Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea

Bovine tuberculosis (TB) is a major zoonosis that''s caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.     

Anaplasma sp. and hemoplasma infection in leopard cats (Prionailurus bengalensis euptilurus) from Korea

This study examined the occurrence of Anaplasma spp. and hemoplasma infection in leopard cats, Prionailurus bengalensis euptilurus, in Korea. Twenty-nine biological samples were tested by molecular analysis. Two (6.9%) and eight (27.6%) tested specimens were positive for Anaplasma bovis and hemoplasma infection, respectively. Based on our results, Anaplasma/Ehrlichia spp. and hemoplasma are regularly infecting leopard cat populations of Korea. Considering their endangered status, regular monitoring of infection by arthropod-borne pathogens known to cause clinical symptoms in feline hosts such as Anaplasma/Ehrlichia spp. and hemoplasma would be crucial as part of ongoing conservation efforts.     

Antimicrobial susceptibility and molecular characterization of macrolide resistance of Mycoplasma bovis isolates from multiple provinces in China

Mycoplasma bovis has spread widely throughout the world via animal movement and has become an important pathogen of bovine respiratory disease. However, the minimum inhibitory concentrations of antimicrobials for Mycoplasma bovis have not been studied in China. The objective of this study was to determine the prevalence and antibiotic resistance of Mycoplasma bovis isolated from young cattle with respiratory infection in China. Mycoplasma bovis was detected in 32/45 bovine respiratory infection outbreaks at beef farms in 8 provinces in China. The isolates were susceptible or had medium sensitivity to ciprofloxacin, enrofloxacin and doxycycline, but were frequently resistant to macrolides (13/32, 41%). An A2058G (Escherichia coli Numbering) mutation located in the rrnA operon in domain V of 23S rRNA was observed in strains that were resistant to macrolides. This single mutations at the rrnA operon in domain V of 23S rRNA may play an important role in the resistance of Mycoplasma bovis strains to macrolides.     

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn''t show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heat-killed oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.     

Prevalence of Anaplasma,Bartonella and Borrelia Species in Haemaphysalis longicornis collected from goats in North Korea

North Korea is located on the northern part of the Korean Peninsula in East Asia. While tick-borne pathogens of medical and veterinary importance have been reported from China and South Korea, they have not been reported from North Korea. To screen for zoonotic tick-borne pathogens in North Korea, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis (H.) longicornis were collected and assayed individually for selected tick-borne pathogens. A total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella (B.) grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%) based on 16S ribosomal RNA and ITS species-specific nested polymerase chain reaction. Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae, respectively. All products were sequenced and demonstrated 100% identity and homology with previously reported sequences from other countries in GenBank. This is the first report of the detection of tick-borne pathogens in the North Korea and suggests that farm animals may act as reservoirs for zoonotic tick-borne pathogens.     

Molecular findings and approaches spotlighting Mycobacterium bovis persistence in cattle

Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (M. bovis) are the etiological agents of human and bovine tuberculosis (TB, bTB) respectively, and share genetic identity over 99% at the whole genome level. Progress has been made towards explaining how mycobacteria and their infected hosts remain in balance without producing clinical symptoms of disease, a phenomenon referred to as latency or persistence, which can be mimicked by certain in vitro conditions. Latency/persistence has mainly been studied using Mtb, where the two-component signalling system, dosRS, has been assigned an instrumental role, and even constitutes the current basis for development of new diagnostic methods and treatment addressing this particular stage of TB. M. bovis conserves homolog genes that in Mtb play a role in human latent TB infection and that, by analogy, would allow it to enter a persistent state in infected cattle; nevertheless, little attention has been paid to this stage in bovine hosts. We suggest that many of the advances acquired through the study of Mtb can and should be taken into consideration by research groups and veterinary professionals dealing with bTB. The study of the infection in bovines, paying particular attention to defining the molecular and cellular markers of a M. bovis persistent infection in cattle, presents great opportunities for the development and trial of new diagnostic tests and vaccines, tools that will surely help in promoting eradication of bTB in high-burden settings.     

Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay

The interferon-gamma (IFN-γ) assay is employed as a complementary diagnostic test for bovine tuberculosis (BTB) in many countries. To simplify this assay, we established a 96-well plate format using the ESAT-6 and CFP-10 antigens and then employed it to determine the extent of Mycobacterium (M.) bovis infection in dairy herds with a history of BTB outbreaks in a country where only selective culling is practiced. The sensitivity and specificity of this IFN-γ assay were 85.9% and 100%, respectively, based on comparison with the conventional single intradermal tuberculin test (SIDT). The IFN-γ assay was also positive in 30.4% and 36.8% of SIDT-negative animals from herds with recent and remote BTB outbreaks, respectively. Of 14 SIDT-negative, IFN-γ positive cattle, five (35.7%) were culture positive and an additional six were positive based on a polymerase chain reaction-based test for M. bovis. Therefore, the IFN-γ assay has the potential to serve as a specific and sensitive test for M. bovis infection in dairy cattle. Further, the results indicated that a substantial portion of SIDT-negative animals in herds with previous BTB outbreaks were actually infected with M. bovis. Accordingly, the present selective-culling strategy may require modifications to include this more sensitive assay.     

Epidemiological characteristics of pulmonary pneumocystosis and concurrent infections in pigs in Jeju Island, Korea

Epidemiological characteristics of swine pulmonary Pneumocystis (P.) carinii and concurrent infections were surveyed on Jeju Island, Korea, within a designated period in 172 pigs submitted from 54 farms to the Department of Veterinary Medicine, Jeju National University. The submitted cases were evaluated by histopathology, immunohistochemistry, PCR/RT-PCR, and bacteriology. P. carinii infection was confirmed in 39 (22.7%) of the 172 pigs. Histopathologically, the lungs had moderate to severe lymphohistioctyic interstitial pneumonia with variable numbers of fungal organisms within lesions. Furthermore, porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2) co-infection was a common phenomenon (12.8%, 20.5%, and 48.7% were positive for PRRS, PCV-2, or both, respectively, as determined by PCR/RT-PCR). Infection was much more concentrated during winter (December to March) and 53.8% of the infected pigs were 7- to 8-weeks old. In addition, three pigs showed co-infection with bacteria such as Pasteurella multocida and Streptococcus suis. The results of the present study suggest that the secondary P. carinii infection is common following primary viral infection in swine in Korea. They further suggest that co-infection of P. carinii might be enhanced by the virulence of primary pathogens or might have synergistic effects in the pigs with chronic wasting diseases.     

A survey of Prototheca bovis infection in dairy farms of the Hubei province,China

Bovine mastitis, caused by Prototheca bovis, has received much attention worldwide. To investigate the status of P. bovis infection in dairy farms of Hubei, we collected 1,158 milk samples and 90 environmental samples from 14 dairy farms of Hubei, China. The isolates were identified with traditional biological methods and molecular biological techniques, and their pathogenicity was tested through mice infection experiments. Isolates from 57 milk and 20 environmental samples were identified as P. bovis. The mice infection tests proved that the isolated P. bovis could cause mastitis in mice, manifesting as severe red swelling of the mammary glands. Histopathological analysis of tissue sections showed necrosis and nodules lesions formed in the infected mice mammary tissue, accompanied by macrophage and neutrophil infiltration. These results suggested the existence of pathogenic P. bovis in dairy farms of the Hubei province, China, with brewer’s grains and fresh feces possibly playing important roles in the spread of this disease.     

Exendin-4 improves resistance to Listeria monocytogenes infection in diabetic db/db mice

The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b⁺ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) from cattle in Korea between 2010 and 2011

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.     

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10⁷ C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.     

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle from farms in China

Fecal samples of 2,056 dairy cattle from 14 farms were collected in three geographical regions of China and stained using a modified acid-fast staining technique to identify Cryptosporidium oocysts. A total of 387 (18.82%) positive samples were identified and further analyzed by polymerase chain reaction (PCR) using primers designed to amplify DNA fragments from the small subunit ribosomal RNA. The PCR products were sequenced and the sequences were deposited in the GenBank database under accession numbers EU369377-84 and GU070730-33. Phylogenetic analysis was performed and a distances matrix generated from these sequences confirmed the existence of Cryptosporidium (C.) parvum ''mouse'' genotype, C. bovis, C. andersoni, C. hominis, and C. serpentis in cattle. These results represent the first report on the prevalence and genetic identification of Cryptosporidium species, and may contribute to a better understanding of the epidemiology of Cryptosporidium in cattle in China.     

Abundance of biting midge species (Diptera: Ceratopogonidae,Culicoides spp.) on cattle farms in Korea

Culicoides biting midges were collected on three cattle farms weekly using light traps overnight from May to October between 2010 and 2011 in the southern part of Korea. The seasonal and geographical abundance of Culicodes spp. were measured. A total of 16,538 biting midges were collected from 2010 to 2011, including seven species of Culicoides, four of which represented 98.42% of the collected specimens. These four species were Culicodes (C.) punctatus (n = 14,413), C. arakawae (n = 1,120), C. oxystoma (n = 427), and C. maculatus (n = 318). C. punctatus was the predominant species (87.15%).     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.     

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n = 215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).