Wen 丨 Xi talks about Suzaku
Editor丨 Xi talks about Suzaku
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●—Preface—●
Ticks cause significant economic and health losses in the reproductive production of cattle and are the main vector of Babesia bidentis (Babesia bifibrids). Babesia bibuds is a tick-borne disease called babesiosis, which can cause hemolytic anemia, fever, and death.
In the study reported here, we investigated the relationship between tick numbers per animal and the number of copies of Bifidobacterium cytochrome B gene (CBISG) in the blood of untreated Blangues and Nellore cattle in the Brazilian Cerrado biome.
●—"Experiment"—●
Tick counts were performed on 19 animals (9 Blangues and 10 Nelor) every 18 days, and blood was collected every 36 days for 12 months. Serological specimens were detected by indirect enzyme-linked immunosorbent assay, genomic DNA was detected by conventional PCR and quantitative PCR, and PCR products were sequenced by Sanger.
The result is that the Blangues and Nelore cattle breeds show similar weight development without clinical signs of babesiosis. There were statistically significant differences in the number of ticks and the gene copy number of Bifidobacterium bifidobacteria between varieties.
Although Nelor cattle had fewer tick numbers than Blangues cattle, and Nelor cattle had a higher CBSG copy number than Blangues cattle, there was no correlation between tick numbers and the circulating copy number of the Bifidobacterium cytochrome B gene.
●—"Background"—●
In the Cerrado biome, the bull tick is the main vector of transmission of bovine tick fever (CTF), a disease complex caused by three blood parasites: marginal anaplasmosis (Order: Ricketisales), a gram-negative bacterium that causes anaplasmosis in wild and domestic ruminants, and Bababesia bobis and Bisbophyllum bifurcate (Order: Caterpillar family), protozoa that causes Babesiosis.
These blood parasites are mainly present in tropical and subtropical regions and may cause clinical signs such as haemolytic anaemia, fever, occasional haemoglobinuria, and death.
A single host can sustainably be infected with one or more blood parasites that can cause similar clinical symptoms, making it difficult to determine the etiology of CTF in large herds and leading to similar but not fully effective comprehensive treatments for disease.
Brazil's Cerrado biome provides a favourable environment for cattle breeding and for the development of microticks. This tick has a strong preference for cattle, causing huge direct and indirect economic losses associated with cattle farming.
Brahman cattle have been found to have fewer congestive ticks than other types of cattle and are known as tick-resistant breeds. They also exhibit lower levels of babesia parasitaemia than other taurine varieties and their hybrids, however, the degree of parasitism may vary depending on the age of the animal and the reproductive system.
●—"Experimental Process"—●
Animals used in the experiment
Nineteen (9 Brangus cattle and 10 Nellore cattle) were studied in pasture-raised animals during the growing period, aged 8~10 months, with an average weight of 219.5 kg. The animals are naturally infected and are housed together at a density of 0.6 animals per hectare. The ground is composed of sandy soil and a pasture of the cercariae (Brachiaria) decubens. Acaricidal treatment and preventive control of tick-borne diseases are not carried out.
Tick count and blood sampling
Data were collected from June 2016 to June 2017, and tick counts were counted at 18 days intervals according to Wharton and Utech's methods. Briefly, ticks with a length of >4.5–8 mm on both sides of each animal were counted, and tick classification was performed according to the system of Pereira et al.
In addition, every 36 days, animals are weighed with a digital scale and blood is taken from the tail vein using a sterile vacuum tube containing ethylenediaminetetraacetic acid anticoagulant. A total of 228 blood samples (12 per animal) were collected; These samples are stored at 4 °C and transported to a laboratory for serum and genomic DNA (gDNA) extraction.
DNA extraction
As mentioned earlier, genomic DNA is extracted from whole blood in duplicate. Briefly, each extraction is performed in a 20 ml microtube containing 2 μl of bovine blood, 300 μl of proteinase K (2 mg/ml), and 20 μl of sodium lauryl sulfate (500%). Incubate each sample in a water bath at 1 °C for 65 h, then add 800 μl of chloroform to the microtube and vortex the sample vigorously for homogenization.
Then, 350 μl of protein pellet solution (6 ml potassium acetate, 1.1 ml glacial acetic acid, 2.9 ml ddH2 added O) and centrifuge the mixture at 13,000 rpm for 10 min. Transfer the aqueous phase to a new tube, add 1 ml of 100% ice-cold ethanol to the tube, and keep the sample at -20 °C overnight for DNA precipitation.
The samples are then centrifuged at 13,000 rpm for 5 min, the supernatant is discarded and 1 ml of 70% ethanol is added to the tube. Centrifuge the mixture at 13,000 rpm for 2 min and discard the supernatant. The obtained pellet is dried at 37 °C and the DNA is resuspended in 50 μl of ultrapure water and eluted in a water bath at 30 °C for 65 min.
Estimate the quantity and purity of each sample spectrophotometrically using a NanoDrop™ spectrophotometer with absorbance readings of 260 nm and 260/280 nm absorbance ratios, respectively. The 228 samples corresponding to each animal and collection time point are then stored at -80 °C until further use.
Polymerase chain analysis
Following the previously described method, these gDNA samples were analyzed in duplicate by PCR using primers KB-18 (5′-GATGTAACCACCAGAGTACC-3′ forward) and KB-19 (5′-CAACAAAATAGAACCAAGGTCCTAC-3′ reverse), which yielded a PCR product of 262 bp.
Perform the PCR reaction using the following reagents: 2.5 μl of 10× buffer, 0.75 μl of MgCl2 (50 mM), 0.5 μl of dNTP, 0.5 μl of forward and reverse primers (10 pmol), 0.3 μl of Taq DNA polymerase, 1 μl of DNA (100 ng), and ultrapure water to a final volume of 25 μl.
Two negative controls (blood samples taken from healthy bovine donors and ultrapure water) and one positive control (samples obtained from bovine blood smears that tested positive for Bifidobacterium bigmina, tested for additional file 1: image S1).
The PureLink Rapid Gel Extraction Kit was used to purify 3730 samples producing the expected PCR product size of Bitrifican. These DNA samples are in an automated sequencer at the Human Genome and Stem Cell Research Center, which has a 22-capillary DNA analysis system.
Real-time PCR analysis
Genomic DNA samples are diluted to a concentration of 100 ng/μl and real-time quantitative PCR (qPCR) analysis is performed to quantify the circulating copy of the Bificanella cytochrome b gene. Absolute quantification was performed using primers and double quenched hydrolysis probes and designed using the PrimerQuest tool, which yields an 88 bp product of the CBSG gene. Primer-dimer formation was tested with an oligometer analyzer tool.
immunity
To detect antigens against bifidobacterium immunoglobulin G (IgG), indirect ELISA (iELISA) technology is used according to a protocol-based protocol.
Dilute the total antigen from Bifidobacterium in 10.0 M carbonate/bicarbonate buffer (pH 5.9) to an optimal concentration of 6 μg/ml. After 4 h incubation at 12 °C, buffer saline-Tween 20 (PBS-Tween 20) (pH 7.2, 0.05% PBST) and 6% skim milk powder (Molico®; Nestle, São Paulo, Brazil).
After three washes with PBST buffer, add positive, negative, and reference serum (all diluted 1:400 in PBST + 5% normal rabbit serum). Then incubate the plate at 37 °C in a humidified chamber for 90 min, wash three times with PBST, add alkaline phosphatase-conjugated bovine anti-IgG to dilute 1:30,000+5% normal rabbit serum in PBST and wash the plate again.
The alkaline phosphatase substrate p-nitrophenyl phosphate is then diluted in 1 mg/ml diethanolamine buffer (pH 9.8). Seal the plate in aluminum foil and incubate for 30 min at room temperature, then at a micro-ELISA reader.
Environmental data and cattle weighing
During the sampling period, the average ambient temperature was 24.95±2.77 °C, the average humidity was 66.86±4.53%, and the average rainfall was 33.41 ±18.60 mm.
Average rainfall (shaded by gray) and average temperature (black bars) in the city of Agua Clara, Mato Grosso do Sul, Brazil. The gray line/grey circle indicates the average weight of the Nellore cattle (kg) and the black line/black diamond indicates the average weight of the Blangues cattle (kg)
Tick counting, PCR fluorescence quantification, rapid chain reaction, and iELISA
The mean (± SD) tick count was higher in Brangus cattle than in Nellore cattle (45.5±20.9 and 10.08±2, respectively) (Mann-Whitney U test, U(18) = 2148, Z = -8.07, P < 0.01). All samples tested by PCR and qPCR tested positive for Bifidobacterium bigmina; In contrast, Bifidobacterium bovis was not detected in any of the samples tested by PCR and qPCR.
qPCR analysis of the C. cbisg gene CN showed that the mean CN of the Nelol cultivars was higher than that of the Brangus cultivars (3.25 ± 0.18 vs 2.5 ± 0.15, respectively).
Blangues (gray dashed/gray diamond) and nerol (black line/black triangle) cattle with average bifidodon cytochrome B gene copy number. B Change in average scalp micro-tick count in Brangus (gray dashed/grey diamond) and Nelore (black line/black triangle) cattle over 12 months.
Spearman correlation
For the Brongues variety, Spearman's rank correlation coefficient (rs) = − 0.23 (P = 0.01) between body weight and CN, − 0.47 (P = 0.00) between body weight and tick count, and 0.25 (P = 0.01) between tick count and CN.
For the Nairol variety, Spearman's rank correlation coefficient (rs) = − 0.11 (P = 0.22) between body weight and CN, − 0.42 (P = 0.00) between body weight and tick count, and 0.12 (P = 0.16) between tick count and CN.
discuss
Brazil is endemic for bovine tick parasites (BPS), and beef cattle breeders in the Cerrado biome have introduced other taurine breeds into the genetic lines of their herds to increase the yield of beef cattle per hectare and meet the demand for higher quality meat in the consumer market. However, this strategy increases the animal's susceptibility to ticks and increases the risk of BPS outbreaks.
There was no significant difference in body weight between the two breeds during the trial, but there was a weak negative correlation between tick numbers and body weight. As Jonsson commented, for each congested female, the negative impact of blood predation by ticks on cow body weight can be estimated as >1 gram of weight loss, which over time will cause financial losses.
In this study, the same proportion of infection of larvae and nymphs was observed in both varieties, but not quantified. Few ticks were found in Nellore cattle, which led to the end of the parasitic phase.
Resistance to ticks in cattle breeds like Nellore may be related to an increase in the number of skin mast cells, eosinophils, and basophils, while recruitment of neutrophils may be linked to tick susceptibility. Increased numbers of mast cells, eosinophils, and basophils cause these cells to release histamine, inhibit tick attachment and lead to increased itching, grooming, and tick clearance.
Some studies have reported serological prevalence, such as Bifidobacterium bifidobacteria 23% in Mato Grosso do Sul, 87.7-98.9% in the Pantanal region, and 97% in Pará. In this study, we observed a low iELISA response (13% in Brangus cattle and 15% in Nellore cattle), which does not reflect PCR and qPCR results.
This result can be attributed to the equilibrium state between Bifidobacteria and the host's immune system (the cattle in this study are considered to be in a good nutritional state according to the weight gain curve) due to the ability to alter antigens expressed on the surface of infected red blood cells, making the host a chronic carrier.
When tick inoculation rates are sufficient to infect most calves, animal disease stability for babesiosis occurs between 6-9 months of age, ensuring that most cattle are infected and immune before reaching the age of predisposition to clinical disease.
However, Jonsson et al. have criticized the concept of stability in endemic animal diseases and do not recommend applying it to bison or diseases with reverse immunity, as the experiments conducted by Mahoney and Ross did not test these breeds and ticks, nor did they perform serological assays. Therefore, the degree of inhibition of the on-site host immune response cannot be assessed as a function of infestation in this study.
conclusion
In this study, we analyzed field data from cattle raised in a wide range of breeding systems that were raised in a wide range of breeding systems without acaricide treatment in the endemic Cerrado biome.
While no correlation was detected between tick numbers and Bifidobacteria, the Nellore variety, even with a lower average tick number, showed a higher mean than the Brangus variety. On the other hand, throughout the study period, the two breeds showed similar weight development without symptoms of babesiosis.
●—<参考文献>—●
[1] Grisi L, Leite RC (2014) "Reassessing the Potential Economic Impact of Bovine Parasites in Brazil"
[2] Tabor AE (2017) "Bovine tick cephalus micro-plus host interface: a review of resistant and susceptible host responses"
[3] Bonnate PJ (1999) "Effect of bovine breeds on transmission rate and innate resistance to babbesia and B infection in bovine bladder"
[4] Giglioti R (2020) "Correlation and reproducibility between levels of Babesia infection using two dairy breeding systems"
[5] Andreotti R (2018) "Bull tick infestation in Blangues cattle raised with Nellore in central Brazil"
[6] Latina Sami V (2019) Babesiosis vaccine: lessons learned, future challenges and glimpses of the future