Listeria monocytogenes is a zoonotic food-borne pathogen that is associated with serious public health and economic implications. In animals, L. monocytogenes can be associated with clinical listeriosis, which is characterised by symptoms such as abortion, encephalitis and septicaemia. In human beings, listeriosis symptoms include encephalitis, septicaemia and meningitis. In addition, listeriosis may cause gastroenteric symptoms in human beings and still births or spontaneous abortions in pregnant women. In the last few years, a number of reported outbreaks and sporadic cases associated with consumption of contaminated meat and meat products with L. monocytogenes have increased in developing countries. A variety of virulence factors play a role in the pathogenicity of L. monocytogenes. This zoonotic pathogen can be diagnosed using both classical microbiological techniques and molecular-based methods. There is limited information about L. monocytogenes recovered from meat and meat products in African countries. This review strives to: (1) provide information on prevalence and control measures of L. monocytogenes along the meat value chain, (2) describe the epidemiology of L. monocytogenes (3) provide an overview of different methods for detection and typing of L. monocytogenes for epidemiological, regulatory and trading purposes and (4) discuss the pathogenicity, virulence traits and antimicrobial resistance profiles of L. monocytogenes.

Listeria monocytogenes is a zoonotic food-borne pathogen that is associated with serious public health and economic implications. In animals, L. monocytogenes can be associated with clinical listeriosis, which is characterised by symptoms such as abortion, encephalitis and septicaemia. In human beings, listeriosis symptoms include encephalitis, septicaemia and meningitis. In addition, listeriosis may cause gastroenteric symptoms in human beings and still births or spontaneous abortions in pregnant women. In the last few years, a number of reported outbreaks and sporadic cases associated with consumption of contaminated meat and meat products with L. monocytogenes have increased in developing countries. A variety of virulence factors play a role in the pathogenicity of L. monocytogenes. This zoonotic pathogen can be diagnosed using both classical microbiological techniques and molecular-based methods. There is limited information about L. monocytogenes recovered from meat and meat products in African countries. This review strives to: (1) provide information on prevalence and control measures of L. monocytogenes along the meat value chain, (2) describe the epidemiology of L. monocytogenes (3) provide an overview of different methods for detection and typing of L. monocytogenes for epidemiological, regulatory and trading purposes and (4) discuss the pathogenicity, virulence traits and antimicrobial resistance profiles of L. monocytogenes.

The non-invasive monitoring of physiological stress can provide conservation and wildlife managers with an invaluable tool for assessing animal welfare and psychological health of captive and free-ranging populations. A significant decrease in free-ranging primate populations globally and an increase in captive-housed primates have led to a need to monitor the stress and general welfare of these animals. We examined the suitability of three enzyme immunoassays (EIAs) for monitoring stress-related physiological responses in the samango monkey, Cercopithecus albogularis erythrarchus. We conducted an adrenocorticotropic hormone (ACTH) challenge on a male and female at the National Zoological Garden, Pretoria, South Africa. Individual faecal samples were collected 8 days pre- and post-ACTH administration and subsequently analysed for faecal glucocorticoid metabolite (fGCM) concentrations. During the study, biological stressors occurred for both the male and female. Two of the three EIAs tested (11-oxoetiocholanolone I and II) were able to reliably monitor fGCM alterations throughout the study period in both sexes. The 11-oxoetiocholanolone I EIA, however, had the lowest mean deviation from the calculated baseline value and was thus chosen as the preferred assay. Both the physiological activation of the stress response and the biological response to a stressor could be monitored with the chosen assay. The successful establishment of a reliable, non-invasive method for monitoring adrenocortical activity in C. albogularis erythrarchus will now allow conservationists, scientific researchers and wildlife managers to evaluate the level of stress experienced, and general welfare, by animals in captivity as well as free-ranging populations.

The non-invasive monitoring of physiological stress can provide conservation and wildlife managers with an invaluable tool for assessing animal welfare and psychological health of captive and free-ranging populations. A significant decrease in free-ranging primate populations globally and an increase in captive-housed primates have led to a need to monitor the stress and general welfare of these animals. We examined the suitability of three enzyme immunoassays (EIAs) for monitoring stress-related physiological responses in the samango monkey, Cercopithecus albogularis erythrarchus. We conducted an adrenocorticotropic hormone (ACTH) challenge on a male and female at the National Zoological Garden, Pretoria, South Africa. Individual faecal samples were collected 8 days pre- and post-ACTH administration and subsequently analysed for faecal glucocorticoid metabolite (fGCM) concentrations. During the study, biological stressors occurred for both the male and female. Two of the three EIAs tested (11-oxoetiocholanolone I and II) were able to reliably monitor fGCM alterations throughout the study period in both sexes. The 11-oxoetiocholanolone I EIA, however, had the lowest mean deviation from the calculated baseline value and was thus chosen as the preferred assay. Both the physiological activation of the stress response and the biological response to a stressor could be monitored with the chosen assay. The successful establishment of a reliable, non-invasive method for monitoring adrenocortical activity in C. albogularis erythrarchus will now allow conservationists, scientific researchers and wildlife managers to evaluate the level of stress experienced, and general welfare, by animals in captivity as well as free-ranging populations.

In current research guidelines, much focus is placed on ethical management of animals and the application of principles of reduction, refinement and replacement. Of these refinements through environmental enrichment is an important aspect when housing primate to prevent behavioural problems. In this study, we investigated the co-housing of domestic cats and vervet monkeys as a novel method of enrichment based on the cohabitation and stress alleviation effect of horses housed with goats and from seeing cats cohabitating with vervet monkeys in an animal sanctuary. The study used a habituation method whereby the cats were stepwise introduced to the monkeys by sight and smell but with physical separation. Assessment included changes in behaviour, weight and faecal glucocorticoid metabolite (fGCM) concentrations over time. On the first day of housing, the vervets whilst inquisitive kept their distance. The vervets housed in cages that were closest to the cats were the most active and during the first minute of introduction made more alarm calls, which stopped a few days later. The fGCMs were non-significantly different. The results of this study provide evidence that vervet monkeys and domestic cats could potentially be housed together without overt aggression. We thus suggest further observations to ascertain if the co-housing could have long-term benefits for vervet monkeys, from the companionship that would be offered by the cats.

In current research guidelines, much focus is placed on ethical management of animals and the application of principles of reduction, refinement and replacement. Of these refinements through environmental enrichment is an important aspect when housing primate to prevent behavioural problems. In this study, we investigated the co-housing of domestic cats and vervet monkeys as a novel method of enrichment based on the cohabitation and stress alleviation effect of horses housed with goats and from seeing cats cohabitating with vervet monkeys in an animal sanctuary. The study used a habituation method whereby the cats were stepwise introduced to the monkeys by sight and smell but with physical separation. Assessment included changes in behaviour, weight and faecal glucocorticoid metabolite (fGCM) concentrations over time. On the first day of housing, the vervets whilst inquisitive kept their distance. The vervets housed in cages that were closest to the cats were the most active and during the first minute of introduction made more alarm calls, which stopped a few days later. The fGCMs were non-significantly different. The results of this study provide evidence that vervet monkeys and domestic cats could potentially be housed together without overt aggression. We thus suggest further observations to ascertain if the co-housing could have long-term benefits for vervet monkeys, from the companionship that would be offered by the cats.

The first known severe disease caused by a coronavirus (CoV) in humans emerged with the severe acute respiratory syndrome (SARS) epidemic in China, which killed 774 people during its 2002/2003 outbreak. The Middle East respiratory syndrome (MERS) was the second human fatal disease, which started in 2012 in Saudi Arabia and resulted in 858 fatalities. In December 2019, a new virus, SARS-CoV-2 (COVID-19), originating from China, began generating headlines worldwide because of the unprecedented speed of its transmission; 5.2 million people were infected and 338 480 had been reported dead from December 2019 to May 2020. These human coronaviruses are believed to have an animal origin and had reached humans through species jump. Coronaviruses are well known for their high frequency of recombination and high mutation rates, allowing them to adapt to new hosts and ecological niches. This review summarises existing information on what is currently known on the role of wild and domesticated animals and discussions on whether they are the natural reservoir/amplifiers hosts or incidental hosts of CoVs. Results of experimental infection and transmission using different wild, domesticated and pet animals are also reviewed. The need for a One Health approach in implementing measures and practices is highlighted to improve human health and reduce the emergence of pandemics from these zoonotic viruses.

The first known severe disease caused by a coronavirus (CoV) in humans emerged with the severe acute respiratory syndrome (SARS) epidemic in China, which killed 774 people during its 2002/2003 outbreak. The Middle East respiratory syndrome (MERS) was the second human fatal disease, which started in 2012 in Saudi Arabia and resulted in 858 fatalities. In December 2019, a new virus, SARS-CoV-2 (COVID-19), originating from China, began generating headlines worldwide because of the unprecedented speed of its transmission; 5.2 million people were infected and 338 480 had been reported dead from December 2019 to May 2020. These human coronaviruses are believed to have an animal origin and had reached humans through species jump. Coronaviruses are well known for their high frequency of recombination and high mutation rates, allowing them to adapt to new hosts and ecological niches. This review summarises existing information on what is currently known on the role of wild and domesticated animals and discussions on whether they are the natural reservoir/amplifiers hosts or incidental hosts of CoVs. Results of experimental infection and transmission using different wild, domesticated and pet animals are also reviewed. The need for a One Health approach in implementing measures and practices is highlighted to improve human health and reduce the emergence of pandemics from these zoonotic viruses.

The study aimed to investigate the presence of peste des petits ruminants (PPR) in pneumonic lung tissues from clinically apparently healthy sheep and goats and further demonstrating its prevalence in Gezira state, central Sudan. During March 2019, 99 pneumonic lung samples were collected from apparently healthy sheep (80) and goats (19) from Al-Hasaheisa slaughterhouse located in Al-Hasaheisa locality, Gezira state. Using the haemagglutination (HA) test for the detection of peste des petits ruminants virus (PPRV) antigen, an overall antigenic prevalence of 86.9% was demonstrated in sheep and goats lung tissue homogenate. Of note, the prevalence of PPRV is higher in goats (100%) compared to sheep (83.7%). In this study, the reported increasing prevalence of PPR in central Sudan might be because of insufficient vaccination of animals. The findings of the present study indicated the widespread of PPR amongst sheep and goats in Al-Hasaheisa, Gezira state. Detection of PPRV antigen in the pneumonic lung samples is an indication of exposure of these animals to PPRV or presence of PPR viral infection and demonstrates the role of PPR as the cause of pneumonia in small ruminants. In fact, the circulation of the virus in clinically apparently healthy animals poses a threat for other in-contact susceptible animals and could play a significant role in the spread of the disease.

The study aimed to investigate the presence of peste des petits ruminants (PPR) in pneumonic lung tissues from clinically apparently healthy sheep and goats and further demonstrating its prevalence in Gezira state, central Sudan. During March 2019, 99 pneumonic lung samples were collected from apparently healthy sheep (80) and goats (19) from Al-Hasaheisa slaughterhouse located in Al-Hasaheisa locality, Gezira state. Using the haemagglutination (HA) test for the detection of peste des petits ruminants virus (PPRV) antigen, an overall antigenic prevalence of 86.9% was demonstrated in sheep and goats lung tissue homogenate. Of note, the prevalence of PPRV is higher in goats (100%) compared to sheep (83.7%). In this study, the reported increasing prevalence of PPR in central Sudan might be because of insufficient vaccination of animals. The findings of the present study indicated the widespread of PPR amongst sheep and goats in Al-Hasaheisa, Gezira state. Detection of PPRV antigen in the pneumonic lung samples is an indication of exposure of these animals to PPRV or presence of PPR viral infection and demonstrates the role of PPR as the cause of pneumonia in small ruminants. In fact, the circulation of the virus in clinically apparently healthy animals poses a threat for other in-contact susceptible animals and could play a significant role in the spread of the disease.

This study was conducted from January to October 2018 with the objective to determine the prevalence and genetic diversity of Eimeria species in broiler and free-range chickens in KwaZulu-Natal province, South Africa. A total of 342 faecal samples were collected from 12 randomly selected healthy broiler chicken farms and 40 free-range chickens from 10 different locations. Faecal samples were screened for the presence of Eimeria oocysts using a standard flotation method. The species of Eimeria isolates were confirmed by amplification of the internal transcribed spacer 1 (ITS-1) partial region and sequences analysis. Among broiler and free-ranging chickens, 19 out of 41 pens (46.3%) and 25 out of 42 faecal samples (59.5%) were positive for Eimeria infection. Molecular detection revealed the following species: Eimeria maxima, Eimeria tenella, Eimeria acervulina, Eimeria brunetti and Eimeria mitis in all the samples screened. Similarly, polymerase chain reaction assays specific for three cryptic Eimeria operational taxonomic units were negative for all the samples. Phylogenetic analysis of the ITS-1 sequences supported species identity with the greatest variation detected for E. mitis. This study provides information on the range and identity of Eimeria species, and their genetic relatedness, circulating in commercially reared broilers and free-ranging chickens from different locations in KwaZulu-Natal province.

This study was conducted from January to October 2018 with the objective to determine the prevalence and genetic diversity of Eimeria species in broiler and free-range chickens in KwaZulu-Natal province, South Africa. A total of 342 faecal samples were collected from 12 randomly selected healthy broiler chicken farms and 40 free-range chickens from 10 different locations. Faecal samples were screened for the presence of Eimeria oocysts using a standard flotation method. The species of Eimeria isolates were confirmed by amplification of the internal transcribed spacer 1 (ITS-1) partial region and sequences analysis. Among broiler and free-ranging chickens, 19 out of 41 pens (46.3%) and 25 out of 42 faecal samples (59.5%) were positive for Eimeria infection. Molecular detection revealed the following species: Eimeria maxima, Eimeria tenella, Eimeria acervulina, Eimeria brunetti and Eimeria mitis in all the samples screened. Similarly, polymerase chain reaction assays specific for three cryptic Eimeria operational taxonomic units were negative for all the samples. Phylogenetic analysis of the ITS-1 sequences supported species identity with the greatest variation detected for E. mitis. This study provides information on the range and identity of Eimeria species, and their genetic relatedness, circulating in commercially reared broilers and free-ranging chickens from different locations in KwaZulu-Natal province.

Despite the availability of Newcastle disease (ND) vaccines for more than six decades, disease outbreaks continue to occur with huge economic consequences to the global poultry industry. The aim of this study is to develop a safe and effective inactivated vaccine based on a recently isolated Newcastle disease virus (NDV) strain IBS025/13 and evaluate its protective efficacy in chicken following challenge with a highly virulent genotype VII isolate. Firstly, high titre of IBS025/13 was exposed to various concentrations of binary ethylenimine (BEI) to determine the optimal conditions for complete inactivation of the virus. The inactivated virus was then prepared in form of a stable water-in-oil emulsion of black seed oil (BSO) or Freund’s incomplete adjuvant (FIA) and used as vaccines in specific pathogen-free chicken. Efficacy of various vaccine preparations was also evaluated based on the ability of the vaccine to protect against clinical disease, mortality and virus shedding following challenge with highly virulent genotype\VII NDV isolate. The results indicate that exposure of NDV IBS025/13 to 10 mM of BEI for 21 h at 37 °C could completely inactivate the virus without tempering with the structural integrity of the viral hemagglutin-neuraminidase protein. More so, the inactivated vaccines adjuvanted with either BSO- or FIA-induced high hemagglutination inhibition antibody titre that protected the vaccinated birds against clinical disease and in some cases virus shedding, especially when used together with live attenuated vaccines. Thus, genotype VII-based NDV-inactivated vaccines formulated in BSO could substantially improve poultry disease control particularly when combined with live attenuated vaccines.

Despite the availability of Newcastle disease (ND) vaccines for more than six decades, disease outbreaks continue to occur with huge economic consequences to the global poultry industry. The aim of this study is to develop a safe and effective inactivated vaccine based on a recently isolated Newcastle disease virus (NDV) strain IBS025/13 and evaluate its protective efficacy in chicken following challenge with a highly virulent genotype VII isolate. Firstly, high titre of IBS025/13 was exposed to various concentrations of binary ethylenimine (BEI) to determine the optimal conditions for complete inactivation of the virus. The inactivated virus was then prepared in form of a stable water-in-oil emulsion of black seed oil (BSO) or Freund’s incomplete adjuvant (FIA) and used as vaccines in specific pathogen-free chicken. Efficacy of various vaccine preparations was also evaluated based on the ability of the vaccine to protect against clinical disease, mortality and virus shedding following challenge with highly virulent genotype\VII NDV isolate. The results indicate that exposure of NDV IBS025/13 to 10 mM of BEI for 21 h at 37 °C could completely inactivate the virus without tempering with the structural integrity of the viral hemagglutin-neuraminidase protein. More so, the inactivated vaccines adjuvanted with either BSO- or FIA-induced high hemagglutination inhibition antibody titre that protected the vaccinated birds against clinical disease and in some cases virus shedding, especially when used together with live attenuated vaccines. Thus, genotype VII-based NDV-inactivated vaccines formulated in BSO could substantially improve poultry disease control particularly when combined with live attenuated vaccines.

Tuberculosis (TB) is a global health concern of zoonotic importance, and Mycobacterium bovis and Mycobacterium tuberculosis are the most common causes of TB in animals and humans, respectively. Integral to TB control strategies are the communities affected by this epidemic. Tuberculosis awareness by the community is an effective TB control strategy as education empowers people to make informed choices with regard to mitigating TB risk factors in their daily lives. We conducted a knowledge, attitude and perceptions survey in Mnisi pastoral community in South Africa using a semi-structured questionnaire to evaluate the level of bovine TB (bTB) awareness, and provided informed feedback to the community on the outcome of the study. Although participants were aware of TB, the knowledge of the zoonotic potential of bTB and about susceptible hosts was limited. The study findings showed knowledge gaps regarding common risk factors, including coughing while herding cattle, unsupervised/uninspected communal slaughter and improper disposal of infected meat. In contrast, it was noted that the majority of participants discarded meat with visible lesions and consumed pasteurised milk; thus, the risk of TB transmission via the ingestion route is low. Tuberculosis knowledge gaps were evident in the community, and public health and veterinary authorities need to improve relationships with stakeholders and implement awareness programmes that use a one health approach.

Tuberculosis (TB) is a global health concern of zoonotic importance, and Mycobacterium bovis and Mycobacterium tuberculosis are the most common causes of TB in animals and humans, respectively. Integral to TB control strategies are the communities affected by this epidemic. Tuberculosis awareness by the community is an effective TB control strategy as education empowers people to make informed choices with regard to mitigating TB risk factors in their daily lives. We conducted a knowledge, attitude and perceptions survey in Mnisi pastoral community in South Africa using a semi-structured questionnaire to evaluate the level of bovine TB (bTB) awareness, and provided informed feedback to the community on the outcome of the study. Although participants were aware of TB, the knowledge of the zoonotic potential of bTB and about susceptible hosts was limited. The study findings showed knowledge gaps regarding common risk factors, including coughing while herding cattle, unsupervised/uninspected communal slaughter and improper disposal of infected meat. In contrast, it was noted that the majority of participants discarded meat with visible lesions and consumed pasteurised milk; thus, the risk of TB transmission via the ingestion route is low. Tuberculosis knowledge gaps were evident in the community, and public health and veterinary authorities need to improve relationships with stakeholders and implement awareness programmes that use a one health approach.

Tuberculosis (TB) is a global health concern of zoonotic importance, and Mycobacterium bovis and Mycobacterium tuberculosis are the most common causes of TB in animals and humans, respectively. Integral to TB control strategies are the communities affected by this epidemic. Tuberculosis awareness by the community is an effective TB control strategy as education empowers people to make informed choices with regard to mitigating TB risk factors in their daily lives. We conducted a knowledge, attitude and perceptions survey in Mnisi pastoral community in South Africa using a semi-structured questionnaire to evaluate the level of bovine TB (bTB) awareness, and provided informed feedback to the community on the outcome of the study. Although participants were aware of TB, the knowledge of the zoonotic potential of bTB and about susceptible hosts was limited. The study findings showed knowledge gaps regarding common risk factors, including coughing while herding cattle, unsupervised/uninspected communal slaughter and improper disposal of infected meat. In contrast, it was noted that the majority of participants discarded meat with visible lesions and consumed pasteurised milk; thus, the risk of TB transmission via the ingestion route is low. Tuberculosis knowledge gaps were evident in the community, and public health and veterinary authorities need to improve relationships with stakeholders and implement awareness programmes that use a one health approach.

All effects taken together, bovine tuberculosis (bTB) has a long-term detrimental effect on bovine herds and many wildlife species in South Africa. The disease is not only found in domestic cattle but also in African buffaloes and has to date been diagnosed in 21 wildlife species, including several rare and endangered species, thus having a potentially serious effect on conservation and biodiversity. In cattle, bTB is mostly characterised by sporadic outbreaks, but bovine herds chronically infected with the clinical disease are not uncommon. Presently, the recognised bTB control strategy in South Africa is based on ‘test and slaughter’, using the intradermal tuberculin test, followed by the slaughter of animals that have tested positive. Affected herds are placed under veterinary quarantine with movement restrictions until the outbreak is eradicated; this can take several years or last indefinitely if the outbreak cannot be eradicated. The same measures apply to infected buffalo populations, often with no prospect of ever being eradicated. This strategy is neither practical nor viable in the context of a communal farming system and becomes unethical when dealing with valuable wildlife reservoir hosts. Transmission of bTB between wildlife and cattle has been demonstrated and emphasises the need for an effective, affordable and culturally acceptable control strategy to curb the spread of bTB in South Africa. In countries with similar challenges, vaccination has been used and found to be promising for treating wild and domestic reservoir species and may hence be of value as a complementary tool for bTB control in South Africa.

All effects taken together, bovine tuberculosis (bTB) has a long-term detrimental effect on bovine herds and many wildlife species in South Africa. The disease is not only found in domestic cattle but also in African buffaloes and has to date been diagnosed in 21 wildlife species, including several rare and endangered species, thus having a potentially serious effect on conservation and biodiversity. In cattle, bTB is mostly characterised by sporadic outbreaks, but bovine herds chronically infected with the clinical disease are not uncommon. Presently, the recognised bTB control strategy in South Africa is based on ‘test and slaughter’, using the intradermal tuberculin test, followed by the slaughter of animals that have tested positive. Affected herds are placed under veterinary quarantine with movement restrictions until the outbreak is eradicated; this can take several years or last indefinitely if the outbreak cannot be eradicated. The same measures apply to infected buffalo populations, often with no prospect of ever being eradicated. This strategy is neither practical nor viable in the context of a communal farming system and becomes unethical when dealing with valuable wildlife reservoir hosts. Transmission of bTB between wildlife and cattle has been demonstrated and emphasises the need for an effective, affordable and culturally acceptable control strategy to curb the spread of bTB in South Africa. In countries with similar challenges, vaccination has been used and found to be promising for treating wild and domestic reservoir species and may hence be of value as a complementary tool for bTB control in South Africa.

Heartwater is a tick-borne disease caused by the intracellular rickettsial parasite Ehrlichia ruminantium and transmitted by Amblyomma hebraeum ticks. Heartwater is problematic in endemic areas because it causes high mortality in ruminants and leads to economic losses that threaten productivity and food security. This may indicate that there is augmented genetic diversity in the field, which may result in isolates that are more virulent than the Ball3 and Welgevonden isolates. The genetic diversity of E. ruminantium was investigated in this study, focussing on the pCS20 gene region and four polymorphic open reading frames (ORFs) identified by subtractive hybridisation. The 16S ribosomal ribonucleic acid gene confirmed E. ruminantium in brain, blood and tick genomic deoxyribonucleic acid samples (n = 3792) collected from 122 farms that were randomly selected from seven provinces of South Africa where heartwater is endemic. The conserved E. ruminantium pCS20 quantitative polymerase chain reaction (qPCR) assay was used to scan all collected field samples. A total of 433 samples tested positive with the qPCR using the pCS20 gene region, of which 167 were sequenced. The known stocks and field samples were analysed, and phylogenetic trees were generated from consensus sequences. A total of 25 new clades were identified; of these, nine isolates from infected blood could be propagated in cell cultures. These clades were not geographically confined to a certain area but were distributed amongst heartwater-endemic areas in South Africa. Thus, the knowledge of strain diversity of E. ruminantium is essential for control of heartwater and provides a basis for further vaccine development.

Heartwater is a tick-borne disease caused by the intracellular rickettsial parasite Ehrlichia ruminantium and transmitted by Amblyomma hebraeum ticks. Heartwater is problematic in endemic areas because it causes high mortality in ruminants and leads to economic losses that threaten productivity and food security. This may indicate that there is augmented genetic diversity in the field, which may result in isolates that are more virulent than the Ball3 and Welgevonden isolates. The genetic diversity of E. ruminantium was investigated in this study, focussing on the pCS20 gene region and four polymorphic open reading frames (ORFs) identified by subtractive hybridisation. The 16S ribosomal ribonucleic acid gene confirmed E. ruminantium in brain, blood and tick genomic deoxyribonucleic acid samples (n = 3792) collected from 122 farms that were randomly selected from seven provinces of South Africa where heartwater is endemic. The conserved E. ruminantium pCS20 quantitative polymerase chain reaction (qPCR) assay was used to scan all collected field samples. A total of 433 samples tested positive with the qPCR using the pCS20 gene region, of which 167 were sequenced. The known stocks and field samples were analysed, and phylogenetic trees were generated from consensus sequences. A total of 25 new clades were identified; of these, nine isolates from infected blood could be propagated in cell cultures. These clades were not geographically confined to a certain area but were distributed amongst heartwater-endemic areas in South Africa. Thus, the knowledge of strain diversity of E. ruminantium is essential for control of heartwater and provides a basis for further vaccine development.