The effect of fertility control on the transmission of bovine tuberculosis in wild brushtail possums

AIM: To determine the effect of fertility control on the rate of transmission of bovine tuberculosis (Tb), caused by Mycobacterium bovis, in brushtail possums (Trichosurus vulpecula). METHODS: At two study sites with a history of Tb infection in the resident possum population, a sample of adult male and female possums (n=50), determined by palpation to be Tb-free, was surgically sterilised by gonadectomy. A sample of untreated Tb-free male and female possums (n=54) served as controls. Each study site was trapped every 2 months over a 3-year period, and the Tb status of the individuals in the trial assessed. At the conclusion of the trial, all remaining experimental individuals were killed, necropsied and examined for characteristic lesions typical of Tb. The rate of transmission of Tb infection was estimated using the incidence of tuberculous cases in the experimental animals and comparing it between sites, sex and sterilisation treatment. RESULTS: Sterilisation by gonadectomy resulted in a reduction in the rate of transmission of Tb in male possums by 53%, but a corresponding increase in sterilised females for reasons that are still unclear. However, this interaction was statistically weak (p=0.10). When the sexes were combined, there was no overall effect of sterilisation on the rate of transmission of Tb (p=0.43). Sterility treatment notwithstanding, there was a highly significant difference in the rate of transmission between the sexes (p=0.01), being almost one order of magnitude higher in untreated males compared with untreated females. CONCLUSIONS: Although lacking strong statistical support, these results suggest that fertility control that targets endocrine control of fertility may provide some additional benefits for disease control over that achieved by reductions in population density, by reducing the rate of transmission of M. bovis in male possums. However, additional studies are needed to confirm the validity of these results. The large difference in the rate of transmission of M. bovis in untreated males compared with untreated females suggests that transmission of Tb among males may be an important driver of the dynamics of Tb infection in possums.     

Hormonal induction of lactation in nonpregnant ewes

Exogenous hormone treatments designed to induce nonpregnant ewes to lactate at levels sufficient to rear orphan lambs were investigated in three breeds (Blackface, Dorset and Greyface) and in a total of 14 groups. The hormone treatments consisted of an induction phase lasting 1–6 weeks during which most ewes received daily subcutaneous injections of 75 mg of progesterone and 100 µg of oestradiol-17β and a trigger phase lasting 1 or 2 weeks during which 5 mg of oestradiol-17β and/or 10 mg of dexamethasone were given daily and the daily dose of progesterone was reduced to zero. From the end of the trigger phase for at least 2 weeks, milk production was determined three times daily by hand milking after prior intravenous injections of 5 IU of oxytocin. The linear dimensions of the udder were measured in each ewe at 5–6 day intervals throughout the induction and trigger phases.

Udder sizes increased in response to hormone treatment in all cases, and the rate of increase was usually 1.75–11.3 times greater during the trigger phase than during the induction phase. Accumulation of milk in the udder during the trigger phase resulted in mean milk yields of 212–763 ml on the first day of milking, which were higher than the mean yields of 130–354 ml on the second day. Thereafter the mean daily milk yields increased progressively to reach 579–1301 ml after 14 days of milking. Group comparisons revealed the following: an induction phase of at least 4 weeks duration was required to ensure that all hormone-treated ewes produced 800 ml or more of milk/day by 14 days of milking; during the trigger phase, oestadiol-17β alone was a more effective lactogenic agent than dexamethasone alone, and dexamethasone apparently hindered the lactogenic actions of oestradiol-17β when both hormones were given together; extending the duration of the trigger phase from 1 to 2 weeks did not improve subsequent milk yields; and there were no significant breed differences in milk yield responses to similar hormone treatments.

It is concluded that a 4–6 week induction phase followed by a l-week trigger phase using the progesterone and oestradiol-17β doses noted above but excluding dexamethasone would induce in most nonpregnant ewes lactation at levels sufficient to rear orphan lambs. Compared to the compositions of normal colostrum and milk, the milk from some of the present ewes had lower dry matter contents, fat concentrations and immunoglobulin-G concentrations and generally similar lactose concentrations. These differences were not considered to be sufficient to jeopardise the survival of lambs reared by such ewes.     

Examination and control of lameness in dairy herds

Extract

Lameness is a significant problem in dairy herds in New Zealand. It affects production and causes cows to lose weight. If antibiotics are used, milk has to be withheld from the vat. Cows may go into extended anoestrus and some which fail to conceive have to be culled. Even in its mildest form lameness is an inconvenience to the normal management of the herd.     

Prolactin assay for fescue toxicity in sheep

Fungal endophyte (Acremonium spp.) toxicity affects sheep and cattle grazing a number of forages, including ryegrasses (Lolium spp.) and tall fescue (Festuca nrundinacea). The endophyte in tall fescue is responsible for ill thrift, as indicated by low weight gains or weight loss⁽¹⁾ and decreased reproductive efficiency⁽²⁾, and is associated with increased body temperatures. Extensive research has been undertaken in the USA to determine ways of reducing the impact of fescue toxicity on the beef industry⁽³⁾, whilst New Zealand research is directed toward both ryegrass and fescue endophyte toxicity and its impact on sheep⁽⁴⁾⁽⁵⁾ and cattle⁽⁶⁾ production. Much of this work involves field evaluations of productive performance in animals fed toxic and non-toxic forage, so that it is important to be sure that treatment effects are due to the endophyte.     

The pathogenesis of experimental endo-bronchial Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula)

Aim. To study the nature and development of experimentally induced respiratory tuberculosis in possums and compare the lesions observed with those seen in the natural disease.

Methods. Thirty-three adult possums were inoculated via the endo-bronchial route with 20–100 colony forming units of Mycobacterium bovis. The possums were killed at 1, 2, 3 and 4 weeks after inoculation and the nature and distribution of the lesions studied in detail histopatbologically. Alveolar macrophages recovered from the infected possums were also studied ultrastructurally.

Results. Macroscopic lesions were largely confined to the respiratory tract, increasing in size and number with time. Histology greatly increased the detection of the total number of lesions. The most common sites affected outside the respiratory tract were the liver and hepatic lymph nodes, but lesions were less common in peripheral lymph nodes than is observed in the natural disease. Intra-pulmonary lesions were centred on blood vessels and their associated lymphatics. Peripheral blood lymphocyte blastogenic responses to M. bovis antigens were first detected at 3 weeks after inoculation, which was 1 week after lymphocyte infiltrations were detected in the lungs, but 1 week before the majority of infections became generalised.

Conclusions. Differences in the nature of pulmonary lesions and the distribution of lesions were observed between experimentally induced and the natural disease. Rapid haematogenous and lymphatic spread occurs early in the experimentally induced disease.     

牛结核病病原体研究进展

牛结核病是一种人畜共患传染病,人畜结核病的交叉传播是造成结核病广泛流行的重要原因之一。本病的传播流行直接影响着畜收业的发展,而且还会通过各种途径传染给人,给人类的健康也带来很大威胁。作者对结核病病原体的特点及分类进行了概述,并主要对牛分枝杆菌和结核分枝杆菌进行了基因组比较以及它们之间的区分和鉴定进行了综述。     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection.

METHODS: Possums were inoculated with 5 x 10⁶ colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7), orally (n=10), via the endo-bronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 10⁶ cfu and half of which received 5 x 10⁷ cfu of BCG). The possums were humanely killed between 1–4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically.

RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endo-bronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i.

CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intra-dermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: a first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n = 207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09–22.99, P < 0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002–2003, O.R. 3.69; 95% CI 1.27–11.9, P < 0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.     

A behaviour study on the potential for direct transmission of tuberculosis from possums (Trichosura vulpecula) to alpacas (Lama pacos), and the converse from alpacas to possums

Aims. To evaluate the potential for the direct transmission of tuberculosis from possums to alpacas, and vice versa.

Methods. A field study was conducted on an alpaca farm in Northland, New Zealand on 7 January 1999. Observations were recorded on the interaction of one group of male alpacas with a simulated dead possum, one male and one female alpaca group with a simulated terminally tuberculous possum, and one group of male alpacas with a normal possum in an enclosure from which the animals could not escape. The possum was sedated with ketamine as hydrochloride to simulate death (inactive; no movement), and terminal illness (active, into-ordinated movement around the paddock). The observations were based on the focal animal sampling technique, they were un-replicated, and recorded visually and manually with intermittent still photography.

Results. Both male and female alpacas showed strongly inquisitive interaction with the possum. They clustered around the possum (focal animal) very soon after it was observed by the first member of the group. The interest of the majority of both sex groups remained high for the observation periods of approximately 30 minutes, and most individuals remained within 5 metres of the possum for that time. Approximately 50% of the alpacas were within possible aerosol transmission distance of 2 metres from the sedated, erratically mobile possum with their heads towards it for approximately 50% of the two observation periods. Aggressive behaviour was recorded for a young male with stamping on the moving possum. Similar, but more vigorous and prolonged stamping behaviour was recorded for a female with a young (<1 week) offspring (cria).The stamping behaviour was accompanied by very close nose to nose contact of the alpaca and possum. At one point the female threw the possum approximately 1.5 metres in the air with her teeth. The group of male alpacas placed in an enclosure with an unsedated normal possum generally moved away from the possum during its rapid active attempts to escape. When it became inactive their approaches were cautious and only once elicited a defence reaction from the possum, from which they recoiled. One male made one attempt to stamp on the active possum. Soon after the possum became inactive in a small loose hay pile, the alpacas lost interest in it.

Conclusions. The alpaca / possum behavioural interactions show there is potential for direct aerosol transmission of tuberculosis from possums to alpacas, but probably not from alpacas to possums.     

Individual animals of a cattle herd infected with the same Mycobacterium bovis genotype shows important variations in bacteriological, histopathological and immune response parameters

Cattle are the host and main reservoir of the etiologic agent of bovine tuberculosis, Mycobacterium bovis; although other mammalian species, including humans, are susceptible. The tuberculin test and/or slaughterhouse surveillance is the diagnostic method used by control programs all around the world to control and eradicate the disease. In order to compare different tuberculosis diagnostic tests and to reach disease confirmation, a study was performed in a group of 14 steers of Friesian breed, reacting positively to tuberculin test. Three ante-mortem assays were performed according to the type of sample: the gamma interferon (IFN-gamma) test (which quantifies the release of this cytokine by sensitized lymphocytes in whole blood in response to purified protein derivative (PPD) and recombinant ESAT-6 and CFP10 proteins); PCR and bacteriologic culture from nasal swab and intradermal tuberculin test. These assays were taken at different times to assess the evolution of clinical parameters. Post-mortem examination showed macroscopic and microscopic tuberculosis lesions with acid-fast bacillus and positive cultures. By spoligotyping, we observed that all the isolates showed the same pattern. The positive results based on comparison to lesions observed ranged from 58% to 75% for the IFN-gamma assays, to 72% for cultures, and ranged from 50% to 90% for PCR in nasal swabs. In conclusion, in a herd infected by the same strain, ante-mortem direct and immune-diagnostic parameters change, suggesting that several tests are needed for a faster control of infection at herd level.     

The impact of badger removal on the control of tuberculosis in cattle herds in Ireland

In Ireland, the herd prevalence of bovine tuberculosis has remained stable for several decades, and in common with several other countries, progress towards eradication has stalled. There is evidence in support of the potential role of infected badgers (Meles meles, a protected species) in bovine tuberculosis in Ireland and Britain. However, this evidence on its own has not been sufficient to prove disease causation. Field trials are likely to offer the best opportunity to define this role. Building on the earlier East Offaly project, our objectives were to assess the impact of badger removal on the control of tuberculosis in cattle herds in Ireland.

The study was conducted from September 1997 to August 2002 in matched removal and reference areas (average area of 245.1 km²) in four counties: Cork, Donegal, Kilkenny and Monaghan. Badger removal was intensive and proactive throughout the study period in the removal areas, but reactive (in response to severe tuberculosis outbreaks in cattle) in the reference areas. Removal intensity in the removal and reference areas during the first 2 years of the study averaged 0.57 and 0.07 badgers/km²/year, respectively.

The outcome of interest was restriction of cattle herds due to confirmed tuberculosis, where tuberculous lesions were detected in one or more animals. Data were analysed using logistic regression (modelling the probability of a confirmed herd restriction) and survival analysis (modelling time to a confirmed herd restriction).

During the study period, there was a significant difference between the removal and reference areas in all four counties in both the probability of and the time to a confirmed herd restriction due to tuberculosis. In the final year of the study, the odds of a confirmed herd restriction in the removal (as compared to the reference areas) were 0.25 in Cork, 0.04 in Donegal, 0.26 in Kilkenny and 0.43 in Monaghan. Further, the hazard ratios (removal over reference) ranged from 0.4 to 0.04 (a 60–96% decrease in the rate at which herds were becoming the subject of a confirmed restriction).     

两种奶牛结核病检测方法的比较

本研究中采用常规皮内变态反应(TST)对广西南宁、柳州共计2 818头黑白花奶牛进行牛结核病检测,采用抗原特异性IFN-γ试验方法对PPD检测结果为阳性、可疑的牛以及部分阴性牛进行复检。将抗原特异性IFN-γ试验与TST检测结果相比,其敏感性为53.33%,特异性为70.49%,符合率为67.11%。本研究证实单独使用PPD皮内变态反应方法在牛结核病的诊断方面存在非特异性强等缺点,易造成假阳性牛的误杀;用抗原特异性IFN-γ试验对TST试验阳性和可疑牛进行复检可以提高特异性,该方法在临床上有重要应用价值。     

抗牛结核分枝杆菌VHH抗体T7噬菌体库的构建与筛选

为获得抗牛结核分枝杆菌的VHH纳米抗体,本研究利用牛结核分枝杆菌Ag85B蛋白免疫双峰驼,免疫效价达到1∶8 000后采外周血分离淋巴细胞,提取RNA反转录成cDNA,利用巢式PCR方法扩增出编码单域抗体VHH的400 bp基因片段。将目的片段用限制性内切酶EcoRⅠ和HindⅢ双酶切,与T7噬菌体载体臂T7Select 10-3RI Arms连接,再通过体外重新包装含目的片段的噬菌体。对建立的VHH噬菌体抗体库进行鉴定,原始文库库容为5.7×10~8 PFU,库阳性率为85.7%。通过4轮生物亲和淘选,筛选并表达出2株高特异性的VHH抗体,均对牛结核分枝杆菌Ag85B蛋白具有特异性。本研究为进一步探讨VHH抗体在牛结核病的诊断和治疗奠定了基础。     

IL-10在结核分枝杆菌感染和免疫中作用的研究进展

白细胞介素10（interleukin 10,IL-10）是一种多功能、多细胞源性的细胞因子,因其具有强抗炎和免疫抑制活性而成为研究热点。在结核病的发生发展中,IL-10的免疫抑制作用受到了研究者的关注,其既能抑制天然免疫,也能影响获得性免疫。研究者普遍认为IL-10与结核分枝杆菌的免疫逃避及潜伏性感染有关。鉴于其在结核分枝杆菌感染中独特的作用,作者对近几年IL-10在结核分枝杆菌免疫逃避、潜伏感染、临床辅助治疗及自身表达调控等方面的研究进展进行了综述。     

Research Progress on Role of IL-10 in Immunodulation during Mycobacterium tuberculosis Infection

Interleukin 10 (IL-10) is a versatile and multi-cell derived cytokine, which has attracted the great attention of researchers because of its strong anti-inflammation and immunosuppression activities. It can not only inhibit the innate but also adaptive immunity. Generally, it is believed that IL-10 is closely related to immune evasion and latent infection by Mycobacterium tuberculosis (MTB). In view of its unique role in MTB infection, progress of role of IL-10 on immune evasion, latent infection, clinical chemotherapies and self-regulation of its expression during MTB infection were reviewed.     

Dental restoration of a canine tooth in a dog

Extract

There was a history of discomfort following fracturc of the lower right canine tooth.     

检测牛结核抗体新型ELISA方法的建立及应用

利用分子克隆和Splicing by overlapping extension(SOE)技术,表达了重组蛋白rM70-83-E6,Western blot分析rM70-83-E6具有很好的特异性。以蛋白rM70-83-E6作为诊断抗原建立了间接ELISA方法,ELISA诊断方法的判定标准,即S/P≥0.5为阳性,S/P<0.4为阴性,0.5>S/P≥0.4为可疑。ELISA方法的特异性为96.0%、敏感性为69.4%。对67份PPD皮试阳性和50份PPD皮试阴性(117份)血清样品进行了检测,并与PPD皮试比较。67份PPD皮试阳性血清样品中有46份为ELISA检测阳性,阳性符合率为68.7%,50份PPD皮试阴性牛血清都为阴性,阴性符合率为100%,本ELISA方法与PPD皮试诊断方法总复合率为82.1%。研究表明,ELISA方法具有很好的开发和应用前景。     

11种牛分枝杆菌抗原在牛结核病诊断中的初步评价

为筛选及评价用于牛结核病诊断的抗原,本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法,比较其对牛结核病的检出率;同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外,将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24 h,检测血浆中的IFN-γ水平,评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示,不同重组蛋白对结核病阳性血清的反应活性不一,MPB70总检出率最高,为59.7%;其次是Ag85B、ESAT-6和MPB83,检出率均在45%以上;MPT51的检出率最低,仅为2.2%。豚鼠和牛皮试试验均显示,单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应（delayed type hypersensitivity,DTH）,而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原,分别与CFP-10或ESAT-6混合,均可特异性地刺激结核病阳性牛产生较强的DTH反应,且与PPD-B无显著差异（P>0.05）。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ,其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛（P<0.05）。因此,这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验,但以CFP-10和ESAT-6为核心,TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原,均能提高检测敏感性,有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。     

Development of gene expression assays measuring immune responses in the spotted hyena (Crocuta crocuta)

As scavengers, spotted hyenas (Crocuta crocuta) are exposed to a wide array of pathogens but exhibit low mortality rates due to infectious disease. This suggests that this species exhibits a unique and robust immune response to pathogens. However, few tools exist to measure cell-mediated immunity (CMI) in hyenas and we aimed to develop a gene expression assay to quantify antigen-specific responses. Whole blood from five Mycobacterium bovis- sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in the expression of IFNG, CXCL8, CXCL9, CXCL10 and CXCL11 in M. bovis-antigen stimulated blood. The expression of YWHAZ and TBP showed greatest stability, and YWHAZ was selected as a reference for further analysis. The expression of CXCL9 and CXCL11 showed greatest upregulation in antigen-stimulated blood and the assay results for these genes were strongly correlated. The measurement of antigen-induced CXCL9 and CXCL11 expression, relative to that of YWHAZ, can be used to measure CMI responses to infectious diseases in spotted hyenas.     

炎性小体的激活机制及其在机体抗结核分枝杆菌中的作用研究进展

由结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染引起的结核病（tuberculosis,TB）是目前危害人类健康的一个重要的公共卫生问题。据估计,目前全球大约有23%的人被Mtb所感染。虽然多数Mtb感染者最终不发病,表现为潜伏期感染（latent TB infection,LTBI）,但全球每年仍有数百万人死于结核病。因此,结核病是一个持续的热门研究领域,研究者试图从不同的角度去揭示Mtb的致病机制,以期最终战胜结核病。炎性小体（inflammasome）是一种聚集在受感染或受损细胞细胞质中的多蛋白复合物,其可识别病原相关分子模式（pathogen-associated molecular patterns,PAMPs）或者宿主来源的危险信号分子（host-derived danger signaling molecules,也称损伤相关分子模式,damage-associated molecular patterns,DAMPs）,在多种感染性疾病中发挥着重要作用。本文将对Mtb感染过程中炎性小体的激活机制及其在机体抗Mtb感染中的作用相关研究进展进行综述。