Segmentation of the Pathophysiological Stages of Diabetic Changes in the db/db Mouse

The db/db mouse is one of the diabetes mellitus animal models and if the pathophysiological stages of diabetic changes in the mouse model could simulate the stages in human diabetes, the db/db mouse could be used to better evaluate drug candidates. Blood insulin, HbA1c levels and morphological features of pancreatic islets in db/db mice were evaluated to determine the pathophysiological stage. At 6 weeks of age, db/db mice showed the highest level of plasma insulin and lowest level of HbA1c, and histopathological examination revealed enlarged islets with a circular shape and hypertrophic islet cells. By 9 and 12 weeks of age, the plasma insulin levels had decreased to mid levels and HbA1c had increased to mid to high levels; histopathological examination at this time revealed two types of islets coexisting, enlarged circular islets and small irregular-shaped islets. By 15 and 22 weeks of age, plasma insulin had decreased further to low levels and HbA1c was at its highest level; the histopathological examination at this time revealed an increase in irregular-shaped and small islets. Based on blood insulin levels, HbA1c levels and histopathology findings in the db/db mice in this study, the clinical staging of diabetic changes were recognized. The pathophysiological stages of diabetes mellitus in this animal model were similar to the stages in humans.     

Histopathological and aquaporin7 mRNA expression analyzes in the skeletal and cardiac muscles of obese db/db mice

The aim of this study is to examine 1) muscle fiber type composition, 2) myofiber diameter, and 3) aquaporin (AQP) 7 and AQP 9 mRNA expressions by quantitative PCR in muscles of obese db/db mice. The myofiber type composition of skeletal muscle was not statistically significantly different between db/db mice and control mice; while the average myofiber diameter ratio showed a decrease in db/db mice. The expression of AQP7 but not AQP9 mRNA in the skeletal and cardiac muscles was significantly upregulated in db/db mice. Thus this study revealed quantitatively that type 2 myofiber atrophy was shown in the skeletal muscles of db/db mice. AQP7 mRNA expression was upregulated in the skeletal and cardiac muscles of db/db mice.     

The ability to enter into an avirulent viable but non-culturable (VBNC) form is widespread among Listeria monocytogenes isolates from salmon, patients and environment

Media-based bacteriological testing will fail to detect non-culturable organisms and the risk of consuming viable but non-culturable (VBNC) Listeria monocytogenes is unknown. We have here studied whether L. monocytogenes obtained from seafoods, processing environment and clinical cases enter the VBNC state and assessed the virulence of the non-culturable forms of the bacteria. A number of 16 L. monocytogenes strains were starved in microcosm water at 4 °C until loss of culturability. Metabolic activity in the VBNC form was measured as ATP generation using a luciferase assay and membrane integrity was examined using the LIVE/DEAD BacLight assay. All tested L. monocytogenes strains entered the VBNC state after starvation in microcosm water. Ongoing mRNA synthesis of hly in VBNC L. monocytogenes cells re-incubated in culture medium indicated a potential virulence of these forms. Sodium pyruvate and replenishment of nutrient were used in attempts to resuscitate VBNC cells. However, VBNC L. monocytogenes were not resuscitated under these conditions. VBNC L. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient RAG1^−/− mice. Inoculation of VBNC L. monocytogenes in immunodeficient mice did not cause morbidity, and plaque assay on HT-29 cells in culture indicated that the VBNC cells were avirulent. The results indicate that the risk of non-culturable L. monocytogenes in foods, when the VBNC state is induced by starvation, is negligible.     

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.     

Immunization of BALB/c mice with Brucella abortus 2308ΔwbkA confers protection against wild-type infection

Brucellosis is a zoonotic disease that causes animal and human diseases. Vaccination is a major measure for prevention of brucellosis, but it is currently not possible to distinguish vaccinated animals from those that have been naturally infected. Therefore, in this study, we constructed the Brucella (B.) abortus 2380 wbkA mutant (2308ΔwbkA) and evaluated its virulence. The survival of 2308ΔwbkA was attenuated in murine macrophage (RAW 264.7) and BALB/c mice, and it induced high protective immunity in mice. The wbkA mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon. Antibodies to 2308ΔwbkA could be detected in sera from mice, implying the potential for use of this protein as a diagnostic antigen. The WbkA antigen would allow serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔwbkA is a potential attenuated vaccine against 16M. This vaccine will be further evaluated in sheep.     

Macrophages, myofibroblasts and mast cells in a rat liver infected with Capillaria hepatica

We trapped a rat (Rattus norvegicus) infected with Capillaria hepatica. At necropsy, grossly yellowish-white nodules (2-3 mm in diameter) were noted to be scattered on the liver''s surface. Microscopically, granulomatous and fibrotic nodules that contained the eggs and/or adult worms of Capillaria hepatica were detected in the liver. Septal fibrosis was diffusely formed throughout the liver. There were a number of ED1-positive macrophages located in the sinusoids of the pseudolobules. On the double staining, myofibroblasts and mast cells were generally observed within the fibrous septa with the mast cells in close proximity to the myofibroblasts. We suggest that the interactions between macrophages, myofibroblasts and mast cells play a role in the septal fibrosis observed in rats infected by Capillaria hepatica.     

Experimental Final Hosts of Metagonimus Hakubaensis (Trematoda: Heterophyidae) and Their Suitability to the Fluke

Seven laboratory mammal and bird species were orally inoculated with 200–1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.     

Comparative clinico-haematological analysis in young Zebu cattle experimentally infected with Trypanosoma vivax isolates from tsetse infested and non-tsetse infested areas of Northwest Ethiopia

Background

Ethiopia, particularly in the Northwest region, is affected by both tsetse and non-tsetse fly transmitted trypanosomosis, with significant impact on livestock productivity. The aim of this study was to determine and compare clinical findings and haematological values between experimental infections induced by Trypanosoma vivax isolates from areas of either transmission mode. Sixteen young (aged between 6 and 12 months) Zebu cattle (Bos indicus), purchased from a trypanosome-free area and confirmed to be trypanosome-negative, were randomly assigned into four groups of four animals. Groups 1, 2 and 3 were infected with an isolate from a tsetse infested or one of two isolates from a non-tsetse infested area, and group 4 was a non-infected control. All animals in the infected groups were inoculated intravenously with 2 × 10⁶ trypanosomes from donor animals. The experimental animals were monitored for eight consecutive weeks post infection for clinical signs, parasitaemia and haematological changes in packed cell volume (PCV), haemoglobin concentration (Hgb), total red blood cell (RBC) and white blood cell (WBC) counts, differential WBC count and blood indices (mean corpuscular volume [MCV], mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration).

Results

Infection was characterized by reduced feed intake, weakness, pyrexia, parasitaemia, rough hair coat, enlarged prescapular lymph nodes, lacrimation, weight loss, pallor mucus membrane and dehydration. Body weight loss in all infected groups was significantly higher than in the non-infected control. Similarly, body weight loss was higher (P < 0.001) in animals infected with the tsetse infested isolate than with the non-tsetse infested isolates. The mean PCV, Hgb, total RBC and WBC counts were lower (P < 0.001), and mean MCV was higher (P = 0.01) in all infected groups than in non-infected control animals at different time points during the study period. Except for minor variations in haematological values, the overall changes were similar in all infected groups.

Conclusion

Clinical signs and significant reduction in haematological values in the infected groups indicated the pathogenicity of the T. vivax parasites. Pathogenicity of T. vivax from the non-tsetse infested area can be considered as nearly as important as that of its counterpart derived from the tsetse infested area.     

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.     

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88^−/−) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88^−/− mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88^−/− mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-γ responses by NKT, CD4⁺ and CD8⁺ T lymphocytes, and production of high levels of IL-10. MyD88^−/− mice replenished with IL-12 and IFN-γ abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-γ-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model

Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 10⁶ tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.     

Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi

Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.     

Fatal cases of Theileria annulata infection in calves in Portugal associated with neoplastic-like lymphoid cell proliferation

This study was carried out to investigate fifteen cases of acute lethal infection of calves (≤ 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79αcy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.     

Efficacy of levamisole alone and in combination with mebendazole against Gongylonema pulchrum infection in rabbits

Gongylonema pulchrum is an important parasite of captive primates. Twelve rabbits were infected with 30 third-stage larvae of G. pulchrum. At 4–7 months post-infection, animals were administered levamisole at a single dose of 12 mg/kg, levamisole at 8 mg/kg three times at 2-day intervals, levamisole at a single dose of 8 mg/kg after administration of mebendazole at 70 mg/kg for 3 days or 8 ml of distilled water for 3 days (control). Necropsy at 14 days after treatment revealed that single and multiple dosages of levamisole reduced nematode burdens by 68.4% and 89.5%, respectively. The combined regimen of mebendazole and levamisole exhibited high efficacy for treating G. pulchrum located widely within the upper digestive tract, with a reduction of 98.2%. These results suggest that this combined chemotherapy treatment may be effective against G. pulchrum infection, including buccal and lingual gongylonemiasis in primates.     

Insulin-like Growth Factor 1 mRNA Expression in the Uterus of Streptozotocin-treated Diabetic Mice

Reproductive functions decline with the onset of diabetes in female mice. Diabetic mice have smaller uteri with an underdeveloped endometrium, suggesting diminished estrogen-induced growth. We aimed to clarify the changes in the estrous cycle and in insulin-like growth factor 1 (IGF1) expression in the uteri of streptozotocin (STZ)-treated diabetic mice, because IGF1 is one of the main growth factors involved in estrogen-induced uterine growth. ICR female mice were intraperitoneally administered STZ (10 mg/100 g BW), and blood glucose levels were determined. Mice with blood glucose levels > 200 mg/dl were classified as diabetic mice. The onset of diabetes was associated with acyclic estrous cycles. Diabetes was also induced with STZ in ovariectomized mice. Uterine Igf1 mRNA levels were reduced in ovariectomized STZ-treated diabetic mice. Estrogen is known to stimulate Igf1 mRNA expression in the uterus, but estrogen action was abolished in the uteri of STZ-treated diabetic mice. mRNA expressions of estrogen receptor α (ERα) and steroid hormone receptor coactivators (SRC-1/Ncoa1, SRC-2/Ncoa2, SRC-3/Ncoa3 and CBP/p300/Crebbp) were reduced in the uteri of ovariectomized STZ-treated diabetic mice. The present study demonstrates that diabetes induces a decline in female reproductive functions in mice. Igf1 expression in ovariectomized diabetic female mice was decreased, and decreased responsiveness to estrogen in the uteri of diabetic mice is probably associated with a reduction in ERα and steroid receptor coactivator mRNA expression.     

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.     

Porphyromonas gulae 41-kDa fimbriae induced osteoclast differentiation and cytokine production

Porphyromonas gulae is considered to be associated with canine periodontitis. We have previously reported that the P. gulae American Type Culture Collection (ATCC) 51700 comprised 41-kDa fimbriae. The purpose of the present study was to demonstrate the roles of 41-kDa fimbrial protein in periodontal disease. In this study, we examined the involvement of the 41-kDa fimbrial protein in osteoclast differentiation and cytokine production in murine macrophages. Furthermore, alveolar bone resorption induced by P. gulae infection in rats was evaluated. To estimate osteoclast differentiation, bone marrow cells and MC3T3-G2/PA6 cells were cultured with or without the 41-kDa fimbrial protein for 7 days. BALB/c mouse peritoneal macrophages were stimulated with the 41-kDa fimbrial protein, and the levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α production were determined by enzyme-linked immunosorbent assay. Osteoclast differentiation was significantly enhanced by treatment with the 41-kDa fimbrial protein in a dose-dependent manner. The total area of pits formed on the dentine slices with osteoclasts incubated with the 41-kDa fimbrial protein was significantly greater than that of the control. The purified 41-kDa fimbrial protein induced IL-1β and TNF-α production in BALB/c mouse peritoneal macrophages after 6 hr of incubation in a dose-dependent manner. The bone loss level in rats infected with P. gulae was significantly higher than that of the sham-infected rats. These results suggest that P. gulae 41-kDa fimbriae play important roles in the pathogenesis of periodontal disease.     

Deletion of the oligopeptide transporter Lmo2193 decreases the virulence of Listeria monocytogenes

BackgroundListeria monocytogenes is a gram-positive bacterium that causes listeriosis mainly in immunocompromised hosts. It can also cause foodborne outbreaks and has the ability to adapt to various environments. Peptide uptake in gram-positive bacteria is enabled by oligopeptide permeases (Opp) in a process that depends on ATP hydrolysis by OppD and F. Previously a putative protein Lmo2193 was predicted to be OppD, but little is known about the role of OppD in major processes of L. monocytogenes, such as growth, virulence, and biofilm formation.ObjectivesTo determine whether the virulence traits of L. monocytogenes are related to OppD.MethodsIn this study, lmo2193 gene deletion and complementation strains of L. monocytogenes were generated and compared with a wild-type strain for the following: adhesiveness, invasion ability, intracellular survival, proliferation, 50% lethal dose (LD₅₀) to mice, and the amount bacteria in the mouse liver, spleen, and brain.ResultsThe results showed that virulence of the deletion strain was 1.34 and 0.5 orders of magnitude higher than that of the wild-type and complementation strains, respectively. The function of Lmo2193 was predicted and verified as OppD from the ATPase superfamily. Deletion of lmo2193 affected the normal growth of L. monocytogenes, reduced its virulence in cells and mice, and affected its ability to form biofilms.ConclusionsDeletion of the oligopeptide transporter Lmo2193 decreases the virulence of L. monocytogenes. These effects may be related to OppD''s function, which provides a new perspective on the regulation of oligopeptide transporters in L. monocytogenes.