Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV‐1), BVDV‐2, border disease virus (BDV), and an atypical pestivirus named HoBi‐like pestivirus. In this study, eighty‐nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5′ untranslated regions (5′UTR), N‐terminal autoprotease (N^pro) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV‐1, 33.7% as BVDV‐2 and 12.4% as HoBi‐like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV‐1a (35.9%), BVDV‐2b (31.4%), BVDV‐1b (10.1%), BVDV‐1d (6.7%), BVDV‐2c (2.2%) and BVDV‐1e (1.1%). BVDV‐2c and BVDV‐1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.     

Clinical Presentation Resembling Mucosal Disease Associated with ‘HoBi’‐like Pestivirus in a Field Outbreak

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV‐1), Bovine viral diarrhoea virus 2 (BVDV‐2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses (‘HoBi’‐like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease‐like clinical presentation (MD) associated with a ‘HoBi’‐like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT‐PCR. The sequence and phylogenetic analysis of 5′NCR, N^pro and E2 regions of the RT‐PCR positive samples showed that four different ‘HoBi’‐like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with ‘HoBi’‐like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different ‘HoBi’‐like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.     

Identification and Epidemiology of a Rare HoBi‐Like Pestivirus Strain in Bangladesh

The genus pestivirus of the family flaviviridae consists of four recognized species: bovine viral diarrhoea virus 1 (BVDV‐1), bovine viral diarrhoea virus 2 (BVDV‐2), classical swine fever virus and border disease virus. A new putative pestivirus species tentatively named as either ‘HoBi‐like pestivirus’ or BVDV‐3 has recently been identified in Brazil, Italy and Thailand. Despite reports of serological evidence of BVDV in Bangladesh, the types of the virus circulating in cattle have not been identified. We conducted surveillance in cattle from May 2009 to August 2010 in three government veterinary hospitals to characterize BVDV in cattle of Bangladesh. We tested serum for BVDV using an antigen‐capture ELISA. Of 638 cattle samples, 3% (16/638) tested positive for BVDV antigen. The ELISA‐positive samples were selected for further molecular detection and characterization of BVDV. Molecular analysis of the partial 5′ untranslated region (UTR) nucleotide sequences of BVDV‐positive samples identified the rare HoBi‐like pestivirus or BVDV‐3 virus circulating in cattle of Bangladesh. The identification of this rare HoBi‐like pestivirus or BVDV‐3 strain in Bangladesh warrants further surveillance to evaluate its impact on livestock production.     

Detection of atypical porcine pestivirus in Brazil in the central nervous system of suckling piglets with congenital tremor

Atypical porcine pestivirus (APPV ) has been detected in piglets with congenital tremor (CT ) from three different continents including North America, Europe and Asia. Thirteen piglets from four farms in two different states in Brazil with CT were sampled. Viral RNA was detected by quantitative real‐time PCR in the cerebellum or cerebellum and spinal cord in the 100% of the piglets with CT , and APPV was not detected in any tissue sample from clinically non‐affected piglets with the exception of the cerebellum of one piglet from Farm A. Piglets with CT had an odds ratio of 99.0 (95% CI 3.4, 2823.8; p  =  .0072) compared to piglets without CT to test positive for APPV by qRT ‐PCR . A subset of positive samples was selected for sequencing of the NS 3 gene. Phylogenetic analysis revealed that Brazilian sequences of the NS 3 formed an independent cluster and had the highest sequence identity with a sequence from the United States. This is the first identification of APPV infection in piglets with CT in South America.     

Molecular detection and genetic diversity of Bartonella species in large ruminants and associated ectoparasites from the Brazilian Cerrado

Currently, five Bartonella species and an expanding number of Candidatus Bartonella species have globally been reported in ruminants. Likewise, different Bartonella genotypes were identified. However, studies relating to ruminant‐associated Bartonella in Brazil are scarce. The current study aimed to assess the prevalence and genetic diversity of Bartonella in cattle, buffaloes and associated ectoparasites in Brazil. For this purpose, EDTA‐blood samples from 75 cattle and 101 buffaloes were sampled. Additionally, 128 Rhipicephalus microplus and one Amblyomma sculptum ticks collected from cattle, and 197 R. microplus, one A. sculptum and 170 lice (Haematopinus tuberculatus) collected from buffaloes were included. Bartonella DNA was initially screened through an HRM real‐time PCR assay targeting the 16S–23S internal transcribed spacer (ITS), and the positive samples were submitted to an additional HRM assay targeting the ssrA gene. The HRM‐positive amplicons were sequenced, and the nucleotide identity was assessed by BLASTn. Bartonella spp.‐positive DNA samples were analysed by conventional PCR assays targeting the gltA and rpoB genes, and then, the samples were cloned. Finally, the phylogenetic positioning and the genetic diversity of clones were assessed. Overall, 21 of 75 (28%) cattle blood samples and 13 of 126 (10.3%) associated ticks were positive for Bartonella bovis. Out of 101 buffaloes, 95 lice and 188 tick DNA samples, one (1%) buffalo and four (4.2%) lice were positive for Bartonella spp. Conversely, none of the ticks obtained from buffaloes were positive for Bartonella. The Bartonella sequences from buffaloes showed identity ranging from 100% (ITS and gltA) to 94% (ssrA) with B. bovis. In contrast, the Bartonella DNA sequences from lice were identical (100%) to uncultured Bartonella sp. detected in cattle tail louse (Haematopinus quadripertusus) from Israel in all amplified genes. The present study demonstrates the prevalence of new B. bovis genotypes and a cattle lice‐associated Bartonella species in large ruminants and their ectoparasites from Brazil. These findings shed light on the distribution and genetic diversity of ruminant‐ and ectoparasite‐related Bartonella in Brazil.     

Molecular Characteristics of Bovine Virus Diarrhoea Virus 1 Isolates from Turkey: Approaches for an Eradication Programme

Forty pestivirus isolates sampled from cattle in Turkey between 2002 and 2007 were characterized according to 5′ untranslated region (5′UTR) sequences and autoprotease (N^pro) gene sequences. The sampling of Bovine virus diarrhoea viruses (BVDVs) from 15 farms in five different regions indicated that BVDV 1‐l (18/40, 45%) was the predominant genotype in Turkey; the samples also contained the genotypes 1‐f (10/40, 25%), 1‐b (7/40, 17.5%), 1‐d (3/40, 7.5%), and 1‐a (2/40, 5%), respectively.     

HoBi‐like is the most prevalent ruminant pestivirus in Northeastern Brazil

The ruminant pestiviral species BVDV ‐1, BVDV ‐2 and BDV , along with the putative species HoBi‐like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT ‐PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi‐like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi‐like viruses. The increasing reports of HoBi‐like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi‐like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi‐like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.     

Evidence for Tunisian‐Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV‐1, BVDV‐2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5′‐UTR and the whole N^pro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV‐1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian‐like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian‐like pestivirus from this species.     

Phylogenetic and genomic characterization of a novel atypical porcine pestivirus in China

Atypical porcine pestivirus (APPV ) has been considered a novel pestivirus and causative agent of congenital tremor type A‐II . An APPV CH ‐GX 2016 strain was characterized from newly born piglets with clinical symptoms of congenital tremor in Guangxi, China. The genome of APPV CH ‐GX 2016 strain was 11,475 bp in length and encoded a polyprotein composed of the 3,635 amino acids. This genome sequence exhibited 88.0% to 90.8% nucleotide sequence homology with other APPV reference sequences in GenBank. Phylogenetic analysis further showed that APPV CH ‐GX is a novel pestivirus compared with previously described classical pestivirus strains. Therefore, APPV is present in pigs in China.     

Analytical Epidemiology of Genomic Variation among Pan Asia Strains of Foot‐and‐Mouth Disease Virus

Genetic data from field isolates of foot‐and‐mouth disease virus (FMDV) have been used to trace the source of recent outbreaks of FMD, to design better vaccines and diagnostic tests for FMDV, and to make conclusions regarding the general variability in the FMDV genome. Though epidemiologic data associated with FMDV isolates are available, these data have been used rarely to explore possible associations of epidemiologic factors with evolution or variation of the FMDV genome. In this study, factors associated with variation in the primary immunogenic peptide gene of FMDV (VP1), for a sample of 147 serotype O, Pan Asia strain sequences were explored using traditional analytical epidemiologic methods: logistic regression and multinomial‐response logistic regression. Hypothesized factors included host type (bovine, ovine, buffalo, or porcine) and geographical region (Middle East, South Asia, East Asia, Southeast Asia, and Europe). Results of two regression analyses suggest that host type and region, considered to be possible surrogates for host management, may be associated with selection in the VP1 amino acid sequence in FMDV. For example, isolates from cattle and sheep in South Asia appear to converge with a proposed ancestor sequence, whereas isolates from the same species in the Middle East and Southeast Asia appear to diverge. The methods demonstrated here could be used on a more detailed dataset to explore the selective pressure of host immunity on the evolution of FMDV antigens in an endemic setting. More broadly, epidemiologic methods could be applied extensively to molecular data to explore the causes of genomic variation in disease‐causing organisms.     

Tuberculosis in Southern Brazilian wild boars (Sus scrofa): First epidemiological findings

Bovine tuberculosis (bTB ) is a zoonosis caused mainly by Mycobacterium bovis that affects domestic and wild animals. In Brazil, there are no epidemiological studies on tuberculosis in wild animal populations and their possible role in the disease maintenance in cattle herds; thus, the aim of this study was to evaluate the occurrence of tuberculosis in wild boars in Rio Grande do Sul, southern Brazil. Tissue samples of animals hunted under government consent were submitted to histopathology and M. bovis polymerase chain reaction (PCR ) as screening tests; the positive samples were subsequently submitted to bacterial isolation, the gold standard diagnosis. Eighty animals were evaluated, of which 27.9% and 31.3% showed histopathological changes and M. bovis genome presence, respectively. Moreover, 23.8% of the animals had at least one organ with isolates classified as Mycobacterium tuberculosis complex (MTC ). Three hunting points were risk factors for positive results on screening tests. This study shows the occurrence of tuberculosis in a wild boars’ population, and raise the possibility of these animals to play a role as disease reservoirs in southern Brazil. These results may help to improve the Brazilian tuberculosis control programme, as well as elucidate the circulation of mycobacteria in this country.     

Presence of atypical porcine pestivirus (APPV) in Brazilian pigs

Recently, a putative new pestivirus species, provisionally named as Atypical Porcine Pestivirus (APPV ), was associated with the congenital tremor in piglets in North America and consequently in Europe and Asia. The present research aimed to describe the detection and characterization of APPV employing NS 5B gene partial sequencing, gross pathology and histologic examination of piglets displaying congenital tremor from two different farms of Southern Brazil. No gross lesions were observed, and the histological findings revealed moderate vacuolization of the white matter of the cerebellum. RT ‐PCR followed by DNA sequencing and a phylogenetic analysis confirmed the presence of APPV in samples from the two farms, which the samples were distinct in nature. Phylogenetic reconstruction reinforced the high genetic variability within the APPV s previously reported. This is the first report of APPV in South America suggesting that this new group of viruses may be widespread in swine herds in other countries as it is in Brazil.     

A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can ‘breakthrough’ the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co‐graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T‐cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long‐term consequences are unknown.     

Multi‐locus sequencing reveals a novel Bartonella in mammals from the Superorder Xenarthra

The superorder Xenarthra consists of sloths, anteaters and armadillos, mammals originated from South America and currently distributed from the south of North America to the south of South America. The present study aimed to investigate the occurrence and genetic diversity of Bartonella spp. in blood and spleen samples from free‐living Xenarthra mammals in the states of São Paulo (SP), Mato Grosso do Sul (MS), Rondônia (RO) and Pará (PA). Based on a quantitative real‐time PCR (qPCR) assay, a Bartonella spp. nuoG gene fragment was detected in 1.51% (5/330) of the samples: 4 six‐banded armadillos (Euphractus sexcinctus) sampled in the MS and 1 southern tamandua (Tamandua tetradactyla) sampled in the PA. Eight sequences (5 ftsZ, 2 gltA and 1 rpoB) were obtained in the conventional PCR assays. In both phylogenetic analyses based on Bayesian and distance (SplitsTree) methods, the obtained ftsZ, gltA and rpoB sequences were positioned in a distinct clade, but related to B. washoensis. The analysis of SplitsTree and genotype networks based on B. washoensis sequences from several hosts from various localities of the world showed that the sequences of the present study were allocated in a group separated from the other sequences, indicating that they probably originated from median vectors and large numbers of mutational events. Additionally, the analyses performed by BLAST showed low percentages of identities of the sequences obtained in the present study when compared to those previously deposited in GenBank. Therefore, we propose a new Candidatus to Bartonella occurring in Xenarthra in Brazil. The present study was the first to report the occurrence of Bartonella sp. in mammals of the superorder Xenarthra in the world, and it was the first to describe a new Candidatus related to B. washoensis in Brazil.     

Serological relationship between a novel ovine pestivirus and classical swine fever virus

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV‐1) and type 2 (BVDV‐2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross‐neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.     

Molecular characterization of whole genome sequence of infectious bronchitis virus 624I genotype confirms the close relationship with Q1 genotype

Infectious Bronchitis virus (IBV) genotype Q1 was detected for the first time in China in 1996, and then spread worldwide. The first report of Q1 genotype in Italy occurred in 2011 and a deep molecular investigation of a Q1 isolated in Italy in 2013 has led to speculation regarding the origin of this genotype. Phylogenetic analysis of the S1 sequence of a Q1 Italian strain revealed a close relationship with sequences of the 624I strains circulating in Italy in the early 1990s and this led to the idea that 624I was an ancestor of the Q1 genotype. Despite the fact that most heterogeneity of IBVs occurs in the S1 gene, the sequence analysis of this gene alone was not sufficient to confirm or deny this hypothesis. In the present study, an Italian 624I (gammaCoV/AvCov/Ck/Italy/IP14425/96) was fully sequenced for the first time and compared to all available complete Q1 genome sequences. This analysis confirmed the genetic correlation between GammaCoV/AvCov/Ck/Italy/IP14425/96 and Q1 strains, suggesting a common origin between 624I and Q1 genotypes.     

Cetacean morbillivirus in Southern Right Whales,Brazil

Cetacean morbillivirus (CeMV) has caused repeated epizootics and interepizootic fatalities in a variety of cetacean species worldwide. Recently, a novel CeMV strain (GD‐CeMV) was linked to a mass die‐off of Guiana dolphins (Sotalia guianensis) in Brazil. Southern right whales (SRWs; Eubalaena australis) migrate to the southern Brazilian coast during austral winter and spring (June through November) for breeding and calving. Because unexplained high calf mortality rates have recurrently been documented in SRWs, we hypothesized they could be infected with CeMV. We developed a novel real‐time RT‐PCR method based on SYBR^® GREEN for detection of CeMV and identified the virus in three out of five stranded SRWs from Santa Catarina state, Brazil. The partial sequences of the morbillivirus phosphoprotein gene suggest that the virus is similar to the GD‐CeMV strain. Our results indicate CeMV can infect SRWs and should be considered in the differential aetiologic diagnosis of infectious diseases in this species. It also raises concern for potential conservation implications for this species in its main coastal breeding area off Southern Brazil.     

Extensive CFTR sequencing through NGS in Brazilian individuals with cystic fibrosis: unravelling regional discrepancies in the country

BackgroundThe Brazilian population has a tri-hybrid composition with a high degree of ethnic admixture. We hypothesized that Brazilian individuals with CF from different Brazilian regions have a specific distribution of CFTR variants.MethodsIndividuals with CF with data available in the Patient Registry and without an established genotype were submitted to CFTR sequencing by Next Generation Sequencing (NGS) methodology, and results were anonymously incorporated to the Registry Database. Genotyping results were expressed as ‘positive’, ‘inconclusive’ or ‘negative’. Logistic regression models were performed to investigate the association between demographic/clinical variables and genotyping results. Mediation analysis was conducted to estimate direct and indirect effects of Brazilian region on a binary positive genotyping response.ResultsIn October 2017, data from 4,654 individuals with CF were available, and 3,104(66.7%) of them had a genotyping result. A total of 236 variants (114 new variants) were identified, with F508del identified in 46% of the alleles tested. Genotyping revealed 2,002(64.5%) individuals positive, 757(24.4%) inconclusive and 345(11.1%) negative. Distribution of genotype categories was markedly different across Brazilian Regions, with greater proportions of negative individuals in the North (45%) and Northeast (26%) regions. Newborn screening (CF-NBS) and age at diagnosis were identified as mediators of the effect of Brazilian region on a positive genotyping result.ConclusionsThis large initiative of CFTR genotyping showed significant regional discrepancies in Brazil, probably related to socio-economic conditions, lack of adequate CF-NBS and poor access to reliable sweat testing. These results may be useful to indicate Regions where CF care demands more attention.     

Identification of Natural Infections in Sheep/Goats with HoBi‐like Pestiviruses in China

The natural infections of HoBi‐like pestiviruses in cattle have been reported in South America, Europe and Asia. In China, although the detections of HoBi‐like pestivirus have been reported, the epidemiological investigation was limited. From January 2014 to October 2015, several flocks of sheep/goats in Henan province in central China suffered respiratory diseases which were recovered slowly after antibiotics treatment. To test whether it is the HoBi‐like pestivirus caused this symptom, 49 serum samples and 22 nasal swabs were then collected for analysis by serology and RT‐PCR. Serological result revealed that prevalence of pestivirus in small ruminants was 12.2% (6/49) in central China. Sequence analysis of partial 5′‐UTR nucleotides of pestivirus‐positive samples suggested that HoBi‐like pestivirus might have circulated in sheep/goats of China for a period and have evolved into new genotype clusters. It is apparent that the study provides the molecular evidence of natural infections in goat/sheep species with HoBi‐like pestiviruses in China.     

The Region of Difference Four is a Robust Genetic Marker for Subtyping Mycobacterium caprae Isolates and is Linked to Spatial Distribution of Three Subtypes

Alpine Mycobacterium caprae isolates found in cattle and red deer display at least three genetic variations in the region of difference four (RD4) that can be used for further differentiation of the isolates into the subtypes ‘Allgäu’, ‘Karwendel’ and ‘Lechtal’. Each genomic subtype is thereby characterized by a specific nucleotide deletion pattern in the 12.7‐kb RD4 region. Even though M. caprae infections are frequently documented in cattle and red deer, little is known about the transmission routes. Hence, robust markers for M. caprae subtyping are needed to gain insight into the molecular epidemiology. For this reason, a rapid and robust multiplex PCR was developed for the simultaneous detection of three M. caprae RD4 subtypes and was used to subtype a total number of 241 M. caprae isolates from animals (145 cattle, 95 red deer and one fox) from Bavaria and Austria. All three subtypes occur spatially distributed and are found in cattle and in red deer suggesting transmission between the two species. As subtypes are genetically stable in both species it is hypothesized that the described genetic variations developed within the host due to ‘within‐host replication’. The results of this study recommend the genomic RD4 region as a reliable diagnostic marker for M. caprae subtype differentiation.