Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.     

Immune response of BALB/c mouse immunized with recombinant MSPs proteins of Anaplasma marginale binding to immunostimulant complex (ISCOM)

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.     

快速凝集试验与补体结合试验检测牛边缘无浆体感染的效果对比

从138份血清样品的比较试验结果显示，快速凝集试验（RCA）比补体结合试验（CF）检测边缘无浆体感染的敏感性高（88．9％：81．5％），假阴性率低（11．1％：18．5％），两者都具有良好的特异性和预测性，检测阳性符合率高，快速凝集试验对一次感染牛的持续检出阳性时间更长久（303天：92天）     

Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.     

The Effect of Management Factors on the Seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican Tropics

A cross-sectional study with a two-stage design and proportional distribution was carried out to determine the effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics. Serum was obtained from 384 cattle aged 1-2 years on 92 farms. The number of samples was proportionally distributed according to the number of farms in eastern Yucatan. Antibody activity against A. marginale was assessed using the card agglutination test. A primary screening using a 2 x K contingency table of the exposed variables with the outcomes was performed. All variables for which p < 0.20 were included in a fixed-effects log regression. The seroprevalence in the cattle was 69.75% (SE +/- 0.02). Sixty-four per cent of the farms had a seroprevalence > or = 75%. The risks related to managemental factors were stocking density ( > or = 1 animal/ha, OR = 10.94), type of acaricide (pyrethroids, OR = 3.8), dipping interval ( > 60 days, OR = 0.13) and type of veterinary instruments used (needles, scalpels, ear tattoos, and dehorners, OR = 0.17).     

Canine vector-borne disease pathogens in dogs from south-east Queensland and north-east Northern Territory

Objectives To determine the prevalence of canine vector‐borne diseases (CVBD: Babesia spp., Anaplasma spp., Ehrlichia spp., haemotropic mycoplasmas and Hepatozoon) in Australian dogs; namely, dogs from pounds in south‐east Queensland and an indigenous Aboriginal community in the north‐east of the Northern Territory. Design and procedure Blood samples were collected from 100 pound dogs and 130 Aboriginal community dogs and screened for the CVBD pathogens using polymerase chain reaction (PCR). All positive PCR products were sequenced for species confirmation. Results In total, 3 pound dogs and 64 Aboriginal community dogs were infected with at least one CVBD pathogen. Overall, B. vogeli was detected in 13 dogs, A. platys in 49, M. haemocanis in 23, Candidatus Mycoplasma haematoparvum in 3 and C. M. haemobos in 1 dog. Co‐infections were detected in 22 Aboriginal community dogs. Conclusions This study found B. vogeli, A. platys and haemotropic mycoplasma infections to be common in dogs in subtropical and tropical areas of Australia. This study also reports for the first time the prevalence and genetic characterisation of haemotropic mycoplasmas in dogs in Australia.     

Congenital infection with Anaplasma phagocytophilum in a calf in northern Germany

Anaplasma phagocytophilum is a Gram-negative, obligate intracellular tick-transmitted bacterium that replicates in neutrophils. It causes tick-borne fever (TBF) in sheep and cattle, but also elicits febrile disease in humans as well as in other domestic animals such as dogs, horses, and cats. Although increasingly recognized in Europe, the first laboratory-confirmed case of TBF in cattle from Germany has been published only recently. We here present the unusual case of an intrauterine transmission of A. phagocytophilum in a calf from northern Germany. To our knowledge, this is the first report of such an event occurring under field conditions in cattle.     

某羊场和田羊与策勒黑羊无浆体季节性感染PCR检测

为了解和田羊和策勒黑羊无浆体季节动态感染情况,分别于2015年冬至、2016年春分、夏至和秋分,在策勒县某牧业合作社采集和田羊和策勒黑羊血液样本共120份,应用PCR对无浆体的MSP4基因和16SrRNA基因进行扩增。结果发现,检测绵羊的无浆体总阳性率为99.2%(119/120),以绵羊无浆体(Anaplasma ovis)和嗜吞噬无浆体(A.phagocytophilum)混合感染为主,占阳性样本比例为61.3%(73/119),A.ovis和A.phagocytophilum占阳性样本比例分别为31.1%(37/119)和7.6%(9/119)。在不同季节,和田羊和策勒黑羊无浆体均普遍感染,差异不显著(P>0.05)。结果显示,新疆策勒县某羊场绵羊的无浆体病全年均可流行,应引起人们重视。     

Prevalence and genetic diversity of Anaplasma marginale strains in cattle in South Africa

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1alpha polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1alpha amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.