Differential diagnosis of tick-borne diseases and population genetic analysis of Babesia bovis and Babesia bigemina

Abstract: Tick-borne diseases are a constraint to livestock production in many developing countries. They are responsible for high morbidity and mortality resulting in decreased production of meat, milk and other livestock by-products. The most important tick-borne diseases of livestock in sub-Saharan Africa are East coast fever (caused by Theileria parva), babesiosis (caused by Babesia bigemina and Ba. bovis), anaplasmosis (caused by Anaplasma marginale) and heartwater (caused by Ehrlichia ruminantium). Despite their economic importance, information on the epidemiology of these diseases in many countries is often lacking or inadequate, resulting in inappropriate disease control strategies being implemented. The availability of specific, sensitive and cost-effective diagnostic methods is important in the design and implementation of effective disease control strategies. In this study PCR assays based on the 18S and 16S rRNA gene sequences, that could identify Theileria / Babesia and Anaplasma / Ehrlichia pathogens of cattle respectively, were developed. In addition, PCR assays based on the β-tubulin gene that could detect T. parva, Ba. bigemina, Ba. bovis and T. taurotragi, and PCR assays based on the cytochrome b gene that could diagnose infection by Ba. bigemina and Ba. bovis were also developed. When the 18S and 16S rRNA gene PCR assays were combined into a multiplex PCR assay, Ba. bigemina and E. ruminantium DNA did not amplify and some non-specific bands were observed following agarose gel electrophoresis. The β-tubulin gene multiplex PCR assay for the diagnosis of T. parva, Ba. bovis and Ba. bigemina worked relatively well when used on laboratory-derived parasite DNA preparations. However, when it was used on field samples collected on FTA cards, multiple non-specific bands were observed after agarose gel electrophoresis of the PCR products. The 18S and 16S rRNA gene PCR assays were used for an epidemiological study of tick-borne diseases of cattle in Central and Eastern Zambia in the wet and dry seasons. All the disease pathogens under study (T. parva, T. mutans, T. taurotragi, Ba. bovis, Ba. bigemina, Anaplasma spp and E. ruminantium) were prevalent in all the regions of the country in both seasons. However, variation was observed in the prevalence of these pathogens between the regions and the seasons. A number of risk factors, associated with the occurrence of tick-borne pathogens in cattle and the tick burdens observed on cattle in the wet season were determined. A negative association was observed between the number of co-infecting pathogens and the erythrocyte packed cell volume (PCV) of carrier cattle. Using recently available genome sequences, mini- and microsatellite markers were developed for population genetic analysis of Ba. bovis and Ba. bigemina parasite populations. Ba. bovis isolates from Zambia and Turkey and Ba. bigemina isolates from Zambia were used in the population genetic analysis. High levels of genetic diversity were observed for both parasites. Population genetic analysis of the Zambian and Turkish Ba. bovis populations, using eight genetic markers showed that the two populations were sub-structured. The Zambian population comprised a single randomly mating population, while the Turkish population comprised two genetically distinct subpopulations. Population genetic analysis of the Ba. bigemina parasites from Zambia showed that this parasite population was in linkage disequilibrium. Further, analysis of the Ba. bigemina population using STRUCTURE showed that it was genetically sub-structured into five distinct subgroups. However, the resulting sample size of each subgroup was too small to definitely determine whether they were panmictic. These results provide an improved understanding of the epidemiology of bovine Babesia parasites in Turkey and Zambia

Foot and mouth disease in Zambia: Spatial and temporal distributions of outbreaks, assessment of clusters and implications for control

Zambia has been experiencing low livestock productivity as well as trade restrictions owing to the occurrence of foot and mouth disease (FMD), but little is known about the epidemiology of the disease in these endemic settings. The fundamental questions relate to the spatio-temporal distribution of FMD cases and what determines their occurrence. A retrospective review of FMD cases in Zambia from 1981 to 2012 was conducted using geographical information systems and the SaTScan software package. Information was collected from peer-reviewed journal articles, conference proceedings, laboratory reports, unpublished scientific reports and grey literature. A space–time permutation probability model using a varying time window of one year was used to scan for areas with high infection rates. The spatial scan statistic detected a significant purely spatial cluster around the Mbala–Isoka area between 2009 and 2012, with secondary clusters in Sesheke–Kazungula in 2007 and 2008, the Kafue flats in 2004 and 2005 and Livingstone in 2012. This study provides evidence of the existence of statistically significant FMD clusters and an increase in occurrence in Zambia between 2004 and 2012. The identified clusters agree with areas known to be at high risk of FMD. The FMD virus transmission dynamics and the heterogeneous variability in risk within these locations may need further investigation

Policy Concerns, Opportunities, Challenges, and Attitude towards One Health Practice in Zambia

One Health in terms of collaboration, particularly between human and animal health sectors to prevent and control zoonoses has been low while the sectors have a lot of things in common. Such common things include aspects of disease causative agents (viruses, bacteria, parasites, etc.) and those of disease occurrence mediator conditions (social, cultural, economic or climatic). Therefore, the research from which this paper is based was done with the objectives to: (a) assess the extent to which human and animal health policies facilitate one health in terms of collaboration; (b) rank opportunities for and challenges to collaboration among medical, and veterinary officers according to the views and experiences of the respondents in the Ministry of Health and Ministry of Agriculture; and (c) determine the attitude of the respondents towards One Health approaches in terms of collaboration in dealing with zoonoses. A cross-sectional research design was used in this study whereby data were collected at a single point in time without repetition. Purposive sampling method was used to make sure that the respondents were only officials who usually participated in policy formulation in the two Ministries. It was found that almost three quarters (73.1%) of the respondents from both ministries agreed that there was no policy which directly facilitated One Health in terms of collaboration. It was also found that 83.6% of the respondents pointed out that human and animal health policy making process was a top-down process. Furthermore, it was found that the main opportunities that could enhance collaboration were sufficient money in budgeting; advocacy for control of neglected zoonotic diseases in human and animal health; and one health policy formulation (71.3%, 68.2% and 65.5% respectively). The overall attitude towards collaboration among respondents was favourable; they scored an average of 62.2 out of 100.0 points on a Likert scale. It is concluded that if opportunities enhancing collaboration were strengthened and challenges to collaboration were overcome, human health and animal health experts could collaborate more in reduction of disease burden in both humans and livestock. Keywords: One health, policy, attitude, opportunities, challenge

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions

Documenting the absence of bovine brucellosis in dairy cattle herds in the southern region of Malawi and the associated knowledge, attitudes and practices of farmers

Source at https://www.jsava.co.za/index.php/jsava/article/view/473.There is paucity of Brucella prevalence data in Malawi. For this reason, a cross-sectional study was conducted, from 06 January 2020 to 27 February 2020, to estimate the seroprevalence of brucellosis in dairy cattle herds amongst smallholder farmers, government and private dairy farms in the southern region. A total of 529 serum samples were screened for anti-Brucella antibodies using the Rose Bengal test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). A pre-tested electronic (Epicollect tool, Wellcome Sanger Institute, United Kingdom) questionnaire was administered to 378 smallholder farmers to assess their knowledge, attitudes and practices towards brucellosis. Descriptive statistics were used to analyse the data in Microsoft Excel® and Statistical Package for Social Sciences (SPSS®) version 21. No animal tested positive for presence of anti-Brucella antibodies, indicating 0% prevalence (individual and herd levels). The majority (94.2%; 95% confidence interval [CI]: 91.8–96.5) of smallholder farmers had never heard about brucellosis. Furthermore, assisting during parturition without protective equipment (41.3%; 95% CI: 36.3–46.2) and using bulls for breeding (75%; 95% CI: 70.2–78.9) were amongst the common risk practices that were identified. We could not detect brucellosis in this study that indicates the disease could be very rare or even absent in the dairy cattle herds of the southern region of Malawi. However, further Brucella studies need to be conducted in cattle, small livestock, wildlife and humans to document the true status of brucellosis in the country. Brucellosis surveillance, monitoring, awareness and preventive measures are required to maintain this favourable situation. Keywords: bovine brucellosis (contagious abortion); dairy cattle herds; seroprevalence; knowledge; attitudes and practices; Malawi

Foot-and-mouth disease control in Zambia: A review of the current situation

Zambia has been experiencing low livestock productivity as well as trade restrictions owing to the occurrence of foot-and-mouth disease (FMD) and contagious bovine pleura pneumonia (CBPP). Foot-and-mouth disease was first recorded in Zambia in 1933 in the Western Province and since then the country has experienced repeated outbreaks. Bearing in mind the pressure that may be existing on the many risk factors for FMD including climate change, there is need to review our knowledge on FMD control. We present the spatial distribution of the FMD outbreaks that have been recorded in Zambia in the last twenty years, and the effect of the vaccinations and movement control that have been applied. We propose further strain characterisation of previous FMD outbreaks, including full sequence of VP1 gene and the 5’UTR site. The data will be geo-coded and populated with risk factor attributes. We also present preliminary findings of the buffalo and cattle probang sampling that was conducted in Lochnivar and Kafue National Park. We further probang sampled 25 buffalo at each interface area in Sioma Ngwezi, Lukusuzi and Lower Zambezi national parks. Villages in close proximity to the buffalo populations as well as those not in close proximity will be multistage cluster sampled for comparison. The data will be geo-coded and populated with risk factor and foot-and-mouth disease virus (FMDV) characterisation attributes. Data collected using a pre-tested structured questionnaire will be geo-coded and populated with identified risk factors and stored in a database and will be spatially modelled to determine their effect on FMD occurrence and control measures. New outbreaks of FMD that may occur will be investigated to find out if there are new strains involved, species affected and predisposing risk factors. The authors conclude that impacts of FMD on livelihoods if appropriate control measures are not put in place are far more devastating especially at community level. Presented with the current poverty levels failure to institute result oriented control measures will exacerbate the already life-threatening situation

Serological Survey of Foot-and-Mouth Disease Virus in Buffaloes ( Syncerus caffer

A study was conducted to determine the serotypes of foot-and-mouth disease viruses (FMDV) circulating in African buffaloes (Syncerus caffer) from selected areas in Zambia. Sera and probang samples were collected between 2011 and 2012 and analysed for presence of antibodies against FMDV while probang samples were used to isolate the FMDV by observing cytopathic effect (CPE). Samples with CPE were further analysed using antigen ELISA. High FMD seroprevalence was observed and antibodies to all the three Southern African Territories (SAT) serotypes were detected in four study areas represented as follows: SAT2 was 72.7 percent; SAT1 was 62.6 percent; and SAT3 was 26.2 percent. Mixed infections accounted for 68.6 percent of those that were tested positive. For probang samples, CPE were observed in three of the samples, while the antigen ELISA results showed positivity and for SAT1 (n=1) and SAT2 (n=2). It is concluded that FMDV is highly prevalent in Zambian buffaloes which could play an important role in the epidemiology of the disease. Therefore livestock reared at interface with the game parks should be included in all routine FMDV vaccination programmes

Antibiogram bakterije Escherichia coli i enterokoka izdvojenih iz goveđega izmeta na području Kafue u Zambiji

Antimicrobial resistance to a panel of ten agents was determined by the disc diffusion technique for 83 Escherichia coli isolates, 29 Enterococcus faecium isolates and 62 Enterococcus faecalis isolates from faecal samples of apparently healthy pastoral cattle in the wildlife/livestock interface areas. Of all the E. coli isolates, 8% were diarrhoeagenic E. coli strains, 6% were enteropathogenic E. coli strains and 2% were enterotoxigenic E. coli strains. A high frequency of E. coli resistance to penicillin, erythromycin, cotrimoxazole and nitrofurantoin was observed. Enterococci showed the highest percentage of resistance to gentamycin, amoxycillin, ampicillin and tetracycline. None of the E. coli strains and Enterococci strains was resistant to tetracycline and vancomycin respectively. The results of this study underscore the presence of an animal reservoir of antibiotic resistant microorganisms that have the potential to enter the food chain.Osjetljivost 83 izolata vrste Escherichia coli, 29 izolata vrste Enterococcus faecium i 62 izolata vrste Enterococcus faecalis izdvojenih iz uzoraka izmeta klinički zdravih goveda u području dodira domaćih i divljih životinja, određena je difuzijskim postupkom prema deset antimikrobnih pripravaka. Od ukupno izdvojenih, 8% sojeva bakterije E. coli bilo je dijarejogeno, 6% enteropatogeno i 2% enterotoksigeno. Ustanovljena je česta otpornost vrste E. coli prema penicilinu, eritromicinu, kotrimoksazolu i nitrofurantoinu. Enterokoki su u najvećem postotku bili otporni prema gentamicinu, amoksicilinu, ampicilinu i tetraciklinu. Nijedan od izolata E. coli nije bio otporan prema tetraciklinu, a nijedan od enterokoka nije bio otporan prema vankomicinu. Rezultati istraživanja upućuju na postojanje životinjskoga rezervoara bakterija otpornih na antibiotike za koje postoji mogućnost da se prenesu u ljudski prehrambeni lanac

Antibiogram bakterije Escherichia coli i enterokoka izdvojenih iz goveđega izmeta na području Kafue u Zambiji

Antimicrobial resistance to a panel of ten agents was determined by the disc diffusion technique for 83 Escherichia coli isolates, 29 Enterococcus faecium isolates and 62 Enterococcus faecalis isolates from faecal samples of apparently healthy pastoral cattle in the wildlife/livestock interface areas. Of all the E. coli isolates, 8% were diarrhoeagenic E. coli strains, 6% were enteropathogenic E. coli strains and 2% were enterotoxigenic E. coli strains. A high frequency of E. coli resistance to penicillin, erythromycin, cotrimoxazole and nitrofurantoin was observed. Enterococci showed the highest percentage of resistance to gentamycin, amoxycillin, ampicillin and tetracycline. None of the E. coli strains and Enterococci strains was resistant to tetracycline and vancomycin respectively. The results of this study underscore the presence of an animal reservoir of antibiotic resistant microorganisms that have the potential to enter the food chain.Osjetljivost 83 izolata vrste Escherichia coli, 29 izolata vrste Enterococcus faecium i 62 izolata vrste Enterococcus faecalis izdvojenih iz uzoraka izmeta klinički zdravih goveda u području dodira domaćih i divljih životinja, određena je difuzijskim postupkom prema deset antimikrobnih pripravaka. Od ukupno izdvojenih, 8% sojeva bakterije E. coli bilo je dijarejogeno, 6% enteropatogeno i 2% enterotoksigeno. Ustanovljena je česta otpornost vrste E. coli prema penicilinu, eritromicinu, kotrimoksazolu i nitrofurantoinu. Enterokoki su u najvećem postotku bili otporni prema gentamicinu, amoksicilinu, ampicilinu i tetraciklinu. Nijedan od izolata E. coli nije bio otporan prema tetraciklinu, a nijedan od enterokoka nije bio otporan prema vankomicinu. Rezultati istraživanja upućuju na postojanje životinjskoga rezervoara bakterija otpornih na antibiotike za koje postoji mogućnost da se prenesu u ljudski prehrambeni lanac