Knockdown of the Rhipicephalus microplus Cytochrome c Oxidase Subunit III Gene Is Associated with a Failure of Anaplasma marginale Transmission

Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.     

Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands

The effect of Anaplasma marginale antibodies ingested with the tick blood meal was tested on infected male ticks that were allowed to feed on cattle immunized with the erythrocytic stage of A. marginale. The experiments were done in two trials. Trial 1 was done using splenectomized calves (two calves per treated and control groups) while ticks in trial 2 were fed on intact yearling cattle (four cattle per treated and control groups). The cattle were immunized with purified outer membrane proteins of erythrocyte-derived A. marginale using saponin (trial 1) or monophosphoryl lipid-A-trehalose dicorynomycolate adjuvant (trial 2). The corresponding control cattle received adjuvant only. All cattle were challenged using Dermacentor andersoni males infected as adults that were allowed to feed for 7 days. In trial 1, the ticks were allowed to feed a second time on susceptible calves to test whether exposure of ticks to immunized cattle affected their ability to transmit anaplasmosis. Infections in fed ticks were monitored by determining the infection rates in salivary glands with an A. marginale-specific RNA probe and light microscopy. Vaccine-derived antibodies ingested with the tick blood meal did not appear to affect the development of A. marginale in previously infected ticks. The infection rates in the salivary glands were not significantly different among ticks fed on immunized versus adjuvant control cattle. When the vaccine-exposed ticks in trial 1 were allowed to feed a second time on susceptible calves, the resulting clinical symptoms of anaplasmosis were similar to those of the controls. There was no statistically significant effect of tick exposure to the anti-erythrocytic stage antibody on the development of salivary gland infection or transmission of A. marginale by ticks.     

Characterization of Anaplasma marginale Isolated from North American Bison

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present study was undertaken to confirm A. marginale infection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of this A. marginale bison isolate for cattle. The results of this study showed that these A. marginale isolates obtained from bison were similar to ones from naturally infected cattle.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Prevalence of Anaplasma marginale in cattle blood samples collected from two important livestock regions in Punjab (Pakistan) with a note on epidemiology and phylogeny of parasite

Anaplasmosis, caused by intracellular gram-negative bacteria Anaplasma marginale is one of the most frequently reported tick-borne disease (TBDs) in tropical and sub-tropical countries, including Pakistan. In the present study, a total of 428 cattle blood samples were collected to examine the prevalence and phylogenetic origin of A. marginale in two important livestock regions of Punjab Province in Pakistan, i.e. Lodhran and Dera Ghazi Khan Districts. In addition, association between occurrence of A. marginale in cattle blood and selected epidemiological factors has been also investigated. The presence of A. marginale genetic material was confirmed in 9% of the tested blood samples taken from cattle in Lodhran and in 17% from Dera Ghazi Khan. Prevalence of A. marginale was significantly higher in cattle from Dera Ghazi Khan. All the cattle breeds from both districts were equally susceptible to A. marginale infection. We reported higher prevalence of A. marginale in cattle living indoors or with other dairy animals in Dera Ghazi Khan district. However, no such relationship was observed in the Lodhran district. Sequencing of the msp1b gene shows 96–99% similarity of A. marginale in the study area to those reported from other parts of Pakistan, South Africa, and Israel. We recommend that large scale tick and tick-borne disease control strategies must be implemented in both districts.     

Adaptations of the Tick-Borne Pathogen, Anaplasma marginale, for Survival in Cattle and Ticks

The tick-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) multiplies within membrane-bound inclusions in host cell cytoplasm. Many geographic isolates of A. marginale occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. A tick cell culture system for propagation of A. marginale proved to be a good model for study of tick-pathogen interactions. Six major surface proteins (MSPs) identified on A. marginale from bovine erythrocytes were conserved on A. marginale derived from tick cells. MSP1a and MSP1b were adhesins for bovine erythrocytes, while only MSP1a was found to be an adhesin for tick cells. The tandemly repeated portion of MSP1a was found to be necessary and sufficient for adhesion to both tick cells and bovine erythrocytes. Infectivity of A. marginale isolates for ticks was dependent on the adhesive capacity of the isolate MSP1a, which was found to involve both the adhesive properties and sequence of the repeated peptides. Cattle immunized with A. marginale derived from bovine erythrocytes or tick cells demonstrated a differential antibody response to MSP1a and MSP1b that resulted from the differential expression of these proteins in cattle and ticks cells. MSP2, derived from a multi-gene family, was found to undergo antigenic variation in cattle and ticks and may contribute to establishment of persistent A. marginale infections. MSP1a has been used as a stable genetic marker for geographic isolates because the molecular weight varies due to differing numbers of the tandem repeats. However, phylogenetic studies of A. marginale isolates from North America using MSP1a and MSP4 demonstrated that MSP4 was a good biogeographic marker, while MSP1a varied greatly among and within geographic areas. Infection and development of A. marginale in cattle and tick cells appears to differ and to be mediated by several surface proteins encoded from the small genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.     

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis

Background  

The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.     

First report regarding molecular epidemiology and novel variant identification of Anaplasma centrale in cattle from Pakistan

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017–18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.     

Babesia bigemina,Babesia argentina, and Anaplasma marginale: Coinfectious immunity in bovines

Forty-eight intact and eight splenectomized cattle were used to evaluate different systems of coinfectious immunization against Babesia bigemina, Babesia argentina, and Anaplasma marginale. Coinfectious immunity was induced by two methods: (1) blood of cattle acutely infected with B. bigemina, B. argentina and A. marginale was used as the source of inoculum and the post vaccination reactions were chemotherapeutically controlled with Imidocarb, Ganaseg, Gloxazone, and Liquamycin, and (2) by artificially inducing babesiosis with the blood of carrier cattle with chronic infections of B. bigemina and B. argentina without chemotherapy. The degree of resistance was determined by bloodborne and tick-borne challenges. Ticks were collected from cattle and identified as Boophilus microplus and Dermacentor nitens. Vaccinated cattle demonstrated a high degree of resistance to babesiosis and anaplasmosis; however, cattle without coinfectious immunity were treated chemotherapeutically to prevent death losses.     

Prevalence and Genotypes of Anaplasma Species and Habitat Suitability for Ticks in a Mediterranean Ecosystem

Anaplasma species are tick-transmitted pathogens that impact veterinary and human health. Sicily is one of the locations where these pathogens are endemic. Sicily represents a typical Mediterranean ecosystem to study Anaplasma infection and tick habitat suitability. The aims of this study were (i) to characterize by 16S rRNA and species-specific msp4 gene PCR the prevalence and genotypes of A. marginale, A. phagocytophilum, and A. ovis in the most abundant host species in Sicilian provinces and (ii) to correlate differences between hosts and between western and eastern Sicily with the habitat suitability for ticks in these regions. Differences were found in the prevalence of Anaplasma spp. between different hosts and between western and eastern provinces. The differences in Anaplasma prevalence between different hosts may be explained by pathogen host tropism. The differences between western and eastern provinces correlated with the tick habitat suitability in these regions. The analysis of Anaplasma genotypes suggested a higher host and regional specificity for A. phagocytophilum than for A. marginale and A. ovis strains, a finding probably associated with the broader host range of A. phagocytophilum. The presence of identical A. marginale genotypes in the two regions may reflect cattle movement. The results for A. ovis suggested the possibility of some genotypes being host specific. These results provide information potentially useful for the management of tick-borne diseases caused by Anaplasma spp. in Sicily and other Mediterranean regions and may contribute to the development of models to predict the risks for these tick-borne pathogens.     

First Evaluation of an Outbreak of Bovine Babesiosis and Anaplasmosis in Southern Brazil Using Multiplex PCR

Outbreaks of tick-borne disease cases in Santa Catarina, Brazil are known, but the presence of the pathogen DNA has never been determined. In this study, the first survey of Anaplasma marginale, Babesia bigemina, and Babesia bovis DNA on blood samples of 33 cattle from an outbreak in Ponte Alta Municipality, Santa Catarina, Brazil, has been carried out. A multiplex PCR detected 54.5% of animals were co-infected with 2 or 3 parasites, while 24.2% were infected with only 1 species. The most prevalent agent was B. bigemina (63.6%) followed by A. marginale (60.6%). This is the first report of tick-borne disease pathogens obtained by DNA analysis in Southern Brazil.     

Red deer (Cervus elaphus) as a host for the cattle tick Rhipicephalus microplus (Acari: Ixodidae) in Yucatan, Mexico

Rhipicephalus microplus is the most economically important cattle tick in the Mexican tropics. Wild ungulate species, including red deer (Cervus elaphus), are gaining popularity in diversified livestock ranching operations in Mexico. However, there is no information available on the susceptibility of red deer to infestation with the cattle tick, R. microplus, under hot, subhumid tropical conditions in Mexico. Biological data on R. microplus as an ectoparasite of cattle and red deer in a farm in the Mexican tropics are presented here. Ticks collected from red deer were identified as R. microplus (97 %) and Amblyomma cajennense (3 %), and tick species infesting cattle included R. microplus (95 %) and A. cajennense (5 %). Standard counts of R. microplus engorged females on red deer were 11 times higher than on cattle (428 ± 43 vs. 40 ± 18; p < 0.001). The reproductive efficiency index and larval hatching of R. microplus collected from cattle and red deer were similar (p > 0.05). Hemolymph samples of R. microplus collected from cattle were positive for Babesia spp. (10 %, 2/50) and all the samples from ticks infesting red deer were negative. Seventeen and ten percent of the blood samples from cattle and red deer were positive for Anaplasma marginale, respectively. The role of red deer as a host of R. microplus in Yucatan, Mexico and the importance of this host-parasite relationship relative to the epidemiology of R. microplus-borne diseases are discussed.     

Epidemiology and risk factors associated with Anaplasma marginale infection of cattle in Peninsular Malaysia

Bovine anaplasmosis is a major concern to cattle farming in most parts of the world. Anaplasmosis negatively impacts the profitability of cattle farming by reducing the production, reproduction, and draft ability of cattle. Here, we report results from a one-year cross sectional study to determine the epidemiology and the risk factors for Anaplasma marginale infection of cattle in Peninsular Malaysia. Examination of one thousand and forty five blood samples of apparently healthy cattle from forty-three farms in all the states of Peninsular Malaysia by polymerase chain reaction (PCR) assay revealed an overall prevalence of A. marginale infection of cattle of 72.6%, showing high endemicity of this heamoprotozoan among cattle in the country. Cattle breeds, production type, herd owner, herd size, management system, farm size, farm age, prophylactic treatment against blood parasites, presence of ticks, frequency of deticking, zones, closeness to forest, closeness to waste area, closeness to human settlement and closeness to body of water were the risk factors significantly associated (P?<?0.05) with the detection of A. marginale in cattle. Results of this first molecular study on the epidemiology and risk factors for A. marginale infection of cattle from all the states of Peninsular Malaysia suggest policies and strategies for the prevention and control of the parasite to improve profitability of cattle farming in the country.     

First detection of Anaplasma ovis in sheep and Anaplasma platys-like variants from cattle in Menoufia governorate,Egypt

Tick-borne diseases are of global economic importance, especially due to the costs associated with disease treatment and productivity losses in livestock. In this study, 244 livestock animals (cattle N = 92, buffaloes N = 86 and sheep N = 66) from Menoufia, Egypt were tested for Anaplasma, Ehrlichia and Babesia species using PCR. Results revealed detection of A. ovis (9.1%) in sheep while Anaplasma spp. (14.1%), A. marginale (15.2%), B. bigemina (6.5%) and B. bovis (5.4%) in cattle. On the other hand, Anaplasma spp. (1.2%), A. marginale (1.2%) and B. bovis (1.2%), were detected in buffaloes. Significantly higher detection rates were observed in cattle for Anaplasma spp. (P = .020), A. marginale (P = .001) and B. bigemina (P = .022) than in buffaloes. Sequence analysis of Anaplasma spp. isolates from cattle, revealed A. platys-like strains. Phylogenetic analyses of the A. platys-like isolates revealed variation among the strains infecting cattle. The A. marginale buffalo isolate, on the other hand, showed some level of divergence from the cattle isolates. This study reports the first detection of A. ovis in sheep and A. platys-like strains in cattle in Menoufia and Egypt at large. The results of the current study provide valuable information on the epidemiology and genetic characteristics of tick-borne pathogens infecting livestock in Egypt.     

Bovine babesiosis and anaplasmosis: Control by premunition and chemoprophylaxis

Experiments were carried out to evaluate two systems: (1) premunition and (2) chemoprophylaxis for the control of bovine babesiosis and anaplasmosis in the Cauca River Valley, Colombia. Control of these diseases was achieved by inoculating cattle with virulent Babesia bigemina, Babesia argentina, and Anaplasma marginale and subsequent treatment with Imidocarb and Gloxazone to moderate the postpremunition reactions. Chemoprophylactic treatment with Imidocarb and Gloxazone was administered to cattle before and during field exposure. Premunized cattle were highly resistant to tick-borne (Boophilus microplus) challenge. Imidocarb had therapeutic and chemoprophylactic properties against babesiosis, but appeared toxic. Gloxazone moderated the A. marginale postpremunition reaction, but failed to prevent clinical anaplasmosis under the conditions of this investigation.     

PCR-diagnosis of <Emphasis Type="Italic">Anaplasma marginale</Emphasis> in cattle populations of Ecuador and its molecular identification through sequencing of ribosomal 16S fragments

Background

Bovine anaplasmosis is an endemic disease in tropical and subtropical areas. It is caused by a bacterium named Anaplasma marginale, and represents an economic problem for cattle farmers due to the losses it generates, such as: mortalities, reduced production, quarantine measures, treatments and control of vectors. The method most often used to diagnose this haemotrophic bacterium is direct examination on blood smear, which sensitivity and specificity are limited compared to other methods such as PCR. The present study aimed at investigating the presence of A. marginale in dairy cattle of Luz de América commune, province of Santo Domingo de los Tsachilas. Two PCRs were used to amplify specific regions of the Rickettsia for its molecular identification.

Results

At first, 151 blood samples were tested: msp5 specific gene of A. marginale was identified in 130 samples, meaning 86.1% of them were infected by the rickettsia. Two positive samples were further randomly selected to confirm the presence of A. marginale through amplification, cloning and sequencing of the conserved region of gene 16S rRNA. The analysis of sequences obtained through cloning revealed a 100% identity between both samples and those registered in GenBank for A. marginale.

Conclusion

This is the first report and molecular identification of A. marginale in the bovine population of Ecuador and its prevalence was high at the level of farms and animals. These results demonstrate the importance of proceeding to evaluate and characterize bovine Anaplasmosis in Ecuador in order to establish control measures and reduce their impact.

     

Blood parasites of livestock in certain Regions in Saudi Arabia

Blood samples from camels, sheep, goats and cattle from six Regions in Saudi Arabia were examined for blood parasites. Asir Region camels were disinfected while those of the Eastern, Jazan, Northern Frontiers, Riyadh and Tabouk Regions were infected with Trypanosoma evansi (5–40%), those of Riyadh and the Eastern Regions were infected with Dipetalonema evansi (1–6%) and those of the Eastern, Jazan and Riyadh Regions were infected with Eperythrozoon species (8–20%). Sheep and goats of all tested regions were infected with Theilaria hirci (4–20% and 6–14%, respectively), Theilaria ovis (5–19% and 6–24%, respectively) and Eperthrozoon ovis (2–9% and 2–8%, respectively). Sheep of the Eastern and Northern Frontiers Regions were also infected with Anaplasma ovis (2%) and also those of the Eastern Region were infected with Babesia motasi (4%) as well. Cattle of Asir and Eastern Regions were infected with Anaplasma marginale (1–3.4%) and those of the Eastern, Jazan and Riyadh Regions were infected with Theileria annulata (11.3–25%) and Eperthrozoon wenyoni (1–4%). Moreover, Jazan cattle were infected with Babesia bigemina (6%) and a benign Theileria species (27%). Some of these parasites are recorded in new localities indicating that they are spreading in the country. Also, this is the first report in Saudi Arabia of D. evansi in camels, A. ovis and B. motasi in sheep and A. marginale and B. bigemina in cattle. These parasites may be introduced into the country with infected livestock infested with the vectors of these parasites. The suspected vectors of the detected parasites in Saudi Arabia is discussed. Follow up surveys of blood parasites are recommended to assess their distribution and infection rates in the livestock of all Regions of Saudi Arabia, to make plans for control measures against their vectors.     

Identification of Anaplasma marginale type IV secretion system effector proteins

Background

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.

Results

By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.

Conclusions

The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.