Diagnosis of paratuberculosis

Diagnostics of paratuberculosis infection is a difficult and complex field, that causes confusion, lack of understanding and frustration in practitioners, veterinary officers and last but not least farmers. In this review the various diagnostic approaches with their potentials, advantages and disadvantages and their limits are discussed.     

Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.     

Mycobacterium paratuberculosis heat shock protein 70 as a tool in control of paratuberculosis

Paratuberculosis in cattle is a chronic intestinal disease in which a distinctive cellular reactivity of a Th1-type preceeds the phase in which antibody titers are easily detectable and the animal becomes clinically ill. During infection with Mycobacterium avium ssp. paratuberculosis (M.a.p), a decrease in CD4 T-helper cells has been observed in the clinical phase. Our ultimate aim is to elicit a cytotoxic reaction against infected macrophages, using recombinant Hsp70 (rHsp70) of M.a.p. as a tool to shuttle antigen into the MHC class I antigen presentation pathway. To investigate the mechanism of rHsp70 as a carrier for antigen into the cell, we studied the interaction between APC and Fitc-labelled rHsp70, using FACS analysis and confocal microscopy. Interaction of rHsp70 with the cell surface of bovine APC, presumably via a receptor, was shown on monocytes, monocyte derived macrophages and dendritic cell (DC). The interaction is detectable on the complete population of freshly derived monocytes, although peak intensity of fluorescence is lower on these cells than on macrophages and DCs. DCs show interaction on a high percentage of the cells, with high intensity, while in the case of macrophages only a subpopulation interacts with rHsp70. Efficient uptake of rHsp70 as compared to OVA is shown. Preincubation of DC with unlabelled rHsp70 leads to a decreased interaction with rHsp70-FITC. DC interacting with rHsp70 in addition showed high expression of MHC I, MHC II, Myd-1 (CD172a) and CD40. Further research will focus on loading of the rHsp70 with M.a.p. antigen for presentation in MHC class I.     

Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis

OBJECTIVE: To assess the protective value of a live-attenuated vaccine in sheep already exposed to Mycobacterium avium subsp paratuberculosis and to investigate the progression of a systemic immune response in experimentally infected sheep. STUDY DESIGN: Twenty-eight lambs, aged 1 to 1.5 months, were dosed via stomach tube with approximately 4.4 x 10(8) M a paratuberculosis organisms. Two weeks later, 14 of these 28 animals received subcutaneous injections of 1 mL of a live-attenuated vaccine. Thirteen additional lambs were neither dosed nor vaccinated (negative controls). Antigen-induced production of IFN-gamma in blood, and antibody concentrations in serum were sequentially monitored in vaccinated, unvaccinated and control animals for 1 year. Each sheep was examined for infection by an IS900-based PCR test on samples of ileum and ileocaecal lymph node and histological examination at the time of necropsy. RESULTS: Seven of 14 unvaccinated and two of 14 vaccinated sheep developed clinical paratuberculosis that was later confirmed by histological examination and/or the IS900-based PCR test. The granulomatous inflammation in the jejunal and ileal mucosa was less severe in vaccinated than in unvaccinated sheep. Acid-fast organisms were detected only in the unvaccinated group. The PCR assay on ileal samples gave positive reactions in two vaccinated and eight unvaccinated sheep. Both the antibody response and IFN-gamma response were detected earlier and were more substantial in vaccinated than in unvaccinated sheep. Furthermore, in experimentally infected but unvaccinated sheep, the IFN-gamma concentrations were higher in those animals without acid-fast organisms than in those with them. CONCLUSIONS: Vaccination of lambs with live-attenuated vaccine 2 weeks after oral inoculation with M a paratuberculosis stimulated the host response against the organism and led to a reduced mycobacterial burden. The diminished IFN-gamma responses in experimentally infected sheep with acid-fast organisms suggest a positive relationship between the magnitude of the systemic cell-mediated immune response and an animal's ability to control infection.     

Isolation of Mycobacterium paratuberculosis from the uterine flush fluids of cows with clinical paratuberculosis

Mycobacterium paratuberculosis was isolated from the uterine flush fluids obtained from 3 to 4 cows with clinical paratuberculosis. Four cows with clinical paratuberculosis were subjected to uterine lavage, using an established embryo recovery technique, and the recovered fluids were cultured for M paratuberculosis. Mycobacterium paratuberculosis has previously been demonstrated to adhere to bovine ova. Embryos within the uterus of a superovulated cow infected with M paratuberculosis could be exposed to the organism.     

Mycobacterium paratuberculosis and milk

The possibility that milk from cattle with Johne's disease could be a potential vehicle of transmission of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) to humans has been the focus of a UK government-funded research programme at Queen's University, Belfast since 1993. The main findings of this research programme are reported and practical advice about the most appropriate methods for the isolation/detection of this organism in milk is given. The findings of several milk surveys during which optimised sensitive detection methods were employed (decontamination with 0.75% cetyl pyridinium chloride for 5 h prior to culture and a novel immunomagnetic PCR technique) have revealed that detectable levels of M. paratuberculosis are present in bulked raw cows' milk in the UK at both the farm level and at dairy processing plants prior to pasteurisation. Furthermore, results of three different experimental approaches to assess the effect of pasteurisation time/temperature conditions on the viability of M. paratuberculosis (laboratory pasteurisation studies, a national survey of commercially pasteurised milk, and processing of naturally infected milk through commercial-scale pasteurising plant) provide firm evidence that this organism is capable of surviving commercial milk pasteurisation on occasion. Hence, both raw and pasteurised cows' milk are potential vehicles of transmission of M. paratuberculosis to humans.     

Diagnosis of paratuberculosis in sheep

Paratuberculosis in sheep usually is manifested as emaciation and decreased wool production. Diarrhea occurred in only 18% of affected animals. Significant hematologic changes included decreased RBC count, hemoglobin level and hematocrit. Necropsy revealed pallor, cachexia and serous fluid in body cavities. Staining of intestinal mucosal scrapings and mesenteric lymph node impression smears for acid-fast organisms revealed bright-red clumps of Mycobacterium paratuberculosis bacilli. Fecal examination identified 70% of affected animals, intradermal injection of johnin 60%, and avian tuberculin 39%.     

Detection methods of Mycobacterium paratuberculosis

Mycobacterium paratuberculosis is receiving increasingly wider interest of scientific groups worldwide. This slow-growing mycobacterium does not only evoke paratuberculosis--an infectious cattle disease that brings huge economic losses--but it is also regarded as a potential cause of human Crohn;s disease. It is very difficult to diagnose precisely this kind of animal infection in its very early stages, as well as to detect occurrence of M. paratuberculosis cells in the environment, including food of animal origin. This paper reviews currently known and employed diagnostic techniques for M. paratuberculosis cell detection and identification.     

肉鸡低血糖-尖峰死亡综合征

肉鸡低血糖--尖峰死亡综合征(Hypoglycemiaspiking mortality syndrome of broiler chickens,HSMS)是一种主要侵害肉仔鸡的疾病。10~18日龄为发病高峰期,有报道称42日龄的商品代肉鸡也发生本病。临床表现为突然出现的高死亡率,至少持续3~5d,同时伴有低血糖症。病鸡头部震颤、运动失调、昏迷、失明、死亡。有些病鸡可以自然恢复,但常会出现生长发育不良、矮小和气囊炎。HSMS最早报道于1986年,发生在美国的Delmarva半岛地区。1991年报道了41个自然发病鸡群和3个实验感染鸡群。之后,该病向美国东南部地区发展。目前在加拿大、欧洲、马来西亚和南非均有本病发生。自1998年以来,我国华北地区多次发生肉鸡突发性高致死性疾病,现已证实是HSMS。1999年在东北的辽宁省也出现了肉鸡群的突然死亡病例,临床症状与病理变化与HSMS极其相似。     

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.