Nuttalliellidae in Burmese amber: implications for tick evolution.

Ticks are composed of 3 extant families (Argasidae, Ixodidae and Nuttalliellidae) and 2 extinct families (Deinocrotonidae and Khimairidae). The Nuttalliellidae possess one extant species (Nuttalliella namaqua) limited to the Afrotropic region. A basal relationship to the hard and soft tick families and its limited distribution suggested an origin for ticks in the Afrotropics. The Deinocrotonidae has been found in Burmese amber from Myanmar and Iberian amber from Spain, suggesting a wider distribution of the lineage composed of Deinocrotonidae and Nuttalliellidae. The current study describes 8 fossils from mid-Cretaceous (ca. 100 Ma) Burmese amber: 2 Deinocroton species (Deinocroton bicornis sp. nov.; Deinocroton lacrimus sp. nov.), 5 Nuttalliella species (Nuttalliella gratae sp. nov., Nuttalliella tuberculata sp. nov., Nuttalliella placaventrala sp. nov., Nuttalliella odyssea sp. nov., Nuttalliella tropicasylvae sp. nov.) and a new genus and species (Legionaris nov. gen., Legionaris robustus sp. nov.). The argument is advanced that Deinocroton do not warrant its own family, but forms part of the Nuttalliellidae comprising 3 genera, Deinocroton, Legionaris nov. gen. and Nuttalliella). Affinities of Burmese tick fossils to the Australasian region, specifically related to rifting of the Burma terrane from northern Australia ~150 million years ago, suggest that Nuttalliella had a much wider distribution than its current limited distribution. The distribution of Nuttalliella likely stretched from Africa over Antarctica and much of Australia, suggesting that extant members of this family may still be found in Australia. Considerations for the geographic origins of ticks conclude that an Afrotropic origin can as yet not be discarded.

Mitochondrial genome and nuclear ribosomal RNA analysis place Alveonasus lahorensis within the Argasinae and suggest that the genus Alveonasus is paraphyletic.

Two major families exist in ticks, the Argasidae and Ixodidae. The Argasidae comprise 2 sub-families, Argasinae and Ornithodorinae. The placement into subfamilies illuminate differences in morphological and molecular systematics and is important since it provides insight into evolutionary divergence within this family. It also identifies fundamental gaps in our understanding of argasid evolution that provide directions for future research. Molecular systematics based on mitochondrial genomics and 18S/28S ribosomal RNA confirmed the placement of various genera and subgenera into the Argasinae: Argas (including Argas and Persicargas), Navis, Ogadenus, Otobius lagophilus, Proknekalia, Secretargas and the Ornithodorinae: Alectorobius, Antricola (including Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (including Microargas, Ornamentum, Ornithodoros sensu strictu, Pavlovskyella), Otobius sensu strictu, Reticulinasus and Subparmatus. The position of Alveonasus remains controversial since traditional taxonomy placed it in the Ornithodorinae, while cladistic and limited molecular analysis placed it in the Argasinae. The current study aimed to resolve the systematic position of Alveonasus using mitochondrial genomic and 18S/28S ribosomal RNA systematics by sequencing the type species Alveonasus lahorensis from Pakistan. In addition, the mitochondrial genomes for Argas reflexus and Alectorobius kelleyi are reported from Germany and the USA, respectively. The systematic data unambiguously place Alveonasus in the Argasinae and also suggest that Alveonasus may be another paraphyletic genus.

The host-specificity of Theileria sp. (sable) and Theileria sp. (sable-like) in African Bovidae and detection of novel Theileria in antelope and giraffe.

Tick-borne diseases caused by Theileria are of economic importance in domestic and wildlife ruminants. The majority of Theileria infects a limited number of host species, supporting the concept of host specificity. However, some Theileria seem to be generalists challenging the host specificity paradigm, such as Theileria sp. (sable) reported from various vertebrate hosts, including African buffalo, cattle, dogs and different antelope species. We tested the hypothesis that T. sp. (sable) uses Bovidae as hosts in general using a real-time polymerase chain reaction assay specific for T. sp. (sable) and a closely related genotype: T. sp. (sable-like). Various antelope species from the Tragelaphini (black wildebeest, blesbuck, blue wildebeest, gemsbuck, sable and waterbuck) tested positive for either T. sp. (sable) or T. sp. (sable-like). However, no African buffalo (n = 238) or cattle (n = 428) sampled in the current study tested positive, suggesting that these latter species are not carrier hosts. The results were confirmed using next-generation sequencing which also indicated at least 13 new genotypes or species found in various antelope and giraffes. Genotypes were found in single host species or in evolutionarily related hosts, suggesting that host specificity in Theileria may be a lineage specific phenomenon likely associated with tick-host-parasite co-evolution.

Geographic distribution of Theileria sp. (buffalo) and Theileria sp. (bougasvlei) in Cape buffalo (Syncerus caffer) in southern Africa: implications for speciation.

Strict control measures apply to movement of buffalo in South Africa including testing for Theileria parva, the causative agent of Corridor disease in cattle. The official test is a real-time hybridization PCR assay that amplifies the 18S rRNA V4 hyper-variable region of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). Mixed infections with the latter organisms affect diagnostic sensitivity due to PCR suppression. While the incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, little information is available on the specific distribution and prevalence of T. sp. (buffalo) and T. sp. (bougasvlei). Specific real-time PCR assays were developed and a total of 1211 samples known to harbour these parasites were screened. Both parasites are widely distributed in southern Africa and the incidence of mixed infections with T. parva within the endemic region is similar (∼25-50%). However, a significant discrepancy exists in regard to mixed infections of T. sp. (buffalo) and T. sp. (bougasvlei) (∼10%). Evidence for speciation between T. sp. (buffalo) and T. sp. (bougasvlei) is supported by phylogenetic analysis of the COI gene, and their designation as different species. This suggests mutual exclusion of parasites and the possibility of hybrid sterility in cases of mixed infections.

The host preferences of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae): a generalist approach to surviving multiple host-switches.

Nuttalliella namaqua has been described as a "living fossil" and the closest extant species to the ancestral tick lineage. It was previously proposed that the Nuttalliella lineage parasitized reptile-like mammals in the Permian and had to switch hosts several times due to mass or host lineage extinctions. Extant hosts include girdled lizards and murid rodents. The current study extends knowledge on the extant host range of N. namaqua using gut meal analysis of field collected specimens. Nymphs and females can parasitize a variety of reptiles that includes skinks, geckos and girdled lizards. Blood-meal from a hyrax was also detected in a specimen suggesting that N. namaqua could parasitize a broader range of mammals than the previously suggested murid rodents. Rather than being host specific, N. namaqua is proposed to be a generalist and the ability to switch and parasitize multiple hosts allowed it to survive multiple mass and host lineage extinctions.

Description of a new tick species, closely related to Amblyomma javanense (Supino, 1897), associated with Varanus bengalensis (Squamata: Varanidae) in Pakistan.

The genus Amblyomma contains the highest percentage of reptile-associated ticks, and comprises approximately nine subgenera. One of these subgenera is Adenopleura, which also encompasses Amblyomma javanense, and its type species Amblyomma compressum. This study describes a new Amblyomma species associated with Bengal monitor lizards (Varanus bengalensis) based on morphology and its mitogenome in Khyber Pakhtunkhwa, Pakistan. Reptiles belonging to different genera were examined for Amblyomma ticks and only the monitor lizard was infested with ticks in the District Bajaur. Collected Amblyomma cf. javanense ticks were analyzed and formally described as a new species. Overall, 57 A. cf. javanense ticks were collected on monitor lizards (4/27) with a 15% prevalence of infestation, 2.1 mean abundance, and 14.3 mean intensity. Ticks comprised males (n = 23, 40%), females (n = 14, 25%) and nymphs (n = 20, 35%), while no larvae were found. BLAST analysis of A. cf. javanense sequences showed the following maximum identities; 98.25% with undetermined Amblyomma species based on 12S rRNA, 96.07% with A. javanense based on 16S rRNA, 99.56% and 90.95% with an Amblyomma sp. and A. javanense, respectively, based on ITS2. Moreover, the mitochondrial genome of A. cf. javanense showed maximum identities of 80.75%, 80.48% and 79.42% with Amblyomma testudinarium, A. javanense, and Amblyomma sp., respectively. The phylogenetic analysis of A. cf. javanense revealed that its 12S rRNA and 16S rRNA are closely related to an Amblyomma sp. and A. javanense, respectively, from Sri Lanka, its ITS2 is closely related to A. javanense from China and an Amblyomma sp. from Sri Lanka, and its mitogenome is closely related to A. javanense and Amblyomma sp. from China. The pairwise distance analysis resulted in divergence of 0-1.71% (12S rRNA), 0-17.5% (16S rRNA), 0-9.1% (ITS2) and 0-20.5% (mitochondrial genome). We also contributed the full-length mitochondrial genome sequence of A. compressum and showed that this species does not share a most recent common ancestor with A. javanense. As the subgenus Adenopleura is paraphyletic, this study could help to understand the systematics and phylogeny of this taxon.

Molecular and serological prevalence of corridor disease (buffalo associated Theileria parva infection) in cattle populations at the livestock/game interface of KwaZulu-Natal province, South Africa.

Theileria parva are intracellular protozoal parasites responsible for three disease syndromes in cattle, namely East Coast fever (ECF), Corridor disease (CD) and Zimbabwean theileriosis. The increase in reports of CD outbreaks in recent years has raised questions about the probability of adaptation of buffalo-derived T. parva strains in cattle herds adjacent to game reserves. A cross-sectional study was conducted from March 2016 to December 2018 to investigate the extent of occurrence of T. parva infections in cattle in the CD-controlled area of KwaZulu-Natal Province. Blood samples were collected from 1137 cattle from 14 herds and analysed by quantitative real-time PCR (qPCR) and indirect fluorescent antibody test (IFAT) to determine the prevalence of T. parva. A total of 484 samples from 4 of the 14 herds were further tested on qPCR for the presence of T. taurotragi infections. The data were analysed using descriptive statistics and a chi-square test was used to assess association between variables. The overall prevalence of T. parva was 1.3% (95%CI:1-2%) and 19.9% (95%CI:17-22%) on qPCR and IFAT, respectively. The qPCR positive samples were detected in March and May while IFAT positive samples were detected in all seasons sampled, with higher numbers during summer months. The Pearson Chi-squared test showed that T. parva prevalence rates based on both qPCR and IFAT were positively associated with herds with previous history of CD outbreaks (χ2 = 8.594, p = 0.003; χ2 = 69.513, p < 0.001, respectively). The overall prevalence of T. taurotragi was 39.4% (95% CI: 35-44%) with the herd-level prevalence ranging between 35.0% and 43.4%. Possible cross-reaction of T. parva IFAT to T. taurotragi was detected on few samples, however, there was no significant association between T. taurotragi infections and IFAT positivity (χ2 = 0.829, p = 0.363). Results from this study demonstrated the extent of occurrence of subclinical carriers and the level of exposure to T. parva infections in cattle populations at a livestock/game interface area of KwaZulu-Natal Province. The molecular and seroprevalence rates were low when compared with other areas where cattle-adapted T. parva infections are endemic. The adaptation of buffalo-derived T. parva in cattle population resulting in cattle-cattle transmissions seem to be unlikely under the current epidemiological state.

A comparison of Illumina and PacBio methods to build tick salivary gland transcriptomes confirms large expression of lipocalins and other salivary protein families that are not represented in available tick genomes.

Tick saliva helps blood feeding by its antihemostatic and immunomodulatory activities. Tick salivary gland transcriptomes (sialotranscriptomes) revealed thousands of transcripts coding for putative secreted polypeptides. Hundreds of these transcripts code for groups of similar proteins, constituting protein families, such as the lipocalins and metalloproteases. However, while many of these transcriptome-derived protein sequences matches sequences predicted by tick genome assemblies, the majority are not represented in these proteomes. The diversity of these transcriptome-derived transcripts could derive from artifacts generated during assembly of short Illumina reads or derive from polymorphisms of the genes coding for these proteins. To investigate this discrepancy, we collected salivary glands from blood-feeding ticks and, from the same homogenate, made and sequenced libraries following Illumina and PacBio protocols, with the assumption that the longer PacBio reads would reveal the sequences generated by the assembly of Illumina reads. Using both Rhipicephalus zambeziensis and Ixodes scapularis ticks, we have obtained more lipocalin transcripts from the Illumina library than the PacBio library. To verify whether these unique Illumina transcripts were real, we selected 9 uniquely Illumina-derived lipocalin transcripts from I. scapularis and attempted to obtain PCR products. These were obtained and their sequences confirmed the presence of these transcripts in the I. scapularis salivary homogenate. We further compared the predicted salivary lipocalins and metalloproteases from I. scapularis sialotranscriptomes with those found in the predicted proteomes of 3 publicly available genomes of I. scapularis. Results indicate that the discrepancy between the genome and transcriptome sequences for these salivary protein families is due to a high degree of polymorphism within these genes.

Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis.

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be cost-prohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies' Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twenty-six of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community.

Diversity in the 18S SSU rRNA V4 hyper-variable region of Theileria spp. in Cape buffalo (Syncerus caffer) and cattle from southern Africa.

Sequence variation within the 18S SSU rRNA V4 hyper-variable region can affect the accuracy of real-time hybridization probe-based diagnostics for the detection of Theileria spp. infections. This is relevant for assays that use non-specific primers, such as the real-time hybridization assay for T. parva (Sibeko et al. 2008). To assess the effect of sequence variation on this test, the Theileria 18S gene from 62 buffalo and 49 cattle samples was cloned and ∼1000 clones sequenced. Twenty-six genotypes were detected which included known and novel genotypes for the T. buffeli, T. mutans, T. taurotragi and T. velifera clades. A novel genotype related to T. sp. (sable) was also detected in 1 bovine sample. Theileria genotypic diversity was higher in buffalo compared to cattle. Polymorphism within the T. parva hyper-variable region was confirmed by aberrant real-time melting peaks and supported by sequencing of the S5 ribosomal gene. Analysis of the S5 gene suggests that this gene can be a marker for species differentiation. T. parva, T. sp. (buffalo) and T. sp. (bougasvlei) remain the only genotypes amplified by the primer set of the hybridization assay. Therefore, the 18S sequence diversity observed does not seem to affect the current real-time hybridization assay for T. parva.

Mixed Theileria infections in free-ranging buffalo herds: implications for diagnosing Theileria parva infections in Cape buffalo (Syncerus caffer).

Buffalo-adapted Theileria parva causes Corridor disease in cattle. Strict control measures therefore apply to the movement of buffalo in South Africa and include mandatory testing of buffalo for the presence of T. parva. The official test is a real-time hybridization PCR assay that amplifies the V4 hypervariable region of the 18S rRNA gene of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). The effect that mixed T. parva and T. sp. (buffalo)-like infections have on accurate T. parva diagnosis was investigated in this study. In vitro mixed infection simulations indicated PCR signal suppression at 100 to 1000-fold T. sp. (buffalo) excess at low T. parva parasitaemia. Suppression of PCR signal was found in field buffalo with mixed infections. The T. parva-positive status of these cases was confirmed by selective suppression of T. sp. (buffalo) amplification using a locked nucleic acid clamp and independent assays based on the p67, p104 and Tpr genes. The incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, while the prevalence in buffalo outside the endemic area is currently low. A predicted increase of T. sp. (buffalo)-like infections can affect future diagnoses where mixed infections occur, prompting the need for improvements in current diagnostics.

The Hybrid II assay: a sensitive and specific real-time hybridization assay for the diagnosis of Theileria parva infection in Cape buffalo (Syncerus caffer) and cattle.

Corridor disease is an acute, fatal disease of cattle caused by buffalo-adapted Theileria parva. This is a nationally controlled disease in South Africa and strict control measures apply for the movement of buffalo, which includes mandatory testing for the presence of T. parva and other controlled diseases. Accurate diagnosis of the T. parva carrier state in buffalo using the official real-time hybridization PCR assay (Sibeko et al. 2008), has been shown to be affected by concurrent infection with T. sp. (buffalo)-like parasites. We describe the Hybrid II assay, a real-time hybridization PCR method, which compares well with the official hybridization assay in terms of specificity and sensitivity. It is, however, not influenced by mixed infections of T. sp. (buffalo)-like parasites and is as such a significant improvement on the current hybridization assay.

De novo assembled salivary gland transcriptome and expression pattern analyses for Rhipicephalus evertsi evertsi Neuman, 1897 male and female ticks.

Ticks secrete proteins in their saliva that change over the course of feeding to modulate the host inflammation, immune responses, haemostasis or may cause paralysis. RNA next generation sequencing technologies can reveal the complex dynamics of tick salivary glands as generated from various tick life stages and/or males and females. The current study represents 15,115 Illumina sequenced contigs of the salivary gland transcriptome from male and female Rhipicephalus evertsi evertsi ticks of early, mid and late feeding stages from 1320 separate assemblies using three short read assemblers. The housekeeping functional class contributed to the majority of the composition of the transcriptome (80%) but with lower expression (51%), while the secretory protein functional class represented only 14% of the transcriptome but 46% of the total coverage. Six percent had an unknown status contributing 3% of the overall expression in the salivary glands. Platelet aggregation inhibitors, blood clotting inhibitors and immune-modulators orthologous to the ancestral tick lineages were confirmed in the transcriptome and their differential expression during feeding in both genders observed. This transcriptome contributes data of importance to salivary gland biology and blood feeding physiology of non-model organisms.

Transcriptomic Analysis of Salivary Glands of Ornithodoros brasiliensis Aragão, 1923, the Agent of a Neotropical Tick-Toxicosis Syndrome in Humans.

Tick salivary glands produce and secrete a variety of compounds that modulate host responses and ensure a successful blood meal. Despite great progress made in the identification of ticks salivary compounds in recent years, there is still a paucity of information concerning salivary molecules of Neotropical argasid ticks. Among this group of ticks, considering the number of human cases of parasitism, including severe syndromes and hospitalization, Ornithodoros brasiliensis can be considered one of the major Neotropical argasid species with impact in public health. Here, we describe the transcriptome analysis of O. brasiliensis salivary glands (ObSG). The transcriptome yielded ~14,957 putative contigs. A total of 368 contigs were attributed to secreted proteins (SP), which represent approximately 2.5% of transcripts but ~53% expression coverage transcripts per million. Lipocalins are the major protein family among the most expressed SP, accounting for ~16% of the secretory transcripts and 51% of secretory protein abundance. The most expressed transcript is an ortholog of TSGP4 (tick salivary gland protein 4), a lipocalin first identified in Ornithodoros kalahariensis that functions as a leukotriene C4 scavenger. A total of 55 lipocalin transcripts were identified in ObSG. Other transcripts potentially involved in tick-host interaction included as: basic/acid tail secretory proteins (second most abundant expressed group), serine protease inhibitors (including Kunitz inhibitors), 5' nucleotidases (tick apyrases), phospholipase A2, 7 disulfide bond domain, cystatins, and tick antimicrobial peptides. Another abundant group of proteins in ObSG is metalloproteases. Analysis of these major protein groups suggests that several duplication events after speciation were responsible for the abundance of redundant compounds in tick salivary glands. A full mitochondrial genome could be assembled from the transcriptome data and confirmed the close genetic identity of the tick strain sampled in the current study, to a tick strain previously implicated in tick toxicoses. This study provides novel information on the molecular composition of ObSG, a Brazilian endemic tick associated with several human cases of parasitism. These results could be helpful in the understanding of clinical findings observed in bitten patients, and also, could provide more information on the evolution of Neotropical argasids.

Nuclear (18S-28S rRNA) and mitochondrial genome markers of Carios (Carios) vespertilionis (Argasidae) support Carios Latreille, 1796 as a lineage embedded in the Ornithodorinae: re-classification of the Carios sensu Klompen and Oliver (1993) clade into its respective subgenera.

Argasid systematics remains controversial with widespread adherence to the Hoogstraal (1985) classification scheme, even though it does not reflect evolutionary relationships and results in paraphyly for the main genera of soft ticks (Argasidae), namely Argas and Ornithodoros. The alternative classification scheme, proposed by Klompen and Oliver (1993), has problems of its own: most notably paraphyly of the subgenus Pavlovskyella and the controversial grouping together of the subgenera Alectorobius, Antricola, Carios, Chiropterargas, Nothoaspis, Parantricola, Reticulinasus and Subparmatus into the genus Carios. Recent phylogenetic analyses of 18S/28S rRNA sequences and mitochondrial genomes agree with the scheme of Klompen and Oliver (1993), with regard to the paraphyly of Pavlovskyella, placement of Alveonasus, Ogadenus, Proknekalia and Secretargas in the Argasinae and placement of Carios and Chiropterargas in the Ornithodorinae (Mans et al., 2019). The Carios clade and its constituent subgenera remain controversial, since the phylogenetic position of its type species Carios (Carios) vespertilionis Latreille, 1796 (formerly Argas vespertilionis) has not been determined with confidence. The current study aimed to resolve Carios sensu lato Klompen and Oliver, 1993, and Carios sensu stricto Hoogstraal, 1985, by determining and analysing phylogenetic nuclear and mitochondrial markers for C. (C.) vespertilionis. Both the nuclear and mitochondrial markers support placement of Carios s.s. within the subfamily Ornithodorinae, but to the exclusion of the clade that includes the 6 other subgenera that are part of Carios s.l. Klompen and Oliver (1993), namely Alectorobius, Antricola, Nothoaspis, Parantricola, Reticulinasus and Subparmatus. These 6 subgenera form a monophyletic clade that might be placed as new subgenera within the genus Alectorobius, or elevated to genera. Given the substantial differences in biology among these subgenera, we propose that these 6 subgenera be elevated to genera. Thus, we propose to modify the classification scheme of Mans et al. (2019) so that the subfamily Argasinae now has six genera, Alveonasus, Argas (subgenera Argas and Persicargas), Navis, Ogadenus, Proknekalia and Secretargas, and the subfamily Ornithodorinae has nine genera, Alectorobius, Antricola (subgenera Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (subgenera Microargas, Ornamentum, Ornithodoros, Pavlovskyella and Theriodoros), Otobius, Reticulinasus and Subparmatus (genera indicated in bold).

Investigations into the carrier-state of Theileria sp. (buffalo) in cattle.

The Theileria are apicomplexan parasites transmitted by ticks to vertebrate hosts. Most Theileria species exhibit some form of host or vector specificity, since under endemic conditions only a limited number of tick species act as vectors and not all vertebrate hosts are able to maintain a persistent carrier state. Data for Theileria sp. (buffalo) suggest host specificity for African buffalo (Syncerus caffer). However, T. sp. (buffalo) infections in cattle co-grazing with African buffalo have been reported in Kenya and schizonts were cultured from these infected cattle, raising questions regarding host specificity. A Corridor disease outbreak in 2013 on a ranch in South Africa where cattle co-grazed with Theileria parva and T. sp. (buffalo) infected buffalo presented the opportunity to investigate the possible carrier-state of T. sp. (buffalo) in cattle using real-time PCR analysis. Almost all buffalo (n = 19, 95%) were infected with T. sp. (buffalo) and showed CP values (22-20) indicative of high parasitemia similar to that observed for buffalo in endemic areas. Conversely, only ~14-27% cattle (n = 69, 100, 96) were positive with CP values (31-40) suggesting low parasitemia and a carrier state epidemiology different from African buffalo. Long term monitoring of T. sp. (buffalo) positive cattle showed that most cattle lost their parasitemia or presented fluctuating parasitemia around the PCR assay detection limit. A single splenectomized animal showed a persistent carrier state. The general trends and epidemiology observed in cattle infected with T. sp. (buffalo) are similar to that seen for buffalo-adapted T. parva, for which a defined carrier state in cattle has not yet been proven. The study suggests that cattle may be infected by T. sp. (buffalo) but are not definitive hosts that play an important part in the epidemiology of this parasite.

Theileria-infected cell line from an African buffalo (Syncerus caffer).

Mononuclear cells were isolated from the peripheral blood of a buffalo infected with a Theileria sp. using density gradient centrifugation, and the cells were put into culture flasks covered by a monolayer of bovine endothelial cells. Twenty days after culture initiation, cells containing macroschizonts were detected in Giemsa-stained smears. The first subculture was carried out on day 45 of culture propagation. Subsequently, infected cells were subcultured twice a week, and each time 1 to 2 x 10(6) per milliliter cells were harvested. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. Sequence data and phylogenetic analysis using the 18S rRNA gene indicated a close relationship to Theileria sp. buffalo, previously described in literature. Here, the first successful attempt to establish a macroschizont-infected lymphoblastoid cell line of Theileria sp. (buffalo) from an African buffalo is described.

Investigation into limiting dilution and tick transmissibility phenotypes associated with attenuation of the S24 vaccine strain.

BACKGROUND: Babesia bovis is the causal agent of Asiatic redwater, transmitted by the pandemic tick Rhipicephalus (Boophilus) microplus. Disease control may target the tick vector using acaricides or anti-tick vaccines, or the parasite using chemoprophylaxis or anti-parasite vaccines. Current anti-parasite vaccines comprise live blood vaccines using attenuated B. bovis strains. Attenuation is attained by rapid passage that may result in different phenotypes such as reduced virulence, non-transmissibility by the tick vector, inability to sequester in the host (lack of limiting dilution) and limited genetic diversity. Attenuation and phenotypes may be linked to selection of subpopulations during rapid passage. The South African B. bovis S24 vaccine strain comprise a subpopulation that present low virulence, non-transmissibility, lack of limiting dilution phenotype and the presence of a single A558 Bv80 allele. The S24 strain could be co-transmitted with a field strain (05-100) suggesting sexual recombination. The present study investigated the change in phenotype for the S24 vaccine strain during rapid passage and co-transmission. METHODS: Vaccine phenotype change during passage as well as co-transmissibility was monitored using Bv80 allele specific PCR, limiting dilution and Illumina-based genome sequencing. RESULTS: The S24 population could not be rescued from the S16 passage as previously attained suggesting that selection of the S24 vaccine strain was a serendipitous and stochastic event. Passage from S16 to S24 also resulted in loss of the limiting dilution phenotype. Genome sequencing indicated sexual recombination during co-transmission with the 05-100 field strain. Analysis of the recombinant strain indicate that VESA1, smORF and SBP2 family members are present and may be responsible for the limiting dilution phenotypes, while various regions may also be responsible for the tick transmission phenotype. CONCLUSIONS: The molecular basis for tick transmission and limiting dilution phenotypes may be defined in future using selection based on these traits in combination with sexual recombination.

Corridor disease (buffalo-associated Theileria parva) outbreak in cattle introduced onto a game ranch and investigations into their carrier-state.

East Coast fever (Theileria parva infection in cattle) was eradicated from South Africa in the mid-1900. However, another form named Corridor disease (CD), associated with T. parva carrier buffaloes exists and outbreaks have increased in endemic areas. The occurrence of a CD carrier state in cattle under field situations has not been demonstrated but remains a subject of controversy. The current study investigated the T. parva carrier state following a severe outbreak in cattle introduced onto a game ranch. Monitoring of the outbreak included clinical signs, mortality, microscopy, serology, real-time PCR and xenodiagnoses. The herd of cattle received block treatment using oxytetracyclines (OTC) by the farmer during the outbreak. Cattle were sampled early during the outbreak and twice within the following 75 days. All buffaloes were tested for a T. parva carrier state. Two batches of questing adult R. appendiculatus were collected at the time of disease occurrence and a year later. These ticks were fed on susceptible cattle under controlled conditions and monitored for disease transmission. Ticks infected with a buffalo-derived stock of T. parva were fed on one bovine under controlled conditions and simultaneously injected with OTC, simulating the infection and treatment method of vaccination and was used as a positive control. Clean R. appendiculatus nymphs were fed on four recovered PCR positive cattle from the outbreak and on the positive control animal. The adult ticks were tested for infectivity by xenodiagnoses on susceptible bovines. For the initial outbreak the CD prevalence was 62.3% with a mortality rate of 29.5%. However, the outbreak was contained by block OTC treatment of the herd since only 3.4% cattle subsequently died until the end of the investigations. Adult ticks fed on one field bovine and the laboratory established T. parva carrier both transmitted fatal infections to susceptible cattle. Ticks fed on two field cattle transmitted T. taurotragi and one failed to transmit any infection. Questing adult R. appendiculatus collected during the outbreak transmitted fatal CD to two bovines while ticks collected a year later transmitted T. taurotragi. These findings demonstrated the effectiveness of disease control either by cattle treatment using OTC simulating the ITM or by intensive cattle dipping following the outbreak or by both interventions. The potential risk of creating carrier cattle by OTC treatment during CD outbreaks should be considered, supporting the continued control measures of segregation of cattle and buffalo herds.

Risk of establishment of canine leishmaniasis infection through the import of dogs into South Africa.

Canine leishmaniasis is a vector-borne disease caused by protozoa of the genus Leishmania that affect dogs, humans and wildlife. Sandflies of the genera Phlebotomus and Lutzomyia are the primary vectors. Canine leishmaniasis is an exotic and controlled disease in South Africa. The main purpose of our risk assessment study was to evaluate the likelihood that this exotic disease could enter and be established in South Africa through importation of live dogs. Risk analysis to the spread of the disease follows the World Organization for Animal Health (OIE) formal method of quantitative risk assessment documented as a step-by-step process. We have identified and discussed 11 possible risk factors involved in three steps for final assessment. The annual average number of diagnostic tests performed on imported dogs from 44 countries for 2011-2015 was 1158. Leishmania is reported to occur in 21/44 (47.7%) exporting countries. A total of 71.1% of Leishmania positive dogs were imported from these endemic countries. The yearly percentage of Leishmania positive dogs ranged from 0.2% to 2%. Three confirmed clinical and fatal cases of leishmaniasis in dogs of unidentified origin have been reported by our laboratory and the state veterinarians. The disease has been reported in neighbouring countries as well as the putative sandfly vectors. This study concluded that the risk for the introduction and degree of uncertainty of Leishmania in imported dogs in South Africa are moderate. Risk mitigation and recommendations such as investigations into possible occurrence of autochthonous leishmaniasis in the country, surveillance in its wildlife reservoirs and systematic surveillance of sandfly populations are discussed.