Investigation of bovine brucellosis in the Northeastern Turkey

Bovine brucellosis, caused by Brucella abortus, is a significant problem for both public and animal health in Turkey. This study was conducted on the calving seasons between 2001 and 2006. A total of 626 serum samples of cattle obtained from 27 herds with a history of abortions was examined for Brucella antibodies by RBPT, SAT and ELISA. Of the cattle sera analysed, 221 (35,30%) and 206 (32, 92%) and 247 (39,45%) were found to be positive by RBPT , SAT and ELISA, respectively. B. abortus was isolated from 48 (32,21%) of 149 lung samples and stomach contents of the aborted fetuses. Based on the biochemical tests and the agglutination tests with monospecific A and M antisera, only 3 of the isolates were found to be B. abortus biotype 1 and the remaining 45 were biotype 3. This study also revealed that the dominant biotype of B. abortus was biotype 3 in this region. The determination of the agents responsible for bovine brucellosis and serosurvey of this disease are expected to help better understanding of this zoonotic infection in this region and neighbouring countries.     

A comparison of the potency of several Brucella allergens used to detect brucellosis in cattle

The potency of Brucella allergens prepared from a smooth Brucella abortus strain S-99, mucoid strain Leewarden, rough strain 45/20, and rough Brucella melitensis strain B-115 was assessed. The potency of these allergens was compared with that of a standard allergen prepared from smooth Brucella abortus S-99 that efficiently detected bovine brucellosis in other studies. Eight cattle experimentally inoculated with Brucella abortus 544 were tested with the allergens 4 and 10 weeks after infection, and again 8 months after infection. All the allergens effectively detected infection but there was a clear distinction in the mean skin reactions 48 and 72 h after injection of the allergens. The skin reactions provoked by the allergens prepared from smooth or mucoid strains of Brucella were most pronounced 48 h after injection. Skin reactions provoked by allergens prepared from rough strains of Brucella were strongest 72 h after injection. Allergens prepared from smooth or mucoid Brucella strains were more potent in detecting brucellosis than those prepared from rough strains of Brucella.Abbreviations Bruc/OCB Brucellergen OCB - cfu colony-forming units - CFT complement fixation test - ID-DLO Institute voor Dierhouderij en Diergezondheid-Dienst Landbouwkundig Onderzoek - ICFTU international complement fixation units - IU international units - LPS lipopolysaccharide - SAT serum agglutination test - SDTH skin delayed-type hypersensitivity     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.     

Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.     

Prevalence of antibodies to Brucella spp. and individual risk Factors of Infection in Traditional Cattle, Goats and Sheep Reared in Livestock–Wildlife Interface Areas of Zambia

A cross-sectional study was performed in the livestock–wildlife interface areas of Lochinvar and Blue Lagoon National Parks and the non-interface area of Kazungula to determine the prevalence of antibodies to Brucella spp. in domestic ruminants and identify individual animal risk factors of infection. A total of 1245 cattle from 124 herds and 280 goats and sheep from 29 flocks were tested sequentially for Brucella antibodies using the Rose Bengal test (RBT) and competitive ELISA. In cattle, individual seroprevalence ranged from 14.1% to 28.1%, while herd sero–prevalence ranged from 46.2% to 74.0% in the three study areas. No goat or sheep tested positive for Brucella antibodies. Three types of cattle grazing strategies were encountered: locally grazed herds (LGH), transhumantly grazed herds (TGH) and river flood plain grazed herds (FGH). Brucella seroprevalence was seen to vary according to area and grazing strategy: Lochinvar and transhumant grazed herds recorded the highest figures, respectively. Age, sex and history of abortion were found to have independent effects on individual seroprevalence. This study establishes that brucellosis is endemic in domestic animals in the livestock–wildlife interface areas of Blue Lagoon and Lochinvar national parks and the disease is also present in Kazungula. We observed that type of grazing strategy had significant impact on cattle Brucella seroprevalence and that transhumant herds were at high risk of being infected.     

布鲁氏菌LPS对小鼠巨噬细胞中NLRP3炎症小体转录水平的影响

为研究布鲁氏菌LPS对巨噬细胞中NLRP3炎症小体的影响,本试验提取布鲁氏菌2308、RB51和△WbkA的LPS,以不同浓度与小鼠巨噬细胞相互作用,荧光定量PCR检测其对NLRP3、ASC、Caspase1、IL1-β、IL18转录水平的影响。结果显示RB51LPS和△WbkA LPS上调NLRP3炎症小体相关基因的转录水平,且呈浓度依赖性,而浓度对2308LPS调节NLRP3炎症小体相关基因转录的作用不大;且同一浓度下,RB51LPS和2308LPS比△WbkA LPS更好的调节Caspase1、IL1-β、IL18的转录水平。     

Outbreak of Brucellosis in Domestic Elk in Korea

Seven of 18 elk on a deer farm were found by the official Rose‐Bengal agglutination test (RBT) and tube agglutination test to be brucellosis reactors/suspects. Evaluation with the competitive ELISA (C‐ELISA) and the fluorescence polarization assay (FPA) tests revealed that six and five sera were positive respectively. The seven reactors/ suspects were slaughtered and their blood and tissues were collected. Brucella species could be isolated from three of the slaughtered animals, with nine isolates being obtained from the popliteal, supramammary and submandibular lymph nodes, vaginal discharge, mammary tissue and spleen. Brucella genus‐specific PCR based on 16S rRNA and AMOS‐PCR, which is specific for differential Brucella species, revealed that all nine isolates were Brucella abortus. These nine were further confirmed to be B. abortus biovar 1 by classical biotyping scheme assays. This is the first report of an outbreak of brucellosis in domestic elk in Korea. Our observations suggest that deer should be included in the routine Brucella surveillance programme for the effective control and prevention of brucellosis in Korea.     

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.     

Brucellosis associated with stillbirth in a bottlenose dolphin in Australia

A captive adult female bottlenose dolphin presented with stillbirth. The placenta appeared oedematous. No other gross lesions were evident in the placenta or the stillborn calf. Histopathology revealed mild multifocal placentitis and foetal encephalitis. Brucella sp. was isolated from lung, liver, spleen and kidney. Sequence and phylogenetic analysis demonstrated this organism to be most similar to Brucella ceti sequence type (ST) 27. Brucella sp. DNA was detected in formalin-fixed paraffin-embedded placenta and brain by real-time PCR using primers targeting the IS711 gene. Immunohistochemical staining revealed Brucella sp. antigen in placental inflammation. This is the first report of isolation of Brucella sp. from a marine mammal in the Southern Hemisphere and the first report of marine Brucella-associated disease in Australia.     

Brucella-associated cervical bursitis in cattle

Bovine brucellosis poses a risk to human health and causes serious economic losses for the animal industry. This report describes the use of different diagnostic methods for the diagnosis of brucellosis in cattle affected by cervical bursitis from a slaughterhouse located in São Luís, Maranhão, Brazil. Serum samples from a total of 47 cattle with bursitis were collected and submitted to the Rose Bengal Test (RBT), and RBT-positive samples were further confirmed by the 2-mercaptoethanol (2-ME) assay. RBT indicated 85.1% (40/47) of positive samples, from which 78.7% (37/47) were confirmed by 2-ME. Immunohistochemistry detected Brucella spp. in 34.0% (16/47) of tissues with bursitis. PCR and/or bacterial isolation demonstrated that 63.8% (30/47) of samples were positive and morphologically compatible with Brucella sp. All colonies suggestive of Brucella sp. were confirmed by PCR. Isolates were further characterized by PCR Multiplex AMOS-ENHANCED, which indicated that the isolates corresponded to biovar 1, 2, 4 (43.33%). This study evidences an association between cervical bursitis and Brucella spp. infection in cattle, and that different biovars of Brucella circulate in bovine herds in Maranhão.

     

Investigation of abortions in brucellosis tested herds

The mandatory reporting and investigation of bovine abortions provides a system for the surveillance of the progress of the Brucellosis Eradication Scheme and a warning of resurgence of brucellosis in tested herds. Before the scheme was introduced in 1971, about a quarter of all abortions were caused by Brucella abortus. By 1976, the percentage of abortions due to Brucella in herds under test for brucellosis had been reduced to 3.4%. Most Brucella abortions occurred during the seventh and eighth months of gestation. Cows in the 4- to 6-year-old age group were at greater risk of aborting from Brucella infection than heifers. Approximately 94% of all aborting cows were in herds which experienced three or fewer abortions. Less than 4% of herds experienced four or more abortions annually. In most cases of abortion due to brucellosis, more than one diagnostic test yielded positive results. However, 13.3% of cases were recognised by positive bacteriological results alone.     

The Effect of Brucella abortus on Hydrogen Peroxide and Nitric Oxide Production by Bovine Polymorphonuclear Cells

The effect of Brucella on the generation of microbicidal reactive oxygen and nitrogen metabolites by bovine peripheral polymorphonuclear cells (PMNs) was investigated. The PMNs were recovered from the peripheral blood of control calves and experimental calves previously vaccinated against brucellosis. Significantly larger quantities of NO and H₂O₂ were generated by PMNs from control and experimental calves following activation by heat-killed whole cells or outer membrane protein of Brucella abortus than by non-activated cells (p<0.05–0.01). In contrast, generation of H₂O₂ and NO decreased when PMNs were exposed to the lipopolysaccharide of Brucella. However, the generation of H₂O₂ and NO by activated PMNs from the control and experimental calves did not differ significantly.     

Type IV secretion and Brucella virulence

The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate.     

Brucellosis among smallholder cattle farmers in Zambia

A cross-sectional study was performed in Southern and Lusaka provinces of Zambia between March and September 2008 to estimate Brucella seroprevalence in cattle kept by smallholder dairy farmers (n = 185). Rose Bengal test (RBT) was used as a screening test followed by confirmation with competitive ELISA (c-ELISA). We investigated 1,323 cattle, of which 383 had a history of receiving vaccination against brucellosis and 36 had a history of abortion. Overall seroprevalence was 6.0% with areas where vaccination was practiced having low seroprevalence. Age was associated with Brucella seropositivity (P = 0.03) unlike cattle breed (P = 0.21) and sex (P = 0.32). At area level, there was a negative correlation (Corr. coeff = −0.74) between percentage of animals with brucellosis vaccination history (vaccination coverage) and level of brucellosis; percentage of animals with history of abortion (Corr. coeff. = −0.82) and brucellosis vaccination coverage. However, a positive correlation existed between brucellosis infection levels with percentage of animals having a history of abortion (Corr. coeff. = 0.72). History of vaccination against brucellosis was positively associated with a positive Brucella result on RBT (P = 0.004) whereby animals with history of vaccination against brucellosis were more likely to give a positive RBT test results (OR = 1.52). However, the results of c-ELISA were independent of history of Brucella vaccination (P = 0.149) but was positively associated with history of abortion (OR = 4.12). Our results indicate a relatively low Brucella seroprevalence in cattle from smallholder dairy farmers and that vaccination was effective in reducing cases of Brucella infections and Brucella-related abortions. Human exposure to Brucella through milk from smallholder farmers could result through milk traded on the informal market since that milk is not processed and there no quality and safety controls.     

Seroprevalence of brucellosis and its contribution to abortion in cattle,camel, and goat kept under pastoral management in Borana,Ethiopia

The involvement of Brucella infection in causing abortion was investigated in a breeding female subpopulation of 283 cattle, 756 camels, and 757 goats. Serum samples were serially tested using the Rose Bengal test and complement fixation test. The study showed that anti-Brucella antibodies were prevalent in 10.6% (95% confidence interval (CI), 7.4, 14.9), 2.2% (95%CI, 1.4, 3.7), and 1.9% (95%CI, 1.1, 3.2) of cattle, camel, and goats, respectively. Abortion was more commonly reported in camels (23.4%) than cattle (13.8%) and goats (12.4%). The results of this study suggested that Brucella infections contribute significantly to abortion in cattle (odds ratio (OR), = 4.7; 95%CI, 2.0, 10.8) and goats (OR = 6.9; 95%CI, 2.2, 21.7) but not in camels. The number of young animals produced by breeding females seems to be apparently reduced in seropositive groups. Keeping more than two animal species at household level was found to be the risk factor for cattle (OR = 3.1; 95%CI, 1.2, 7.9) and camel (OR = 5.3; 95%CI, 1.2–23.5) seropositivity to Brucella infection when compared to those animals from households that keep only two animal species. This may suggest a possibility of cross species transmission of Brucella infection under such mixed herding. Wet season (OR = 4.8; 95%CI, 1.3, 18.1) was found to be associated with seropositivity in goats, linked to a coincidence of increased deliveries in flocks with possible excretion of Brucella organisms. The study results suggest that Brucella infection is the likely cause of abortion in cattle and goats while other causes largely outweigh brucellosis as a cause of abortion in camels in Borana, hence, contributing to reproductive loss.     

Improving the specificity of immunodiagnosis for porcine brucellosis

This report describes the use of cell mediated immunity to improve specificity of current diagnosis for Brucella suis. Diagnosis is problematic due to cross reactions that lead to false positive serological reactions (FPSR) in the standard diagnostic tests. A common cause of this cross reactivity is infection with the organism Yersinia enterocolitica O:9. Gottingen™ mini-pigs were experimentally infected with B. suis biovar I field strain or Y. enterocolitica serotype O:9 biotype 3. Infection was followed for 70 days. During this time whole blood stimulation assays were set up using Brucella specific antigen. IFNγ was measured in the supernatants (SN) from these assays by ELISA. Concurrent standard serological tests were carried out. The results indicate that the IFNγ assay is specifically able to distinguish Y. enterocolitica O:9 infection from a B. suis infection in experimentally infected mini-pigs. These results represent an improvement in diagnostic specificity compared to currently used serological tests. Thus suggesting that in a surveillance setting this test could be applied as a confirmatory test in the face of FPSR. The authors are British Civil Servants and as such their work is subject to British Crown Copyright. This means the exclusive copyright for the article cannot be transferred.     

Risk factors associated with camel brucellosis in Jordan

During the period between February, 2004 and December, 2006, a cross-sectional study was performed to investigate some epidemiological aspects related to camel brucellosis in Jordan. Four hundred twelve camel sera from 37 herds were randomly collected and analyzed using Rose Bengal plate test and complement fixation test. A structured pre-tested questionnaire was administered to collect information on camel herd health and management. A multivariable logistic regression model was constructed to investigate risk factors associated with seropositivity to Brucella antigens. Moreover, the incidence of Brucella-specific abortion was investigated in 7 camel herds located in different locations in Southern Jordan. The true prevalence of Brucella-seropositive in camels was 12.1%. Thirteen (35.1%) herds had at least one positive camel. The seroprevalence of brucellosis in camels was significantly higher in the southern part of Jordan than that in central or northern Jordan. The multivariable logistic regression model on both individual and herd levels revealed large herds and contact with small ruminants as risk factors for Brucella seropositivity. On the other hand, using disinfectants was identified as a protective factor (OR = 0.8; 95% CI: 0.1, 0.9) only on the camel herd level. The incidence of Brucella-caused abortion was 1.9%. Brucella melitensis biotype 3 was isolated from 4 aborted camel fetuses.     

Meta-analysis of Brucella seroprevalence in dairy cattle of Ethiopia

This meta-analysis estimates a single-group summary (effect size) for seroprevalence of Brucella spp. exposure in dairy cattle of Ethiopia. It also attempts to identify study-level variables that could explain the variation in apparent seroprevalence. The literature search was restricted to studies published in English language from January 2000 to December 2013. A template was designed to retrieve the most biologically plausible and consistent variables from the articles. A total of 29 published papers containing 40 animal-level studies were used in the analyses. The single-group summary of Brucella seroprevalence in cattle was estimated to reach 3.3 % with 95 % confidence interval (CI) (2.6–4.2 %). Of all the variables considered, region was the only specific factor identified to explain about 20 % of between-study variation. Accordingly, the region-based meta-analysis forest plot revealed the highest prevalence in central Ethiopia followed by southern part. The lowest prevalence estimate was observed in the western part of the country. The visual inspection of the funnel plot demonstrated the presence of possible publication bias which might dictate shortage of studies with higher prevalences or variance inflation due to infectiousness of Brucella. In conclusion, the quantitative review showed the seroprevalence to be low but widely distributed. More importantly, the review underscores the need for isolation and characterization of the circulating Brucella spp. to capture the type of Brucella spp. involved and its distribution in cattle in Ethiopia.     

The use of skin delayed‐type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: A review

Summary

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella. Therefore, the use of the skin delayed‐type hypersensitivity (SDTH) test, which is independent of circulating antibodies, might improve the diagnosis of brucellosis. In the literature, however, there are conflicting reports as to the value of the SDTH test for the diagnosis of brucellosis. Some studies consider the test unreliable, whereas others advocate its use because it detects brucellosis earlier than serological tests. The objectives of this study were therefore to assess the characteristics of the SDTH test, to select a Brucella strain that will yield a suitable brucellin for use in the field, and to determine whether the use of serological tests in combination with the SDTH test improves the detection of brucellosis. The results of this study clearly show that the SDTH test detects latent carriers of Brucella and confirms brucellosis in cattle with ambiguous serological test results. Brucellins prepared from smooth or mucoid strains of Brucella are better suited for use in the field than brucellins prepared from rough strains because they detect brucellosis in cattle with acute as well as chronic infection. The SDTH test is highly specific (99.3% specificity), and repeated testing of naive cattle or cattle infected with microorganisms that serologically cross‐react with Brucella does not sensitize cattle to subsequent SDTH tests. However, it is possible that some naive cattle may serologically react to the injection of brucellin. The effect of these serological reactions on the sero‐diagnosis of brucellosis is limited, because cattle may only now and then react serologically either with the serum agglutination test (SAT) or the complement fixation test (CFT). Nevertheless, cattle infected with microorganisms that serologically cross‐react with Brucella may test seropositive for brucellosis 4 to 7 weeks after injection of brucellin, depending on the cross‐reacting microorganism. The value of the SDTH test for the diagnosis of brucellosis was demonstrated after an outbreak of brucellosis. When the SDTH test was used in combination with SAT and CFT at diagnostic threshold ≥2 mm or ≥1 mm (increase in skinfold thickness), respectively, 39/44 (88%) or 42/44 (95%) of the infected cattle were detected compared with only 27/44 (61%) when SAT and CFT were used. When cattle in areas of low prevalence or in areas free from brucellosis are tested with the SDTH test an increase ≥2 mm in skinfold thickness should be considered indicative of infection. When the control and eradication of brucellosis is based on test‐and‐slaughter, an increase of ≥1 mm in skinfold thickness should be considered indicative of infection. Repeated serological testing complemented with the SDTH test in this programme will shorten the quarantine (movement control) period of a suspect herd, limiting the financial loss incurred during outbreaks of the disease. Consequently, since the SDTH test usually does not interfere with the serological diagnosis and can safely be used to establish the infection status of cattle in a suspect herd, it is opportune to consider adding the SDTH test to the procedure currently used to diagnose brucellosis in individual animals.     

Molecular typing of Brucella species isolates from livestock and human

Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus–melitensis–ovis–suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.