Pro-inflammatory cytokines and reproductive hormone responses in bucks post-challenge with Mannheimia haemolytica A2 and its outer membrane protein.

The lipopolysaccharide (LPS) endotoxin and outer membrane protein (OMP) are among the virulence factors of Gram-negative bacteria responsible for inducing pathogenicity in the infected host. OMP and LPS occur on the outer membrane of M. haemolytica A2, the primary aetiological agent of pneumonic mannheimiosis in small ruminants. While the LPS is known to mediate Gram-negative bacterial infection by activating downstream inflammatory pathways, the potential role of OMP during inflammatory responses remained unclear. Hence, this study determined the effect of the OMP of M. haemolytica A2 on the serum concentration of pro-inflammatory cytokines and the male reproductive hormones (testosterone and Luteinizing Hormone). We randomly assigned twelve bucks to three groups (n = 4 bucks each): Group 1 was challenged with 2 mL PBS buffer (pH 7.0) intranasally; Group 2 received 2 mL of 1.2 X 109 CFU/mL whole M. haemolytica A2 intranasally; and Group 3 received 2 mL of OMP extract obtained from 1.2 X 109 CFU/mL M. haemolytica A2 intramuscularly. Serum samples collected at pre-determined intervals were used for the quantitative determination of the pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα) and reproductive hormones (testosterone and LH) using commercial sandwich enzyme-linked immunosorbent assay (ELISA). The serum concentration of IL1ß was initially increased within the first-hour post-challenge in Groups 2 and 3, followed by a significant decrease in concentration at 21d and 35d (p < 0.05) in Group 3. Only mild fluctuations in IL-6 occurred in group 2, as opposed to the 1.7-fold rapid increase in TNFα within 2 h post-challenge before decreasing at 6 h. An increase in pro-inflammatory cytokines was accompanied by an acute febrile response of 39.5 ± 0.38 °C (p < 0.05) at 2 h and 40.1 ± 0.29 °C (p < 0.05) at 4 h in Group 2 and Group 3, respectively. Serum testosterone decreased significantly (p < 0.05) in both treatment groups but remained significantly (p > 0.05) lower than in Group 1 throughout the study. There was a moderate negative association between testosterone and IL1ß (r = -0.473; p > 0.05) or TNFα (r = -0.527; p < 0.05) in Group 2. Serum LH also showed moderate negative associations with TNFα in Group 2 (r = -0.63; p < 0.05) and Group 3 (r = -0.54; p > 0.05). The results of this study demonstrated that M. haemolytica A2 and its OMP produced marked alterations in serum levels of pro-inflammatory cytokines and male reproductive hormones. The negative correlations between serum testosterone and inflammatory cytokines would suggest the potential role of OMP in causing male infertility by mediating innate inflammatory responses to suppress testosterone production in bucks.

Seminal and histopathological alterations in bucks challenged with Mannheimia haemolytica serotype a2 and its LPS endotoxin.

Pneumonic mannheimiosis is a widespread respiratory bacterial disease of small ruminants caused by Mannheimia haemolytica serotype A2. The disease is known to affect the respiratory organs of infected animals, but its effect on other vital and reproductive organs has not been fully explored. Previous studies have demonstrated increased serum pro-inflammatory cytokine concentration post-challenge with M. haemolytica A2 and its LPS, indicating systemic inflammation in the host. This study determined the potential tissue changes and alterations of sperm parameters due to infection of M. haemolytica A2 and its LPS endotoxin. In this study, twelve experimental bucks were randomly assigned to three groups of four bucks each: group 1 (control group) were intranasally inoculated with 2 mL of PBS pH 7.0, group 2 received 2 mL of 1.2 × 109 CFU/mL M. haemolytica A2 intranasally, and group 3 received 2 mL of LPS extracted from 1.2 × 109 CFU/mL of M. haemolytica A2 intravenously. Semen samples were collected at pre-determined intervals using an electro-ejaculator and analysed immediately after collection. All experimental bucks were slaughtered via exsanguination on day 60 to collect their vital and reproductive organs at necropsy, and the samples were processed and analysed for histopathological changes. The current study has revealed that bucks challenged with M. haemolytica A2 and its LPS exhibited alterations in semen parameters such as motility, wave pattern, viability, and morphological abnormalities. Mild to moderate histopathological changes of the lung, liver, testis, epididymis, vas deferens, prostate, and lymph nodes were also observed in both challenged groups. Therefore, this study revealed the potential harmful effects of respiratory mannheimiosis on the reproductive organs of the infected bucks and sheds light on the expanse of systemic effects of this disease.

Changes in selected cytokines, acute-phase proteins, gonadal hormones and reproductive organs of non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its LPS endotoxin.

Previous investigations have revealed that lipopolysaccharide (LPS) endotoxin from certain Gram-negative bacteria could adversely affect the reproductive system of female animals. However, it is unknown whether LPS endotoxin of Mannheimia haemolytica serotype A2, the principal causative bacteria that cause pneumonic mannheimiosis in small ruminants, may also induce similar insidious effects. Therefore, this study aimed to investigate the effects of M. haemolytica serotype A2 and its LPS endotoxin on the responses of female gonadal hormones (progesterone and oestrogen), pro-inflammatory cytokines (interleukin-1ß, interleukin-6), acute-phase proteins (haptoglobin and serum amyloid A) and cellular changes via histopathology study of female reproductive organs of the treatment does. Twelve clinically healthy, non-pregnant, crossbred does were randomly allocated into three equal groups. Group 1 was administered intranasally with 2 ml of sterile phosphate-buffered saline (PBS) and served as a negative control group. Group 2 was challenged intranasally with 2 ml of bacterial inoculum containing 109 colony-forming units (CFU)/ml of M. haemolytica serotype A2, while Group 3 was challenged intravenously with 2 ml of LPS endotoxin extracted from 109 CFU/ml of M. haemolytica serotype A2. Following that, blood samples were collected serially at pre-determined intervals for serological analyses. All does were euthanised 60 days post-challenges, and tissue samples from the ovaries, oviducts, uterine horns, uterine body, cervix and vagina were collected for histopathological study. The serological result revealed a significant increase (p < 0.05) in the mean concentrations of progesterone, oestrogen, interleukin-1ß, interleukin-6, haptoglobin and serum amyloid A for both challenged groups. Histopathologically, all reproductive organs (except the cervix and vagina) from both challenged groups displayed significant cellular alterations (p < 0.05) characterised by haemorrhage and congestion, necrosis and degeneration, inflammatory cell infiltration and oedema. This study provides new information that elucidates the potential role of pneumonic mannheimiosis in the pathogenesis of female infertility amongst small ruminants.

Responses of testosterone hormone and important inflammatory cytokines in bucks after challenge with Mannheimia haemolytica A2 and its LPS endotoxin.

Previous studies have shown that Mannheimia haemolytica A2 is the principal microorganism causing pneumonic mannheimiosis, a major bacterial respiratory disease among sheep and goats. The effect of this bacteria on the respiratory system is well-established. However, its effect on the reproductive physiology remains unclear. Therefore, this study aimed to determine the alterations in the level of pro-inflammatory cytokines and testosterone hormone post-inoculation with M. haemolytica serotype A2 and its lipopolysaccharide (LPS) endotoxin which were hypothesized to affect the reproductive functions of bucks. Twelve clinically healthy adult male goats were divided equally into three groups. Goats in group 1 were treated with 2 ml of sterile phosphate-buffered saline (PBS) pH 7.0 intranasally (negative control), group 2 with 2 ml of 109 colony-forming unit (CFU) of M. haemolytica serotype A2 intranasally (positive control), and group 3 were treated with 2 ml of lipopolysaccharide extracted from 109 CFU of M. haemolytica serotype A2 intravenously. Following inoculation, blood samples were collected via jugular venipuncture into plain tubes at pre-determined intervals for serum collection to determine the concentration of interleukin (IL)-1ß, IL6, tumor necrosis factor (TNF)-α, and testosterone hormone by using commercial ELISA test kits. Results from this study demonstrated that the inoculation of M. haemolytica A2 and its LPS increases the concentration of pro-inflammatory cytokines but decreases the concentration of testosterone hormone in challenged animals at most time points throughout the 56 days experimental period (p < 0.05). This study suggests that the M. haemolytica A2 and its LPS could alter the concentration of pro-inflammatory cytokines and testosterone hormone, which in turn, may negatively affect the reproductive functions of bucks.

Histopathological changes of reproductive organs of goats immunized with Corynebacterium pseudotuberculosis killed vaccine.

Caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis is characterized by the development of abscesses, mainly in superficial and internal lymph nodes, visceral and reproductive organs in small ruminants. This study aims to examine the histopathological changes in reproductive organs of goats immunized with killed vaccine of C. pseudotuberculosis. In this study, twenty four (24) clinically healthy bucks and does were divided into four groups A, B, C and D. Animals in groups A and B were immunized with 0.5 and 1% formalin killed vaccine, respectively; followed by a booster dose. After the booster dose of immunization, groups A, B and C were challenged with C. pseudotuberculosis at 106 cfu/ml. Goats in group D were immunize and unchallenged and left as control group. All C. pseudotuberculosis infected animals were euthanized humanely 12 weeks post-challenged. Tissue samples such as testes, epididymis, spermatic cord, penis, pituitary gland, mammary gland, vulva, vagina, cervix, uterus, fallopian tube and ovaries were collected for histopathology study. Microscopic examination of all tissues (testes, seminiferous tubules, spermatic cord, penile tissues and the pituitary gland) in the male reproductive organs of the bucks that were inoculated with 2 ml of 0.5% and 1.0% of C. pseudotuberculosis killed vaccine showed normal (animals inoculated with 1.0%) to mild (animals inoculated with 0.5%) histopathological changes when compared with those from group C which showed varying degrees of histopathological changes (p < 0.01) in their various tissues. For the female does, similar histopathological changes were observed for the various tissues examined (ovaries, fallopian tubes, uterine horns, uterine tissues, cervix, vaginal, vulva, mammary glands and the pituitary glands) in which the vaccinated groups A &B showed a significantly (p < 0.001) less histopathological changes when compared with those in group C that showed varying degrees of histopathological changes in the reproductive organs investigated. This study showed the efficacy of C. pseudotuberculosis killed vaccine protecting against reproductive tissue damages cause by the active infection with the live bacteria in both bucks and does in the study area.

A case of aspergillosis outbreak in a broiler duck farm in Kelantan, Malaysia.

OBJECTIVE: This case report aims to discuss the veterinary approach taken to control a case of aspergillosis outbreak on a duck farm. MATERIALS AND METHODS: A broiler duck farm with a population of 900 Muscovy ducks was having a complaint of a 5% mortality rate in their 3-week-old ducklings. Upon presentation, 10% of the ducks appeared to be listless, dyspneic, ruffled feathers, and cyanotic. Postmortem examination of the dead birds was conducted. The collected samples were subjected to isolation and identification of the associated Aspergillus fumigatus under the microscope using the scotch tape method. RESULTS: Postmortem examination revealed whitish to creamy caseous nodules in the lungs, thoracic air sacs, gizzard, proventriculus, and intestines. Granuloma lesions and infiltration of inflammatory cells were observed in the lung and liver tissues. As for therapeutic management, all ducks were treated with copper sulfate, erythromycin, and multivitamins as the fungicide, antibiotic, and supplement, respectively, via drinking water. CONCLUSION: There is no effective treatment for Aspergillosis as the spores are difficult to destroy completely. Nonetheless, the disease can be controlled and prevented effectively with proper farm sanitation and providing a suitable feed storage environment to inhibit the growth of this opportunistic fungus.

Responses of pro-inflammatory cytokines, acute phase proteins and cytological analysis in serum and cerebrospinal fluid during haemorrhagic septicaemia infection in buffaloes.

Sudden death is usually the main finding in field animals during haemorrhagic septicaemia outbreaks caused by Pasteurella multocida type B:2 that causes acute, fatal and septicaemic disease in cattle and buffaloes. This situation may be due to failure in early detection of the disease where early treatment of antibiotics may improve the prognosis of the animal and other surviving animals. Thus, there is a grey area on the knowledge on the potential usage of pro-inflammatory cytokines and acute phase proteins as early biomarkers in the diagnosis of haemorrhagic septicaemia. In addition, exploration of the cerebrospinal fluid during infection has never been studied before. Therefore, this study was designed to fill up the grey areas in haemorrhagic septicaemia research. Twenty-one buffalo calves were divided into seven treatment groups where group 1 was inoculated orally with 10 mL of sterile phosphate-buffered saline pH 7 which act as a negative control group. Groups 2 and 3 were inoculated orally and subcutaneously with 10 mL of 1012 colony-forming unit of P. multocida type B:2. Group 4 and 5 buffaloes were inoculated orally and intravenously with 10 mL of lipopolysaccharide broth. Groups 6 and 7 were administered orally and subcutaneously with 10 mL of outer membrane protein broth. During the post-infection period of 21 days, blood and cerebrospinal fluid were sampled for the analyses of pro-inflammatory cytokines, acute phase proteins and cytological examination. Buffalo calves infected with P. multocida and its immunogens via different routes of inoculation showed significant changes (p < 0.05) of pro-inflammatory cytokines, acute phase proteins and cytological changes in both the serum and cerebrospinal fluid. Buffalo calves from groups 3 and 7 showed the highest pro-inflammatory cytokines, whereas group 6 had the highest acute phase protein concentration and group 5 revealed the highest value for cytology changes. In summary, results obtained in this study could be used as a profiling study to add novel knowledge to the haemorrhagic septicaemia research as well as the development of biomarkers.

Reproductive hormonal variations and adenohypophyseal lesions in pre-pubertal buffalo heifers inoculated with Pasteurella multocida type B: 2 and its immunogens.

BACKGROUND: Hemorrhagic septicemia is a fatal disease of cattle and buffaloes caused by P. multocida. Although the pathogenesis of the bacteria has been well established in literature, there is a paucity of information on the possible role of the bacteria and its immunogens; lipopolysaccharide (LPS) and outer membrane proteins (OMPs) on the reproductive capacity of buffalo heifers. METHODS: In this study, twenty one healthy prepubertal female buffaloes aged 8 months were divided into seven groups of 3 buffaloes each (G1-G7). Group 1 (G1) served as the negative control group and were inoculated orally with 10 mL sterile Phosphate Buffer Saline (PBS), groups 2 (G2) and 3 (G3) were inoculated orally and subcutaneously with 10 mL of 1012 colony forming unit (cfu) of P.multocida type B: 2, while groups 4 (G4) and 5 (G5) received 10 mL of bacterial LPS orally and intravenously, respectively. Lastly, groups 6 (G6) and 7 (G7) were orally and subcutaneously inoculated with 10 mL of bacterial OMPs. Whole blood was collected in EDTA vials at stipulated time points (0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 120, 168, 216, 264, 312, 360, 408, 456 and 504 h), while tissue sections of the pituitary glands were collected and transported to the histopathology laboratory in 10% buffered formalin for processing and Hematoxylin and eosin staining. Plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (PG), estradiol (EST) and gonadotrophin releasing hormone (GnRH) were determined. RESULTS: The histopathological lesions observed in the pituitary gland included hemorrhage, congestion, inflammatory cell infiltration, hydropic degeneration, necrosis and edema. These changes were higher (p < 0.05) in distribution and severity in G3, G6 and G7. Hormonal concentrations of LH, FSH, PG, EST and GnRH declined in all inoculation groups as time elapsed and were lower (p < 0.05) than that of the control group. CONCLUSION: Based on these findings, P.multocida B: 2 and its immunogens can be said to negatively affect the hypothalamic-pituitary-gonadal axis, resulting in decreased levels of reproductive hormones which may predispose to infertility in buffalo heifers.

Clinico-pathology and hemato-biochemistry responses in buffaloes infected with Pasteurella multocida type B:2 immunogen outer membrane protein.

The aim of this study was to investigate the clinico-pathology and haemato-biochemistry alterations in buffaloes inoculated with Pasteurella multocida type B:2 immunogen outer membrane protein via subcutaneous and oral routes. Nine buffalo heifers were divided equally into 3 treatment groups. Group 1 was inoculated orally with 10 mL of phosphate buffer saline (PBS); Group 2 and 3 were inoculated with 10 mL of outer membrane protein broth subcutaneously and orally respectively. Group 2 buffaloes showed typical haemorrhagic septicaemia clinical signs and were only able to survive for 72 h of the experiment. However, Group 3 buffaloes were able to survive throughout the stipulated time of 21 days of experiment. There were significant differences (p < 0.05) in the rectal temperature between the experimental and control group. In the hematology and biochemistry findings, there were significant differences (p < 0.05) in packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin concentration, leukocytes, band neutrophils, segmented neutrophils, lymphocytes, eosinophils, basophils, gamma glutamyl transferase, total protein, and globulin between Group 2 and control group. In contrast, Group 3 and control group revealed significant differences (p < 0.05) in erythrocytes, haemoglobin, mean corpuscular haemoglobin concentration, segmented neutrophils, lymphocytes, monocytes, eosinophils, basophils, thrombocytes, gamma glutamyl transferase, total protein, globulin, and albumin:globulin ratio. In Group 2 buffaloes, there were gross lesions observed in the lung, trachea, heart, liver, spleen, kidney and submandibulae lymph nodes. In contrast, lesions were only observed in the lung, and liver of Group 3 buffaloes. There were significant differences (p < 0.05) in hemorrhage and congestion; necrosis and degeneration; and inflammatory cells infiltration between experimental groups and control group. However, there were no significant differences (p > 0.05) in edema between groups except for the lung. This study was a proof that oral route infection of Pasteurella multocida type B:2 immunogen outer membrane protein can be used to stimulate host cell.