical to understand the patterns of anti-malarial drug use to ascertain that the currently applied new combination therapy to malaria treatment, will achieve sustained cure rates and protection against parasite resistance. Therefore, this cross-sectional study was designed to determine the patterns of use of anti-malarial drugs in households (n = 397) in peri-urban location of Manyatta-B sub-location in Kisumu in western Kenya., Methods: Household factors, associated with the pattern of anti-malarials use, were evaluated. Using clusters, questionnaire was administered to a particular household member who had the most recent malaria episode (within <2 weeks) and used an anti-malarial for cure. Mothers/caretakers provided information for children aged <13 years., Results: Stratification of the type of anti-malarial drugs taken revealed that 37.0% used sulphadoxine/pyrimethamine (SP), 32.0% artemisinin-based combined therapy (ACT), 11.1% anti-pyretics, 7.3% chloroquine (CQ), 7.1% quinine, 2.5% amodiaquine (AQ), while 3.0% used others which were perceived as anti-malarials (cough syrups and antibiotics). In a regression model, it was demonstrated that age (P = 0.050), household size (P = 0.047), household head (P = 0.049), household source of income (P = 0.015), monthly income (P = 0.020), duration of use (P = 0.029), dosage of drugs taken (P = 0.036), and source of drugs (P = 0.005) significantly influenced anti-malarial drug use. Overall, 38.8% of respondents used drugs as recommended by the Ministry of Health., Conclusion: This study demonstrates that consumers require access to correct and comprehensible information associated with use of drugs, including self-prescription. There is potential need by the Kenyan government to improve malaria care and decrease malaria-related morbidity and mortality by increasing drug affordability, ensuring that the recommended anti-malarial drugs are easily available in all government approved drug outlets and educates the local shopkeepers on the symptoms and appropriate treatment of malaria. Following a switch to ACT in national drug policy, education on awareness and behaviour change is recommended, since the efficacy of ACT alone is not sufficient to reduce morbidity and mortality due to malaria.

Background: Endemic Burkitt's lymphoma (BL) is an extranodal tumor appearing predominantly in the jaw in younger children while abdominal tumors predominate with increasing age. Previous studies have identified elevated levels of antibodies to Plasmodium falciparum schizont extracts and Epstein-Barr virus (EBV) viral capsid antigens (VCA) in endemic BL relative to malaria exposed controls. However, these studies have neither determined if there were any differences based on the site of clinical presentation of the tumor nor examined a broader panel of EBV and P. falciparum antigens., Methods: We used a suspension bead Luminex assay to measure the IgG levels against EBV antigens, VCA, EAd, EBNA-1 and Zta as well as P. falciparum MSP-1, LSA-1, and AMA-1 antigens in children with BL (n = 32) and in population-based age-and sex-matched controls (n = 25) from a malaria endemic region in Western Kenya with high incidence of BL. EBV viral load in plasma was determined by quantitative PCR., Results: Relative to healthy controls, BL patients had significantly increased anti-Zta (p = 0.0017) and VCA IgG levels (p < 0.0001) and plasma EBV viral loads (p < 0.0001). In contrast, comparable IgG levels to all P. falciparum antigens tested were observed in BL patients compared to controls. Interestingly, when we grouped BL patients into those presenting with abdominal tumors or with jaw tumors, we observed significantly higher levels of anti-Zta IgG levels (p < 0.0065) and plasma EBV viral loads (p < 0.033) in patients with abdominal tumors compared to patients with jaw tumors., Conclusion: Elevated antibodies to Zta and elevated plasma EBV viral load could be relevant biomarkers for BL and could also be used to confirm BL presenting in the abdominal region.

Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies. The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0. 0001) than those of the controls. Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Chi2 = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates. Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells.

Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P < 0.0001) and produced pupae that were of significantly less weight in milligrams (P < 0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P < 0.00001).

The distribution of peptides thought to be involved in pain modulation--substance P, calcitonin gene-related peptide (CGRP), and enkephalin--were studied in the spinal cord and dorsal root ganglia of polyarthritic rats and in rats with one sciatic nerve sectioned prior to induction of arthritis. In arthritic rats there was a bilateral increase of CGRP- and substance P-immunoreactive fibers and appearance of enkephalin-immunoreactive cell bodies in the dorsal horn of the lumbar (L4) spinal cord when compared to controls. In the corresponding dorsal root ganglia there were significant increases of CGRP- (P less than 0.02) and substance P- (P less than 0.001) immunoreactive cell bodies compared to controls. In the ventral horn of the control rats CGRP-immunoreactive motoneurons were abundant but were significantly (P less than 0.001) reduced in the arthritic spinal cord. Less pronounced changes were seen in the contralateral L4 spinal cord of arthritic rats with unilateral sciatic nerve section. In the ipsilateral dorsal horn, however, CGRP- and substance P-immunoreactive fibers were markedly depleted, and no enkephalin cell bodies were present. Furthermore, a number of CGRP-immunoreactive motoneurons were observed. In the ipsilateral L4 ganglia CGRP- (P less than 0.02) and substance P- (P less than 0.02) immunoreactive cells were significantly decreased compared to the contralateral side. The data suggest that pain perception is linked to complex interactions between CGRP, substance P, and enkephalin in sensory pathways and an intact peripheral input. The loss of CGRP-immunoreactive motoneurons may reflect muscular dysfunction associated with the arthritic condition.

Theileria lawrencei tick-derived stabilate infection of 8 cattle resulted in the development of panleukopenia and hypoproteinemia. In addition to these changes, T. parva infection caused mild normocytic, normochromic, non-responsive anemia at either of two dose rates. Disseminated intravascular coagulation, as indicate by positive protamine paracoagulation tests, prolonged prothrombin and partial thromboplastin times, and thrombocytopenia, developed in cattle infected with either of the Theileria spp., and was probably an important intermediary mechanism leading to death. Infection of calves with a high dose of T. parva stabilate resulted in more rapid onset of clinico-pathologic changes than did the low dose infection. Theileria lawrencei infection produced a severe, acute syndrome, the clinico-pathologic alterations of which varied in time of onset and severity between those of the T. parva high dose and low dose groups.

The interaction, in vitro, between bovine peripheral blood lymphocytes and sporozoites of Theileria annulata (Ankara) was studied by light and electron microscopy. Beginning five minutes following incubation, samples were taken for Giemsa-stained smears and glutaraldehyde-fixed pellets, for light and electron microscopy, respectively. Sporozoites of T. annulata measure an average of 0.9 microns long, 0.8 microns broad and possess a limiting unit membrane, the pellicle; a round-to-ovoid, eccentrically situated, non-chromocentric nucleus; double-membraned, tubular, acristate mitochondria; varying numbers of anisocytic, densely osmiophilic and pleomorphic organelles, the rhoptries which together with the polar ring form the apical complex; and numerous, loosely scattered, electron-dense ribosomal particles. As early as 5 min of incubation, sporozoites had made contact with, and penetrated, lymphocytes. Sporozoites consistently attached to the lymphocyte plasmalemma by their basal end, possibly at specific receptor sites. Apparently only a proportion of lymphocytes (up to 40% and more commonly 10-20%) were susceptible. Two subpopulations of the susceptible lymphocytes were observed; one which appeared to have receptor sites localized on one pole of the plasmalemma and the other subpopulation in which the receptor sites were distributed evenly around the plasmalemmal surface. Within individual susceptible lymphocytes, the number of interiorized sporozoites increased from 1 to 3 at 5-10 min to as many as 15 or more parasites at around 60 min of incubation. Theileria annulata sporozoites were interiorized by the invagination of the host cell plasmalemma which remained intact throughout the process but later fragmented. Within 30 min of interiorization, each sporozoite underwent dedifferentiation by the loss of its rhoptries and transformed into a trophozoite. Around 24 h, the trophozoite, a uninucleate, motile and feeding stage of the parasite, developed into a schizont by an acentric, closed mitosis.

Interactions between Theileria annulata sporozoites and lymphoblastoid cell lines already transformed by the Hissar and Ankara strains of T. annulata [T. a. (H) and T.A. (A), respectively] and the Muguga strain of T. parva [T.P. (M)] were studied in vitro. Although sporozoites of the Hissar strain of T. annulata attached to and entered peripheral blood lymphocytes (PBL) and lymphoblastoid cell lines transformed by T. a. (H) and T. a. (A), they neither attached to nor entered the T. p. (M) cell line. Whether the superinfecting T. a. (H) sporozoites developed intracellularly was studied by monitoring daily changes of mean schizont nuclear numbers and by determining electrophoretic mobilities of schizont glucose phosphate isomerase in each cell line using thin-layer starch gel electrophoresis. While the mean schizont nuclear number in freshly-infected PBL underwent a steady increase to the level of those in long standing T. annulata cultures, analysis of variance of similar data in T. a. (H) and T. a. (A) cell lines in which superinfection was demonstrated revealed no significant differences between them and their respective control counterparts, i.e., T. a. (H) and T. a. (A) cultures with no superinfection. Enzyme polymorphism studies showed the formation of uncontaminated species- or strain-specific bands of glucose phosphate isomerase (GPI) isoenzyme activity in the T. p. (M) and in the superinfected T. annulata cell lines.

Cyclical interactions between intracellular schizonts of the Ankara and Hissar strains of Theileria annulata and the Muguga strain of T. parva and the parasitised host lymphoblasts have been studied autoradiographically by following the incorporation of (3H) thymidine into parasite and host cell nuclei, and also by quantitating the number of schizont nuclei per lymphoblast, at various stages and phases of host cell cycle. The synthesis of DNA by Theileria schizonts and the parasitised host lymphoblasts was found to be asynchronous and to occur at different phases of the host cell interphase stage. While the lymphoblast nuclear DNA incorporates (3H) thymidine during the S phase, schizont nuclei were labelled during the G2 phase of the host lymphoblast interphase stage. The replication of schizont nuclei took place before the metaphase stage of host cell cycle, viz, prometaphase, so that the mean schizont nuclear number at host prometaphase and at anaphase--telophase was consistently more or less double the mean nuclear number at interphase.

The number of T. annulata sporozoites invading bovine peripheral blood lymphocytes (PBL) under different conditions (in vitro) was determined. Heat-inactivation of T. annulata sporozoites for 45 min, in a thermostatically controlled, shaking water bath preset and stabilised at 60 degrees C resulted in an almost total lack of invasion of fresh, normal PBL by the sporozoites, indicating that the interiorization process is parasite-effected. The mean number of T. annulata sporozoites interiorization (per 1000 lymphocytes) in cultures set up using sporozoites and PBL, mixed and incubated at 0 degrees C for 1 h in melting ice, was highly significantly reduced (P less than 0.01), indicating the invasion of bovine lymphocytes by T. annulata sporozoites is an active process dependent on active metabolism which is markedly affected by temperature. Pre-treatment of PBL with trypsin significantly reduced the number of invading sporozoites thus incriminating proteins or glycoproteins as constituents of receptors involved in sporozoite-lymphocyte recognition.

The fate of developing intracellular Theileria annulata sporozoites within bovine peripheral blood lymphocytes (PBL) was investigated in vitro using acid phosphatase ultracytochemistry. The intracellular sporozoites, while they developed into trophozoites, fed, and transformed into schizonts, provoked intense host lysosomal activity as early as 30 min after they were mixed with lymphocytes. Although viable developing trophozoites failed to fuse with lysosomal vesicles, thereby avoiding enzymatic digestion, the dead parasites readily formed 'phagolysosomes', resulting in lysosomal degranulation and trophozoite digestion. Possible factors responsible for the observed lack of lysosomal fusion with viable trophozoites are discussed.

The existence of metabolic energy pathways has been studied in extracellular T. annulata sporozoites using chemicals known to inhibit specific energy-generating pathways, and their role during invasion of bovine peripheral blood lymphocytes (PBL) by the sporozoites determined in an in vitro system. An inverse relationship was depicted between the dose of various chemicals and the number of T. annulata sporozoites invading PBL: as the concentrations of the inhibitor drugs increased, the number of T. annulata sporozoites within the lymphocytes decreased. An ultracytochemical study demonstrated the presence of the respective pathway marker enzymes, i.e., lactic dehydrogenase (LDH) in the cytosol and within mitochondria, succinic dehydrogenase (SDH) on the mitochondrial membranes and in the contiguous matrix, and cytochrome oxidase (CO) between the inner and outer mitochondrial membranes, in infective T. annulata sporozoites fixed in situ within whole salivary glands of 3-day fed Hyalomma anatolicum anatolicum ticks.

Host-parasite relationships have been studied by electron microscopy using glutaraldehyde-OsO4-fixed pellets of lymphoid cultures infected in vitro by Theileria annulata and T. parva. Intracellular presence of the parasite resulted in a progressive and marked lymphoblastoid transformation. The schizont stage periodically provoked the formation of, and adopted an intimate association with, cytoplasmic annulate lamellae in the interphase cell. Annulate lamellae developed from the outer nuclear membrane of the host cell by a delamination process and were taken into the cytoplasmic matrix of the schizont by phagotrophy through the cytostome. Schizont nuclei themselves were seen to divide at the prometaphase stage of host cell mitosis, the division being characterized by the development of intranuclear spindle microtubules anchored in spindle pole bodies. A hypothesis is propounded that Theileria parasites, consequent on interiorization, provoke the blastoid transformation and the formation of annulate lamellae through the influence of components of their genomic material on host cell deoxyribonucleic acid (DNA) and that the annulate lamellae represent a species of messenger ribonucleic acid (mRNA) and serve as a monitoring device for the schizont, facilitating the accurate timing of the host cell cyclical events.

The integrity of host lymphocyte plasma membrane during its invasion by Theileria annulata sporozoites was examined in an in vitro system using ruthenium red staining. It was demonstrated that during interiorisation of the sporozoites, the dye was completely excluded from the intracellular milieu, indicating that the continuity of the host-cell plasmalemma was not disrupted throughout the process of penetration, and confirmed a previous observation that the fragmentation of the host lymphocyte membrane only occurs later, after the parasite is intracellular.