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A field survey was conducted to evaluate susceptibility of Rhipicephalus (Boophilus) microplus to cypermethrin on 49 farms in three areas of Yucatan, Mexico. The modified larval packet test was used to evaluate larval mortality at different cypermethrin concentrations. Dose-mortality regressions, lethal concentrations (LC(50)-LC(99)), confidence intervals and slope were estimated by probit analysis. Phenotype was defined as susceptible, tolerant or resistant when the resistance factor (RF) derived from both LC(50) and LC(99) determinations were3, 3-5 or5, respectively. An allele specific PCR (AS-PCR) was used to determine the frequency of a sodium channel mutation (F1550I, Phe→Ile) associated with pyrethroid resistance. Overall, 26.5%, 40.8% and 32.6% of tick populations were susceptible, tolerant and resistant to cypermethrin, respectively. A substantial inter-population variation in the level of cypermethrin response was evident (resistance factors ranged from 0.3 to 2599 and from 0.7 to5000 when were indicated by the LC(50) and LC(99), respectively). The F1550I mutation (R allele) in R. microplus was present in all studied areas. The increasing presence of the R allele correlated well with increased levels of response indicated by both the LC(50) (r(2)=0.659, p=0.001) and LC(99) (r(2)=0.688, p=0.001) to cypermethrin. These results indicated that the F1550I mutation is a major common mechanism responsible for pyrethroid resistance in field populations of R. microplus ticks in the Mexican tropics. Both bioassay and AS-PCR showed that the prevalence of cypermethrin-resistant/tolerant R. microplus is high in Yucatan, Mexico and the relationship between the RF and the frequency of the R allele supports the role of F1550I as one of the most important mechanisms conferring pyrethroid resistance in these R. microplus populations.

There have been few, if any, studies of arthropod field populations quantifying the kinetics of evolution of phenotypic and genotypic resistance to chemicals in response to the presence or absence of selection pressure. A prospective intervention study was undertaken over 2 years in Mexico to measure changes in resistance phenotype and genotype in the presence or absence of pyrethroid selection pressure on field populations of Rhipicephalus (Boophilus) microplus ticks on 11 farms with varying degrees of pyrethroid resistance. The resistance phenotype was evaluated by bioassay in a larval packet test expressed as the resistance factor (RF) derived from probit analysis of dose mortality regressions, and resistance genotype by an allele-specific PCR (AS-PCR) to determine the frequency of a sodium channel mutation (F1550I) associated with pyrethroid resistance. To validate the AS-PCR, a Pyrosequencing™ method was developed to detect the F1550I mutation. There was good concordance with the genotypes identified by both Pyrosequencing™ and AS-PCR (Kappa: 0.85). On five farms cypermethrin (CY) was exclusively used at intervals and on six farms amitraz was used. On two of the five CY-treated farms, the experiment was prematurely terminated due to unacceptably high levels of tick resistance. For all five farms, after 8-24 months of continued selection pressure with CY, the RF had increased 2-125-fold. The frequency of the resistance allele increased on all five farms from a starting range of 5-46% to a range of 66-95% after 8-24 months. On six farms treated with amitraz neither the RF nor the frequency of the resistance allele changed. A clear correlation between the phenotype and genotype was found in three of four treated farms confirming that the F1550I mutation is a major cause of synthetic pyrethroid resistance in Mexico. These results show that the pyrethroid resistance trait is stable (> 2 years) and that resistance is acquired much faster than it is lost. Hence, alternation of pyrethroid acaricide with other chemicals is likely to lead to the stepwise acquisition of synthetic pyrethroid resistance but not additional prolongation of its efficacious lifespan.

SUMMARYVertical transmission of the protozoan parasite, Neospora caninum is highly efficient and can take two forms – endogenous transplacental transmission resulting from activation of the quiescent bradyzoite stage during pregnancy or exogenous transplacental transmission resulting from ingestion of oocysts during pregnancy. Calves born carrying infection derived from either endogenous or exogenous transplacental transmission are capable of infecting their offspring when they start to breed. This review considers firstly the frequency with which exogenous and endogenous transmission occur, secondly the role of the immune response in controlling N. caninum infection and thirdly how the parasite persists in an immune-competent host and is re-activated during pregnancy.

The protozoan parasite Neospora caninum is the most frequently diagnosed abortifacient in the UK and a leading cause of abortion worldwide but the mechanisms leading to abortion are not fully understood. The distribution of parasites and the histopathological changes in the placenta and foetus were compared in 12 cows following experimental infection of cattle with N. caninum in early (n=6) and late (n=6) gestation, by PCR, immunohistology, light microscopy and transmission electron microscopy. Twelve uninfected pregnant cattle were used as controls. Infection in early gestation led to foetal death. In the placentae of cattle immediately following foetal death, N. caninum DNA was detected and there was evidence of widespread parasite dissemination. This was associated with extensive focal epithelial necrosis, serum leakage and moderate maternal interstitial mononuclear cell infiltration. In the foetuses, parasites were evident in all tissues examined and were associated with necrosis. In the placenta of cattle infected in late gestation, N. caninum DNA was detected sporadically but parasites were not evident immunohistologically. Small foci of necrosis were seen associated with mild interstitial mononuclear cell infiltration. Detection of N. caninum DNA in the foetuses was sporadic and parasites were demonstrated immunohistologically in brain and spinal cord only, with an associated mononuclear cell infiltration. This data is consistent with uncontrolled parasite spread in an immunologically immature foetus and could, via multiparenchymal necrosis of foetal tissues or the widespread necrosis and inflammation observed in the placenta, be the cause of Neospora-associated abortions.

Neosporosis by Neospora caninum causes losses to livestock production through abortion in cattle while, in dogs, it induces neuromuscolar disease. This study investigated neosporosis seroprevalence associated risk factors (including the seropositivity to leishmaniosis) in dogs of southern Italy, determined the prevalence of N. caninum oocyst shedding and examined the relationship between seroprevalence of neosporosis in farm dogs and cattle. Using an inhibition ELISA, 20.9% of 306 dogs had percent inhibition values >10 (indicative of exposure) and farm dogs had a significantly (p < 0.001) higher seroprevalence than dogs in a rescue kennel. Whilst N. caninum seroprevalence was associated with increasing age in dogs (p

Although vector control and ivermectin chemotherapy have led to a dramatic reduction in the incidence of river blindness (onchocerciasis), there is a consensus that additional control tools are required to sustain and extend this success. The recognition of endosymbiotic bacteria (Wolbachia) in filariae and their targeting by antibiotics constitutes the most significant and practicable opportunity for a macrofilaricidal therapy in the short-to-medium-term. Using Onchocerca ochengi in cattle, an analog of human onchocerciasis, we have previously shown that oxytetracycline is macrofilaricidal, and protective immunity exists naturally in a subset of animals termed putatively immune. Here, we report that although 24 weeks of weekly oxytetracycline treatment eliminated adult worms, cured animals remained susceptible to re-infection by natural challenge when compared with putatively immune cattle. However, their susceptibility was not significantly different from that of concurrently exposed, heavily infected animals. Thus, cattle cured by oxytetracycline are neither hypo-susceptible nor hyper-susceptible.

Development of a drug lethal to adult Onchocerca volvulus (i.e., macrofilaricide) is a research priority for the control of human onchocerciasis. Using bovine O. ochengi infections, we investigated the effects of oxytetracycline administered in a short intensive regimen (SIR; 10 mg/kg daily for 14 days), compared with a prolonged intermittent regimen (PIR; 20 mg/kg monthly for 6 months) or a combination of both (COM), on the viability of adult worms and their endosymbiotic bacteria (Wolbachia species). The long-term treatments eliminated >80% (COM) or >60% (PIR) of adult female worms (P

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was ≤35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 ( n = 3) or week 9 ( n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.

Ivermectin administration is now the major tool in the control of human onchocerciasis (caused by Onchocerca volvulus) based on its suppression of microfilariae and hence the prevention of disease. However, in Africa, transmission is not eliminated and treated populations continue to be exposed to infective larval (L(3)) challenge, albeit at reduced levels. We have investigated whether protective immunity might develop under such conditions using the analogous host-parasite system Onchocerca ochengi in cattle, based on our previous findings in cattle exposed to challenge, that in vivo ivermectin attenuates the development of adult infections and that irradiation-attenuated L(3) induce significant protection. In a two-phase prospective study over 4 years, groups of cattle were exposed to severe natural challenge. In the first phase, 38/40 animals treated either with ivermectin or with moxidectin at either monthly or 3-monthly intervals had not developed detectable infections after 22 months of exposure whereas, in a non-treated control group (n = 14) nodule prevalence was 78.6% and the geometric mean (range) nodule load was 4.8 (0-33). In the second phase, all drug treatments were withdrawn, a new control group (n = 8) introduced, and exposure continued at the same site. After 24 months, all groups had developed patent infections, with geometric mean (range) nodule loads of 17.4 (4-99), 38.4 (10-111), 50.7 (26-86), 14.3 (0-69) and 14.7 (0-55) for the control, monthly-ivermectin, 3-monthly ivermectin, monthly moxidectin and 3-monthly moxidectin groups, respectively. There was no evidence of protection-indeed the 3-monthly ivermectin group was significantly (P < 0.05) hyper-susceptible. In addition, microfilarial densities and the rate of increase in microfilarial load were significantly higher (P < 0.05) in the ivermectin-treated groups than in control animals. These results have important implications for ivermectin-based control of human onchocerciasis and suggest that humans exposed to ongoing transmission in endemic areas whilst receiving ivermectin are unlikely to develop immunity and will be highly susceptible should drug distribution cease.

IT gives us no pleasure to say that the recent report of four cases of canine babesiosis from Essex (one of them fatal) is not a surprise ( VR , February 13, 2016, vol 178, p 172; March 5, 2016, vol 178, p 243). Since 2000 when quarantine regulations were changed and the Pet Travel Scheme (PETS) introduced, there has been a huge increase in the number of dogs entering the UK – either returning travellers or new importations. Globalisation brings many economic gains and improvements in quality of life but comes at a price, and that price is a reduction in biosecurity. In April 2000, just a few weeks after the introduction of PETS, the first travelling dog died in the UK of babesiosis contracted shortly before in France. In 2005, a fatal case of babesiosis in an untravelled dog occurred in Kent, the first apparent autochthonous case of canine babesiosis in the UK (Holm and others 2006). Now, in 2016, there are four cases in Essex within a …

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3–5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection—which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.

On the basis of a systematic review of the literature, this paper provides the first evidence-based maps of the distribution of the major vector-borne parasitic infections of dogs and cats in Europe. From an initial survey of 894 publications, data from 268 were analysed, summarised and collated. Prevalence data were used to calculate the force of infection to provide distribution and incidence maps for canine infections with Leishmania infantum, Dirofilaria immitis, Dirofilaria repens, Babesia canis and Ehrlichia canis. There was little information from some areas but, in spite of its acknowledged incompleteness, the study provides an evidence-based framework upon which to assess the risks of infection, and will provide a basis, by correlation with climatic and vegetation data, to derive more comprehensive risk assessment maps for Europe. To clinicians in both endemic and non-endemic countries, it will be of assistance in decision-making with respect to diagnosis and preventive measures.

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums. Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc.

This review summarizes research using Onchocerca spp. in chimpanzees, cattle and mice to gain insight into the protective immune response to the filarial worm Onchocerca volvulus in humans. In addition, Acanthocheilonema viteae has been evaluated as a surrogate filarial worm for studying immunity to the infection. Immune mechanisms controlling these infections are described and initial success using recombinant antigen vaccines in these models is reviewed.

Onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus , is a major cause of visual impairment and dermatitis in sub-Saharan Africa. As O. volvulus contains an obligatory bacterial symbiont ( Wolbachia ), it is susceptible to antibiotic chemotherapy, although current regimens are considered too prolonged for community-level control programs. The aim of this study was to compare the efficacies of oxytetracycline and rifampin, administered separately or in combination, against a close relative of O. volvulus ( Onchocerca ochengi ) in cattle. Six animals per group were treated with continuous or intermittent oxytetracycline regimens, and effects on adult worm viability, dermal microfilarial loads, and Wolbachia density in worm tissues were assessed. Subsequently, the efficacies of 3-week regimens of oxytetracycline and rifampin alone and a combination regimen were compared, and rifampin levels in plasma and skin were quantified. A 6-month regimen of oxytetracycline with monthly dosing was strongly adulticidal, while 3-week and 6-week regimens exhibited weaker adulticidal effects. However, all three regimens achieved >2-log reductions in microfilarial load. In contrast, rifampin monotherapy and oxytetracycline-rifampin duotherapy failed to induce substantive reductions in either adult worm burden or microfilarial load, although a borderline effect on Wolbachia density was observed following duotherapy. Dermal rifampin levels were maintained above the MIC for >24 h after a single intravenous dose. We conclude that oxytetracycline-rifampin duotherapy is less efficacious against O. ochengi than oxytetracycline alone. Further studies will be required to determine whether rifampin reduces oxytetracycline bioavailability in this system, as suggested by human studies using other tetracycline-rifampin combinations.

Nine cows which were naturally and persistently infected with Neospora caninum were housed and observed intensively throughout pregnancy. No recrudescence of a latent infection was detected by PCR tests on maternal blood but fetal infection, implying a recrudescence of maternal parasitosis, was associated with a marked increase in maternal antibody. The increase occurred in the second half of pregnancy in five cows which infected their calves, and before mid-pregnancy in one cow which aborted. There was no change in the avidity of the antibody, which remained high and characteristic of long-term infection. In three infected cows that gave birth to uninfected calves there was no marked increase in maternal antibody. Antigen-specific interferon gamma responses of peripheral blood mononuclear cells were observed in all the infected cattle but they did not vary significantly either during pregnancy, or whether the cows did or did not infect their calves, although the responses were consistently higher in the latter. There was no change in the plasma concentrations of cortisol or acute phase proteins associated with the recrudescence of the parasite. Three uninfected cows housed with the infected cows remained uninfected throughout the experiment. No immunosuppressive event was detected which might have provoked parasite recrudescence but the acute antibody rise associated with transplacental infection provides a valuable, non-invasive marker for further studies to investigate the cause and consequences of parasite recrudescence in N caninum infection in cattle.

The presence of intracellular bacteria in the body of various species of filarial nematodes, including important parasites such as Brugia malayi, Dirofilaria immitis, and Onchocerca volvulus, was observed as early as the mid-1970s. These bacteria were shown to be transovarially transmitted (from the female worm to the offspring) and to be present in significant amounts in the body of the nematode. As highlighted by their discoverers, the potential importance of these bacteria is fairly obvious: (1) bacteria-derived molecules should be considered as having an immunological and pathological role in filarial diseases; (2) the interaction between the bacteria and the filarial host deserves investigation, in view of the possibility that the bacteria are needed by the host nematode and could thus represent a target for therapy. Other authors, independently from the discovery of these intracellular bacteria, showed that the antibiotic tetracycline (which is well known for its efficacy on intracellular bacteria) had detrimental effects on two species of filarial nematodes (Brugia pahangi and Litomosoides sigmodontis). It is therefore surprising that for more than 20 years, no further investigations focused on the bacteria of filarial nematodes, nor on the anti-filarial properties of tetracycline. Recently, the bacteria of filarial nematodes have been independently "rediscovered" by research groups from the schools of Hamburg, Liverpool and Milan. These bacteria are now classified as Wolbachia, and the basic aspects of their phylogenetic history and relationship with the Wolbachia of arthropods have been reconstructed. In addition, their implications for the pathogenesis and treatment of filarial diseases have started to be uncovered. This paper, which is authored by representatives of the three European schools who reopened this research area, reviews our present knowledge of these fascinating microorganisms, highlighting the complexity of a symbiotic system which involves, in addition to the nematode and its bacterium, the vertebrate host.

Arthropod-transmitted (filarial) nematodes are important causes of disease in humans in tropical countries, yet no safe drug appropriate for mass delivery kills the adult worms. However, most filarial nematodes contain rickettsia-like bacteria of the genus Wolbachia, and related bacteria also occur in insects. There is increasing evidence that these bacteria have significant functions in the biology of filarial nematodes. They are thus important targets in the search for antifilarial drugs and experiments in animals and humans have suggested that antibiotic therapy has potential in treating filarial infections. To optimize future clinical trials there is a need for a fast and simple in vitro drug screen to compare drug efficacies against Wolbachia. In the absence of Wolbachia-infected nematode cell lines, we have utilized an Aedes albopictus insect cell line, naturally infected with Wolbachia, to test the activity of antimicrobial agents. Of the five antibiotics tested, doxycycline, oxytetracycline and rifampicin showed good activity (MICs of 0.0625, 4 and 0.0625 mg/L, respectively) whereas ciprofloxacin and penicillin were shown to have no effect.

From blood collected from 94 cattle at 12 locations in the eastern and northeastern areas of Zimbabwe, DNA was extracted and analysed by polymerase chain reaction with primers previously reported to be specific for Babesia bigemina and Babesia bovis . Overall, DNA of Babesia bigemina was detected in the blood of 33/94 (35 %) cattle and DNA from B. bovis was detected in 27/58 (47 %) of cattle. The prevalence of DNA of B. bigemina was significantly higher in young animals (

Filarial nematodes are important and widespread parasites of animals and humans. We have been using the African bovine parasite Onchocerca ochengi as a chemotherapeutic model for O. volvulus , the causal organism of 9river blindness9 in humans, for which there is no safe and effective drug lethal to adult worms. Here we report that the antibiotic, oxytetracycline is macrofilaricidal against O. ochengi . In a controlled trial in Cameroon, all adult worms (as well as microfilariae) were killed, and O. ochengi intradermal nodules resolved, by nine months9 post–treatment in cattle treated intermittently for six months. Adult worms removed from concurrent controls remained fully viable and reproductively active. By serial electron–microscopic examination, the macrofilaricidal effects were related to the elimination of intracellular micro–organisms, initially abundant. Analysis of a fragment of the 16S rRNA gene from the O. ochengi micro–organisms confirmed them to be Wolbachia organisms of the order Rickettsiales, and showed that the sequence differed in only one nucleotide in 858 from the homologous sequence of the Wolbachia organisms of O. volvulus . These data are, to our knowledge, the first to show that antibiotic therapy can be lethal to adult filariae. They suggest that tetracycline therapy is likely to be macrofilaricidal against O. volvulus infections in humans and, since similar Wolbachia organisms occur in a number of other filarial nematodes, against those infections too. In that the elimination of Wolbachia preceded the resolution of the filarial infections, they suggest that in O. ochengi at least, the Wolbachia organisms play an essential role in the biology and metabolism of the filarial worm.

The transmission and control of Neospora caninum infection in dairy cattle was examined using deterministic and stochastic models. Parameter estimates were derived from recent studies conducted in the UK and from the published literature. Three routes of transmission were considered: maternal vertical transmission with a high probability (0.95), horizontal transmission from infected cattle within the herd, and horizontal transmission from an independent external source. Putative infection via pooled colostrum was used as an example of within-herd horizontal transmission, and the recent finding that the dog is a definitive host of N. caninum supported the inclusion of an external independent source of infection. The predicted amount of horizontal transmission required to maintain infection at levels commonly observed in field studies in the UK and elsewhere, was consistent with that observed in studies of post-natal seroconversion (0.85-9.0 per 100 cow-years). A stochastic version of the model was used to simulate the spread of infection in herds of 100 cattle, with a mean infection prevalence similar to that observed in UK studies (around 20%). The distributions of infected and uninfected cattle corresponded closely to Normal distributions, with S.D.s of 6.3 and 7.0, respectively. Control measures were considered by altering birth, death and horizontal transmission parameters. A policy of annual culling of infected cattle very rapidly reduced the prevalence of infection, and was shown to be the most effective method of control in the short term. Not breeding replacements from infected cattle was also effective in the short term, particularly in herds with a higher turnover of cattle. However, the long-term effectiveness of these measures depended on the amount and source of horizontal infection. If the level of within-herd transmission was above a critical threshold, then a combination of reducing within-herd, and blocking external sources of transmission was required to permanently eliminate infection.

A case control study was conducted to evaluate the significance of Neospora caninum infections in cattle in England and Wales. The prevalence of N. caninum in normally calving cattle (the control group; n = 418) and aborting cattle (n = 633) was estimated using a commercial antibody-detection ELISA. Prevalence estimates for bovine virus diarrhoea virus, infectious bovine rhinotracheitis virus and Leptospira hardjo were also obtained by serology. The prevalence of N. caninum was significantly higher (P < 0.0001) in the aborting group (18%; 95% confidence interval: 15%, 21%) than in the control group (6%; 95% confidence interval: 4%, 8%); the latter is the first estimate, to date, of the national seroprevalence of N. caninum in dairy cattle in England and Wales. Prevalence estimates for bovine virus diarrhoea virus, infectious bovine rhinotracheitis virus and L. hardjo were not found to be higher in the aborting cattle than in the control group. With N. caninum, a strong association between seropositivity and abortion was found, with seropositive cows being 3.5-times more likely to abort than seronegative cows (odds ratio = 3.49; 95% confidence interval: 2.16, 5.69). Furthermore, 12.5% of abortions in dairy cattle in England and Wales may be attributable to N. caninum, as indicated by estimation of the population aetiological fraction.

All the cattle in 14 dairy herds in England were tested for Neospora caninum-specific antibodies with a commercial ELISA. Three of the herds had had sporadic abortions, eight had had endemic abortions and three had had epidemic abortions associated with N caninum before the study. Of 4295 cattle tested, 17.1 per cent were seropositive and the herd-specific prevalence ranged from 7.3 per cent to 44.8 per cent. No significant effect of either herd size (P = 0.988), endemic (P = 0.869) or epidemic (P = 0.138) patterns of abortion on herd-specific prevalence was found by using logistic regression analysis. There was no evidence in any herd of a significant increase in prevalence with age; the prevalence in seven-to 12-month-old cattle was not significantly different (P0.400) from the prevalence in older cattle, except that there was a significantly lower prevalence (P = 0.017) in 13-to 24-month-old cattle. The results of this study are consistent with vertical transmission being the major route of N caninum transmission in these herds.

Onchocerciasis (‘River Blindness’), caused by the filarial nematode Onchocerca volvulus is of major public health importance in West Africa. Ivermectin, a drug originally developed for veterinary use, is now being incorporated in control strategies but whilst it has potent efficacy against L1 larvae (microfilariae), ivermectin is not lethal to adult (L5) O. volvulus, nor to adults of the related cattle parasite O. ochengi. We have exploited this model to determine if ivermectin has prophylactic activity against naturally transmitted, O. ochengi infections in a controlled, prospective study in northern Cameroon. Calves were treated monthly with ivermectin at either 200 μg/kg or 500 μg/kg for 21 months. None of 15 treated calves developed adult worm infection, whereas 5/6 untreated controls became infected (PO. ochengi nodules, and all 5 developed patent microfilaridermia. These results have significant implications for the use of ivermectin in humans, and suggest that strategic chemotherapy at times of maximal transmission will confer prophylactic as well as therapeutic benefits.

Retrospective testing of 3,232 serum samples from the general population and 518 serum samples from a high-risk group showed no evidence of human exposure to Neospora caninum in England. Results were obtained by using immunofluorescence antibody testing and ELISA to analyze frequency distribution.

Ivermectin is not lethal to the adult worms of Onchocerca volvulus or to those of O. ochengi, a cattle parasite closely related to O. volvulus. Although ivermectin penetrates the nodules in which the adults of these nematodes live, it is not known what levels of the drug enter the worms. Adult male O. ochengi were incubated in [3H]ivermectin in a saturated solution of unlabelled ivermectin (11.44 microM), to measure uptake by the oral and transcuticular routes, and in [3H]inulin, to ascertain if oral ingestion occurs in vitro. Uptake of [3H]ivermectin was high [1040 disintegrations/min (d.p.m.) at 3 h, representing a mean total of 86 pmoles ivermectin/worm] and occurred predominantly by the transcuticular route. Viability of worms was not reduced by this exposure, and uptake continued for up to 12 h. Only low levels of [3H]inulin (four d.p.m.) were detected in worms, indicating that the gut is probably not functional in vitro. Scanning and transmission electron microscopy revealed that the epicuticle of both sexes had an irregular surface which was pitted with a honeycomb structure in males, and rough and abundantly folded in females. These structures greatly increased the absorptive surface of the worms. In conclusion, ivermectin is able to enter adult O. ochengi males at concentrations sufficient to kill non-filarial nematodes.

(1998). Animal models—Onchocerca ochengi and the development of chemotherapeutic and chemoprophylactic agents for onchocerciasis. Annals of Tropical Medicine & Parasitology: Vol. 92, No. sup1, pp. S175-S179.

Eighteen oestrus-synchronised ewes were infected experimentally with 1500 sporulated oocysts of Toxoplasma gondii between 80 and 90 days of gestation. The infection induced pyrexia and specific antibody in all the ewes. One ewe resorbed its fetus, five ewes aborted and 12 delivered live, congenitally-infected lambs whose pre-colostral serum was antibody-positive. Tissues from the aborted fetuses and placentae from the live lambs were examined for toxoplasma infection by polymerase chain reaction (PCR) amplification of the Bi gene and by mouse inoculation. Using a simple protocol of tissue preparation without DNA extraction and a nested format, PCR was as sensitive as mouse inoculation. Placental cotyledon gave a higher sensitivity of detection than brain, lung or liver, and 16 of 19 placentae were positive by PCR compared with 13 of 18 by mouse inoculation. In mock-infected tissues, as few as 10 tachyzoites could be detected. The PCR could be applied to tissues unfit for mouse inoculation.

Vertical transmission of Neospora caninum was studied in naturally infected, privately owned dogs, using antibody detection by IFAT to identify infected individuals. Retrospective studies were undertaken in litters from six bitches of the Hamiltonstövare breed, and on litters from seven bitches of other breeds--in both cases following diagnosis of clinical disease in puppies--and prospective studies were carried out on 17 seropositive bitches of other breeds. Puppies were classified as infected if either pre-colostral sera, or serum taken ator = 5 weeks, had a titre ofor = 1:50 in the IFAT. The frequency of putative congenital transmission was variable; three seropositive dams produced successive litters, each containing seropositive puppies; four other seropositive bitches, which each whelped twice, produced six litters in which all pups were seronegative. Bitches born to seropositive dams themselves produced litters containing seropositive pups. In the retrospective studies, 32/61 (52%) of pups tested born to 13 seropositive dams were themselves seropositive and 22/88 (25%) of all pups born developed clinical signs compatible with a diagnosis of neosporosis. However, in the prospective study, only 4/118 (3%) pups tested from 17 seropositive dams were seropositive and 4/122 (3%) of all pups born developed signs consistent with neosporosis. This difference reflected levels of IFAT titre in the two groups of bitches and overall the proportion of pups seropositive was strongly and positively correlated (r = 0.980, P0.05) with bitch IFAT titre. Overall, 80% of pups born to seropositive dams were not infected as determined serologically. This study shows that the frequency of vertical transmission of naturally acquired Neospora infection in dogs is variable, but much too low to sustain infection alone. Post-natal infection must occur to maintain infection at seroprevalence rates reported in dog populations.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Neospora species in cattle was developed. Whole formalin-fixed Neospora caninum (NC-Liverpool) tachyzoites were used as antigen and a monoclonal antibody to bovine immunoglobulin light chain and an anti-mouse horseradish peroxidase conjugate were used to reveal bound antibody. A panel of 46 sera, negative by the immunofluorescent antibody test (IFAT), were used in the ELISA at a serum dilution of 1:500 to calculate the negative cut-off value of OD 405 = 0.77. There was a 95 per cent agreement between the results of the ELISA and the IFAT with 104 serum samples. The specificity and sensitivity of the ELISA were 96 per cent and 95 per cent, respectively, when compared with the IFAT. No significant cross-reaction was observed with sera from cattle infected experimentally with Toxoplasma gondii, Cryptosporidium parvum, Babesia divergens, Sarcocystis cruzi, Eimeria alabamensis or E bovis . A significantly modified version of the test is now commercially available.

The equine intestinal cestode Anoplocephala perfoliata has been the subject of recent epidemiological and immunological studies because of its suspected association with intestinal disease in the horse. We have previously shown that the IgG(T) subtype antibody response to the 12/13 kDa component of the parasite excretory/secretory (E/S) antigen is positively correlated with parasite intensity. In this study, we utilize that correlation to examine the changes in natural infection intensity with age. Infection intensity based on IgG(T) responses showed a triphasic age-dependency pattern with peak mean worm burden in the 6 months–2 years age group, falling to a lower plateau level from 3 to 15 years, and rising again in older age groups. Anti-E/S total IgG was found to have a convex age-dependency curve, with maximal response in the 6 months–2 years old age group. IgG(a) showed a triphasic response similar to the age-intensity profile of IgG(T); IgG(c) showed steadily increasing levels of antibody with age. The IgG(b) age-dependency profile was intermediate between IgG(a) and IgG(c). Age-specific correlation coefficients between anti-12/13 kDa IgG(T) (as a measure of infection intensity) and IgG(a) and IgG(b) revealed statistically significant values for many age groups. The relative importance of exposure to infection and the development of acquired immunity as determinants of the observed age-intensity pattern is considered.

Twenty-seven cases of neosporosis in European dogs are described. The disease was confirmed by immunohistochemistry, electron microscopy, or a favourable response to treatment in the dogs with appropriate clinical signs, and by the presence of antibodies to Neospora caninum but not to Toxoplasma gondii. The affected dogs were two days to seven years old, and of 13 different breeds. Both sexes were affected and in most cases littermates remained normal. Twenty-one cases had an initial hindlimb paresis or ataxia, in which muscle atrophy was the most consistent clinical sign. Rigid hyperextension developed in approximately half of the cases. Anorexia and pyrexia were rare. Other clinical signs included forelimb ataxia, head tremors with tetraparesis and sudden collapse due to myocarditis. Titres of > or = 1:800 in the N caninum indirect fluorescent antibody test were detected in the 20 cases from which serum samples were taken. Such high titres are rare in healthy dogs and strongly suggest a diagnosis of neosporosis. Sixteen of the dogs received appropriate antiprotozoal treatment with clindamycin, potentiated sulphonamides and/or pyrimethamine; 10 made a full or functional recovery. Recovery was less likely in peracute cases with severe clinical signs, and when the treatment was delayed.

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P

A South African stock of Babesia bovis was successfully resuscitated from liquid nitrogen, and cultured in microaerophilous stationary phase. The in vitro susceptibility of the B. bovis stock to titrated concentrations of imidocarb dipropionate was observed and the 50% inhibitory concentration (IC) was determined (8.7 x 10(7) g ml-1). A drug-adapted line was developed by culture in the presence of sub-inhibitory concentrations of imidocarb dipropionate and it had an IC50 eight times higher than that of its original stock (6.6 x 10(-6) g ml-1). The drug-adapted line was cryopreserved and resuscitated from liquid nitrogen. Continuous culture of the non-drug adapted line through 15 subcultures did not change the IC50 (8.3 x 10(-7) g ml-1).

Whole worm extract (WWE) and excretory/secretory (E/S) antigens of Anoplocephala perfoliata were characterised by SDS-PAGE and their use in the serodiagnosis of equine cestodosis was evaluated. An enzyme-linked immunosorbent assay (ELISA) was used to compare WWE and E/S antigen as the capture layer in an antibody capture ELISA. E/S antigen gave the best differentiation between sera from tapeworm-positive and tapeworm-negative horses. The E/S-ELISA was optimised and validated against sera from horses of known tapeworm status. This assay gave a diagnostic sensitivity of 68% (n = 38) and a specificity in helminth-naive horses of 95% (n = 20). Western blot analysis of tapeworm-positive and tapeworm-negative sera revealed the immunoreactive proteins of E/S antigen to be those of 12 and 13 kDa.

We have previously described the presence of haemagglutinins in tissues of the tick, Rhipicephalus appendiculatus and determined their sugar specificities by inhibition experiments. In this study, haemagglutination inhibitory sugars are shown to have an effect in vivo on the abundance of Theileria parva infected salivary gland acini in Rhipicephalus appendiculatus. A significant increase (P < 0.05) was observed in T. parva acinar infection rates in the salivary glands of R. appendiculatus fed on ears of rabbits infused with melibiose and raffinose. In contrast, mannose and turanose (non-haemagglutination inhibitory sugars) did not cause elevation of T. parva acinar infection rates. The effect of melibiose in elevating acinar infections was observed when used only during T. parva maturation in the salivary glands but not during parasite pick-up from an infected bovine host. Stabilates produced from ticks with elevated acinar infections did not differ from control stabilates in infectivity to cattle, by comparison of prepatent periods to pyrexia, or parasitosis, or in the severity of reactions.

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species., Author Summary Coccidian parasites have a major impact on human and animal health world-wide and are among the most successful and widespread parasitic protozoa. They include Neospora caninum which is a leading cause of abortion in cattle and one of its nearest relatives, Toxoplasma gondii. Despite its close phylogenetic relationship to Toxoplasma, Neospora has a far more restricted host range, does not infect humans and its epidemiology depends predominantly on efficient vertical transmission. The divergent biology of these two closely related species provides a unique opportunity to study the mechanisms of host specificity, pathogenesis and zoonotic potential not only in these, but other Coccidia. We have sequenced the genome of Neospora and the transcriptomes of both species to show that despite diverging some 28 million years ago, both genome and gene expression remain remarkably conserved. Evolution has focused almost exclusively on molecules which control the interaction of the parasite with the host cell. We show that some secreted invasion-related proteins and surface genes which are known to control virulence and host cell interactions in Toxoplasma are dramatically altered in their expression and functionality in Neospora and propose that evolution of these genes may underpin the ecological niches inhabited by coccidian parasites.

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

SUMMARY An indirect enzyme linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from dogs is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen. A mixture of a monoclonal antibody to dog immunoglobulin G and a horse radish peroxidase conjugated antibody to mouse Ig was used to detect bound antibody. When the iscom preparation was analysed by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis it appeared to consist of a restricted number of proteins compared with whole parasite homogenates. In immunoblot analysis, using N. caninum positive sera from rabbits and dogs as probes, the major antigens recognized had approximate molecular weights between 30 and 45 and 17 to 19kDa. Compared with an ELISA using a crude solubilized tachyzoite antigen, the iscom ELISA substantially improved the sensitivity and specificity (to 97·6% and 95·6%, respectively, against an immunofluorescence test, IFAT, as indicator of true status). There was a statistically significant positive correlation between IFAT titres and iscom ELISA OD450 values. The iscom ELISA absorbances (and the IFAT titres) of dogs with proven clinical infections were not higher than those from nonclinically affected, putatively infected dogs.

Incubation of fluorescein-conjugated and biotinylated lectins with Rhipicephalus appendiculatus salivary glands revealed differences between the basal laminae of the haemocoelic surfaces of the three acinal types. There were some similarities in the lectin binding characteristics of the surfaces of type II and type III acini when Con A, PNA and TVA were applied, indicating the presence of galactose, mannose and glucose moieties on the acinal surfaces. The binding of BPA, HPA and HAA lectins, specific for galactosyl and glycosyl ligands, which occurred only on the surface of type III acini, was moderate to intense. The remaining galactose-reactive lectins (GMA, DBA and VVA) also bound only to type III acini and the level of binding was weak to moderate. Although the relationship between the haemocoelic surface of the R. appendiculatus salivary gland acini and Theileria kinetes is not known, the consistent differences in the surface carbohydrate composition of the acini confirm the existence of differences in the basic physiology of the acini which may correlate with the specific susceptibility of the type III acinus to Theileria parva infection.

Onchocerca ochengi , a filarial parasite of cattle, represents the closest relative of the human pathogen, Onchocerca volvulus . Both species harbour Wolbachia endosymbionts and are remarkable in that adult female worms remain viable but sessile for many years while surrounded by host cells and antibodies. The basis of the symbiosis between filariae and Wolbachia is thought to be metabolic, although a role for Wolbachia in immune evasion has received little attention. Neutrophils are attracted to Wolbachia , but following antibiotic chemotherapy they are replaced by eosinophils that degranulate on the worm cuticle. However, it is unclear whether the eosinophils are involved in parasite killing or if they are attracted secondarily to dying worms. In this study, cattle infected with Onchocerca ochengi received adulticidal regimens of oxytetracycline or melarsomine. In contrast to oxytetracycline, melarsomine did not directly affect Wolbachia viability. Eosinophil degranulation increased significantly only in the oxytetracycline group; whereas nodular gene expression of bovine neutrophilic chemokines was lowest in this group. Moreover, intense eosinophil degranulation was initially associated with worm vitality, not degeneration. Taken together, these data offer strong support for the hypothesis that Wolbachia confers longevity on O. ochengi through a defensive mutualism, which diverts a potentially lethal effector cell response.

Investigations into the role of mucosal IgA in the protective immune response to alimentary tract parasites could be facilitated by the production of specific IgA monoclonal antibodies. To this end, we sought to generate IgA hybridomas against sporozoites of the protozoan, Eimeria tenella using different protocols. Of the methods investigated, intra-enteric priming followed by intravenous boosting of germ-free mice, using splenic lymphocytes in fusions, optimised the yield of IgA hybridomas. Using this protocol, 7 27 specific anti- Eimeria hybridoma antibodies isolated were of IgA isotype. When lymphocytes from mesenteric lymph nodes (MLN) were used in fusions more non-specific IgA secretors were produced than when splenic lymphocytes were used, but the yield of specific anti- Eimeria IgA secreting hybridomas was not improved. By all protocols, a total of nine IgA secreting hybridomas were identified of which eight have been cloned for further studies.

Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund's adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate., Author Summary River blindness, or onchocerciasis, is caused by a parasitic worm (Onchocerca volvulus) that is transmitted by blood-feeding blackflies, which breed in fast-flowing rivers. More than 37 million people are infected and may experience visual impairment and/or severe dermatitis. Control of onchocerciasis is largely dependent on a single drug, ivermectin. Whilst this is extremely effective at killing the worms' offspring (microfilariae) and preventing symptoms, ivermectin does not eliminate the long-lived adult parasites or always stop transmission. Consequently, treatments must be repeated for many years, and drug resistance may be emerging. Against this background, a vaccine against onchocerciasis would provide an important additional tool to sustain effective control. In this study, we evaluated eight worm antigens as vaccine components in cattle, which are often parasitized by O. ochengi (the closest relative of O. volvulus) in West Africa. Twelve uninfected animals received all eight antigens and were exposed to natural transmission of O. ochengi alongside 13 unvaccinated cattle. After almost two years, 92% of vaccinated animals had acquired adult worms, but only 58% were positive for microfilariae; whereas 100% of unvaccinated animals harboured both parasite stages. This suggests that a vaccine against microfilariae to prevent development of disease in humans may be achievable.

Trap perches were utilised to quantify mite recoveries during efficacy tests of pyrethrin formulations against experimental infestations of Dermanyssus gallinae (De Geer, 1778) in budgerigars and pigeons, and to monitor mite recoveries from untreated canaries. Pyrethrins applied topically before infestation significantly reduced the mean numbers of poultry red mites found in both budgerigar and pigeon trap perches, over those in untreated controls (P = less than 0.01). In untreated control budgerigars and pigeons, and in untreated canaries, the percentage recoveries after 48 h were 12.6 +/- 1.6, 4.1 +/- 1.9, and 32.6 +/- 1.6%, respectively (geometric mean +/- SD), and the blood engorgements rates were 71.5 +/- 1.7, 11.9 +/- 5.1, and 90.1 +/- 1.0% respectively. The recovery in trap perches of mites from canaries declined exponentially; 85% of the cumulative total recovered was collected 24 h after infestation, but small numbers were recovered daily up to 96 h (limit of observation). The use of the trap perches is discussed as a way of assessing the efficacy of acaricides, and as a laboratory tool in studies on mite aggregation behaviour or immune responses to mite infestation.

Single-oocyst-derived field strains of Eimeria tenella isolated from Rugby in the United Kingdom (E tenella R) and from Mymensingh and Dhaka in Bangladesh (E tenella M and D, respectively) and a laboratory strain (E tenella, Houghton, H) were compared by isoenzyme electrophoresis, reactivity with antisporozoite monoclonal antibodies and, for some pairs of strains, cross-protection in vivo. The three field strains conformed to one zymodeme with respect to six isoenzymes. For glucose phosphate isomerase (GPI) all field strains were characterised by GPI-9. A panel of six different monoclonal antibodies raised against sporozoites of E tenella H did not discriminate between strains by titration in an immunofluorescence assay against air-dried, acetone fixed sporozoites. In cross-protection experiments involving immunisation and challenge of young chickens, two immunisation schedules were used which, after homologous challenge, provided complete immunity either by the criterion of oocyst output, or by the criterion of weight gain (and more than 94 per cent protection by the criterion of oocyst output). While strain heterogeneity was minimal in the former situation, there was poor cross protection between some strains in the latter case. Under those conditions, heterologous challenge with E tenella M resulted in dysentery and in significantly (P less than 0.05) increased oocyst output and decreased weight gain. The results suggested that E tenella M was immunologically superior to E tenella R and H strains. The results show that a limited degree of immunogenic variability exists between these strains of E tenella and that, unless homologous strain immunity is complete by the criterion of oocyst output, challenge with heterologous strains may result in depressed weight gain.

Background Human onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is currently controlled using the microfilaricidal drug, ivermectin. However, ivermectin does not kill adult O. volvulus, and in areas with less than 65% ivermectin coverage of the population, there is no effect on transmission. Therefore, there is still a need for a macrofilaricidal drug. Using the bovine filarial nematode O. ochengi (found naturally in African cattle), the macrofilaricidal efficacy of the modified flubendazole, UMF-078, was investigated. Methods Groups of 3 cows were treated with one of the following regimens: (a) a single dose of UMF-078 at 150 mg/kg intramuscularly (im), (b) 50 mg/kg im, (c) 150 mg/kg intraabomasally (ia), (d) 50 mg/kg ia, or (e) not treated (controls). Results After treatment at 150 mg/kg im, nodule diameter, worm motility and worm viability (as measured by metabolic reduction of tetrazolium to formazan) declined significantly compared with pre-treatment values and concurrent controls. There was abrogation of embryogenesis and death of all adult worms by 24 weeks post-treatment (pt). Animals treated at 50 mg/kg im showed a decline in nodule diameter together with abrogated reproduction, reduced motility, and lower metabolic activity in isolated worms, culminating in approximately 50% worm mortality by 52 weeks pt. Worms removed from animals treated ia were not killed, but exhibited a temporary embryotoxic effect which had waned by 12 weeks pt in the 50 mg/kg ia group and by 24 weeks pt in the 150 mg/kg ia group. These differences could be explained by the different absorption rates and elimination half-lives for each dose and route of administration. Conclusion Although we did not observe any signs of mammalian toxicity in this trial with a single dose, other studies have raised concerns regarding neuro- and genotoxicity. Consequently, further evaluation of this compound has been suspended. Nonetheless, these results validate the molecular target of the benzimidazoles as a promising lead for rational design of macrofilaricidal drugs.

Five species of Eimeria, namely E. acervulina, E. tenella, E. maxima, E. brunetti and E. necatrix were identified in chickens in Bangladesh on the basis of lesions seen at post-mortem examinations of naturally infected birds, and on the dimensions of oocysts and the lesions seen in chicks experimentally infected with single oocyst derived strains. The use of filter top, polycarbonate cages permitted the isolation of strains without sophisticated animal isolators and is appropriate for use in laboratories throughout the developing world. Responses to a questionnaire sent to all known intensive poultry farms suggested that coccidiosis is a major disease. For control, producers rely mostly on management procedures and the tactical use of sulphonamides; in-feed chemoprophylaxis is not widely used. These control measures are unsatisfactory and recent coccidiosis outbreaks were reported from seven of 16 farms. There was evidence of a seasonal incidence in clinical coccidiosis.

Whilst it is presumed that infection of pregnant cattle with Neospora caninum oocysts can provoke abortion and is the likely cause of epidemic abortion outbreaks, only two previous experiments have involved inoculation of pregnant cows with oocysts (and only one abortion was provoked in 22 pregnancies). Here, we describe the oral oocyst challenge of 18 cows synchronously bred and inoculated precisely at 70 (n = 6), 120 (n = 6) and 210 (n = 6) days in pregnancy with a nominal dose of 40,000 oocysts. Only one abortion occurred (at the 120 days challenge) which could be definitively ascribed to N. caninum and no transplacental infection (TPI) was detected in any of the other 11 calves born in the 70 and 120 day challenge groups. In contrast, 4/5 live calves born to cattle challenged at 210 days were transplacentally infected. When cows which had transplacentally infected their calves in the first pregnancy were rebred, no TPI occurred. The results show that the timing of challenge influences clinical and parasitological outcomes and that cattle in late pregnancy are exquisitely sensitive to oocyst challenge leading to exogenous TPI and congenitally infected calves. However, cattle which were indisputably systemically infected in their first pregnancy did not induce endogenous TPI in their subsequent pregnancy. This confirms previous results with experimental tachyzoite challenge and suggests that post-natal infection does not lead to persisting infections which can recrudesce in pregnancy.

Neospora caninum is an obligate intracellular protozoan parasite that causes abortion in cattle. It is normally found as a latent infection controlled by a T-helper-cell type 1 response involving CD4 + cytotoxic T cells and gamma interferon. Cattle may be infected by two different routes: transplacentally as a result of activation of the latent infection in the mother causing congenital infection or abortion and by ingestion of oocysts, which, if it occurs during gestation, can also result in abortion. Here, for the first time, we establish proof that live vaccination protects against fetal death, whereas immunization using whole-tachyzoite lysate in different adjuvants fails to protect against fetal death. Strong antibody responses were induced in all the vaccinated groups, and the quality and magnitude of these responses were similar in the live- and the lysate-vaccinated groups. In contrast, only the group immunized with live tachyzoites had strong cellular and gamma interferon responses prior to challenge, and these responses correlated with protection against fetopathy. These results suggest that a cellular immune response may be important in the mechanisms involved in protection against N. caninum -associated abortions.