Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Artificial Feeding of All Consecutive Life Stages of Ixodes ricinus.

The hard tick Ixodes ricinus is an obligate hematophagous arthropod and the main vector for several zoonotic diseases. The life cycle of this three-host tick species was completed for the first time in vitro by feeding all consecutive life stages using an artificial tick feeding system (ATFS) on heparinized bovine blood supplemented with glucose, adenosine triphosphate, and gentamicin. Relevant physiological parameters were compared to ticks fed on cattle (in vivo). All in vitro feedings lasted significantly longer and the mean engorgement weight of F0 adults and F1 larvae and nymphs was significantly lower compared to ticks fed in vivo. The proportions of engorged ticks were significantly lower for in vitro fed adults and nymphs as well, but higher for in vitro fed larvae. F1-females fed on blood supplemented with vitamin B had a higher detachment proportion and engorgement weight compared to F1-females fed on blood without vitamin B, suggesting that vitamin B supplementation is essential in the artificial feeding of I. ricinus ticks previously exposed to gentamicin.

Suspected autochthonous Thelazia callipaeda infection in a dog in northern Germany.

A 12-year old Elo dog was presented with recurring symptoms of conjunctivitis in November 2019. A single whitish nematode was found upon inspection of the eye and identified as a Thelazia callipaeda male. The morphological identification of the eye worm was supported by analysis of a partial cytochrome c oxidase I (cox1) gene sequence. The dog lived in Lower Saxony, northwestern Germany, and had not visited regions known to be endemic for T. callipaeda. This suggests that a local transmission cycle of this zoonotic nematode may exist in Germany.

Monitoring vaccinated cattle for induction and longevity of persistent tick-transmissible infection: Implications for wider deployment of live vaccination against East Coast fever in Tanzania.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

The protection afforded to cattle immunized with Theileria annulata infected cell line is enhanced by subunit vaccine candidate TaSP.

Tropical theileriosis constraints the development of the dairy industry in the Sudan and vaccination using live attenuated schizont vaccines is considered a promising measure for its control. The present study was carried out to investigate the ability of recombinant T. annulata surface protein (TaSP) to improve the efficacy of the attenuated Atbara cell line in protecting calves against field challenge. To this end, 23 cross-bred (Friesian × Kenana) calves were divided into four groups. Animals in group 1 (n = 5) were left unvaccinated. Group 2 (n = 6) received the Atbara cell line, animals in group 3 (n = 6) were immunized with three doses of TaSP on days 21, 49 and 77, while animals in group 4 (n = 6) received the cell line vaccine on day 0 and three doses of TaSP in Freund's incomplete adjuvant at days 21, 49 and 77. Twenty-eight days after the last TaSP boost, all groups were challenged by exposing them to natural field tick infestation in a region known to be endemic for tropical theileriosis. No thermal reactions, piroplasms or schizonts were observed in the immunized animals following immunization. Upon challenge, all animals showed a range of symptoms of clinical theileriosis with variable degrees of severity. The application of TaSP alone appeared to have no effect in terms of protection. The efficacy of the cell line alone was lower than the 100% level of protection against mortality observed in the group that received the combined cell line vaccine and TaSP, suggesting a synergistic effect of this combination.

Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south-western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci.

Theileria parva is a tick-transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti-transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south-western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen-encoding genes that are targets of CD8+T-cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south-western Uganda. Cattle-derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever.

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co-graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T-cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long-term consequences are unknown.

Relative Distribution, Diversity, and Bloodmeal Sources of Mosquitoes and Known Vectors of Rift Valley Fever Phlebovirus in Three Differing Ecosystems in Bura, Tana River County, Kenya.

Environmental modifications disturb the equilibrium of mosquito populations, altering the risk of mosquito-borne diseases. Mosquito distribution, diversity, and bloodmeal sources were examined to compare Rift Valley fever (RVF) risk among irrigated, riverine, and pastoral ecosystems in Bura, Tana River County, Kenya, between September 2014 and June 2015. Thirty-eight households and 21 irrigation fields were selected for the study. Mosquitoes were trapped with carbon dioxide-impregnated CDC traps, one trap per household and three traps per irrigated field, and morphologically identified using taxonomic keys. Host DNA was extracted from engorged females and cytochrome b genes amplified by PCR to identify sources of bloodmeals. A total of 21,015 mosquitoes were collected; 5742 within households in the 3 ecosystems and 15,273 within irrigated fields. Mosquitoes collected within irrigated fields belonged to 8 genera and 37 species, while those from households within the irrigation scheme belonged to 6 genera and 29 species. Collections from riverine and pastoral households belonged to five and four genera, respectively. The most abundant genera in the irrigated fields were Aedes (21%) and Mansonia (22%), while Anopheles (43%) was the most abundant within households. Most mosquitoes in riverine and pastoral households belonged to Anopheles (76%) and Aedes (65%) genera, respectively. Seasonal variation driven by rainfall was evidenced by spikes in mosquito numbers within irrigated and riverine ecosystems. Host species identification revealed that goats and humans were the main sources of bloodmeal. There was an overall increase in mosquito abundance and diversity as a result of the presence of the irrigated ecosystem in this county, and an increased availability of highly RVF-susceptible hosts as a result of the establishment and concentration of residential areas, promoting potential vector-host contacts. These results highlight the impact of anthropogenic changes on mosquito ecology, potentially heightening the risk of transmission and maintenance of RVF in this region.

Molecular detection of tick-borne pathogens in bovine blood and ticks from Khentii, Mongolia.

Recent studies reported the detection of DNA from tick-borne pathogens (TBPs) of veterinary relevance such as Anaplasma marginale, Babesia bigemina, Babesia bovis and Theileria orientalis in bovine blood samples from Mongolia. These findings were unexpected, as the known tick vectors of these pathogens are not known to occur in Mongolia. We therefore conducted a study in May and June 2013 in six districts of Khentii province where DNA of the said TBPs was previously found. Ticks collected from the vegetation and rodents, as well as blood samples from cattle, were screened for the presence of TBPs by reverse line blot (RLB) hybridization. Tick larvae collected from rodents were pooled. A total of 310 adult ticks were collected from the vegetation, and 249 tick larvae were collected from 24 rodents. Adult ticks (n = 2,318) and blood samples were collected from 481 heads of cattle. All adult ticks were identified as Dermacentor nuttalli. DNA from Rickettsia raoultii (252/310; 81.3%), an uncharacterized Anaplasma species preliminary named Anaplasma sp. Mongolia (26/310; 8.4%), Candidatus Midichloria sp. (18/310; 5.8%), Theileria equi (16/310; 5.2%), Babesia caballi (5/310; 1.6%), T. orientalis (1/310; 0.3%), Borrelia afzelii (1/310; 0.3%) and Candidatus Neoehrlichia mikurensis (1/310; 0.3%) was detected in ticks collected from the vegetation. DNA of R. raoultii (27/28; 96.4%) and Midichloria sp. (2/28; 7.1%) was detected in the pooled tick larvae. Anaplasma sp. Mongolia, a species related to Anaplasma ovis based on a multi-locus analysis, was also detected in 153/481 (31.8%) of the bovine blood samples. DNA of B. bovis, B. bigemina and A. marginale was not detected in the ticks or bovine blood samples from Khentii district.

Insecticide resistance in stable flies (Stomoxys calcitrans) on dairy farms in Germany.

Stable flies (Stomoxys calcitrans Linnaeus, 1758) can have a considerable negative impact on animal well-being, health, and productivity. Since insecticides constitute the mainstay for their control, this study aimed at assessing the occurrence of insecticide resistance in S. calcitrans on dairy farms in Brandenburg, Germany. First, the susceptibility of stable flies from 40 dairy farms to a deltamethrin-impregnated fabric was evaluated using the FlyBox® field test method. Then, S. calcitrans strains from 10 farms were reared in the laboratory, and the offspring was tested against the adulticides deltamethrin and azamethiphos and the larvicides cyromazine and pyriproxyfen. The FlyBox® method indicated 100% resistance in stable flies against deltamethrin. Later, to the offspring of those 10 established laboratory strains previously caught on suspected dairy farms, these field findings could be confirmed with mortalities well below 90% 24 h following topical application of the calculated LD95 of deltamethrin and azamethiphos. The ten strains could therefore be classified as resistant to the tested insecticides. In contrast, exposure to the insect growth regulators cyromazine and pyriproxyfen at their recommended concentrations demonstrated 100% efficacy. Both larvicides inhibited the moulting process of the stable fly larval stages completely, showing that the stable fly strains tested were susceptible to them. The intensive use of insecticides in recent decades has probably promoted the development of insecticide resistance. Systematic surveys in different livestock production systems and vigilance are therefore deemed necessary for estimating the risk of insecticide resistance development on a nationwide scale.

First report of a Hypoderma diana infestation in alpaca (Vicugna pacos) in Germany.

A Hypoderma larva was removed from a painful swelling in the lumbar region of a 17-month-old male alpaca kept on a farm in the Brandenburg district, eastern Germany. Morphological analysis and sequencing of the 18S rRNA gene demonstrated it was a second instar larvae of Hypoderma diana. The main host of H. diana is the roe deer (Capreolus capreolus). This is the first description of hypodermosis caused by H. diana in a camelid species.

How rational drug use reduces trypanosome infections in cattle in chemo-resistance hot-spot villages of northern Togo.

The study assessed an integrated trypanosomosis control strategy in drug-resistant hotspot villages of northern Togo. This strategy comprised (i) rational trypanocidal drug use in symptomatic cattle, (ii) vectors and ticks control by targeted bi-monthly insecticidal spraying of the lower body parts of cattle and (iii) strategic deworming with Albendazole in the beginning and the end of the rainy season. The program was implemented between June 2014 and October 2015 in four villages in northern Togo, which had been previously identified as drug resistant hotspots for diminazene diaceturate (DA) and isometamidium chloride (ISM). The integrated control strategy was implemented in eight cattle herds at risk of the disease from two villages. Twelve herds from two other villages served as controls where trypanosomosis management and deworming remained under control of the farmers. Trypanocidal drug use during the study period was recorded by the intervention team based on the farmers' reports and own observations. Cattle herds were followed-up for trypanosomosis symptoms which were recorded at 3 to 4-month intervals, while extensive trypanosome diagnostics and recording of the packed cell volume were done before and after the intervention. Intervention herds had a significantly lower risk of trypanosome infection with a risk ratio of 0.18 (95% CI: 0.04, 0.91; p = 0.03), but no significant effect on mean packed cell volume was observed. However, trypanocidal treatments per animal per year were lower in intervention herds compared to control herds (0.3 vs 5 for DA and 0.8 vs 2 for ISM). This study demonstrates that the implementation of an integrated best-bet strategy leads to a reduced trypanosome prevalence under lowered trypanocidal use.

Epidemiological study on tropical theileriosis (Theileria annulata infection) in the Egyptian Oases with special reference to the molecular characterization of Theileria spp.

Theileria annulata infection is a tick-borne disease known as Egyptian fever since 1947. It is a destructive obstacle for the livestock production in the Egyptian Oases (EL-Wady EL-Geded Province). The present study was conducted on 1068 cattle, ranged from below one year to more than eight years old; belonged to different farms and villages in EL-Wady EL-Geded Province. The infection was confirmed by blood smears, Tams-1 target based polymerase chain reaction (Tams-1 PCR), 18Ss rRNA polymerase chain reaction and semi nested-polymerase chain reaction (nPCR) followed by DNA sequencing and phylogenetic analyses, in addition to tick identification. Molecular techniques confirmed the infection in 63.6% (679/1068) of the examined animals while Giemsa-stained blood smears confirmed it in 36.8% (393/1062). Male and female animals showed molecular confirmed infection rates of 64.5 and 62.7%, respectively. Animals less than one year old were more infected (83.33%, 400/480) followed by animals less than three years (57.31%, 149/260) and animals less than five years (42.45%, 90/212), respectively. On the other hand, animal of five years old or above were less infected and the infection rate in this group was estimated to be 34.48% (40/116). Two tick species were identified during the present study: Hyalomma anatolicum and Rhipicephalus annulatus. Theileria annulata was the only Theileria species found in the Egyptian oases in respect to phylogenetic analysis of the obtained sequences.

Established and novel approaches for teaching and learning of veterinary parasitology in Berlin.

The teaching of veterinary parasitology to the large number of students at the Freie Universität Berlin is mainly limited to conventional face-to-face lectures, supplemented by practical classes. Extensive parasite descriptions and diagnostic techniques are at the core of the practical classes, which are also intended to emphasise key biological and veterinary aspects covered in lectures. Further in-depth and specific learning is achieved within a detailed framework of elective courses, with defined learning outcomes for small groups of students, focusing on themes such as 'diagnosis and treatment of ectoparasites in companion animals' or 'zoonotic parasites'. Additionally, structured excursions are designed to offer experience through collaborative international investigations. Organ-based approaches are also an integral part of our veterinary parasitology teaching, done in collaboration with the clinical and para-clinical departments, either via face-to-face interactions or online. Wide-ranging themes, such as 'causes of colic in horses' or 'atopic dermatitis in dogs' are covered. Recently, diverse blended learning elements were introduced into the curriculum (e.g., QuerVet), which makes teaching and learning more flexible, in terms of time and space, and fosters self-directed learning and participation among the students. A new platform to provide online lectures for students, termed VET Talks, was launched in 2015 by the International Veterinary Student's Association (IVSA), and is as a publicly available educational support system for students. Provided free to veterinary students throughout the world, this platform offers students the opportunity to access lectures on interesting topics by outstanding speakers who are nominated by their students. Finally, continuing education (CE) opportunities are provided through specific Masters courses (Master of Equine Medicine, Master of Small Animal Sciences), classical seminars and recent webinars.

Evaluation of a semi-automated in vitro feeding system for Dermacentor reticulatus and Ixodes ricinus adults.

The long feeding duration of ixodid ticks and need for regular blood changes turns the artificial feeding of ticks into a tedious process. To reduce the number of blood changes, a semi-automated system (SAS) for the artificial feeding of hard ticks was developed and evaluated. It consisted of a glass feeding reservoir that can accommodate six tick feeding chambers. A peristaltic pump was used to pump blood through the feeding reservoir, which was changed once daily. Groups of Dermacentor reticulatus and Ixodes ricinus adults were fed simultaneously in both the SAS and a conventional in vitro feeding system. In the conventional system, feeding chambers were hung inside a glass beaker filled with blood that was replaced twice daily. Dermacentor reticulatus adults fed in the SAS obtained significantly higher engorgement weights. Although engorgement rates between both systems were comparable, significantly more SAS-fed females laid fertile egg batches. The egg batch weight of SAS-fed females was also significantly higher. In contrast, the engorgement rate and fecundity of SAS-fed I. ricinus were significantly reduced in comparison to ticks fed in the conventional system. This reduction was likely to be caused by fungal infestation, which could spread between feeding chambers in the SAS. Although the SAS reduced the workload compared to the conventional feeding system and showed promising results for the in vitro feeding of D. reticulatus adults, measures to prevent fungal infestations in the SAS should be considered in future studies.

Resistance to trypanocidal drugs in cattle populations of Zambezia Province, Mozambique.

African animal trypanosomosis is a debilitating tsetse-transmitted parasitic disease of sub-Saharan Africa. Therapeutic and prophylactic drugs were introduced more than 50 years ago, and drug resistance is increasingly reported. In a cross-sectional study, 467 cattle were microscopically screened for trypanosomes. Samples were collected in May-July 2014 from five villages (Botao, Mungama, Zalala-Electrosul, Zalala-Madal, and Namitangurine) in Nicoadala district, Zambezia province. To evaluate treatment efficacy, trypanosome-positive animals in each village were randomly assigned to two groups, one treated with 0.5 mg/kg b.w. isometamidium (Inomidium®), the second with 3.5 mg/kg b.w. diminazene (Inomazene®). Cattle were microscopically monitored at days 0, 14, and 28 post-treatment. At day 28, trypanocides were swapped to investigate single or multiple resistance. Microscopically negative samples from the monitoring days were tested using 18S-PCR-RFLP. 22.9% (107/467) was found positive on day 0. On day 14, nine animals in Botao and seven in Mungama were positive. On day 28, in Botao, four animals from the diminazene group and four from the isometamidium group were positive. In Mungama, four animals from the diminazene group were positive on day 28. On day 42, six animals (9%) in Botao and two (9.5%) in Mungama remained positive after drug swap. No relapses occurred in Namitangurine. The 18S-PCR-RFLP consistently detected more positive than microscopy: indeed, positives reached 12, 13, and 8 in Botao and 9, 7, and 4 in Mungama, at days 14, 28, and 42, respectively. Single- and multi-drug resistance in Nicoadala district, Zambezia province, is thus here confirmed. This should be considered when choosing control options.

Molecular detection of tick-borne pathogens in cattle from Southwestern Ethiopia.

Tick-borne diseases (TBDs) cause significant losses among livestock and impact the livelihoods of resource-poor farming communities worldwide. In Ethiopia, detailed studies on the epidemiology of tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to determine the prevalence and species composition of bovine TBPs of veterinary significance in local cattle populations. A comprehensive cross-sectional epidemiological study was conducted in cattle populations of Illubabor zone in Southwestern Ethiopia from June to August 2013. For this purpose, blood samples were collected from 392 cattle. A combination of polymerase chain reaction (PCR) and a Reverse Line Blot (RLB) hybridization assay was employed for the detection of TBPs in these samples. The PCR/RLB results of the 392 blood samples indicated a high overall prevalence of 96.9% for TBPs, including Theileria mutans (66.1%), Theileria orientalis (51.8%), Anaplasma sp. Omatjenne (25.5%), Anaplasma marginale (14.5%), Babesia bigemina (14.0%) and Theileria velifera (13.0%) and minor occurrences of Ehrlichia ruminantium (0.5%) and Ehrlichia minasensis (0.26%). Moreover, three novel Anaplasma genotypes were detected in bovine blood samples. A phylogenetic analysis revealed that they most likely represent three, but at least two, new species. The prevalence of the three novel Anaplasma species, preliminary designated as Anaplasma sp. Hadesa, Anaplasma sp. Saso and Anaplasma sp. Dedessa, was 12.5%, 14.3% and 5.6%, respectively. Overall, a total of 227 cattle (57.9%) were found to be co-infected with two or more TBPs simultaneously and 86 different species combinations were observed. The findings show a very high burden of infection of cattle with TBPs in Ethiopia. The high frequency of co-infections suggests that clinical manifestations might be complex. Further research is required to determine the pathogenicity, host cell types and vector of the three novel Anaplasma species identified in this study.

The impact of insecticide-treated material to reduce flies among pork outlets in Kampala, Uganda.

Synanthropic flies have adapted to the mass of decaying organic matter near human settlements. As such, they feed and breed on food, faeces and other organic material and are known vectors for various diseases. Many of these diseases are associated with food, and foodborne diseases are of growing concern in developing countries where human population and food consumption increase. This pilot study aims at investigating the impact of a novel application of insecticide-treated material (ZeroFly®) to reduce flies among pork outlets in Kampala, Uganda. A cross-sectional survey randomly selected 60 of 179 pork outlets in Kampala. A controlled longitudinal trial followed in which 23 out of the 60 pork outlets were recruited for an intervention with insecticide-treated material. The pork outlets were randomly allocated to a group of 18 netted pork outlets (intervention) and five non-netted pork outlets (control). Monitoring took place over 15 weeks including 2 weeks as the baseline survey. The units were monitored for fly abundance using non-attractant sticky traps, which were placed within the pork outlet once per week for 48 consecutive hours. Medians of fly numbers before and after the intervention indicated a decrease of fly numbers of 48% (p = 0.002). Fly bioassays showed that the insecticidal activity of the netting remained active over the entire intervention period and led to a total paralysis of flies within at least 6 h after exposure. Insecticide-treated material provides a practical and sustainable solution in controlling flies and is therefore recommended as a complementary strategy for an integrated vector control and hygiene management.