Serodiagnosis of cystic echinococcosis in naturally infected camels.

An ELISA was developed for serological detection of Echinococcus granulosus infection in dromedary camels. Antigen B (AgB) partially purified from hydatid cyst fluid of camels or sheep naturally infected with cystic echinococcosis (CE) due to E. granulosus, as well as a recombinant antigen B product (r-AgB) were used in an ELISA to screen panels of serum samples from slaughtered camels naturally infected with CE. Native hydatid cyst fluid antigen preparations were able to detect antibodies in sera from a significant proportion of camels with CE, as confirmed at post-mortem. Seroreactivity however, was variable. ELISA specificity for sera from naturally infected camels versus inspection-negative animals ranged from 90 to 99%. Native antigen B gave the highest sensitivity (97%) in ELISA for camel CE confirmed at slaughter. In contrast, r-AgB gave lower sensitivity for camel (84%) and sheep (28%) CE. The r-AgB-ELISA was, however, highly specific (90 and 95%) respectively for both camel and sheep natural CE infection. These results indicate that an ELISA based on serum antibody detection to AgB could be developed for immunodiagnosis of cystic echinococcosis in camels.

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4.

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.

Comparative studies on the sensitivity of polymerase chain reaction and microscopic examination for the detection of Trypanosoma evansi in experimentally infected mice.

Trypanosoma evansi, a protozoan parasite in the blood of camels is routinely diagnosed by finding the flagellates in the wet films or stained smear of peripheral blood, examined under a microscope. Although specific, this method is not sensitive at early stages of infection. We have tested the use of polymerase chain reaction (PCR) in the identification of T. evansi in different stages of infection in mice and compared its sensitivity with that of the standard microscopic examination method. Using a specific pair of primers, it was possible to identify T. evansi in the blood of infected mice. Experimentally, groups of mice were infected with T. evansi, isolated from a naturally infected local camel and blood samples were collected every day for 30 days post-infection. Direct microscopy or PCR was applied to detect parasitaemia. Results showed that during the acute phase of infection, parasites were detected by PCR three days earlier than by microscopy. Furthermore, the infected mice were consistently positive by PCR during the chronic phase while the parasites could not be demonstrated during this period using microscopic examination. These findings suggest that PCR may be applied to camel trypanosomosis during both acute and chronic phase of infection. Furthermore, it would provide an excellent tool in the evaluation of treatment of anti-trypanocidal drugs.

The prevalence of Toxoplasma gondii specific IgG and IgM antibodies in blood donors in Al Ain, United Arab Emirates indicates a potential risk to recipients.

The results of a prospective cross-sectional study on the anti-toxoplasma IgG and IgM specific antibody profile among blood donors in Al Ain United Arab Emirates are presented. The overall infection rate was 34%. Based on IgM specific antibody positive rate, acute toxoplasmosis was evident among 3% of the blood donors studied. The theoretical implications of these findings are discussed.

Toxoplasma antibody prevalence among healthy adults in United Arab Emirates.

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Public health aspects of cystic echinococcosis in the Arab countries.

Human cystic echinococcosis (CE) caused by larval Echinococcus granulosus is a zoonosis of major public health importance throughout the region comprising Arab North Africa and the Middle East. Prevalence rates are determined by epizootiological factors related to the size of stray dog population and its worm burden and to the infection rates in the intermediate host reservoir livestock population. Socio-economic development and socio-cultural practices are considered important determinants in the continued transmission of the disease. The reasons why CE remains a significant public health problem in the region are summarized.

Gestational and neonatal toxoplasmosis: regional seroprevalence in the United Arab Emirates.

Paired maternal/cord blood samples were tested for anti-Toxoplasma IgG or IgM antibodies using Biomerieux Micro-EIA2 IgG and IgM test kits. Of the 1503 women tested at the time of delivery, 344 (22.9%) were IgG seropositive. Three hundred and one maternal sera, including 265 that were IgG positive, were tested for IgM antibodies: 47 were found positive, indicating a gestational toxoplasmosis incidence of 31 per 1000 pregnancies over one year. All but one of the IgM positive maternal sera had tested IgG positive. Cord blood IgG seropositivity was similar to the maternal rate but 18 of the 301 babies had significant levels of anti-Toxoplasma IgM antibodies. As these 18 babies were all born to mothers also positive for IgM antibodies, the calculated rate of transplacental transmission was 38.3% with the estimated prevalence of congenital toxoplasmosis of 12 per 1000 live births. There was no statistically significant positive correlation between maternal seroprevalence and such well-known risk factors as consumption of raw meat and milk, or proximity of cats and other animals. One baby was born with the classical stigmata of congenital toxoplasmosis.

Effect of previous chloroquine intake on in vivo P. falciparum drug sensitivity.

Investigation of the in vivo response of P. falciparum malaria parasites to chloroquine was conducted during 1993/94 in Al-Ain District of Abu Dhabi Emirate, UAE. Sixty seven expatriates who developed falciparum malaria on their return from Pakistan, Oman and Sudan were recruited for the WHO in vivo tests. Of the 67 patients, eight were classified as having RII and RIII responses, while 59 remained aparasitaemic at the end of the seven-day WHO standard test. On continuation into the 28-day WHO extended test, a further 34 patients exhibited RI resistance. Resistance of parasites to chloroquine was confirmed by measurement of plasma chloroquine using High-Performance Liquid Chromatography. In all 67 patients, the level of chloroquine was well above the minimum therapeutic level. The outcome of the in vivo test in patients treated for the first time was significantly different from that in patients who were previously on chloroquine. Among patients treated for the first time, 36 out of 41 (88%) had a resistant response, whereas, among those previously on chloroquine only six out of 26 (23%) had a resistant response. The difference is probably due to the higher initial plasma level of chloroquine among patients who were previously on the drug. Curing more patients with higher plasma chloroquine implies that chloroquine shall continue to be useful, particularly if resistance is at the RI level. Appropriate higher therapeutic levels of chloroquine should be defined for such patients.

Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4.

A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein. The reaction of anti-peptide serum with infectious virus neutralized infectivity thereby supporting the biological importance of this site. Another interesting characteristic of this peptide was its ability to bind to the nucleocapsid protein resulting in a laddering effect on the nucleocapsid monomer (45 k), dimer (90 k) and trimer (135 k) [Gorzilia et al., J. Gen. Virol. 66, 1889-1900 (1985); Sabara et al., J. Virol. 53, 58-66 (1985); Sabara et al., J. Gen. Virol. 67, 201-212 (1986)]. Definitive proof of binding was provided by the fact that the increments in the ladder corresponded to the molecular weight of the synthetic peptide and that anti-peptide serum specifically reacted with the ladder formations. The laddering of the nucleocapsid could be eliminated by incubation with trypsin thus further supporting the formation of a synthetic peptide-nucleocapsid complex. Due to the ability of the peptide to bind to trypsin and to the nucleocapsid protein its biological activity was investigated. It appeared that increasing concentrations of the peptide reduced the rate of virus plaque formation, thereby suggesting that virus replication was inhibited. These results illustrate two features of this synthetic peptide which warrant further investigation; (1) its capacity to mimic an enzyme cleavage site and, (2) its ability to complex tightly to another protein. In protection-challenge experiments performed using a murine model, animals immunized with VP4-peptide provided protection passively, to neonates suckling on the immune dams, against a virulent rotavirus. The potential applications of this peptide in rotavirus diagnosis, therapy and synthetic peptides based vaccine is discussed.

Suppression of transplant immunity in experimental trichinellosis.

Skin allograft rejection in Balb/c and C57BL/6J mice following experimental infection with 300 larvae of Trichinella spiralis or Trichinella pseudospiralis was studied. Skin grafts from normal C57BL/6J mice were transplanted to infected Balb/c mice and vice versa at days 3, 10, 20 and 30 post-infection. The clinical criteria for graft rejection, scarring and graft falling, were followed. The results indicated that T. spiralis and T. pseudospiralis infections induced a significant delay in graft rejection when compared to the control groups. A maximum rejection time of 24 days was observed in T. spiralis infected C57BL/6J mice which received skin grafts from Balb/c mice on day 3 post-infection. The rejection in the uninfected control group was on day 7 post transplant. The mean rejection times for transplants on various days post-infection, with both species were very similar. Also, the rejection profiles in Balb/c mice were comparable to that observed in C57BL/6J mice, with a maximum delay of 26 days to rejection again obtained in mice transplanted on day 3 post-infection, for both species. When the skin grafts were performed 5 or 10 days prior to infection, the rejection occurred on day 7, as in the control group. The effect of T. spiralis and T. pseudospiralis soluble larval extracts (TSE or TPE) on graft rejection was also examined. Four intraperitoneal injections of 50 micrograms each of TSE or TPE every 48 h for 7 days did not induce any significant delay in graft rejection. In contrast, secretory antigens prepared from cultured larvae in vitro induced significant delays in graft rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Priming and induction of anti-rotavirus antibody response by synthetic peptides derived from VP7 and VP4.

Synthetic peptides derived from bovine rotavirus C-486 (BRV) outer capsid (VP7 and VP4) and inner capsid (VP6) proteins were tested to evaluate their ability to prime and induce an anti-rotavirus antibody response. Peptides corresponding to the amino acid residues 232-255 of VP4 (VP4-peptide), 275-295 of VP7 (VP7-peptide) and 40-60 of VP6 (VP6-peptide) of BRV were chemically synthesized. These peptides were coupled to carrier proteins (either keyhole limpet haemocyanin (KLH) or recombinant rotavirus inner capsid protein-VP6 assembled into virus-like particles (VP6-carrier) were used as carrier to link the synthetic peptides under study), and the resulting conjugates were used to immunize rotavirus seronegative mice. An enzyme-linked immunosorbent assay (ELISA) was used to determine anti-peptide and anti-rotavirus antibody titres in serum samples collected after immunization. All peptides were immunogenic in mice and induced the production of anti-peptide antibodies, but with the exception of VP6-peptide they were not able to induce anti-rotavirus antibodies as measured by ELISA. Western blot analysis indicated that antibodies against each peptide were able to react with the respective authentic viral proteins of various rotavirus serotypes. To determine if a peptide-primed animal would respond to native viral proteins, animals were subsequently injected with purified BRV. A rapid and high anti-rotavirus antibody titre, in addition to a rise in anti-peptide antibody titre, was observed in peptide-primed mice. Furthermore, the sera obtained from these mice neutralized the virus under in vitro conditions. The significance of these results in relation to a potential rotavirus synthetic peptide-based vaccine is discussed.

Studies on the rate of selective uptake of amino acids by Trichinella larvae in vivo.

Groups of C57BL/6J mice, orally infected with 300 larvae each of Trichinella spiralis or T. pseudospiralis were injected with [3H]-alanine, tyrosine, tryptophan or glycine. The incorporation of isotope labelled amino acids into larval proteins was measured at 2, 6, and 12 months post-infection. It was shown that there is a significant increase in the in vivo uptake of isotope labelled amino acids with time by the larvae of T. spiralis and T. pseudospiralis. The level of uptake was highest for tyrosine followed by tryptophan, alanine and then glycine, for both species. The in vivo uptake of amino acids by T. pseudospiralis larvae was always higher than T. spiralis or the host at 6 and 12 months post-infection. At 2 months post-infection, T. spiralis uptake of these amino acids was higher, except for tyrosine. This may be related to the special needs of these larvae during the process of encystation. The higher metabolic requirements of T. pseudospiralis may be related to the higher energy needs of these non encapsulated, highly motile and mobile muscle larvae.

Contractile characteristics of the flexor muscle of mice infected with Trichinella spiralis, T. nativa or T. pseudospiralis.

A comparative analysis of skeletal muscle isometric contractile characteristics was performed in vivo on the flexor muscle of mice infected 6 mo earlier with 400 larvae of Trichinella pseudospiralis, Trichinella spiralis, or Trichinella nativa. The control group consisted of age- and sex-matched uninfected mice. The mice were injected with 0.1 ml of 50% urethane in saline, and the skin of the left hind limb was cut open longitudinally. The exposed flexor muscle was freed from the adjacent tissue and left attached freely to the knee joint while the tendon was hooked to a transducer. The signals were amplified with an amplifier connected to a chart recorder. The sciatic nerve was exposed and attached to an electrode. Impulses were generated and muscle contraction recorded. The exposed muscle and nerve were bathed in normal Krebs solution at all times and the animals were kept alive during the experiment. The normal muscle twitch tension of uninfected mice reached an average of 2.26 +/- 0.24 (SD) g. Tetany was achieved at 15 Hz. Low-Ca2+ Krebs depressed the twitch tension to 2.0 +/- 0.08 g while tetany remained at 15 Hz. Muscle twitch tension in mice infected with T. pseudospiralis reached 2.47 +/- 0.17 g and tetany at 15 Hz. Low Ca2+ depressed twitch tension to 1.14 +/- 0.12 g. Tetany was achieved at 20 Hz. In contrast, the muscle twitch of mice infected with T. nativa was significantly reduced to 1.4 +/- 0.09 g and tetany at 15 Hz. Low Ca2+ depressed twitch tension to 0.9 +/- 0.16 g and tetany at 15 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the survival of aerosolized bovine rotavirus (UK) and a murine rotavirus.

The effect of relative humidity (RH) and temperature on the survival of airborne bovine rotavirus UK isolate (BRV-UK) and a murine rotavirus (MRV) was studied. In any one experiment, the virus under test was suspended in tryptose phosphate broth (TPB) supplemented with uranine (physical tracer) and an antifoam, was aerosolized using a Collison nebulizer into the rotating drum with the RH at either low (30 +/- 5%), medium (50 + 5%) or high (80 +/- 5%) level at 20 +/- 1 degrees C. Following a 15-min period of viral aerosol stabilization, sequential samples of drum air were collected using an All-Glass Impinger (AGI) for 24 h post-aerosolization. Both of the rotavirus isolates were found to survive best at medium RH level and high RH was found least favorable for the survival of these aerosolized rotaviruses. The survival pattern of aerosolized MRV was found to be the best when compared with survival pattern of all animal and human rotavirus isolates studies performed under aerosolized conditions in our laboratory. The findings of these experiments confirm and extend our previous reports on the survival of other animal and human aerosolized rotaviruses and emphasize the fact that air may be one of the vehicles for their dissemination and could explain why it is difficult to control nosocomial outbreaks of rotavirus gastroenteritis and to keep animal colonies rotavirus-free.

Molecular determinants of rotavirus virulence: localization of a potential virulence site in a murine rotavirus VP4.

The molecular basis of pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared under in vitro and in vivo conditions. Obvious differences in the mobility of several genomic RNA segments were observed in one-dimensional gels. Under in vitro conditions, partial proteolytic peptide mapping identified differences between the two outer capsid proteins of these virus and no difference in inner capsid protein was observed. Since it has been observed by us and others that the gene coding for VP4 protein plays a significant role in determining virulence, the variability observed in the present study between the 84 k proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant. A comparison of the carboxypeptidase digests of the MRV- and BRV-VP4 revealed an area of variability between amino acids 307 and 407, which may represent a site of virulence determinant. Under in vivo conditions the virulence of both parenteral BRV and MRV isolates and their corresponding reassortants (with replaced gene 4) were studied in murine and bovine hosts. Like their parents, BRV and MRV isolates, reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus isolate used and the clinical outcome in vivo was determined by gene segment 4. The implications of these findings to elucidate the molecular basis of pathogenesis of rotaviruses are discussed.

Echinococcus multilocularis: effect of size of inoculum and route of infection on metastasis, amyloidogenesis and alveolar hydatid cysts mass in mice.

Alveolar hydatid disease (AHD) in mice, caused by Echinococcus multilocularis, is characterized by restrictive and metastasizing progressive growth phases. In experimentally induced infections, neither inoculum size (5, 50 or 250 viable cysts) nor the route (intraperitoneal/subcutaneous) of infection altered the course of disease as measured by the size of the larval cyst mass (LCM) produced. Spleen weight and amyloid deposition were also shown to be independent of the route or size of inoculum. Inoculation of a soluble parasite protein extract (AHC-EXT) induced amyloid deposition, with a dose-dependent threshold. These results support our postulate that soluble component(s) of the LCM are the major factor in the pathogenesis of AHD.

Characterization of two rotaviruses differing in their in vitro and in vivo virulence.

The proteins, genomic RNA and disassembly conditions and pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared. An obvious difference in the mobility of several genomic RNA segments were observed in one-dimensional gels. Reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus used and the clinical outcome in vivo was determined by gene segment 4. Under in vitro conditions, a comparison of the inner capsid proteins by partial proteolytic peptide mapping did not reveal any difference between corresponding proteins. However, this technique did identify differences between the two corresponding outer capsid proteins of these viruses. These differences, in turn, may account for the increased stability of MRV, as compared to BRV, when subjected to calcium-chelating and chaotropic agents and may be one of the mechanisms involved in conferring virulence on the virus. The observed variability between the 84K proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant, since it has been observed by us and others that the gene coding for this protein plays a role in determining virulence. A comparison of the carboxypeptidase digests of the MRV and BRV VP4 revealed an area of variability between amino acids 307 and 407, which may represent the site of a virulence determinant.

Status of imported malaria in a control zone of the United Arab Emirates bordering an area of unstable malaria.

A concerted malaria eradication programme in the United Arab Emirates has reduced local transmission to only a very few small foci in the country. The Al Ain district is now a consolidation zone. However, transmission across the undemarcated border with Oman continues. Malaria imported by the large immigrant work force from major disease endemic areas remains a large burden. An added threat is the appearance of chloroquine-resistant Plasmodium falciparum principally from Sudan and Pakistan but increasingly amongst Omani cases seen in the hospitals and clinics in Al Ain. The implications of re-introduction of malaria and the establishment of chloroquine resistance, particularly for non-immune residents and visitors, are emphasized.

PIP: In Al Ain District located in the Abu Dhabi Emirate of the United Arab Emirates (UAE), clinicians at the Al Ain General Hospital outpatient clinic conduct a medical examination of and take blood samples from all new immigrants applying for resident or work permits. All persons with malaria receive 600 mg chloroquine phosphate at the clinic (day 0) and a prescription for another 600 mg for day 1 and 300 mg on day 2. The malaria cases are to return to the clinic on day 3. Entomologists record mosquito larval counts at all field sides and conduct susceptibility tests in the laboratory. The malaria control program applies a larvicide on breeding sites and uses pyrethroids to eliminate adult flying insects. In 1981, local transmission of malaria ceased. During 1988-1991, 4.7-9.1% of the population was slide-positive for malaria. This incidence rate is high enough to introduce imported malaria into the local anopheline mosquito population, should malaria control activities be reduced. Pakistanis, UAE nationals, and Omanis comprise most malaria cases (36.3-56.6%, 12.8-21.9%, and 15.7-25.3%, respectively). The immigrants tend to visit their home countries, where they acquire malaria. All UAE nationals acquire it while on holiday in or traveling to endemic areas, especially Oman. In 1991, out of 1150 cases, the leading sources of malaria were clearly Pakistan (576) and Oman (526). Most slide-positive children are either Omanis or part of UAE families with relatives across the border in Oman. Plasmodium falciparum, mostly from Pakistan and Oman, is responsible for 69.5% of malaria cases. The Indian subcontinent is the source of most P. vivax cases. Beginning in 1987, the number of chloroquine-resistant P. falciparum cases increased from 9 to 302. The leading sources of resistant cases originate from Oman (160), Pakistan (100), and Sudan (26). All chloroquine-resistant cases except one responded to Fansidar. Factors that may disturb effective malaria control efforts in this new agriculture area are rapid development of water resources and the undemarcated border with Oman.