Bovine anaplasmosis as a risk factor for retained placenta, mastitis, and abomasal displacement in dairy cattle.

This study aimed to evaluate the frequency of IgG antibodies against A. marginale, the occurrence of this bacterium by qPCR, and the effect of bovine anaplasmosis as a risk factor for clinical cases of retained placenta, mastitis, and abomasal displacement in dairy cattle. For that 179 Holstein cows out of three dairy herds, in the municipality of Sertão, Rio Grande do Sul, Brazil. These cows were on farms that were vulnerable to risk factors that are crucial to susceptibility among these animals to this intracellular hemoparasite. The mean seropositivity for A. marginale from the periods evaluated was 54% on farm A, 69.4% on farm B, and 27.3% on farm C. Molecular diagnosis was performed with qPCR and the mean positivity for A. marginale among the cows on farms A, B, and C in December 2017 was 34.6% (67/179). Infected animals showed clinical cases of retained placenta (6.1%), mastitis (6.1%), and abomasal displacement (0.5%). The association between positivity for anaplasmosis and these clinical cases was assessed through the odds ratio. Our results show that females with a positive qPCR assay for A. marginale had 52.48 times increased probability (OR) to develop clinical cases of retained placenta and mastitis (P < 0.001). These clinical cases negatively impact the productivity of positive females. Thus, implementing preventive and prophylactic control measures to ensure the sanitary quality of the herds is needed to avoid losses due to morbidity and mortality and diminish the economic losses suffered by farmers.

The co-infection with Ehrlichia minasensis, Anaplasma marginale and Anaplasma platys is not associated with anemia in beef cattle in the Brazilian Pantanal.

The Anaplasmataceae family is composed of obligatory intracellular Gram-negative bacteria transmitted by arthropod vectors. In Brazil, with the exception of Anaplasma marginale, little is known about the occurrence of other Anaplasma and Ehrlichia species infecting cattle. The present study aimed at investigating the occurrence of Anaplasma spp. and Ehrlichia spp. in beef cattle (Bos indicus) sampled in the Brazilian Pantanal, an area prone to periodic flooding and endemic for bovine trypanosomiasis. Blood samples from 400 cattle were collected and screened by PCR assays based on rrs and dsb genes from Anaplasma spp. and Ehrlichia spp., respectively. Positive samples for Anaplasma spp. were subjected to qPCR assays based on the msp-2 gene and nPCR based on the groEL gene. As a result, 4.75% (19/400) and 48.12% (167/347) were positive for Anaplasma platys and Ehrlichia minasensis, respectively. Besides, positivity of 56.75% (227/400) for A. marginale and seropositivity of 90.75% (363/400) for Trypanosoma vivax were found. A high rate of co-infection was observed (67.25%), from which the co-infection by A. marginale and E. minasensis was more frequently found in calves than cows. Interestingly, none of the animals presenting co-infection showed anemia or other clinical signs. The present study showed, for the first time, the occurrence of A. platys and E. minasensis in beef cattle in the southern Pantanal, as well as a high rate of co-infection by A. marginale, E. minasensis and T. vivax in the sampled animals.

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.

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal, an endemic area for bovine trypanosomiasis in South America.

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.

Genetic diversity of Babesia bovis in beef cattle in a large wetland in Brazil.

Babesia bovis is the etiological agent of bovine babesiosis, a disease transmitted by Rhipicephalus microplus, which affects cattle herds in tropical and subtropical regions of the world, causing significant economic losses due to decreasing meat and milk yield. This study used molecular techniques to determine the occurrence and genetic diversity of B. bovis, based on the genes encoding the spherical body protein (sbp-2) and the merozoite surface antigens (MSAs) genes, in a herd of 400 Nellore (Bos indicus) sampled from beef cattle farms in the Pantanal region, state of Mato Grosso do Sul, Midwestern Brazil. The results of the nested PCR assays based on the sbp-2 gene indicated that 18 (4.5%) calves were positive for B. bovis; out of them, while 77.7% (14/18) were positive for the B. bovis msa-2b fragment, 66.6% (12/18) were positive for the msa-2c fragment. The phylogenetic analysis based on the maximum likelihood method using 14 sequences from msa-2b clones and 13 sequences from msa-2c clones indicated that the sequences detected in this study are clearly distributed in different cladograms. These findings corroborated the diversity analysis of the same sequences, which revealed the presence of 14 and 11 haplotypes of the msa-2b and msa-2c genes, respectively. Furthermore, the entropy analyses of amino acid sequences revealed 78 and 44 high entropy peaks with values ranging from 0.25 to 1.53 and from 0.27 to 1.09 for MSA-2B and MSA-2C, respectively. Therefore, the results indicate a low molecular occurrence of B. bovis in beef cattle sampled in the Brazilian Pantanal. Despite this, a high degree of genetic diversity was found in the analyzed B. bovis population, with possibly different haplotypes coexisting in the same animal and/or in the same studied herd.

Genetic diversity of Anaplasma marginale in beef cattle in the Brazilian Pantanal.

There are few studies on the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially about beef cattle. The objective of this study was to evaluate the genetic diversity of A. marginale, based on the msp1α gene in Bos taurus indicus sampled from the Brazilian Pantanal. Aliquots of blood with and without EDTA were taken from 400 cattle (200 cows and 200 calves) across five extensive farms. The samples were submitted to the indirect immunoenzymatic assay (iELISA), quantitative real-time PCR (qPCR) for the msp1ß gene and to the semi-nested (sn) PCR for the msp1α gene. Positive samples were sequenced by the Sanger method and subjected to diversity analysis using the RepeatAnalyser software. The percentage of positive animals by iELISA, qPCR and (sn) PCR was 72.2% (289/400), 56.7% (227/400) and 23% (52/227), respectively. Cows (154/200) showed to be significantly more seropositive than calves (135/200). In qPCR, the number of calves and average quantification value (138/200; 1.3 × 106) A. marginale msp1α copies per µL proved to be higher when compared to that found for the cows (89/200; 3.9 × 104). The microsatellite analysis of the 26 sequences obtained from the msp1α gene revealed the presence of E (77%), C (15.4%) and B (7.7%) genotypes. Fourteen A. marginale strains were identified in the studied region, with eight that have never before been described in the literature (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-ß-ß-ß-100; EV7-11-10-15; τ-11-11-27-18; τ-11-10-15; τ-27-13-18). Beef cattle are highly exposed to A. marginale in the Brazilian Pantanal. Moreover, a high genetic diversity of A. marginale, with eight new strains, was found in the studied region. While cows may act as chronic carriers, perpetuating the pathogen within the herd, male beef calves sold to other regions may disperse these strains.

Genetic diversity and hematological and biochemical alterations in Alouatta primates naturally infected with hemoplasmas in Brazil.

Mycoplasma spp. and Bartonella spp. are Gram-negative bacteria transmitted by arthropod vectors that infect red blood cells of several mammal species. This study investigated the occurrence and genetic diversity of hemoplasmas and Bartonella spp. in 68 howler monkeys kept in captivity in São Paulo, a southeastern state in Brazil. In addition, possible hematological, biochemical and electrophoretic changes of serum proteins associated with the occurrence of hemoplasmas and Bartonella spp. in captive primates were also investigated. The cPCR results showed that all sampled howler monkeys were negative for Bartonella spp. based on the gltA gene. The cPCR results indicated that 18 (26.47%) non-human primates (NHP) were positive for hemoplasmas based on the 16S rRNA gene. Monocyte and lymphocyte counts were higher in hemoplasma-positive howlers (P < 0.05). Platelet counts decreased in nonhuman primates (NHP) positive for hemoplasmas (P < 0.05). The results from the blood serum proteinogram and biochemistry analyses were not significantly different between NHPs positive and negative for hemotrophic mycoplasmas. Phylogenetic analysis using Bayesian Inference (BI) based on the 16S rRNA gene positioned the obtained sequences close to 'Candidatus Mycoplasma kahanei'. The analysis of sequence diversity of the 16S rRNA gene showed that 5 different genotypes are circulating in NHP in Brazil and in the world; besides, a clear separation between the sequences of hemoplasmas that infect NHP of the Sapajus and Alouatta genus in Brazil was found, probably corresponding to two different species. The pathogenic potential of this hemoplasma species in NHP should be further investigated.

Diversity of Anaplasma species in cattle in Mozambique.

Although species of Anaplasma are highly prevalent Rickettsiales agents in domestic and wild ruminants with a wide distribution worldwide, few studies have been conducted so far to detect and/or investigate the diversity of these agentsin cattle in Mozambique. In the present study, serological and molecular assays were used to investigate the occurrence of Anaplasma spp. in 219 bovines sampled in the districts of Boane, Magude, Matutuíne, Moamba and Namaacha in Maputo, Mozambique. In the iELISA test for detection ofIgG antibodies to A. marginale, 86.3% (189/219) of the samples were positive. In qPCR assays for the gene msp1ß for A. marginale and msp2 for A. phagocytophilum, 97.3% (213/219) and 2.7% (6/219) of the animals were positive, respectively. Two different cPCR protocols based on the 16S rRNA gene showed that 100% of the samples were positive for Anaplasma spp. The DNA sequences obtained were phylogenetically related to A. platys, A. phagocytophilum, Candidatus Anaplasma boleense, A. centrale, A. marginale and A. ovis. Phylogenetic inference based on the msp4 and msp5 genes positioned the obtained sequences in the clade of A. marginale, with evidence of occurrence of 8 and 5 different haplotypes for each gene, respectively. Anaplasma sp. phylogenetically associated with A. platys was evidenced in phylogenetic analyzes based on 16S rRNA and groEL genes. It is concluded that a high diversity of species of Anaplasma spp. occurs in cattle in Mozambique.

Molecular detection and characterization of Ehrlichia ruminantium from cattle in Mozambique.

Heartwater caused by Ehrlichia ruminantiumis a disease of domestic and wild ruminants and one of the most economically important tick-borne diseases in Africa. The present study aimed to investigate the occurrence and genetic diversity of E. ruminantium in blood samples from 210 cattle sampled in five districts of Maputo Province, Mozambique. DNA blood samples were initially submitted to PCR assays targeting E. ruminantium pCS20 gene fragments. Additionally, in order to assess the genetic diversity of E. ruminantium, the positive samples were submitted to a PCR assay targeting the E. ruminantium map1 gene. Finally, the amplicons were sequenced and phylogenetic position was inferred using the Maximum Likelihood method. PCR results revealed that the overall prevalence in Maputo Province was 15% of the animals sampled. E. ruminantium map1 sequences showed not to be conserved. In the phylogenetic analysis, E. ruminantium map1 genotypes were positioned into multiple-clades. This study provides information on the prevalence and genetic diversity of E. ruminantium in five localities of Maputo Province. The future immune control strategies against local E. ruminantium must be designed in the light of the genetic diversity of this parasite.