Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.     

Effects of branched-chain amino acids on immune status of young racing horses

High-intensity exercise and competition are associated with depressed immune function. Young horses, which participate in high-intensity exercise and competitions, are at increased risk for the development of infectious disease due to depression of immune function. The effects of branched-chain amino acid (BCAA) supplementation on the immune status of young racing horses were evaluated, determining whether BCAA might help to avoid or reduce immune suppression during exercise and competitions. Twenty horses (10 male and 10 female) were treated with BCAA supplementation; another twenty untreated horses (10 male and 10 female) constituted control group. Peripheral blood was collected from each animal and evaluated for lymphocyte subsets, phagocytosis analysis of monocytes and granulocytes, lymphocyte proliferative response, and expression of cytokine-encoding messenger ribonucleic acids (mRNAs). The numbers of CD4⁺, CD8⁺, and major histocompatibility complex (MHC) class II⁺ cells in females of the treated group were significantly higher than those in females of the control group. The lymphocyte proliferative response in female of the treated group also was significantly higher than that in females of the control group. In addition, expression of mRNAs encoding interleukin-1β (IL-1β) and interferon-γ (IFN-γ) in females of the treated group was significantly higher than that in females of the control group. There were no significant differences between males of the treated and control groups. The results of this study indicated the positive effects of BCAA supplementation in counteracting immunosuppression in young female racing horses during and following high-intensity exercise.     

Studies on the immune status of calves with chronic inflammation and thymus atrophy

The thymus is a primary lymphoid organ where the primary T cell repertoire is generated. Thymus atrophy is induced by various conditions, including infectious diseases, glucocorticoid treatment, and poor breeding management. Cattle with thymus atrophy tend to exhibit weak calf syndrome, a condition in which approximately half of neonates die shortly after birth. Calves with thymus atrophy that survive the first month typically contract chronic inflammatory diseases. In this study, we analyzed the populations of the peripheral blood mononuclear cells and thymocytes in calves with thymus atrophy. In addition, we evaluated polarization of master gene and cytokine mRNA expression in peripheral blood CD4⁺ cells in the calves. The population of CD4⁺CD8⁺ cells in thymus of the calves with thymus atrophy was lower than that of control calves. IL10 mRNA expression in peripheral blood CD4⁺ cells of calves with thymus atrophy was significantly lower than that of control calves. TBX21 mRNA expression in peripheral CD4⁺ cells of thymus atrophy calves was tended to be higher than that of the control group. In addition, FOXP3 mRNA expression in peripheral CD4⁺ cells of the thymus atrophy calves was tended to be lower than that of the control calves. Thymus atrophy calves exhibited chronic inflammatory disease leading, in severe situations, to conditions such as pneumonia with caseous necrosis. These severe inflammatory responses likely are due to decreases in IL10 mRNA expression, impairing control of macrophages, one of the main cell fractions of natural immunity.     

Genetic and antigenic analysis of Chlamydia pecorum strains isolated from calves with diarrhea

Chlamydia pecorum (designated 22–58) was isolated in 2010 in HmLu-1 cells from the jejunum of a calf which died of necrotizing enterocolitis in Yamaguchi Prefecture, Japan. Immunohistochemical staining identified C. pecorum positive reactions in the jejunal villi. C. pecorum, designated 24–100, was isolated from the feces of a calf with diarrhea in another farm in Yamaguchi Prefecture in 2012. A significant increase in neutralizing antibody titers against C. pecorum was confirmed in paired sera. Nucleotide sequence identities of omp1 genes of the 2 isolates were 100%. The isolates were genetically and antigenically more closely related to C. pecorum Bo/Yokohama strain isolated from cattle with enteritis in Japan than to the other prototype strains, Bo/Maeda isolated from cattle with pneumonia and Ov/IPA isolated from sheep with polyarthritis. These results indicate that C. pecorum strains similar to 22–58 and 24–100 might be endemic in Yamaguchi Prefecture and cause enteric disease in cattle.     

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4⁺ and CD8⁺ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.     

Effect of dietary excess of branched‐chain amino acids on performance and serum concentrations of amino acids in growing pigs

Depressed performance and availability of some amino acids (AA) in pigs fed excess Leu diets appear to be related to lower feed intake. Surplus Ile and Val may help to overcome this effect. An experiment was conducted with 24 pigs (31.8 ± 1.2 kg initial BW) to evaluate the effect of dietary excess of either Leu alone or with surplus Ile and Val on performance and serum concentration (SC) of essential AA. Treatments were as follows: T1, basal diet; T2, basal plus 0.43% L‐Leu (excess Leu); T3, basal added with 0.43% L‐Leu, plus 0.20% L‐Ile and 0.25% L‐Val (excess LIV). The basal diet was formulated to contain 0.90% standardized ileal digestible Lys and added with crystalline L‐Lys, L‐Thr, DL‐Met, L‐Trp, L‐Leu, L‐Ile, L‐His and L‐Val to create essential AA:Lys ratios close to an ideal protein for growing pigs. All pigs were fed the same amount of feed twice a day (average, 3.42× the requirement of NEm). Blood samples were collected at 2.5 (absorptive) and 11.0 h (post‐absorptive) post‐prandial to analyse SC of AA. Excess of either Leu or LIV did not affect growth rate nor feed conversion. Excess Leu increased Leu SC and decreased Ile and Val SC (p < 0.05) at both absorptive and post‐absorptive phases, but excess LIV restored the SC of Ile and Val. The SC of other essential AA was not affected by excess of either Leu or LIV. The SC of all AA during absorptive, on average, was about two times higher than that of post‐absorptive phase. These results suggest that the reduced availability (SC) of Ile and Val in pigs consuming excess Leu diets is attributed to a reduced absorption and increased cellular degradation rates of them.     

Mycoplasma bovis and otitis in dairy calves in the United Kingdom

Signs of severe otitis media in 20% of dairy calves on one farm were associated with Mycoplasma bovis infection, based on isolation from the external ear canal and nares. Affected calves seroconverted to M. bovis and no other significant bacteria were isolated. Infection was considered likely to have originated from cows in the milking herd based on evidence of seroconversion and detection of infection in a milk sample. M. bovis infection should be considered when investigating otitis problems in calves.     

Matricaria chamomilla CH12 decreases handling stress in Nelore calves

Matricaria chamomilla CH₁₂ is a phytotherapeutic or homeopathic product, which has been used to reduce stress. Here, we examined its effect on preventing handling stress in bovines. Sixty Nelore calves were randomly distributed into two equal groups. One group was administered Matricaria chamomilla CH₁₂ in diet and the other the ''control'' was not. Animals in both groups were maintained unstressed for 30 days to adjust to the feeding system and pasture, and were then stressed by constraint on the 31th, 38th, 45th and 60th experimental days. Blood samples were taken on these days after animals had been immobilization in a trunk contention for 5 min. Stress was followed by analyzing serum cortisol levels. These peaked on the 45th day and then decreased, but not to baseline, on the 60th day. On the 45th day cortisol levels were significantly lower in animals fed Matricaria chamomilla CH₁₂, suggesting that this product reduces stress. These effects may be a consequence of its inhibiting cortisol production and its calming and anxiolytic effects.     

Dietary levels of protein and free amino acids affect pancreatic proteases activities,amino acids transporters expression and serum amino acid concentrations in starter pigs

The dietary contents of crude protein and free amino acids (AA) may affect the protein digestion and AA absorption in pigs. Trypsin and chymotrypsin activities, AA serum concentrations and expression of AA transporters in the small intestine of pigs fed a low protein, AA‐supplemented (19.2%, LPAA) or a high protein (28.1%, HP), wheat‐soybean meal diet were measured in two 14‐d trials. The LPAA diet contained free L‐Lys, L‐Thr, DL‐Met, L‐Leu, L‐Ile, L‐Val, L‐His, L‐Trp and L‐Phe. All pigs were fed the same amount of feed (890 and 800 g/d for trial 1 and 2 respectively). In trial 1, samples of mucosa (duodenum, jejunum and ileum) and digesta (duodenum and jejunum) were collected from 14 pigs (17.2 ± 0.4 kg); in trial 2, blood samples were collected from 12 pigs (12.7 ± 0.3 kg). The trypsin and chymotrypsin activities in both intestinal segments were higher in pigs fed the HP diet (p < 0.01). Trypsin activity was higher in jejunum than in duodenum regardless the dietary treatment (p < 0.05). Pigs fed the LPAA diet expressed more b^0,+AT in duodenum, B⁰AT1 in ileum (p < 0.05), and tended to express more y⁺LAT1 in duodenum (p = 0.10). In pigs fed the LPAA diet, the expression of b^0,+AT was higher in duodenum than in jejunum and ileum (p < 0.01), but no difference was observed in pigs fed the HP diet. Ileum had the lowest b^0,+AT expression regardless the diet. The serum concentrations of Lys, Thr and Met were higher in LPAA pigs while serum Arg was higher in HP pigs (p < 0.05). Serum concentrations of AA appear to reflect the AA absorption. In conclusion, these data indicate that the dietary protein contents affect the extent of protein digestion and that supplemental free AA may influence the intestinal site of AA release and absorption, which may impact their availability for growth of young pigs.     

Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island,Kagoshima Prefecture,Japan

Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.     

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 becomean important pathogen of bovine respiratory disease. However, the minimum inhibitory concentrations ofantimicrobials for Mycoplasma bovis have not been studied in China. The objective of thisstudy was to determine the prevalence and antibiotic resistance of Mycoplasma bovis isolatedfrom young cattle with respiratory infection in China. Mycoplasma bovis was detected in 32/45bovine respiratory infection outbreaks at beef farms in 8 provinces in China. The isolates were susceptible orhad medium sensitivity to ciprofloxacin, enrofloxacin and doxycycline, but were frequently resistant tomacrolides (13/32, 41%). An A2058G (Escherichia coli Numbering) mutation located in therrnA operon in domain V of 23S rRNA was observed in strains that were resistant tomacrolides. This single mutations at the rrnA operon in domain V of 23S rRNA may play an important role in theresistance of Mycoplasma bovis strains to macrolides.     

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.     

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral,tonsillar, and nasal fluids

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.     

Development of a Medium-term Animal Model Using gpt Delta Rats to Evaluate Chemical Carcinogenicity and Genotoxicity

In this study, the potential for development of an animal model (GPG46) capable of rapidly detecting chemical carcinogenicity and the underlying mechanisms of action were examined in gpt delta rats using a reporter gene assay to detect mutations and a medium-term rat liver bioassay to detect tumor promotion. The tentative protocol for the GPG46 model was developed based on the results of dose-response exposure to diethylnitrosamine (DEN) and treatment with phenobarbital over time following DEN administration. Briefly, gpt delta rats were exposed to various chemicals for 4 weeks, followed by a partial hepatectomy (PH) to collect samples for an in vivo mutation assay. The mutant frequencies (MFs) of the reporter genes were examined as an indication of tumor initiation. A single intraperitoneal (ip) injection of 10 mg/kg DEN was administered to rats 18 h after the PH to initiate hepatocytes. Tumor-promoting activity was evaluated based on the development of glutathione S-transferase placental form (GST-P)-positive foci at week 10. The genotoxic carcinogens 2-acetylaminofluorene (2-AAF), 2-amino-3-methylimidazo [4,5-f] quinolone (IQ) and safrole (SF), the non-genotoxic carcinogens piperonyl butoxide (PBO) and phenytoin (PHE), the non-carcinogen acetaminophen (APAP) and the genotoxic non-hepatocarcinogen aristolochic acid (AA) were tested to validate the GPG46 model. The validation results indicate that the GPG46 model could be a powerful tool in understanding chemical carcinogenesis and provide valuable information regarding human risk hazards.