Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: a review

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn's disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.     

Genetics and physiology of leptin in periparturient dairy cows

In dairy cattle, the increase in milk yield has been accompanied by a more negative energy balance (EB) during early lactation and a decrease in fertility. As the hormone leptin is involved in regulation of nutritional status and reproductive function this hormone is an interesting protein to investigate during the periparturient period in dairy cattle. This study was performed to get insight into the function of leptin during the periparturient period and to perform an association study between polymorphisms in the bovine leptin gene and leptin receptor gene and fertility as well as production traits. Leptin concentrations in the periparturient cow undergo remarkable changes; leptin concentrations were high during late pregnancy and declined to a nadir at parturition. Genetic analysis of the leptin gene indicated that a combination of three polymorphisms located at a 135 bp region of the leptin promoter explained most of the variance in prepartum leptin concentrations. The two extreme genotype combinations could be used to investigate the function of leptin concentrations in pregnant cows. A polymorphism located on intron 2 of the leptin gene explained a significant part of the variation in milk yield. On the promoter region of the leptin gene an SNP was detected that was associated with first postpartum luteal activity (FPLA). This SNP could be a candidate marker for fertility in dairy cows. Another SNP on the leptin promoter was associated with energy balance and dry matter intake (DMI) where a higher dry matter intake occurred together with a higher energy balance. Two genotype combinations of the aforementioned three associated SNPs were defined which had a good milk yield together with a good energy balance and fertility. Calculations of an economical value per trait have to validate if one of these genotype combinations would be a possible candidate to be used in selection.     

Analysis of the effect of inapparent bovine paratuberculosis.

Inapparent paratuberculosis was studied in a Guernsey herd with a history of paratuberculosis (Johne's disease); the herd was maintained at between 900 and 950 cattle. Fecal specimens and intestinal tissue specimens from any of these that were slaughtered were examined culturally for the presence of Mycobacterium paratuberculosis. The reasons given for culling and slaughtering of cows from this herd were compared with infection status determined by culturing. Less than one-third of the culturally confirmed paratuberculous cattle were culled because of clinically apparent paratuberculosis. Mastitis was the reason for culling 3.6% of the non-infected cattle and 22.6% of the cattle with inapparent paratuberculosis. Infertility was also significantly higher in cows with inapparent paratuberculosis than in noninfected cows in the same herd. Separation of parturient dams and calves from the rest of the herd was shown to materially reduce the level of infection and incidence of clinical paratuberculosis.     

Bovine fetal infection with Mycobacterium paratuberculosis

In utero transmission of Mycobacterium paratuberculosis, the causative agent of paratuberculosis in cattle, has been suggested. Tissue specimens were obtained at a packing plant from pregnant dairy cows and their fetuses and from cows with clinical signs of paratuberculosis and from their fetuses. Specimens were processed according to methods described for isolating M paratuberculosis from bovine tissues and were incubated on Herrold egg yolk medium for 16 weeks. Presumed positive specimens were confirmed to be M paratuberculosis, using acid-fast staining and subculturing. Of 407 lymph nodes from cows, 34 (8.4%) were culture positive for M paratuberculosis; 9 of 34 (26.4%) of these culture-positive cows had fetuses from which specimens were also culture positive. The results estimated the risk of fetal infection with M paratuberculosis to be 26.4% (95% confidence interval between 11.3 and 40.7%).     

Novel SNPs in IL-17F and IL-17A genes associated with somatic cell count in Chinese Holstein and Inner-Mongolia Sanhe cattle

Background: Bovine mastitis is the most common and costly disease of lactating cattle worldwide. Apart from milk somatic cell count(SCC) and somatic cell score(SCS), serum cytokines such as interleukin-17(IL-17) and interleukin-4(IL-4) may also be potential indicators for bovine mastitis. The present study was designed to investigate the effects of single nucleotide polymorphisms(SNPs) in bovine IL-17 F and IL-17 A genes on SCC, SCS and serum cytokines in Chinese Holstein and Inner-Mongolia Sanhe cattle, and to compare the m RNA expression variations of the cows with different genotypes.Results: A total of 464 lactating cows(337 Holstein and 127 Inner-Mongolia Sanhe cattle) were screened for SNPs identification and the data were analyzed using fixed effects of herd, parity, season and year of calving by general linear model procedure. The results revealed that SNP g.24392436 C T in IL-17 F and SNP g.24345410 A G in IL-17 A showed significant effects on SCC and IL-4 in Holstein(n = 337) and on IL-17 and IL-4 in Sanhe cattle(n = 127). The homozygous GG genotype of SNP g.24345410 A G had significantly higher m RNA expression compared with the heterozygous AG genotype.Conclusions: The results indicate that IL-17 F and IL-17 A could be powerful candidate genes of mastitis resistance and the significant SNPs might be useful genetic markers against mastitis in both dairy and dual purpose cattle.     

CARD15基因StyⅠ酶切多态性与奶牛结核病易感性的相关性分析

本试验旨在探讨CARD15基因StyⅠ酶切位点多态性与云南高原奶牛结核病易感性的关联.选择云南昆明、玉溪、大理等地结核病阳性奶牛103头,结核病阴性奶牛98头,共201头为研究对象,应用创造酶切位点原理设计引物,通过PCR-RFLP与PCR直接测序法相结合的检测方法,获得CARD15基因StyⅠ酶切位点的基因型及等位基因频率,分析该位点与云南高原奶牛结核病易感性的关系.结果显示,CARD15基因的StyⅠ酶切位点存在AA、AG、GG 3种基因型,在结核病例组中AA、AG、GG基因型频率为0.03、0.42和0.55,A、G等位基因频率为0.24和0.76;在结核对照组中AA、AG、GG基因型频率为0.01、0.05和0.94,A、G等位基因频率为0.04和0.96;对多态性位点的遗传模式进行分析,最佳遗传模式为显性遗传(dominant),在此遗传模式下基因型AG-AA在病例组的频率44.7%,明显高于对照组的频率6.1%,OR(95% CI)=0.08(OR< 1),在病例组与对照组的分布之间有极显著差异(P< 0.0001).结果表明,云南高原奶牛CARD15基因StyⅠ酶切位点多态性与结核病的发生有显著关联性,且基因型AG-AA可能是结核易感型.     

Correlation Study of CARD15 Gene StyⅠPolymorphism and Susceptibility of Tuberculosis in Dairy Cows

The aim of this study was to investigate the association between CARD15 gene StyⅠpolymorphism and susceptibility to tuberculosis (TB) of dairy cows from Yunnan plateau.A total of 201 dairy cows (103 cases and 98 controls) from Kunming,Yuxi and Dali were used in this study.We designed a specific primer pair for PCR-RFLP analysis.By using PCR-RFLP genotyping and PCR sequencing,We analysized allele frequency and correlation between polymorphism of CARD15 gene and susceptibility to bovine tuberculosis in Yunnan province.The results showed that there were three genotypes in Sty Ⅰ enzyme site,AA,AG and GG.In cases,the genotype frequencies of AA,AG and GG were 0.03,0.42 and 0.55,respectively,while in controls were 0.01,0.05 and 0.94,and the allele frequencies of A and G were 0.24 and 0.76 in cases,while were 0.04 and 0.96 in controls.Then analysizing the genetic pattern of polymorphic loci,we found that the best genetic model was dominant and the frequency of AG-AA in cases (44.7%) was apparently higher than that in controls (6.1%) (OR(95% CI)=0.08 (OR< 1)),so extremely significant difference existed between cases and controls (P< 0.0001).The polymorphism of StyⅠenzyme cut site in CARD15 gene was significantly associated with the susceptibility to tuberculosis of Yunnan plateau dairy cows,and AG-AA might be the susceptible genotype to tuberculosis.     

Experimental infection of a bovine model with human isolates of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease (JD) in ruminants, has been implicated in the pathogenesis of Crohn's disease (CD) in humans. We developed a bovine ileal cannulation model to facilitate comparison of the immune response to Map and the mechanisms of pathogenesis in cattle and humans. Initial studies showed a T cannula could be maintained for up to a year in calves without inducing inflammation or adversely affecting intestinal function. Map introduced through the cannula established a persistent low level of infection without inflammation. Infection elicited an immune response to Map antigens detectable by flow cytometry. Further studies now show the cannulation model can be used with cows during the later stage of infection, affording access to the target tissue at all stages of infection. The studies also revealed no difference in infectivity or immunogenicity of isolates of Map obtained from cattle or humans with CD. Comparison of the immune response to Map during the early and late stages of infection using PCR, flow cytometry and QRT-PCR, showed the immune response early in the disease process is dominated by CD4 T cells. A CD8 response is delayed but comparable at later stages of infection. Genes for pro-inflammatory cytokines IFN-γ and the recently identified genes encoding IL-17 and IL-22 are up regulated in infected animals. These findings reveal that both human and bovine isolates of Map can establish infection and induce similar immune responses in a bovine model. They also reveal the cytokine responses elicited in cattle are similar to those implicated in CD pathogenesis.     

Disseminated Mycobacterium paratuberculosis infection in a cow

A cow with chronic diarrhea and weight loss caused by localization of Mycobacterium paratuberculosis in the intestinal tract (Johne's disease) had gross and microscopic changes indicative of a disseminated infection. A direct association between the remote lesions and the intestinal infection was shown by isolation of M paratuberculosis from renal tissue, detection of intracellular M paratuberculosis antigen(s), using an indirect immunoperoxidase method, and by the characteristic granulomatous nature of the lesions. This case illustrates the potential for extra-intestinal lesions in M paratuberculosis infection of cattle and should cause veterinarians to consider mycobacterial disease when confronted with multinodular lesions of the bovine kidney. The immunoperoxidase method was useful in determining the cause of the inflammatory lesion in which intact organisms were not evident.     

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows.     

新疆北疆某规模化牛场奶牛副结核病的检测与分析

为查明导致新疆北疆某规模化牛场奶牛腹泻、消瘦的主要病原菌,本研究采用微生物学与分子生物学方法对采集的患牛粪便进行检测与鉴定。结果显示:2份粪样抗酸染色阳性,IS900基因及亚型分型基因检测阳性,IS900基因与GenBank中多个副结核分枝杆菌序列同源性在99%以上,亚型分型基因与Ⅱ型同源性为99.81%,从而确定2份样本均被Ⅱ型(牛型)副结核分枝杆菌感染。     

郏县红牛TAFA1基因多态性及其与生长性状的关联分析

本研究旨在探讨TAFA趋化素样家族成员1（TAFA1）基因多态性与郏县红牛生长的关联性。试验共采集了79头郏县红牛成年母牛的血样并提取基因组DNA,利用直接测序法对TAFA1基因上的错义突变SNP rs137516577进行基因型分型,并与郏县红牛体高、体长、胸围、腰角宽、坐骨端宽、尻长、十字部高、荐高、胸深、胸宽、体重等11个生长和体尺性状进行关联分析。同时根据“Animal Omics Datebase”数据库对TAFA1基因的组织表达情况进行分析。结果发现该SNP与体长、腰角宽、坐骨端宽、尻长和体重等性状显著相关（P<0.05）,且GC型个体体长、腰角宽、坐骨端宽、尻长和体重均显著高于GG型（P<0.05）。TAFA1基因在牛大脑组织中表达量最高。结果表明,TAFA1基因上错义突变SNP rs137516577与郏县红牛生长性状相关,可作为郏县红牛生长性状的分子标记。     

Seroprevalence of infection with Mycobacterium avium subspecies paratuberculosis, bovine leukemia virus, and bovine viral diarrhea virus in maritime Canada dairy cattle

The purpose of this study was to survey the seroprevalence of infection with the agents of production-limiting diseases in dairy cattle in New Brunswick, Nova Scotia, and Prince Edward Island. In 30 randomly selected herds per province, 30 cattle per herd were randomly selected and tested for antibodies to bovine leukemia virus (BLV) and Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhea virus (BVDV). For BLV, 20.8% (15.8% to 27.0%) of cows were positive, and 70.0% (60.3% to 79.7%) of herds had at least one positive cow. In BLV-positive herds, the average BLV prevalence was 30.9% (24.8% to 37.2%). For M. paratuberculosis, 2.6% (1.8% to 3.9%) of cows were positive, and 16.7% (8.8% to 24.5%) of herds had at least 2 M. paratuberculosis-positive cows. In M. paratuberculosis-positive herds, the average M. paratuberculosis prevalence was 8.5% (6.9% to 10.1%). For BVDV, 46.1% (35.5% to 56.7%) of herds had at least 1 BVDV-positive animal with a titer greater than or equal to 1:64.     

奶牛副结核病的诊断方法研究

副结核是一种慢性传染性细菌疾病,病原为禽分支杆菌副结核亚种,主要感染牛、羊,造成病畜小肠和其他器官损伤,引发腹泻、体重降低、营养不良、贫血甚至死亡。奶牛副结核病可导致奶牛死淘率增加、产奶量下降、饲料转化率降低、繁殖能力下降、屠宰率降低以及增加其他病原的感染率,从而对奶牛养殖业造成重大的经济损失。副结核分支杆菌主要通过粪口途径进行传播,也可通过子宫进行垂直传播。副结核分支杆菌通过在小肠黏膜下层中的巨噬细胞中持续感染来逃避免疫系统,在大多数情况下,这种隐性感染至少会持续两年,然后细菌开始脱落并出现临床症状。副结核分支杆菌感染进程缓慢,管理不当会使易感犊牛发病率更高,这使得牛副结核病的防控具有很大挑战性,尤其是在季节性产犊牛群中。本文对奶牛副结核病的概念、可造成的经济危害以及诊断方法进行阐述,旨在为奶牛副结核病的临床诊断与新型诊断方法的研制提供参考。     

荷斯坦奶牛STAT5A基因的SNPs检测及其与产奶性状的关联分析

根据GenBank公布的牛STAT5A基因的2个SNPs（AJ237937和AF079568）的引物序列,采用PCR-SS-CP方法对758头中国荷斯坦奶牛进行了STAT5A基因多态性检测,并将其与5个产奶性状进行了关联分析。在SNP1的9501碱基处发现A→G突变;SNP2中发现2个连锁点突变,即12 440位T→C和12 550位的CCT插入/缺失;方差分析结果表明：SNP1对乳蛋白率有显著影响（P〈0.05）;SNP2对产奶量有极显著影响（P〈0.01）,对乳脂量、乳蛋白量有显著影响（P〈0.05）;单倍型组合对产奶量和乳蛋白量有极显著影响（P〈0.01）,对乳脂量有显著影响（P〈0.05）。多重比较分析表明SNP2的AB基因型的产奶量、乳脂量和乳蛋白量的最小二乘均数极显著（P〈0.01）或显著（P〈0.05）高于基因型AA;而单倍型组合H1H2的产奶量、乳脂量和乳蛋白量的最小二乘均数显著（P〈0.05）或极显著（P〈0.01）高于其它4种单倍型组合;H3H4对产奶量和乳蛋白量的效应极显著高于其它4种单倍型组合（P〈0.01）。结果提示：STAT5A基因可以作为奶牛产奶性状的候选分子遗传标记。     

Association of single nucleotide polymorphisms in the leptin gene with body weight and backfat growth curve parameters for beef cattle

Previous research has identified differences in carcass characteristics across SNP in the bovine leptin gene at slaughter, but before feedlot operators implement selection and sorting strategies, more information is needed to determine how carcass characteristics change over time. The objective of this study was to investigate the effect of 2 leptin SNP on growth curve parameters for BW and backfat. Two SNP (UASMS2 and R25C) were genotyped on 1,653 cross-bred steers and heifers in a commercial feedlot. Up to 4 serial measures of BW and ultrasound estimates of backfat thickness were taken for each animal from the time of placement on feed to slaughter. The measures were used to estimate growth models that describe changes in BW and backfat thickness as a function of days on feed. Data analysis was carried out by estimating nonlinear mixed models to determine the individual and joint effect of each SNP on growth curve parameters. Brody growth curves were fit to the BW data. Variations in the R25C SNP did not significantly affect growth parameters individually or in combination with the UASMS2 SNP. Variations in the UASMS2 SNP were significant in Brody growth curve parameters for BW growth (P < 0.001). The genotype UASMS2-CC was the heaviest at the beginning of the feeding period and exhibited the largest asymptotic mature BW, but UASMS2-TT cattle exhibited the fastest rate of BW growth. A modified power function was fit to the serial ultrasound backfat measures. Models that included the combined effect of the R25C and UASMS2 SNP provided the best fit to the data. Genotypes differed significantly in power function parameters for backfat growth (P < 0.001). The R25C-CC/UASMS2-TT cattle had the smallest backfat thickness at placement. The genotype R25C-CC/UASMS2-TT exhibited the fastest backfat growth rate, whereas backfat in R25C-CC/UASMS2-CC cattle grew at the slowest rate. The association between leptin genotype and growth in BW and backfat presents opportunities to identify genetically distinct cattle and to differentially optimize feeding times accordingly.     

Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle

Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.     

HspX is present within Mycobacterium paratuberculosis-infected macrophages and is recognized by sera from some infected cattle

A portion of the gene encoding HspX has been previously identified as a sequence specific to Mycobacterium avium subspecies paratuberculosis (hereafter referred to as M. paratuberculosis) based on DNA hybridization experiments. In this study, rabbit antisera were raised against a recombinant protein of HspX fused to the Escherichia coli maltose binding protein (MBP/HspX). Immunoblots of lysates of M. paratuberculosis-infected macrophages probed with the rabbit antisera showed that HspX was present within infected macrophages of bovine and murine origin. This observation was confirmed by immunofluorescence microscopy of infected macrophages. Lysates of E. coli expressing HspX without the MBP fusion partner were loaded onto preparative SDS-PAGE gels and used to determine whether infected cattle generated a humoral immune response to the antigen. Sera from four of 24 paratuberculous cows (17%) detected HspX. No reactivity was present in sera from control cows. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. Collectively, these data give a preliminary characterization of the first described M. paratuberculosis protein identified within infected macrophages.     

Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method.

The ability of Mycobacterium avium subsp. paratuberculosis to survive in bovine monocytes was studied using radiometric (BACTEC) culture, standard plate counting and microscopic counting of acid-fast stained monocyte monolayers. Results of microscopic counts sharply contrasted with results of viable counts determined both by plate counting and radiometric counting. We observed an early phase (the first 6 d after in vitro infection) of intracellular bacillary growth, followed by a later phase of mycobacteriostasis or killing (up to 12 d after in vitro infection) in monocytes from non-infected cows. The data suggest that multiplication and death of M. avium subsp. paratuberculosis occur simultaneously in bovine monocytes infected in vitro. Using the BACTEC method, we compared the ability of bovine monocytes from normal cows and cows infected with M. avium subsp. paratuberculosis and showing evidence of a strong Thl-like cellular immune response to ingest and inhibit the intracellular growth of M. avium subsp. paratuberculosis. There was a trend toward greater phagocytosis and faster killing of Mycobacterium avium subsp. paratuberculosis by monocytes from the infected, immune responder cows. However, the observed numbers of viable M. avium subsp. paratuberculosis at each time after monocyte infection were not significantly different between normal and infected cows.