Developmental stages and molecular phylogeny of Hepatozoon fitzsimonsi (Dias 1953) (Adeleorina: Hepatozoidae) in tortoises Stigmochelys pardalis (Cryptodira: Testudinidae) and ticks of the genus Amblyomma (Acari: Ixodidae) from South Africa.

BACKGROUND: Hepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species' reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach. METHODS: Specimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database. RESULTS: Peripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi. CONCLUSIONS: The present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.).

Molecular detection of Fasciola, Schistosoma and Paramphistomum species from freshwater snails occurring in Gauteng and Free State provinces, South Africa.

Trematodiases are diseases caused by snail-borne trematode parasites that infect both animals and humans. Fascioliasis, schistosomiasis and paramphistomosis are some of these diseases and they affect millions of livestock, leading to significant economic losses. The aim of the study was to document freshwater snails occurring in selected study sites in the Free State and Gauteng provinces as well as identify and detect larval trematodes that they harbour. Samples were collected from a total of five study sites within two provinces of South Africa. Morphological features were used to identify snail species and were further confirmed genetically by polymerase chain reaction (PCR), sequencing and phylogenetic analysis. The larval trematodes were also detected by PCR, PCR-Restriction Length Fragment Polymorphism (PCR-RLFP), sequencing and phylogenetic analysis. A total of 887 freshwater snails were collected from Free State (n = 343) and Gauteng (n = 544). Five different genera of snails as well as species in the Succineidae family were documented. The snails in descending order of abundance were identified as: Physa (P.) spp. (51%), Succineidae spp. (20%), Galba (G.) truncatula (12%), Pseudosuccinea (Ps.) columella (10%), Planorbella (Pl.) duryi (6%) and Bulinus (B.) truncatus (1%). Approximately 272 DNA pools were created for genetic identification of snails and detection of trematode parasites. Schistosoma species were not detected from any of the snail species. A total prevalence of 46% was obtained for Fasciola hepatica in the identified snail species across all study sites. Overall, the highest prevalence of F. hepatica was obtained in Physa species (24%), whilst the lowest was observed in B. truncatus snails (1%). Forty three percent (43%) of the snail samples were PCR positive for Paramphistomum DNA. This is the first report of P. mexicana in South Africa. Fasciola hepatica was confirmed from all obtained snail species per study site. This is the first reported detection of F. hepatica in Pl. duryi and P. mexicana snails as well as the first confirmation of natural infection from P. acuta in South Africa.

First report of gastrointestinal nematodes and coccidia parasites from free-range chickens in Mafeteng district, Lesotho.

Free-range chickens are an integral part of poultry production in many developing countries. In the Mountain Kingdom of Lesotho, the majority of the population own free-range chickens, which serve a variety of purposes including being a source of meat, eggs and use for cultural rituals amongst others. However, there is lack of scientific studies on occurrence of parasitic infections on free-range chickens in Lesotho. The aim of this study was to document common gastrointestinal parasites infecting free-range chickens in four villages of Mafeteng District in Lesotho. A total number of 462 pooled faecal samples were collected from various households in HaKubutu (n = 114), HaMatjeka (n = 120), HaMpalipali (n = 120) and Thabang Villages (n = 108) which were subjected to microscopic examination using McMaster technique. The prevalence of gastrointestinal parasite infection was as follows: Eimeria tenella (12.8%), Ascaridia galli (10.4%) and Heterakis gallinarum (5%). The prevalence for H. gallinarum and Ascaridia galli were comparatively higher during the hot-wet season (7.1% and 2.8% respectively) than the cold-dry season (3.2% and 1.9% respectively) and varied significantly (P < 0.05). For E. tenella, the oocysts per gram were slightly higher in the cold-dry season than the hot-wet season. Polymerase chain reaction only amplified DNA from six (29%) adult A. galli worms of which two amplicons were successfully sequenced. The obtained cytochrome C oxidase subunit 1 partial gene sequences displayed 98-100% identity with South African A. galli isolates. This is the first scientific study on prevalence and molecular characterization of nematodes and coccidia species infecting free-range village chickens in Lesotho. The findings can be used to review management of gastrointestinal nematodes and protozoal parasites of free-range chickens in Lesotho.

Molecular Detection and Genetic Characterization of Ehrlichia ruminantium Harbored by Amblyomma hebraeum Ticks of Domestic Ruminants in North West Province, South Africa.

Ehrlichia ruminantium (E. ruminantium) is the causative agent of heartwater disease and it is mainly transmitted to livestock by Amblyomma hebraeum (A. hebraeum) tick in South Africa. This study investigated the occurrence of E. ruminantium and its genetic diversity in ticks within Ngaka Modiri Molema district of North West Province in South Africa. Genomic DNA was extracted from whole A. hebraeum ticks totaling 876 and resulted in a total of 292 pooled DNA samples. Firstly, conventional PCR was used to detect Ehrlichia spp. targeting the dsbA gene, followed by nested PCR targeting the Map1 gene performed on DNA pool samples that were positive from the first PCR. One hundred and six tick DNA pool samples were positive by dsbA gene PCR for the presence of Ehrlichia spp. with minimum infection rate (MIR) of 121, while 13/106 were positive by Map1 PCR with MIR of 15. Different E. ruminantium Map1 genotypes (NWUe1, NWUe2, NWUe3, NWUe5, and NWUe6) were detected from tick samples and were closely related to more than 13 gene sequences of E. ruminantium from the NCBI GenBank database. These findings suggest that there is a significant diversity of E. ruminantium infecting ticks in the study area.

Molecular Detection of Tick-Borne Bacteria from Amblyomma (Acari: Ixodidae) Ticks Collected from Reptiles in South Africa.

Reptiles are hosts for various tick species and tick-associated organisms, many of which are zoonotic. However, little is known about the presence and diversity of tick-borne bacteria infecting reptiles and their ticks in South Africa. Amblyomma ticks (n = 253) collected from reptiles were screened for the presence of Coxiella, Anaplasma, Rickettsia, and Borrelia species by amplification, sequencing and phylogenetic analysis of the 16S rRNA, 23S rRNA, gltA, OmpA, and Flagellin genes, respectively. This study recorded the presence of reptile associated Borrelia species and Coxiella-like endosymbiont in South Africa for the first time. Furthermore, a spotted fever group Rickettsia species was observed in 7 Amblyomma marmoreum and 14 Amblyomma sylvaticum from tortoises of genera Kinixys and Chersina. Francisella-like endosymbiont was observed from 2 Amblyomma latum collected from the Mozambique spitting cobra, Naja mossambica. Coxiella burnetii and Anaplasma spp., were not detected from the current samples. Although the direct evidence that reptiles can act as reservoir hosts remains to be determined, observations from this study provide indications that reptilian ticks may play a role in the transmission of pathogenic bacteria to homothermic animals. Furthermore, the absence of Anaplasma spp., and C. burnetii does not mean that these pathogens should be completely neglected.

Molecular survey for tick-borne pathogens and associated risk factors in sheep and goats in Kano Metropolis, Nigeria.

Tick-borne pathogens (TBPs) pose an increased health and productivity risk to livestock in sub-Saharan Africa. Information regarding TBPs infecting small ruminants in Kano metropolis is scarce. Therefore, we investigated the molecular epidemiology of tick-borne pathogens of economic importance from sheep and goats in Kano, Nigeria using Polymerase chain reaction (PCR). A total of 346 blood DNA samples were collected from small ruminants and analyzed for TBPs using PCR and sequencing. Risk of infection was determined for age, sex, breed and animal species. Our results indicate the absence of piroplasmids (Babesia/Theileria) and Rickettsia spp. infections. The overall prevalence for Anaplasma spp. was 9.25% (32/346) with a higher prevalence in goats 13.59% (25/184) compared with sheep 4.32% (7/162). With respect to age of animals, goats >4 years had the highest prevalence of 32.45% (11/37) which differs significantly (P = 0.0059) compared with other age categories. Cross breed goats had a prevalence of 15.63% (5/32) compared with Kano brown breed 14.08 (20/142). Sex significant difference (P = 0.029) was observed in the goats with females having the highest prevalence 20.89% (14/67) compared with males 9.40% (11/117). Furthermore, with regards to sheep, no significant difference (P > 0.05) was observed with respect to age and breed. Finally, no significant difference (P > 0.05) was observed with the prevalence of Anaplasma spp. due to Body condition score (BCS) in both sheep and goats. Conclusively, the occurrence of TBPs in small ruminants is low. Continuous efforts in tick control must be sustained to ensure high productive yield and reduced disease burden associated with TBPs of sheep and goats in Kano metropolis.

Molecular screening of ticks of the genus Amblyomma (Acari: Ixodidae) infesting South African reptiles with comments on their potential to act as vectors for Hepatozoon fitzsimonsi (Dias, 1953) (Adeleorina: Hepatozoidae).

In South Africa, the role of reptilian ticks in the transmission of haemoparasites is lacking, in part, due to limited information on tick diversity and their associated haemoparasites. The aim of this research was to identify tick species parasitizing reptiles and to molecularly screen these ectoparasites for species of the blood apicomplexan genus Hepatozoon. Samples were collected from Ndumo Game Reserve, KwaZulu-Natal, and the Cape Columbine region, Western Cape. Reptiles collected included 2 snakes, 2 monitor lizards of a single species respectively, as well as 17 tortoises of four species. Ticks collected from these were morphologically identified as Amblyomma latum (n = 2) and Amblyomma marmoreum (n = 98), this identification was molecularly confirmed using 16S rRNA and CO1 genes. Screening for Hepatozoon was done by amplifying the 18S rRNA gene. A species of Hepatozoon, Hepatozoon fitzsimonsi, was identified from A. marmoreum ticks, with an overall prevalence of 10%. This Hepatozoon species, has been described parasitizing tortoises from southern Africa, and has been reported from ticks infesting tortoises from Kenya, East Africa. Even though ticks have been suggested to be the likely vector of this Hepatozoon species, with this supported by the findings of Hepatozoon-like developmental stages in ticks collected off of infected tortoises, a recent systematic revision placed this species in a newly erected genus Bartazoon, a genus vectorised by biting insects. The present study thus provides further support for ticks acting as the potential vectors of H. fitzsimonsi.

Parasites of veterinary importance from domestic animals in uMkhanyakude district of KwaZulu-Natal province.

This study investigated the occurrence and phylogenetic relationship of protozoan parasites and Ehrlichia infecting domestic animals from three municipalities in uMkhanyakude district of KwaZulu-Natal province, South Africa. A total of 208 blood samples collected from clinically healthy cattle, sheep, goats and dogs from uMkhanyakude district were examined by polymerase chain reaction (PCR) assays, using either genus or species-specific primers to determine the occurrence and phylogenetic relationship of various protozoan parasites and Ehrlichia of veterinary importance. A total of 5/109 (4.6%) cattle were PCR-positive for the presence of Toxoplasma gondii, 33/109 (30.3%) for Babesia bovis, 24/109 (22.02%) for Babesia bigemina and 20/109 (18.3%) for Trypanosoma sp., while 3/10 (30%) of sheep were PCR-positive for Theileria ovis and none of the goats were positive for any of the detected pathogens. The co-infection of 4/109 (3.7%) B. bovis and B. bigemina was detected in cattle. Only Ehrlichia canis was detected in dogs with infection rate of 20/48 (41.7%). Sequences of PCR-positive isolates (B. bovis, B. bigemina, E. canis, T. ovis and T. gondii) showed that they were closely related to their relevant species from various countries. These findings have expanded our knowledge about the prevalence and phylogenetic similarity between protozoan parasites and Ehrlichia isolates of South African origin. To date, this is the first study in South Africa to detect T. gondii infections from cattle blood using PCR.