Bovine Disease Research Articles

Distal Intravenous Regional Anaesthesia for Claw Diseases in Bovines

The bovine having surgical ailments of claw, were divided in to two groups having six animals/limbs in each group. Tourniquet for standard and modified intravenous regional anesthesia (IVRA), was placed circumferentially at the middle of the metacarpus and just below dewclaw in group I and II, respectively. Lignocaine was injected @ 4 mg/kg and 2 mg/kg b.wt. in group I and group II animals, respectively. In group I, pulse rate increased significantly P andlt; 0.05 at 20 and 30 minutes. Heart rate increased significantly P andlt; 0.05 at 20 minute and 30 minute interval in group I and II animals, respectively. In animal of both groups, oxygen saturation significantly P andlt; 0.05 decreased during study time period. Systolic pressure significantly increased at 15, 20 and 20 minutes in group I animals. It was concluded that modified IVRA technique was safer as compared to standard IVRA..

Assessment of the direct economic losses associated with hydatid disease (Echinococcus granulosus sensu stricto) in beef cattle slaughtered at an Australian abattoir

Bovine hydatid disease, characterised by fluid-filled hydatid cysts, is regularly found in the offal of beef cattle at slaughter. Organs found to be infected at slaughter are removed to preclude them from entering the human food chain. The organs are either downgraded to pet food or condemned. Previous studies have focussed on total economic losses, but have not calculated the cost of disease per animal, which would be useful information for producers when determining how best to manage hydatid disease. This study estimated the direct losses associated with hydatid disease in beef cattle slaughtered at an Australian beef abattoir both at the population (all cattle slaughtered) and individual animal level.Data on annual prevalence of hydatid disease in beef cattle were obtained from an Australian abattoir for the years 2011–2017. The direct losses resulting from the condemnation and downgrading of offal infected with hydatid cysts at the abattoir were estimated using data stratified by age, sex and feed-type. Official and literature-based sources of organ weight and price were used to estimate direct losses associated with hydatid disease in beef cattle slaughtered at the abattoir. Uncertainty and variability in input parameters were represented using uniform distributions and Monte Carlo sampling was used to model output parameter uncertainty.Out of 1,097,958 beef cattle slaughtered between January 2011 and December 2017, 97,832 (8.9%) were reported infected with hydatid disease. The median estimated direct loss to the abattoir for the duration of the study period was AU$655,560 (95% confidence interval [CI] AU$544,366–787,235). This equated to approximately AU$6.70 (95% CI AU$5.56–8.05) lost per infected animal. The annual median estimated direct losses due to hydatid disease at the abattoir were AU$93,651 (95% CI AU$77,767–112,462). Direct losses varied each year of the study and ranged from AU$38,683 in 2016 to AU$163,006 in 2014.This estimate of the direct losses associated with bovine hydatid disease most likely underestimates the true extent of the overall losses because indirect losses such as reduced carcass weights were not estimated in this study. Nevertheless, these estimates illustrate the negative economic impact of bovine hydatid disease and demonstrate that improved surveillance to enable control of hydatid disease should be considered both in Australia and globally. It would be worthwhile to estimate the losses in other beef abattoirs for the same time period to compare results, and to investigate the cost-benefit of control programs for bovine hydatid disease.

The Fabrication of a Highly Sensitive Nano Green Carbon Paste Electrode Modified with Yttrium Doped Manganese Oxide (Mn2O3/Y2O3) for Electrochemical Determination of Marbofloxacin and Its Residues in Bovine Meat and Milk Samples

In the present work, a novel sensitive electrochemical carbon paste electrode chemically modified with yttrium doped manganese oxide Mn2O3/Y2O3 nanostructures was assigned for determination of marbofloxacin (MRB) using square wave voltammetry (SWV) method. MRB has a broad spectrum of bactericidal activity for the treatment of urinary, respiratory and dermatological diseases in bovines and their retention in animal meats and milk leads to adverse side effects for the consumer. Thus a rapid estimation of minor concentrations of MRB has exerted a great concern to ensure food safety. XRD, EDX, Raman spectroscopy, SEM, and TEM techniques were employed to characterize the samples. The electrochemical oxidation behavior of MRB shows irreversible anodic peak at 1.10 V vs Ag/AgCl, in Britton–Robinson buffer (BR) at pH 5.0. The relationship was rectilinear over the range 10 × 10−9–1.0 × 10−4 M between the peak current and its related concentration with a minimum detection limit of 2.4 × 10−9 M. The developed method was green chemistry challenges and successfully applied to assay the drug in its dosage form, bovine meat and milk samples with a good recovery lies between 94.56% and 105.33% with relative standard deviation less than 10%.

Prevalence and Risk Factors Analysis of Bovine Foot Diseases in Certain Milk Pocket Areas of Sirajganj District, Bangladesh

Prevalence and Risk Factors Analysis of Bovine Foot Diseases in Certain Milk Pocket Areas of Sirajganj District, Bangladesh

Antiviral activity of 4-oxoquinoline-3-carboxamide derivatives against bovine herpesvirus type 5.

Bovine herpesvirus type 5 is an important agent of meningoencephalitis in cattle and has been identified in outbreaks of bovine neurological disease in several Brazilian states. In recent years, oxoquinoline derivatives have become an important focus in antiviral drug research. The cytotoxicity and anti BoHV-5RJ42/01 activity of a set of synthetic 4-oxoquinoline derivatives 4a-k were assayed on Madin-Darby Bovine Kidney cell and antiviral activity by plaque reduction assay. The most promising substance (4h) exhibited CC50 and EC50 values of 1,239 µM ±5.5 and 6.0 µM ±1.5, respectively, with an SI =206. Two other compounds 4j (CC50 = 35 µM ±2 and EC50 = 24 µM ±7.0) and 4k (CC50= 55 µM ±2 and EC50 = 24 µM ±5.1) presented similar inhibitory profile and selectivity indexes of 1.4 and 2.9, respectively. The results of the time-of-addition studies revealed expressive reduction of virus production (≥80%) in different stages of virus replication cycle except for compound 4h that slightly inhibited virus yield in the first 2 h post infection, but it showed expressive virus inhibition after this time. All three compounds slightly interact with the virus on the virucidal assay and they are not able to block virus attachment and penetration. Antiviral effect of oxoquinoline 4h was more prominent than acyclovir which leads us to suggest compound 4h as a promising molecule for further anti-BoHV-5 drug design.

Seroprevalence of bovine echinococcusis in Pakistan

ABSTRACT: Cystic echinococcosis is a parasitic infection of worldwide distribution, which, despite causing significant loss of health and money, is still a neglected disease. The present study was performed to determine the prevalence of bovine hydatid disease at Research Centre for the Conservation of Sahiwal Cattle, Jahangirabad, Khanewal, Pakistan. Five hundred bovine serum samples were tested for the detection of bovine Echinococcus antibodies (IgG), using the ELISA kits. Antibodies of bovine Echinococcus were detected in 24.2% cattle. Female cattle showed higher disease prevalence (33.2%) compared to (6.02%) in male cattle. There was positive association between sex of cattle and prevalence (P<0.05). Positive correlation between disease prevalence and age was detected. Younger animals (<3 years old) had lower prevalence value of disease than to older animals (> 3 years old). It is concluded that there is high degree of exposure of cattle to Echinococcus at Research Centre for Conservation of Sahiwal Cattle, Khanewal, Punjab. Measures should be taken to reduce risk of disease to avoid its spread to humans as well as great degree of economic losses.

Sensitive screen-printed electrodes with the colorimetric zone for simultaneous determination of mastitis and ketosis in bovine milk samples

Bovine metabolic diseases are silent clinical conditions caused by a disturbance in normal cellular metabolic processes majorly due to the demand for nutrients, physiological stress and inflammation in lactating animals. Even though these are non-devastating diseases, conditions like mastitis and ketosis highly influence cow's lactation and fertility performance that leads to economic losses. Hence, in the present study, a combined detection including a sensitive paper-based colorimetric device for the rapid determination of somatic cells (SCC) and electrochemical detection of β-hydroxybutyrate (BHB) in the milk samples has been developed. The working range was 1 × 105 to 2 × 106 for SCC and 0.05 to 2.5 mM BHB in which the normal and extreme cases of mastitis and ketosis values exist. The detection method is based on reaction using substrate 5-Bromo-indoxyl acetate (5BIA) and enzyme (BHB dehydrogenase) for the enzymes (esterase) and substrate (BHB) present in the milk samples during mastitis and ketosis respectively. An increase in blue color development was associated with an increase in the number of SCC; likewise, the increased voltage denoted the increase concentration of BHB in the samples. The repeatability was evaluated, and the % relative standard deviation (RSD) value <8.0 (n = 12). The limit of detection (LOD) was close to that achieved by a desktop spectrophotometer. The applicability of the developed kit demonstrated good sensitivity that can be deployed for other biological samples of interest. The proposed method provides a rapid, sensitive and promising tool for the regular monitoring of cows as a point of care tests (POC) at the field level.

Mapping of CD4+ T-cell epitopes in bovine leukemia virus from five cattle with differential susceptibilities to bovine leukemia virus disease progression

BackgroundBovine leukemia virus (BLV), which is closely related to human T-cell leukemia virus, is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly prolonged course involving persistent lymphocytosis and B-cell lymphoma. The bovine major histocompatibility complex class II region plays a key role in the subclinical progression of BLV infection. In this study, we aimed to evaluate the roles of CD4+ T-cell epitopes in disease progression in cattle.MethodsWe examined five Japanese Black cattle, including three disease-susceptible animals, one disease-resistant animal, and one normal animal, classified according to genotyping of bovine leukocyte antigen (BoLA)-DRB3 and BoLA-DQA1 alleles using polymerase chain reaction sequence-based typing methods. All cattle were inoculated with BLV-infected blood collected from BLV experimentally infected cattle and then subjected to CD4+ T-cell epitope mapping by cell proliferation assays.ResultsFive Japanese Black cattle were successfully infected with BLV, and CD4+ T-cell epitope mapping was then conducted. Disease-resistant and normal cattle showed low and moderate proviral loads and harbored six or five types of CD4+ T-cell epitopes, respectively. In contrast, the one of three disease-susceptible cattle with the highest proviral load did not harbor CD4+ T-cell epitopes, and two of three other cattle with high proviral loads each had only one epitope. Thus, the CD4+ T-cell epitope repertoire was less frequent in disease-susceptible cattle than in other cattle.ConclusionAlthough only a few cattle were included in this study, our results showed that CD4+ T-cell epitopes may be associated with BoLA-DRB3-DQA1 haplotypes, which conferred differential susceptibilities to BLV proviral loads. These CD4+ T-cell epitopes could be useful for the design of anti-BLV vaccines targeting disease-susceptible Japanese Black cattle. Further studies of CD4+ T-cell epitopes in other breeds and using larger numbers of cattle with differential susceptibilities are required to confirm these findings.

Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

Salmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

Determining an optimal pool size for testing beef herds for Johne's disease in Australia.

Bovine Johne’s disease (JD) is a chronic debilitating disease affecting cattle breeds worldwide. Pooled faecal samples are routinely tested by culture to detect Mycobacterium avium subsp. paratuberculosis (Mptb) infection. More recently, a direct high throughput molecular test has been introduced in Australia for the detection of Mptb in faeces to circumvent the long culture times, however, the optimal pool size for beef cattle faeces is not known. This study aimed to determine the optimal pool size to achieve the highest test sensitivity and specificity for beef cattle. Individual archived faecal samples with low, medium and high quantities of Mptb (n = 30) were pooled with faecal samples from confirmed JD negative animals to create pool sizes of 5, 10, 15 and 20, to assess the diagnostic sensitivity relative to individual faecal qPCR. Samples from JD-free cattle (n = 10) were similarly evaluated for diagnostic specificity. Overall, 160 pools were created, with Mptb DNA extracted using magnetic bead isolation method prior to Mptb-specific IS900 quantitative PCR (qPCR). The pool size of 10 yielded the highest sensitivity 73% (95% CI: 54–88%), regardless of the quantity of Mptb DNA present in the faeces. There was no significant differences between the four different pool sizes for positive pool detection, however, there was statistical significance between low, medium and high quantities of Mptb. Diagnostic specificity was determined to be 100%. The increase in pool size greater than 10 increased the chances of PCR inhibition, which was successfully relieved with the process of DNA dilution. The results of this study demonstrate that the pool size of 10 performed optimally in the direct faecal qPCR. The results from this study can be applied in future simulation modelling studies to provide suggestions on the cost-effective testing for JD in beef cattle.

Characterization of Salmonella Dublin isolated from bovine and human hosts

BackgroundSalmonella enterica subsp. enterica serovar Dublin (S. Dublin), a cattle adapted serovar causes enteritis, and systemic disease in bovines. The invasive index of this serovar far exceeds that of the other serovars and human infections often present as fatal or highly resistant infections. In this, observational study, phenotypic properties of human and bovine-derived isolates of S. Dublin along with antibiogram of common antimicrobials were evaluated. The multiplex PCR confirmed isolates were genotyped using 7-gene legacy MLST. MIC assay was done by broth microdilution method. Previously published protocols were used to assess the motility, biofilm formation and morphotype. Vi antigen was agglutinated using commercial antiserum. Caenorhabditis elegans infection model was used to evaluate the virulence potiential. Phenotyping experiments were done in duplicates while virulence assay was done in triplicates. Whole-genome sequencing was used to predict the genes responsible for acquired resistance and a genotype-phenotype comparison was made.ResultsWe evaluated 96 bovine and 10 human isolates in this study. All the isolates belonged to ST10 in eBG53 and were negative for Vi-antigen. The swarming motility, biofilm formation and morphotype were variable in the isolates of both groups. Resistance to sulfamethoxazole, ampicillin, chloramphenicol, tetracycline was > 90% in animal isolates whereas resistance to sulfamethoxazole was > 70% in human isolates. MDR was also higher in animal isolates. Human isolates were significantly (P < 0.0001) more virulent than animal isolates on C. elegans infection model. The genomic comparison based on the core SNPs showed a high degree of homogeneity between the isolates. The carriage of IncA/C2 plasmid was seen as a typical feature of isolates from the bovine hosts.ConclusionHuman isolates showed more diversity in the phenotypic assays. Animal isolates showed a higher degree of antimicrobial resistance with greater MDR but human isolates formed more biofilm and had greater swarming motility as well as increased virulence to the nematode C. elegans. The carriage of IncA/C2 plasmid could contribute to the distinguishing feature of the bovine isolates. The tandem use of genotypic-phenotypic assays improves the understanding of diversity and differential behaviour of the same serovar from unrelated host sources.

KARAKTERISTIK FISIKOKIMIA GELATIN KULIT IKAN AYAM-AYAM ( Abaliste stellaris ) DENGAN PRA-PERLAKUAN KONSENTRASI ASAM SITRAT

Gelatin is a fibrous protein obtained by partial denaturation of collagen. Traditionally, gelatin derived from mammals’ skins and bones, such as cow and pork. However, both mammals’ gelatin could risk for bovine spongiform encephalopathy (BSE) and foot mouth disease (FMD), besides, gelatin extracted from pork is prohibited in Islam rules. Therefore, fish processing waste is potential as a source of gelatin in terms of starry triggerfish (Abalistes stellaris) skin gelatin. The aims of this study is to characterize physichochemical of gelatin extracted from skin of starry triggerfish. The methods used in this research, experiment with completely randomized design (CDR) by soaking different concentrations of citric acid (0.2; 0.4; and 0.6 M). The results showed that the concentrations of citric acid had significantly different (P&lt;0.05) on the yield, viscosity, gel strength, and fat content of starry triggerfish skin gelatin. On the other hand, the pH, melting point, galling point, protein, moisture, and ash value of starry triggerfish skin gelatin did not perform significantly different (P&gt;0.05). The most properties of starry triggerfish skin gelatin meet the commercial gelatin, and it is able to as a potential alternative of halal gelatin.

Assessing the efficacy of autogenous vaccines in bovine diseases

Vaccination is an important tool for preventing disease. In human medicine and veterinary health, the eradication of smallpox and rinderpest are stellar examples of how effective vaccinations can be. Other outstanding examples include vaccines against measles, mumps, and rubella in humans. In animal health, most vaccines are marketed broadly for use on multiple animals and farms. However, autogenous vaccines, which are intended for use on a single farm, are also common in livestock health worldwide. For this presentation, I discuss concepts related to assessing the efficacy of vaccines and provide a summary of the publicly available evidence from the scientific literature regarding the efficacy of autogenous vaccines.

Follicular structures of cows with cystic ovarian disease present altered expression of cytokines.

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1β, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1β, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1β and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1β and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Isolation and Full-Length Sequence Analysis of a Pestivirus from Aborted Lamb Fetuses in Italy.

Pestiviruses are distributed worldwide and are responsible for a variety of economically important diseases. They are not very host-specific, and thus sheep can be infected by well-known pestiviruses like bovine viral diarrhea virus (BVDV) and border disease virus (BDV), as well as by other recently discovered pestivirus species. The aim of this study is to describe the isolation and characterization of four pestivirus strains detected in aborted lamb fetuses from a single farm in the Brescia province (Northern Italy). A total of twelve aborted fetuses were collected and examined. After necropsy, organs were tested for the presence of infectious agents known as potential causes of abortion (Brucella spp., Listeria spp., Coxiella burnetii, Chlamydophila spp., Mycoplasma spp., Neospora caninum, and Toxoplasma gondii), and submitted to viral identification by isolation on Madin Darby bovine kidney (MDBK) cell culture and by PCR assay for Schmallenberg virus and pan-pestivirus RT-PCR real time assay. Three viral strains (Ovine/IT/1756/2017, Ovine/IT/338710-2/2017, and Ovine/IT/338710-3/2017) were isolated in the absence of cytopathic effects (CPEs) in cell cultures and identified with RT-PCR. Another pestivirus strain (Ovine/IT/16235-2/2018) was detected by PCR, but was not successfully isolated. Complete sequence genomic data of the three isolated viruses showed that they were highly similar, differed genetically from known pestivirus species, and were closely related to classical swine fever virus (CSFV). Beyond the identification of new ovine pestiviruses, this study indicates that a systematic diagnostic approach is important to identify the presence and map the distribution of both known and emerging pestiviruses.

Emergence of Fungal-Like Organisms: Prototheca.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.

Short communication: Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, causing pyogranulomatous mastitis and its cross-reactivity in bovine (para)tuberculosis testing

Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-γ showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.

Risk prioritization as a tool to Guide Veterinary Public Health activities at the regional level in Italy.

In this study we developed a model for risk prioritisation and characterisation focused on zoonoses and food safety for diseases of interest in veterinary public health at a regional level in Italy. A previous model (Discontools) based on scorecards was used as a basis to develop the new model. A Formalised Consensus Process approach involving academics and veterinary officers was used to develop scorecards and relative form and guidelines. Scorecards include several areas of interest, with different categories and coefficient of importance. The following areas were identified: relevance of the disease, socio-economic impact, impact on public health, impact on trade, impact on animal welfare, control tools. A guide and a form were finalised in order to fill scorecards. Scorecards were filled by consulting available data, literature, and expert opinions. Among bovine diseases, mastitis (Salmonella aureus) showed the highest score; Q fever was the highest among small ruminants; among swine diseases the highest was salmonellosis; while among other animal diseases, toxoplasmosis had the highest score. The approach described in this study is designed to aid professionals in risk prioritisation, decision-making, and to improve disease control systems at a regional level in Italy. It also facilitates risk characterisation in different backgrounds and the identification of data holes in specific areas of interest for the diseases considered. This approach is conceived to aid professionals in risk prioritization, decision-making and to improve disease control systems at a regional level. It also allows to perform risk characterization in different backgrounds and to identify lacks of data in specific areas of interest for the diseases considered.

Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin.

Mycoplasma bovis isolates belonging to the sequence type 5 (ST5) group, the dominant group in Japan since 1999, were low susceptible to 16-membered macrolides and tetracyclines and were confirmed to have a guanine-to-adenine transition mutation at position 748 in the 23S rRNA gene (rrl) and adenine-to-thymine transversion mutations at positions 965 and 967 in the 16S rRNA gene (rrs) (Escherichia coli numbering). Moreover, isolates of ST93 and ST155, members of the ST5 group, were low susceptible to lincosamides and azithromycin and showed an adenine-to-guanine transition mutation at position 2059 of rrl Isolates of ST93 were additionally low susceptible to spectinomycin and showed a cytosine-to-adenine transversion mutation at position 1192 of rrs Strains of the ST5 group seem to spread to Japan and Europe from North America with imported cows, while strains of ST93 and ST155 originated in Japan. Melting curve analysis using hybridization probes revealed the existence of point mutations involved in decreased susceptibility to macrolides, lincosamides, and spectinomycin, as demonstrated by changes in the melting curve shape and/or decreases in the melting peak temperature, so the susceptibility to these antimicrobials can be assessed on the same day. For decreased susceptibility to fluoroquinolones to exist, nonsynonymous mutations in the DNA gyrase gene (gyrA) and topoisomerase IV gene (parC) had to coexist. The combination of amino acid substitutions of serine at position 83 in gyrA and serine at position 80 in parC resulted in particularly low susceptibility to fluoroquinolones.IMPORTANCEMycoplasma bovis is the main causal species of bovine mycoplasmal disease and leads to significant economic losses because of its severe symptoms, strong infectivity, and refractoriness. As for mastitis, culling cows with intramammary infections is a general countermeasure to prevent spreading. The conventional antimicrobial susceptibility test for mycoplasma is time-consuming and troublesome, but no quick and easy method for grasping the antimicrobial susceptibility of the causal strain exists at present. Treatment without antimicrobial susceptibility information may be one reason why M. bovis infection is refractory. Detecting a mutation involved in decreased susceptibility to antimicrobial agents of the causal strain makes it possible to easily select suitable antimicrobials for treatment, and this technique will help improve the cure rate and prevent the overuse of ineffective antimicrobial agents. In this study, we developed a technique to quickly and easily assess antimicrobial susceptibility based on the genetic characteristics of M. bovis strains in Japan.

Analysis of the microRNA Expression Profile of Bovine Monocyte-derived Macrophages Infected with Mycobacterium avium subsp. Paratuberculosis Reveals that miR-150 Suppresses Cell Apoptosis by Targeting PDCD4.

M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne’s disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA libraries of MAP-infected and control MDMs. The results showed that a total of 21 miRNAs were differentially expressed in MDMs after MAP infection, and 8864 target genes were predicted. A functional analysis showed that the target genes were mainly involved in the MAPK signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and apoptosis. In addition, using a dual-luciferase reporter assay, flow cytometry, and a small interfering (si)RNA knockdown assay, the role of miR-150 in regulating macrophage apoptosis by targeting the programmed cell death protein-4 (PDCD4) was demonstrated. These results provide an experimental basis to reveal the regulatory mechanism of MAP infection and suggest the potential of miRNAs as biomarkers for the diagnosis of Johne’s disease in bovines.