Epidemiological assessment of Anaplasma marginale, Babesia bigemina, and Babesia bovis infections in Colombian creole cattle breeds: A molecular survey in northeastern Colombia.

Anaplasmosis and babesiosis are globally distributed arthropod-borne diseases known for causing substantial economic losses due to their high morbidity and mortality rates. This study aims to assess the frequency and epidemiological features associated with the infection of Anaplasma marginale, Babesia bigemina, and Babesia bovis in three Creole cattle breeds (Chino Santandereano (Chino), Casanareño (CAS), and Sanmartinero (SM)) in northeastern Colombia. Between June 2019 and March 2020, a total of 252 Creole cattle were sampled, with Chino, CAS, and SM accounting for 42.8%, 29.5%, and 29.5% of the samples, respectively. Blood samples were subjected to molecular analysis to detect the DNA of A. marginale, B. bigemina, and B. bovis, using species-specific primers. Additionally, Packed Cell Volume (PCV), total serum proteins, and body condition were evaluated. Molecular analyses revealed the presence of B. bigemina, A. marginale, and B. bovis in 83.7% (211/252; 95% CI = 79.1%-88.3%), 59.9% (151/252; 95% CI = 53.8%-66.1%), and 40.9% (103/252; 95% CI = 34.7%-46.9%) of the samples, respectively, with 69% (174/252; 95% CI = 57.8%-80.3%) exhibiting coinfections. Notably, in infected animals, no significant alterations in PCV, total serum proteins, or body condition were observed. Multivariate analyses indicated a statistically significant association between the frequency of A. marginale infection and the breed and season, with a higher frequency in SM during the rainy season (P < 0.05). To our knowledge, this is the first molecular survey that evaluates multiple arthropod-borne pathogens in Colombian Creole breeds. The results revel a high frequency of B. bigemina and A. marginale infections, coupled with a notable frequency of coinfections, all without significant alteration in the PCV, total serum proteins and body conditions. Our findings enhance the understanding of the epidemiological aspects of arthropod-borne pathogens in Colombian Creole breed and contribute to the improvement of sanitary programs for these animals.

Molecular epidemiology, phylogenetic analysis and risk assessment of Anaplasma marginale from naturally infected bovines of Punjab (India).

Anaplasmosis, an infectious vector borne rickettsial disease caused by different species of Anaplasma transmitted through mechanical and biological (tick) processes has a great concern to livestock industry due to its associated economic losses. The current cross sectional comprehensive epidemiological study was conducted from August 2020 to November 2021 on 391 (277 cattle and 114 buffaloes) bovines from different districts of five agro climatic zones of Punjab state. Classical microscopic examination of Romanowsky stained blood smears showed an occurrence of 13.00%, while DNA amplification targeting major surface protein (msp5) of Anaplasma marginale revealed the 382 bp amplicon in 32.48% samples. Zone based molecular prevalence of A. marginale was highest in the Undulating zone (41.93%) and least in the Sub-Mountain zone (18.84%). The prevalence in Central plain zone, Western plain zone, and Western zone were 40.15, 30.95 and 29.91%, respectively. An overall molecular prevalence of A. marginale was 34.52, 32.75 and 20.0% in young, adults and calves, respectively, the difference being non-significant. Anaplsma marginale was more prevalent in unorganized farms (38.22%; 60/157) than organized farms (28.63%; 66/234). Risk factors analysis revealed young, female cattle at unorganized farms were more prone to anaplasmosis. Out of 127 positive samples, four samples were customed to sequencing revealed 98-99% homology with published sequences for other available global isolates. Multi-single nucleotide polymorphisms (SNP) were observed in the sequence of two samples when aligned with the reference sequence from the NCBI database (CP023731).

Molecular Assessment of Anaplasma marginale in Bovine and Rhipicephalus (Boophilus) microplus Tick of Endemic Tribal Belt of Coastal South Gujarat, India.

BACKGROUND AND METHODS: This study described the detection, prevalence and phylogeny of Anaplasma marginale in the bovine (cattle and buffaloes) and Rhipicephalus (Boophilus) microplus tick belonged to the tribal area of coastal South Gujarat, India, by amplifying 576 bp of major surface protein (msp) 5 gene using custom designed primers in the polymerase chain reaction (PCR). RESULTS: The PCR detection limit was up to 20 parasites/µl of blood in sensitivity experiment, and observed 100% specificity against Trypanosoma evansi, Babesia bigemina and Theileria annulata. Prevalence rate of the A. marginale in the bovine (n = 211)) was 18.48% and 6.64% (p < 0.05) as per the PCR and Giemsa stained blood smear, respectively. Febrile animals (35%) observed significantly (p < 0.05) higher incidence rate than the non-febrile (14.62%). The amplified msp5 had single cut site for the EcoR1 enzyme, upon digestion yielded two fragments of 365 and 211 bp on 1.0% agarose gel. The current sequence (KC811329) showed 100% homology and 1064 total score with the published nucleotide sequences of msp5 of A. marginale in the NCBI-BLAST study. Monophyletic relationship was observed with high bootstrap proportion (> 76% in Neighbor-Joining/ Maximum Likelihood) between the current and published nucleotide sequences in the phylogeny. Twenty out of 39 A. marginale infected bovine recorded R. (B.) microplus on their body surface, out of which 18 had detected the infection. The rickettsia was in 55%, 65% and 25% of anterior half, posterior half and egg of tick, respectively. CONCLUSION: The test detected A. marginale in a carrier, pre-symptomatic and symptomatic vertebrate hosts (cattle and buffalo) and different body parts of the starved R. (B.) microplus including its egg. The current genotype could be an explanation for the frequent outbreaks of bovine anaplasmosis in the targeted areas.

Differential expression analysis for subolesin in Rhipicephalus microplus infected with Anaplasma marginale.

Rhipicephalus microplus (formerly Boophilus microplus) ticks are potential vectors of several pathogens of livestock especially in tropical and subtropical regions where may have substantial effects on economic development. Among tick-borne pathogens, Anaplasma marginale is considered one of the most important in domestic and wild ruminants worldwide. Different molecular mechanisms have been employed by both ticks and these intracellular pathogens, in order to be able to adapt and survive. Subolesin, originally called 4D8, is an evolutionarily well-preserved protein among ixodid tick species. This new antigen was found to be protective against tick infestations when used as a vaccine, as it has an essential role in tick blood digestion, development and infection of host cells by A. marginale. Recent studies have demonstrated that infection of both tick and vertebrate host cells with this microorganism changed gene expression. Therefore, the main objective of this study was to investigate subolesin expression in uninfected and A. marginale-infected R. microplus salivary glands by real-time reverse transcriptase (RT)-PCR. To analyze the differential expression of the recombinant protein subolesin, the gene was previously expressed from ticks infected with A. marginale. Results from this study revealed that, the expression of subolesin was significantly higher in salivary glands of infected R. microplus in comparison to uninfected ones.

A partially purified outer membrane protein VirB9-1 for low-cost nanovaccines against Anaplasma marginale.

Anaplasma marginale is a devastating tick-borne pathogen causing anaplasmosis in cattle and results in significant economic loss to the cattle industry worldwide. Currently, there is no widely accepted vaccine against A. marginale. New generation subunit vaccines against A. marginale, which are much safer, more efficient and cost-effective, are in great need. The A. marginale outer membrane protein VirB9-1 is a promising antigen for vaccination. We previously have shown that soluble recombinant VirB9-1 protein can be expressed and purified from Escherichia coli and induce a high level of humoral and cellular immunity in mice. In this study, we re-formulated the nanovaccines using the partially-purified VirB9-1 protein as the antigen and hollow nano-size silica vesicles (SV-100) as the adjuvant. We simplified the purification method to obtain the partially-purified antigen VirB9-1 with a six-fold higher yield. The new formulations using the partially-purified VirB9-1 protein achieved higher antibody and cell-mediated immune responses compared to the purified ones. This finding suggests that the partially-purified VirB9-1 protein performs better than the purified ones in the vaccination against A. marginale, and a certain level of contaminants in the protein antigen can be self-adjuvant and boost immunogenicity together with the nanoparticle adjuvant. This may lead to finding a "Goldilocks" level of contaminants. The new nanovaccine formulation using partially-purified antigens along with nanoparticle adjuvants offers an alternative strategy for making cheaper veterinary vaccines.

Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab.

Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was standardized and employed on 219 blood samples collected from cattle (165) and buffaloes (54) from eastern Punjab to evaluate the status of concurrent infection and associated risk factors. The reaction produced 257- and 407-bp amplification products targeting repetitive nucleotide sequence of T. evansi and msp1ß gene of A. marginale, respectively. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used; duplex PCR was 100% sensitive and 92.66 % specific with conventional microscopy for the detection of mixed infections. Among the agro-climatic zones of interest, undulating zone was at higher risk of T. evansi infection (odds ratio (OR) = 1.75, 95% confidence interval (CI) = 0.94-3.27), and submountain zone (OR = 1.89, 95% CI = 1.11-3.33) for A. marginale. For the concurrent infection, the relative risk among the two zones was almost unity. The cross-bred cattle population was at the highest risk of infection, may it be solo infection of T. evansi (OR = ∞, 95% CI = 1.18-∞)/A. marginale (OR = 6.39, 95% CI = 1.14-125.3) or dual infection (OR = ∞, 95% CI = 0.39-∞) of both as the indigenous cattle are resistant to the infection. Cross-bred cattle were at approximately three times the risk than buffaloes. For the dual infection, the cattle calves were at about 2.5 times higher risk than buffalo calves. Results indicate the endemic status of these infections in the region and mark out the commodities at great risk and requiring better surveillance.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle.

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.

Prevalence of livestock diseases and their impact on livelihoods in Central Equatoria State, Southern Sudan.

A participatory epidemiological (PE) study was conducted in Kajo Keji and Yei Counties, Central Equatoria State, southern Sudan to assess the impact of livestock diseases on livelihoods. A serological survey of tick-borne diseases was conducted to supplement the PE study. PE data collection tools consisted primarily of focus group interviews and key informant interviews supplemented by observation. Information was collected on the social context, history and species of livestock kept. Constraints in livestock keeping were explored through description and probing. Proportional piling on the importance of different diseases and relative incidence scoring were also conducted. 243 sera were collected from cattle and tested for antibodies to Anaplasma marginale, Babesia bigemina, B. bovis, Theileria mutans and T. parva by ELISA. Additionally, 173 blood samples were collected for a PCR assay of T. parva. Livestock diseases were ranked as the most important constraint to livestock keeping. While East Coast fever was ranked as the most important disease in Kajo Keji, diarrhoea in small ruminants was reported as the most important disease in Yei. Serological analyses of the sera indicated that A. marginale, B. bigemina, T. mutans and T. parva were most prevalent. Prevalence of B. bovis was found to be low (4.0% and 7.4% in Kajo Keji and Yei, respectively). 35% of the samples screened with the T. parva p104 gene nested PCR assay were positive. The study concludes that while ECF is the most important disease in Kajo Keji, it was not the case in Yei. Additional epidemiological studies are proposed before control strategies are recommended.

Seroprevalence of Anaplasma marginale in 2 Iowa feedlots and its association with morbidity, mortality, production parameters, and carcass traits.

A prospective cohort observational study was conducted to investigate the seroprevalence of Anaplasma marginale in Iowa feedlots and its association with morbidity, mortality, and treatment costs. Blood samples were taken from 659 calves from 31 consigners at processing and classified as seropositive to A. marginale using a competitive enzyme-linked immunosorbent assay (cELISA) with a 30% cutoff. Health and production parameters were modeled by A. marginale serostatus with mixed model regression analysis. The apparent prevalence of seropositive cattle was 15.17% (100/659). When the cELISA positive cutoff was at 42% inhibition, the apparent prevalence was 5.00% (33/659). There was no significant association between A. marginale serostatus and production parameters; however, seropositive status had a weak positive association with undifferentiated fever (P = 0.17). Although prevalence of anaplasmosis in Iowa feedlots is higher than reported in Montana-sourced calves arriving in Canadian feedlots, this was not associated with increased production costs.

Validation and field assessment of a rapid lateral flow assay for detection of bovine antibody to Anaplasma marginale.

The lateral flow assay (LFA) is a rapid diagnostic test which may be performed under most conditions and is especially useful for field applications. This type of assay was applied to the detection of antibody to bovine Anaplasma marginale using sera from endemic areas and from areas which have been free from infection for more than 25 years. Briefly, the test uses recombinant A. marginale major surface protein 5 peptide (Msp5), immobilized on a cellulose acetate membrane. A serum sample is added to a pad containing a monoclonal antibody specific for bovine IgG(1), conjugated with colloidal gold, located at one end of the strip. The sample and gold conjugate are wicked along the membrane and if antibody is present in the serum, a visible line will form between the Msp5-antibody-conjugate immune complex in minutes. An additional band of recombinant protein A/G was added to the membrane as a positive control reaction of the monoclonal antibody conjugate. For comparison, direct examination of blood smears and a nested polymerase chain reaction (PCR) were performed on some of the samples. Using samples from herds in one endemic area, the PCR gave a sensitivity value of 9.2% while a commercial competitive enzyme immunoassay (CELISA) gave a sensitivity value of 17.2% and the LFA values of 20.5%. In a second endemic area, selected samples, all positive by direct examination gave a 71.7% sensitivity values with the PCR, 94.5% with the CELISA and 95.5% with the LFA. Using sera from a disease-free area, the specificity values were 100% for the PCR (testing a proportion of randomly selected samples), 99.5% for the CELISA and 98.0% for the LFA. It is envisaged that the validated LFA will be a useful tool for screening cattle moving from an area with infection to a disease-free area.

Anaplasma marginale Yucatan (Mexico) Strain. Assessment of low virulence and potential use as a live vaccine.

Anaplasma marginale Yucatan strain was found to have low virulence in cattle. We studied the virulence of this isolate by experimental inoculation of 113 susceptible cattle at increasing doses, after which only one animal required treatment for clinical disease. Subsequently, 104 cattle received a live vaccine of this strain by inoculation, which induced immunoprotection after heterologous challenged exposure with a different A. marginale isolate. In this study 14% of the immunized cattle required treatment as compared with the control nonimmunized cattle, in which 56% required treatment. The A. marginale vaccine strains used for the immunization studies had MSP1a variable regions that were different from those used for the challenge exposure.

Prevalence of blood parasites in cattle from wilayates of Annaba and El Tarf east Algeria.

Between June and September 2002, a preliminary study was conducted to assess the prevalence of blood parasites of cattle in eastern Algeria. Fifty-four bovines of different genotypes were submitted to clinical examination. From each animal, blood smears were made and stained by Giemsa. Four species of parasites, namely Theileria annulata, T. orientalis, Babesia bovis and Anaplasma marginale were encountered. Fifty animals carried single or multiple infections with blood parasites and four were found negative. The rate of single infections (72.3%, n = 39) was almost three times higher than multiple infections (20.3%, n = 11). The high percentage of single infections was recorded with T. annulata (53.7%). However single infection with Anaplasma marginale (7.4 %), B. bovis (5.6%) and T orientalis (5.6%) were very low compared to T. annulata infection. The rates of mixed infection were as follows: T. annulata/A. marginale 9.3%, T. annulata/T. orientalis 5.6%, A. marginale/T. orientalis 3.7% and B. bovis/A. marginale 1.9%.

The prevalence of serum antibodies to tick-borne infections in Mbale District, Uganda: the effect of agro-ecological zone, grazing management and age of cattle.

Between August and October 2000, a cross-sectional study was conducted in smallholder dairy farms in Mbale District, Uganda to assess the prevalence of ticks and tick-borne diseases under different grazing systems and agro-ecological zones and understand the circumstances under which farmers operated. A questionnaire was administered to obtain information on dairy farm circumstances and practices. A total of 102 farms were visited and sera and ticks were collected from 478 animals. Sero-prevalence of tick-borne diseases was determined using an enzyme-linked immunoassay. Acaricides were used indiscriminately but the intensity of their use varied with the grazing system and zone. Cattle from different farms mixed for various reasons. During the dry seasons farmers have to get additional fodder from outside their farms that can result in importation of ticks. The prevalence of ticks and serum antibodies to tick-borne infections differed across the grazing systems and zones. The highest serum antibody prevalence (>60%) was recorded in the lowland zone under the free range and tethering grazing systems. The lowest tick challenge and serum antibody levels (<50%) were recorded in the midland and upland zones under a zero-grazing system. These findings suggest that endemic stability to East Coast Fever, babesiosis and anaplasmosis is most likely to have existed in the lowland zone, particularly, under the tethering and free-range grazing systems. Also, endemic stability for babesiosis existed in the upland zones. Endemic instability for East Coast Fever existed in the midland and upland zones. These structured observational studies are instrumental in planning of control strategies for ticks and tick borne diseases since production systems and the cattle population at high risk of the diseases in the district have been identified.

Assessment of a low virulence Australian isolate of Anaplasma marginale for pathogenicity, immunogenicity and transmissibility by Boophilus microplus.

A 14-year-old cow (Dawn) born and kept in a Boophilus microplus-free region gave birth to a calf, which showed the presence of an Anaplasma marginale infection after splenectomy. The calf's grand dam was from a B. microplus infected area and we assume the infection originated via the transplacental route over two generations. An isolate, prepared from the calf, had similar or lower pathogenicity as Anaplasma centrale, and previously exposed steers were resistant to challenge by four A. marginale field isolates. Two attempts to transmit the isolate using B. microplus were unsuccessful. Our results indicate that Dawn A. marginale may be a useful vaccine in Australia and warrants larger scale validation of its safety and potency locally as well as of the protection it affords against African and New World isolates.