Genotyping of Theileria lestoquardi from sheep and goats in Sudan to support control of Malignant Ovine Theileriosis.

Theileriosis, caused by parasitic protozoa of the genus Theileria parasites, are among the major tick-borne diseases of ruminant livestock. The largest economic losses are attributed in particular to those caused by the leukoproliferative species of Theileria: T. parva, T. annulata and T. lestoquardi. Theileria lestoquardi is transmitted by Hyalomma ticks and causes malignant ovine theileriosis (MOT), a disease that is particularly prevalent in Sudan. The disease is considered of a high economic importance in Sudan, where export of sheep is a major component of the national economy. A live vaccine based on a Sudanese isolate of T. lestoquardi (Atbara strain) was previously developed for the control of MOT in Sudan, but not yet deployed in the field. The present study aims to genetically characterize and compare samples of T. lestoquardi circulating in Sudan as well as the live vaccine isolate in order to understand vaccine breakthroughs and failure that may occur. Sheep and goats blood samples were collected from six regions in Sudan that are known to be endemic for T. lestoquardi infection or have experienced outbreaks of MOT. Blood samples infected with T. lestoquardi were identified by PCR or RLB. Genotyping was carried out by (1) sequencing the homologues of two T. parva CD8+ T cell antigen genes, Tp1 and Tp2, and (2) using a panel of seven micro- and mini-satellite markers. A total of 100 T. lestoquardi positive field samples and the T. lestoquardi (Atbara) vaccine were genotyped. The results showed that all samples had mixed genotypes, with several alleles identified at one or more loci. The gene diversity ranged from 0.7840 (TS8) to 0.2133 (TS12) with mean values of 0.5470. PCA revealed three clusters of the parasite in Sudan; interestingly one independent cluster was clearly seen, corresponding to the vaccine isolate. The T. lestoquardi Tp1 homologue showed higher homology with T. annulata than with T. parva sequences included the defined single CD8+ T cell target epitope region. The result indicates that multiple genotypes are a common feature of T. lestoquardi infection in Sudan. Both genotyping and the sequencing results clearly showed that the vaccine isolate is highly distinct from the field samples. This finding raised the question whether vaccination with the prepared lived vaccine will effectively protect animals against challenges by the field isolates of T. lestoquardi. The results of this work will inform on the best approach for controlling MOT in Sudan.

The Emergence of Theileria parva in Jonglei State, South Sudan: Confirmation Using Molecular and Serological Diagnostic Tools.

A cross-sectional survey was carried out in four counties of Jonglei State, South Sudan, between May and June 2012 to determine the distribution and northern limit of Theileria parva, the causative agent of East Coast fever in cattle, and its tick vector Rhipicephalus appendiculatus, as a prerequisite to the deployment of relevant control strategies. A total of 1636 ticks, 386 serum samples and 399 blood samples were collected from indigenous, apparently healthy, cattle of different age groups. Tick species were identified morphologically, and the identity of R. appendiculatus was confirmed by DNA barcoding. Overall, the T. parva infection rate in R. appendiculatus was 25% as shown by nested PCR. ELISA was used to assess antibodies to T. parva, and the overall seroprevalence was 22.8%. PCR of the blood samples showed 55 (13.8%) were positive for T. parva. This is the first molecular confirmation of T. parva DNA in areas north of Juba, where it was previously known and established. The northern limit of T. parva was determined as N°06.17.792, about 242 Km north from Juba. Implication of this limit on the epidemiology and control of ECF is discussed.

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: predominance of P[8] genotype resembling human strains.

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1-29.7%). This study revealed that age, management system and pig density significantly (p<0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811-1604nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4-91.3% and 95.1-96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6-100% to 92-100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6-91.7% and 89.3-96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.

Concentrations of four fecal steroids in wild baboons: short-term storage conditions and consequences for data interpretation.

One source of both bias and "noise" in fecal steroid analysis is temporal change in steroid concentrations resulting from duration or conditions of fecal sample storage. However, no consensus currently exists regarding correct procedures or precautions necessary for fecal sample storage, and conditions vary widely within field endocrinology literature. This study considered the effects of short-term, weeks-long, storage conditions on quantifiable fecal testosterone (fT), glucocorticoids (fGC), estrogens (fE), and progestagen (fP) metabolite concentrations in wild baboons (Papio cynocephalus). Quadruplicate subsamples of fecal samples (n=29) collected at Amboseli National Park and its environs were subjected to four different storage conditions prior to lyophilization, in order to determine the effects of storage on subsequent steroid concentrations, as assessed by 125I radioimmunoassays. As expected, the best alternative to the "initial condition" of lyophilization at three days after collection was to freeze fecal samples at -20 degrees C for two weeks prior to lyophilization. This storage method resulted in no significant change from initial steroid concentrations for fE, fT, or fP, although fGC showed a slight but significant decline. Storage for two weeks in a charcoal refrigerator caused a mean increase in all four steroid concentrations. However, the results from this storage condition were robust in terms of practical questions asked of the data: fE and fP values still reflected pregnant versus non-pregnant states in baboon females; a fGC profile constructed by age class resembled that created from the samples from the initial condition, although slightly inflated across age classes; and there were only moderate changes in relative fT concentrations across adult males. Knowledge of the effects of storage upon each steroid analyzed within one's study is a necessary component in determining the optimal compromise for storage protocol in a particular research project.