An outbreak of caseous lymphadenitis in dairy goats: First report of the disease in the Netherlands

Summary

An outbreak of caseous lymphadenitis in which three herds of goats were involved is described. Corynebacterium pseudotuberculosis was shown to be the causative organism. The outbreak was associated with imported goats. As this first report of C. pseudotuberculosis causing caseous lymphadenitis as a clinical entity in small ruminants in the Netherlands, clinical and epizootiological aspects as well as possible methods of control are discussed.     

Responses of testosterone hormone concentration,semen quality,and its related pro-inflammatory cytokines in bucks following Corynebacterium pseudotuberculosis and its mycolic acid infection

Tropical Animal Health and Production - Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a debilitating chronic disease of sheep and goats. Little is known about...     

Characterization of strains of corynebacterium pseudotuberculosis.

Twenty-five strains of Corynebacterium pseudotuberculosis isolated from lesions of caseous lymphadenitis in goats were examined for their biochemical characteristics, antimicrobial susceptibility, and phospholipase D activity. The strains were uniform in biochemical reactions, cultural characteristics, and susceptibility to antimicrobial agents. Presence of urease and phospholipase D and absence of pyrazinamidase were valuable criteria in the identification of C. pseudotuberculosis.     

Comparison of an interferon-gamma to a phospholipase D enzyme-linked immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats

The optimal method of control of caseous lymphadenitis of goats caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. The objective of this study was to compare detection of C. pseudotuberculosis experimentally infected goats using a commercially available bovine interferon-gamma (IFN-gamma) whole blood enzyme-linked immunosorbent assay (ELISA) to serological response to a recombinant phospholipase D (PLD) ELISA. The tests were assessed repeatedly over 1 year in three infected and three non-infected goats. Using a IFN-gamma optical density cut-off at 0.10 as positive under the conditions used, the test accurately detected C. pseudotuberculosis experimentally infected goats over a 363 day period with a reliability of 89.2% and non-infected goats with a reliability of 97.1%. Using a cut-off value of the mean for negative samples plus two standard deviations, the PLD ELISA detected C. pseudotuberculosis experimentally infected goats over this period with a reliability of 81.0% and non-infected goats with a reliability of 97.0%. The PLD ELISA was however more predictive than the IFN-gamma ELISA of the presence of lesions observed at postmortem examination of infected goats.     

Host associated polymorphisms in the Corynebacterium pseudotuberculosis rpoB gene sequence

Corynebacterium pseudotuberculosis is a widespread facultative intracellular pathogen that causes caseous lymphadenitis disease in sheep and goats, and generates cutaneous abscesses and granulomas in horses and cattle. Although some genes have been studied for diagnostic and phylogenetic analysis within the genus Corynebacterium, at subspecies level the pathogen has been poorly analyzed. The aim of this study was to characterize C. pseudotuberculosis strains isolated from domestic animals, through the sequencing of a hypervariable rpoB gene segment. As result, there were identified host associated rpoB polymorphisms in strains infecting sheep, goats and horses from Chile. These differences suggest the existence of bacterial genotypes, in which the nucleotide similarity values were ranging from 98.8 to 99.8%. In conclusion, the analysis of polymorphisms in the partial rpoB sequence can be used as a diagnostic tool that differentiates C. pseudotuberculosis strains at subspecies level.     

羊伪结核棒状杆菌的分离鉴定及ELISA检测方法的建立

从疑似山羊干酪性淋巴结炎羊的肩前淋巴结脓肿内分离到2株菌,经鉴定均为羊伪结核棒状杆菌。利用羊伪结核棒状杆菌标准菌株(ATCC19410株)制成外毒素作为检测抗原,通过酶标抗体、阳性血清、外毒素抗原最适浓度的选择试验,确定适当的抗原、抗体和酶标抗体稀释浓度,建立了间接ELISA方法用于检测抗体。用建立的ELISA方法检测100份待检羊血清,其中阳性血清4份,阳性检出率为4％。     

Epidemiological, bacteriological and molecular studies on caseous lymphadenitis in Sirohi goats of Rajasthan, India

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CL), a chronic debilitating disease of goats. In the present study, a total of 575 goats of Sirohi breed on an organized farm situated in the semi-arid tropical region of Rajasthan, India were clinically examined. Pus samples from superficial lymph nodes of 27 (4.7%) adult goats presenting clinical lesions suggestive of CL were collected for bacteriological and molecular analyses. Of these goats, 51.9% yielded C. pseudotuberculosis on the basis of morphological, cultural and biochemical characteristics. A polymerase chain reaction (PCR) assay targeting proline iminopeptidase gene specific to C. pseudotuberculosis was developed that confirmed all 14 bacterial isolates. The specificity of the PCR product was confirmed by sequencing of the 551-bp amplicon in both senses, showing 98-100% homology with published sequences. Thus, overall prevalence rate based on clinical, bacterial culture and PCR assay were found to be 4.7%, 2.4% and 2.4%, respectively. The PCR assay developed in this study was found to be specific and rapid, and could be used for confirmation of CL in goats as an alternative method to generally cumbersome, time-consuming and less reliable conventional methods.     

Visceral caseous lymphadenitis in thin ewe syndrome: isolation of Corynebacterium, Staphylococcus, and Moraxella spp from internal abscesses in emaciated ewes

The relationship between the visceral form of caseous lymphadenitis and a chronic debilitating condition of mature sheep designated as the thin ewe syndrome was investigated. Internal abscesses were found during necropsy in 81% of animals with thin ewe syndrome and Corynebacterium pseudotuberculosis (C ovis) was recovered from 86% of the animals with internal abscesses. Other pyogenic bacteria, including C pyogenes, C equi, Staphylococcus epidermis, S aureus, and Pseudomonas aeruginosa were often recovered in association with C pseudotuberculosis. Moraxella sp was recovered in 41% of the animals with internal abscesses. In some abscesses, Moraxella sp was the dominant microorganism isolated and in others, they were outnumbered only by C pseudotuberculosis. Species isolated included M bovis, M osloensis, and M nonliquefaciens. The potential importance of Moraxella sp to the cause and pathogenesis of the thin ewe syndrome is not known. The results of the present study indicate that visceral caseous lymphadenitis is either an important contributing factor to the development of thin ewe syndrome or that the presence of thin ewe syndrome may predispose affected sheep to the development of visceral caseous lymphadenitis. A skin test reagent prepared by sonicating C pseudotuberculosis was of limited value in detecting animals with visceral caseous lymphadenitis. Only 56% of the animals with abscesses caused by C pseudotuberculosis gave positive delayed-type hypersensitivity skin test responses.     

Synergistic hemolysis-inhibition titers associated with caseous lymphadenitis in a slaughterhouse survey of goats and sheep in Northeastern Brazil

A survey of caseous lymphadenitis was conducted at a goat and sheep slaughterhouse in Northeastern Brazil One hundred and fifty-eight goats and 43 sheep were examined for the presence of abscesses, with bacterial culturing of purulent material to define the etiological agent. Blood was collected simultaneously for determination of serological titer via the synergistic hemolysis-inhibition test which measures antibodies to an exotoxin of Corynebacterium pseudotuberculosis. Thirteen and nine-tenths percent of the goats had abscesses, with a high proportion having mediastinal or pulmonary lesions (9.5%). Two sheep had abscesses, both with internal organ involvement. Corynebacterium pseudotuberculosis was the most frequently isolated organism. Of 22 goats with abscesses, 20 were positive via the synergistic hemolysis-inhibition test. Both of the sheep with abscesses had positive synergistic hemolysis-inhibition titers. The proportion of serological reactors was greater than the proportion of animals with abscesses. The synergistic hemolysis-inhibition test may be detecting subclinically infected animals.     

In vitro IFN-gamma production by goat blood cells after stimulation with somatic and secreted Corynebacterium pseudotuberculosis antigens

Corynebacterium pseudotuberculosis is the causal agent of caseous lymphadenitis, a chronic illness that attacks goats and sheep characterized by pyogranulomas formation in lymph nodes and organs. Regarding the current knowledge of the pathogenesis of the caseous lymphadenitis, there is evidence that besides the humoral response the induction of a durable cellular response is fundamental for its control. In this sense, research on antigens of C. pseudotuberculosis that are capable to inducing cellular immunity is an important step for the development of diagnosis tests and more efficient vaccines. In the present study, the interferon-gamma production in cultures of whole blood from infected goats stimulated with secreted bacterial antigen or somatic antigen were used to evaluate the cellular response. The results demonstrated a significant difference in the ability of the two antigens to induce a cellular response. That is, IFN-gamma production was high with cells from infected animals in response to the secreted antigen while IFN-gamma production was low when somatic antigen was used. The concomitant use of these antigens with PWM also showed differences. That is, the secreted antigen increased the IFN-gamma production induced by PWM, while the somatic antigen seems not to have altered the response to PWM.     

陕南白山羊伪结核棒状杆菌分离鉴定及灭活铝胶疫苗研制

羊干酪性淋巴结炎又称羊伪结核病,是由伪结核棒状杆菌感染引起的一种人畜共患慢性接触性传染病,对山羊危害最为严重,目前尚无有效治疗措施。陕西省石泉县陕南白山羊饲养量较大,羊群也存在伪结核棒状杆菌的感染,造成较大的经济损失。为筛选对当地伪结核棒状杆菌敏感的药物和研制灭活疫苗,本研究从疑似伪结核病患羊采取多份新鲜的脓液,进行细菌分离鉴定;对获得的1株分离菌进行药物敏感性试验并用其研制灭活疫苗。结果显示,分离菌在固体培养基上形成白色、干燥、扁平、不透明、边缘整齐的中等大小菌落,革兰染色阳性,经16SrRNA检测和序列分析,证实分离到的菌株为羊伪结核棒状杆菌。药物敏感性试验结果显示,该分离菌对环丙沙星、头孢曲松、红霉素、卡那霉素、头孢噻肟、妥布霉素、强力霉素、庆大霉素、氯霉素高度敏感;对四环素、利福平、链霉素中度敏感。以该分离菌为种子菌制备的灭活铝胶疫苗无菌检验合格,试验接种山羊无不良反应,目前已经在采样羊场进行临床应用,进一步评价免疫效果。     

Immunisation against ovine caseous lymphadenitis: comparison of Corynebacterium pseudotuberculosis vaccines with and without bacterial cells

Sheep were immunised with Corynebacterium pseudotuberculosis vaccines prepared from cell-free toxoid or from toxoid with formalin-killed cells of C pseudotuberculosis added. Resistance of sheep to infection was tested 6 months after immunisation by inoculation with caseous lymphadenitis pus. The outcome was assessed 3 months later by slaughter and inspection of the sheep for lesions of caseous lymphadenitis. immunised sheep were adequately protected against infection as shown by a significant reduction in the number of sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected vaccinated sheep than in affected control sheep. The protective potency of the vaccines was not improved by the inclusion of cells of C pseudotuberculosis.     

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.     

Evaluation of the humoral and cellular immune response to different antigens of Corynebacterium pseudotuberculosis in Canindé goats and their potential protection against caseous lymphadenitis

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freund's incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.     

The effects of caseous lymphadenitis on wool production and bodyweight in young sheep

Two hundred Merino wether hoggets were used to examine the effect of Corynebacterium pseudotuberculosis infection (caseous lymphadenitis) on wool production and bodyweight. Sheep which were challenged with C. pseudotuberculosis (artificially infected) and not vaccinated against this disease produced 0.20 kg less clean wool than unchallenged controls during the following 12 months. The incidence of sheep with lesions in the group that was vaccinated prior to challenge was 55% lower than in unvaccinated challenged sheep but their wool production was not significantly different from either the controls or the unvaccinated challenged sheep. Vaccinated sheep were also heavier than unvaccinated sheep 12 months after challenge. These results indicate that caseous lymphadenitis infection may reduce wool production.     

Serologic response and lesions in goats experimentally infected with Corynebacterium pseudotuberculosis of caprine and equine origins

Fifteen goat kids were experimentally inoculated with Corynebacterium pseudotuberculosis. Five were given a strain of caprine origin (nitrate-negative biotype) intradermally, 5 were given a strain of equine origin (nitrate-positive biotype) intradermally, and 5 were inoculated intranasally with the caprine-origin strain. Animals were monitored for 127 days. The goats given the inocula intradermally developed abscesses; those given caprine-origin strain had multiple lesions both peripherally and in visceral locations (primarily endothoracic abscesses), whereas those given the equine-origin strain had abscesses only at injection sites and draining nodes. The difference in extent of lesions could be due to biotypic bacterial differences or to the individual strains used. Intranasally inoculated goats did not develop abscesses and were essentially no different from controls. The cranial part of the respiratory tract may not be an important portal of entry for C pseudotuberculosis. Serum samples obtained monthly from all animals were subjected to the synergistic hemolysis-inhibition test, which measures antibodies to the exotoxin of C pseudotuberculosis. Animals with abscesses developed titers within 1 month of inoculation. Animals without abscesses remained seronegative. The synergistic hemolysis-inhibition test may be a reliable diagnostic assay for caseous lymphadenitis in goats.     

A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis in sheep and goat flocks.

A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis (CLA) in sheep and goats was performed in one goat herd and one sheep flock over a period of three years. In goats, there was a nonstatistically significant trend for fewer cases of CLA in the vaccinated animals compared to the controls. In sheep, from six months to 36 months postinitial vaccination, the proportion of vaccinated sheep that developed CLA was significantly less (p less than 0.05) than in the control sheep. The antibody titers to Corynebacterium pseudotuberculosis as detected by microagglutination assay were significantly different (p less than 0.0001) at all times except at the initial vaccination. Swellings occurred at the vaccination site at an incidence level of 29.6% in goats and 34.1% in sheep. The vaccine appeared to be efficacious in reducing the proportion of sheep that developed CLA when challenged naturally in a field situation.     

Corynebacterium pseudotuberculosis infection in goats. I. Evaluation of two serological diagnostic tests

The bacterial agglutination test (BAT) and the hemolysis inhibition test (HIT) were employed and evaluated.The threshold value between positive and negative antibody titres in BAT and HIT was determined on the basis of titration results for sera from 25 adult goats infected with Corynebacterium pseudotuber-culosis (positive), and sera from 25 adult goats from a herd in which caseous lymphadenitis did not occur (negative).Antibody titres were expressed as logio to the reciprocal value of the highest positive serum dilution in both tests. Positive titre (T) in BAT was stipulated as T ≥ 2.1 and in HIT as T > 0.6. The sensitivity and specificity was 0.96 in both tests in the material used. Twenty-three of the 25 goats infected with G. pseudotuberculosis were positive in both BAT and HIT.The reproducibility in both tests was described by the use of correlation coefficient and coefficient of variation. The values were estimated using duplicate determination of titres of 155 serum samples. Correlation coefficients were 0.87 for BAT and 0.95 for HIT. Coef-ficients of variation were 26.4 % for BAT and 14.1 % for HIT. The coefficient of variation was related to titres. It was highest for BAT at low titres.It was concluded that both tests can be used for seroepidemio-logical investigations of C. pseudotuberculosis infection in goats.     

Survival of Corynebacterium pseudotuberculosis in axenic purulent exudate on common barnyard fomites

Several inanimate surfaces (eg, plastic, wood, and steel) and particulate fomites (eg, wood shavings, hay, straw, and feces), common to the environment of confined small ruminants, were inoculated with Corynebacterium pseudotuberculosis in axenic purulent exudate that had been surgically removed from a naturally occurring case of caprine caseous lymphadenitis. Each inoculated fomite was incubated at 37, 22, and 4 C, and the length of time that C pseudotuberculosis survived was determined by isolation of bacteria from the fomite. The organism remained viable longer when caseous lymphadenitis abscess contents were mixed with particulate fomites than when spread on surfaces. Incubation at lower temperatures generally extended the survival potential of C pseudotuberculosis. Depending on the particulate fomite and the incubation temperature, viable C pseudotuberculosis organisms were isolated for mean periods ranging from 7 to 55 days, whereas recovery of bacteria from surfaces varied from 1 to 8 days.     

Characterization of lectin-binding lymphocytes in goats with caseous lymphadenitis

Numbers of lymphoid cells bearing markers for peanut agglutinin, wheat germ agglutinin, and anti-immunoglobulin G in healthy goats and in goats with caseous lymphadenitis were determined by use of immunofluorescent techniques. Results indicated that, when compared with healthy goats, diseased goats had normal numbers of anti-immunoglobulin G-positive cells (B cells), had significantly reduced numbers of peanut agglutinin-positive cells (T cells), and had fewer cells with receptor sites for Lens culinaris agglutinin and concanavalin A. Compared with healthy goats, numbers of peanut agglutinin-positive cells in goats with caseous lymphadenitis indicated that the goats may have had a compromised cell-mediated immunity.