Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa.

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

The Nexus between Bovine Tuberculosis and Fasciolosis Infections in Cattle of the Kafue Basin Ecosystem in Zambia: Implications on Abattoir Surveillance.

Bovine tuberculosis (bTB) and fasciolosis are important but neglected diseases that result in chronic infections in cattle. However, in Zambia, these diseases are mainly diagnosed at abattoirs during routine meat inspection. Albeit the coinfection status, these diseases have been reported as nothing more than normal separate findings without an explanatory phenomena. Forthwith, we formulated this study to assess the possible association of the two diseases in a known high prevalence area on the Kafue basin ecosystem. Of the 1,680 animals screened, 600 (35.7%; 95% CI 33.4%-38%) and 124 (7.4%; 95% CI 6.1%-8.6%) had fasciolosis and tuberculous lesions; respectively, whilst 72 had both fasciola and tuberculous lesions representing 12% (95% CI 9.4%-14.6%) and 58.1% (95% CI; 49.3%-66.7%) of the total positives for fasciola and tuberculosis, respectively. Jaundice was seen in 304 animals, 18.1% (95% CI; 16.3%-19.9%) and was significantly correlated to fasciolosis (r = 0.59, P < 0.0001). A significant association (χ(2) = 76.2, df = 1, and P < 0.0001) was found between fasciolosis and tuberculous lesions. Simple logistic regression intimated fasciolosis as a strong predictor for tuberculous lesions with animals that had fasciola being five times more likely to have tuberculous lesions (odds ratio = 4.8, 95% CI: 3.3-7.0). This study indicates that transmission and spatial risk factors of communicable and noncommunicable diseases such as bTB and fasciolosis can be correlated in an ecosystem such as the Kafue flats.

Comparative Intradermal Tuberculin Testing of Free-Ranging African Buffaloes (Syncerus caffer) Captured for Ex Situ Conservation in the Kafue Basin Ecosystem in Zambia.

Bovine tuberculosis (BTB) is endemic in African buffaloes (Syncerus caffer) in some National Parks in Southern Africa, whilst no studies have been conducted on BTB on buffalo populations in Zambia. The increased demand for ecotourism and conservation of the African buffalo on private owned game ranches has prompted the Zambian Wildlife Authority (ZAWA) and private sector in Zambia to generate a herd of "BTB-free buffaloes" for ex situ conservation. In the present study, 86 African buffaloes from four different herds comprising a total of 530 animals were investigated for the presence of BTB for the purpose of generating "BTB free" buffalo for ex-situ conservation. Using the comparative intradermal tuberculin test (CIDT) the BTB status at both individual animal and herd level was estimated to be 0.0% by the CIDT technique. Compared to Avian reactors only, a prevalence of 5.8% was determined whilst for Bovine-only reactors a prevalence of 0.0% was determined. These results suggest the likelihood of buffalo herds in the Kafue National Park being free of BTB.

Detection of Babesia spp. in free-ranging Pukus, Kobus vardonii, on a game ranch in Zambia.

Babesia spp. were detected from 4 asymptomatic pukus captured on a game ranch in central Zambia in October 2008. Blood smears were examined in 4 species of aymptomatic free-ranging antelopes, namely the puku (Kobus vordanii), reedbuck (Redunca arundinum), bushbuck (Tragelaphus sylvaticus), and kudu (Tragelaphus strepsiceros), and showed the presence of Babesia parasites only in the puku. In the puku, the prevalence of babesiosis was estimated at 33.3% (n = 12), while the overall prevalence in all examined animals was 8.5% (n = 47). The parasites showed morphological characteristics of paired ring-like stages with the length varying between 1.61 µm and 3.02 µm (mean = 2.12 µm, n = 27; SD = 0.76 µm). Both the infected and non-infected pukus showed good body condition scores (BCS), while the dominant tick species detected from all animals were Rhipicephalus appendiculatus, Rhipicephalus spp., and Boophilus spp. To our knowledge this is the first report of Babesia spp. infection in pukus in Zambia. These findings suggest that wildlife could play an important role in the epidemiology of babesiosis in Zambia.

Investigating effects of parasite infection on body condition of the Kafue lechwe (Kobus leche kafuensis) in the Kafue basin.

BACKGROUND: The Kafue lechwe (Kobus leche Kafuensis), a medium-sized semi-aquatic antelope, is endemic to the Kafue basin of Zambia. The population of the Kafue lechwe has significantly dropped in the last decades leading to its subsequent inclusion on the red list of endangered species. In order to save the remaining population from extinction, it has become increasingly important that the impact of parasite infection and infestation on the Kafue lechwe is investigated. FINDINGS: Endoparasites accounted for the majority of parasites observed from a study of 40 Kafue lechwe occurring in the the Kafue basin. Amphistoma spp. were present in all animals examined, while Fasciola gigantica had a prevalence rate of 0.525 (95% CI: 0.36 to 0.69) and species of Schistosoma 0.3 (95% CI: 0.15 to 0.45). Among the ectoparasites, Strobiloestrous vanzyli, had a prevalence rate of 0.15 (95% CI: 0.03 to 0.27), while Rhipicephalus appendiculatus had a prevalence of 0.075 (3/40). Our findings indicate that body condition was not influenced by the parasitic infestation in Kafue lechwe. There was no association between sex and parasitic burden (odds ratio = 0.3, 95% CI: 0.8-1.3). However, an association between age and parasitic burden was observed as older animals above 15 years were more likely to get parasite infections than those aged between 1-5 years (odds ratio = 1.5, 95% CI: 1.1-2.4). CONCLUSION: Overall, there was no evidence that parasitic infections and infestations adversely affected the lechwe population on the Kafue basin. These findings indicate that ecto- and endo-parasite infestation might not play a significant role in reducing the Kafue lechwe population on the Kafue basin.

Trypanosoma brucei Infection in asymptomatic greater Kudus (Tragelaphus strepsiceros) on a game ranch in Zambia.

Trypomastogotes of Trypanosoma brucei were detected from 4 asymptomatic kudus (Tragelaphus strepsiceros) on a game ranch located approximately 45 km north east of Lusaka, Zambia. Blood smears examined from 14 wildlife species comprising of the impala (Aepyceros melampus), Kafue lechwe (kobus leche kafuensis), sable antelope (Hippotragus niger), tsessebe (Damaliscus lunatus), warthog (Phacochoerus aethiopicus), puku (Kobus vardoni), zebra (Equus burchelli), waterbuck (Kobus ellipsiprymnus), bushbuck (Tragelaphus scriptus), reedbuck (Redunca arundinum), wilderbeest (Connochaetes taurinus), hartebeest (Alcephelus lichtensteini), African buffalo (Syncerus caffer), and kudu (Tragelaphus strepsiceros) showed that only the kudu had T. brucei. Although game ranching has emerged to be a successful ex-situ conservation strategy aimed at saving the declining wildlife population in the National Parks, our findings suggest that it has the potential of aiding the re-distribution of animal diseases. Hence, there is a need for augmenting wildlife conservation with disease control strategies aimed at reducing the risk of disease transmission between wildlife and domestic animals.

Detection of Theileria parva antibodies in the African buffalo (Syncerus caffer) in the livestock-wildlife interface areas of Zambia.

A serolocigical survey was conducted for the detection of Theileria parva antibodies in 176 African buffaloes (Syncerus caffer) sampled between 1996 and 2005 in livestock-wildlife interface areas of Zambia. Rhipicephalus appendiculatus, Rhipicephalus species, and Amblyomma variegatum were the most abundant tick species identified on buffaloes. T. parva sero-positives were reported in buffaloes sampled from game management areas at Mlanga and Nanzhila bordering the Kafue National Parks and in the Lochnivar National Park while buffaloes sampled from Lower Zambezi National Park were sero-negative. Given that Game Management Areas serve as interface areas that permit the co-existence of livestock and wildlife in similar ecological habitats our findings suggest that buffaloes could play a significant role in the epidemiology of theileriosis in livestock-wildlife interface areas. Thus far, the disease has only been reported in livestock and is herein being reported in the African buffalo for the first time in Zambia.

Quantitative analysis of cytokine mRNA expression and protozoan DNA load in Theileria parva-infected cattle.

Theileria parva (T. parva) causes a highly serious bovine disease called East Coast fever (ECF), which is characterized by pyrexia, dyspnea and cachexia and is of great economic importance in African countries. We hypothesize that the clinical symptoms of ECF could be explained by a cytokine dysregulation. In this study, we investigated the relationship between T. parva DNA load and expression levels of cytokine mRNAs in leukocytes from experimentally infected calves by quantitative PCR. The p104 gene, which encodes the T. parva 104 kDa microneme-rhoptry protein, was detected in cattle blood from day 10 after T. parva-infected tick infestation, and the protozoan DNA load was increased together with severity of disease. The mRNA expressions of pro-inflammatory cytokines, such as interleukin (IL)-1beta and IL-6, were up-regulated with protozoan DNA load increasing. In addition, the level of a type-2 cytokine (IL-10) transcript was also increased during the acute phase. In contrast, the down-regulation or no detectable levels of the expression of type-1 cytokines, such as IL-2 and interferon (IFN)-gamma were observed in T. parva-infected animals. Thus, our observations indicated that high protozoan load and resulting intense inflammatory responses might be involved in the severity of clinical signs observed in T. parva-infection.

PCR-based detection of blood parasites in cattle and adult Rhipicephalus appendiculatus ticks.

To ascertain the infection rate for tick-borne pathogens in Zambia, an epidemiological survey of Theileria parva, Babesia bigemina and Anaplasma marginale in traditionally managed Sanga cattle was conducted using PCR. Of the 71 native Zambian cattle, 28 (39.4%) were positive for T. parva, 16 (22.5%) for B. bigemina and 34 (47.9%) for A. marginale. The mixed infection rate in cattle was 8.5% (6/71), 16.9% (12/71), 7.0% (5/71) and 2.8% (2/71) for T. parva/B. bigemina, T. parva/A. marginale, B. bigemina/A. marginale and T. parva/B. bigemina/A. marginale, respectively. To predict the risk for transmission of tick-borne pathogens from ticks to cattle, a total of 74 Rhipicephalus appendiculatus ticks were collected from a location where cattle had been found positive for T. parva. Of the ticks collected, 10 (13.5%) were found to be PCR-positive for T. parva. The results suggest that the infection rate for tick-borne pathogens was relatively high in Sanga cattle and that adult R. appendiculatus ticks were highly infected with T. parva.

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites.

A loop-mediated isothermal amplification (LAMP) technique has been used as a novel nucleic acid detection method, whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. In this study, we designed two sets of the LAMP primers for rhoptry-associated protein-1 genes of Babesia bovis and B. bigemina, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers to construct a multiplex LAMP (mLAMP) method by combining these two sets totaling eight primers. The mLAMP method was distinguishable between B. bovis and B. bigemina, simultaneously, due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 10(3) and 10(5) times higher on the detection limits for B. bovis and B. bigemina, respectively, than those of the classical PCR methods. Of 40 blood samples collected from cattle living in Ghana, 12 and 27% were positively detected by the mLAMP for B. bovis and B. bigemina, respectively. Furthermore, 14 and 23% of 90 blood samples from cattle in Zambia showed mLAMP-positive reactions to B. bovis and B. bigemina, respectively. These findings indicate that this mLAMP method is a new convenient tool for simultaneous detection of the bovine Babesia parasites.

Attachment duration required for Rhipicephalus appendiculatus to transmit Theileria parva to the host.

Theileria parva, the agent of East Coast fever (ECF), is transmitted to the host during the blood meal feeding of Rhipicephalus appendiculatus ticks. In order to investigate the relationship between the attachment duration of R. appendiculatus and the transmission of T. parva, infected adult ticks were allowed to attach to naive mice for variable lengths of time. Attached ticks and host animal's back skin biopsies from the tick attachment site were collected daily, starting from 24 hours post-tick attachment, and used for seminested polymerase chain reaction (PCR) detection of T. parva. T. parva-infected ticks started to transmit the parasites from 72 hours post-tick attachment. As expected, the transmission of T. parva from ticks to mouse skin increased with duration of tick attachment. Transmission of the parasites was 77.7%, 100%, 85.5%, and 100% on day 4, 5, 6, and 7 post-tick attachment, respectively, as could be detected from mice skin biopsies taken from T. parva-infected ticks' attachment sites. These results have important implications for our understanding of early events in the transmission of T. parva and would help in the development of effective pharmacologic substances and/or vaccines against ticks.

Species-specific loop-mediated isothermal amplification (LAMP) for diagnosis of trypanosomosis.

In this study, we developed loop-mediated isothermal amplification (LAMP) for the specific detection of both animal and human trypanosomosis using primer sets that are designed from 5.8S rRNA-internal transcribed spacer 2 (ITS2) gene for Trypanosoma brucei gambiense, 18S rRNA for both T. congolense and T. cruzi, and VSG RoTat 1.2 for T. evansi. These LAMP primer sets are highly sensitive and are capable of detecting down to 1 fg trypanosomal DNA, which is equivalent to approximately 0.01 trypanosomes. LAMP is a rapid and simple technique since it can be carried out in 1 h and requires only a simple heating device for incubation. Therefore, LAMP has great potential of being used for diagnosis of trypanosomosis in the laboratory and the field, especially in countries that lack sufficient resources needed for application of molecular diagnostic techniques.

Acquisition and transmission of Theileria parva by vector tick, Rhipicephalus appendiculatus.

In order to investigate the transmission dynamics of Theileria parva (T. parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T. parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T. parva mixture became PCR-positive for T. parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T. parva infection. Furthermore, the T. parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T. parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T. parva DNA. However, since the rate of transmission of T. parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures.

Disease constraints for utilization of the African buffalo (Syncerus caffer) on game ranches in Zambia.

Eco-tourism depending on wildlife is becoming increasingly profitable and landowners are beginning to favor game farming and ecotourism. In these areas, large-scale translocation of wildlife involves a diversity of species and large populations. The African buffalo (Syncerus caffer) is one of the major tourist attractions in Zambia. It accounts for 8.7% and 12.4% of the total animal species hunted in the Game Management Areas and the total hunting revenue earned in Zambia, respectively. It is ecologically an important animal species essential for the purpose of habitat control and facilitating the provision of suitable grazing pastures. However, the rearing of the African buffalo on game ranches has been hampered by its carrier state of the Southern Africa Terroritory (SAT) serotypes of foot and mouth disease virus (FMD). The African buffalo is also known to be a carrier of Theileria parva lawrencei, the causative agent of corridor disease (CD) that continues to have devastating effects on the livestock industry in Zambia. In addition, the importation of buffaloes from countries with populations endemic to bovine tuberculosis is highly restricted. Veterinary regulations in Zambia, strongly advocate against the translocation of buffaloes from protected areas to private ranches for disease control purposes thereby mounting a considerable constraint on the economic and ecological viability of the industry. It is hoped that this review will motivate the relevant government authorities in exploiting ways in which this animal species play a central role in eco-tourism.

Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia.

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard PCR. Salmonella invA was detected in 12 of 14 carcass rinses by ICAN, while in 7 of 14 rinses by standard PCR. These results indicate that ICAN is an efficient, sensitive and simple system to detect invA of Salmonella species in developing countries such as Zambia.