Growth of Mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with Mycoplasma dispar

Inoculation of tracheal organ cultures from bovine foetuses with Mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed 20-22 days later by lifting and detachment of overlying epithelium. The effect was associated with large numbers of M. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. Ciliary activity was undiminished for up to 18 days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. In contrast, M. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. The cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma.     

Feeding 5-hydroxy-l-tryptophan during the transition from pregnancy to lactation increases calcium mobilization from bone in rats

An increasing demand for calcium during pregnancy and lactation can result in both clinical and subclinical hypocalcemia during the early lactation period in several mammalian species, in particular the dairy cow. Serotonin (5-HT) was recently identified as a regulator of lactation and bone turnover. The purpose of this study was to determine whether supplementation of the maternal diet with a 5-HT precursor would increase maternal bone turnover and calcium mobilization to maintain appropriate circulating maternal concentrations of ionized calcium during lactation. Female Sprague-Dawley rats (n = 30) were fed either a control diet (n = 15) or a diet supplemented with the 5-HT precursor 5-hydroxytryptophan (5-HTP, 0.2%; n = 15) from day 13 of pregnancy through day 9 of lactation. Maternal serum and plasma (day 1 and day 9 of lactation), milk and pup weight (daily), mammary gland and bone tissue (day 9 of lactation) were collected for analysis. The 5-HTP diet elevated circulating maternal concentrations of 5-HT on day 1 and day 9 of lactation and parathyroid hormone related-protein (PTHrP) on day 9 of lactation (P < 0.033). In addition, 5-HTP supplementation increased total serum calcium concentrations on day 1 of lactation and total milk calcium concentration on day 9 of lactation (P < 0.032). Supplemental 5-HTP did not alter milk yield, maternal body weight, mammary gland structure, or pup litter weights (P > 0.05). Supplemental 5-HTP also resulted in increased concentrations of mammary 5-HT and PTHrP, as well as increased mRNA expression of rate-limiting enzyme in 5-HT synthesis, tryptophan hydroxylase 1, and Pthrp mRNA on day 9 of lactation (P < 0.028). In addition, supplementation of 5-HTP resulted in increased mRNA expression of maternal mammary calcium transporters and resorption of bone in the femur, indicated by increase osteoclast number and diameter as well as mRNA expression of classical markers of bone resorption on day 9 of lactation (P < 0.048). These results show that increasing 5-HT biosynthesis during the transition from pregnancy to lactation could be a potential therapeutic target to explore for prevention of subclinical and clinical hypocalcemia.     

Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus

Three-week-old cesarean-derived colostrum-deprived (CD/CD) pigs were inoculated with porcine circovirus type 2 (PCV2, n = 19), porcine reproductive and respiratory syndrome virus (PRRSV, n = 13), concurrent PCV2 and PRRSV (PCV2/PRRSV, n = 17), or a sham inoculum (n = 12) to compare the independent and combined effects of these agents. Necropsies were performed at 7, 10, 14, 21, 35, and 49 days postinoculation (dpi) or when pigs became moribund. By 10 dpi, PCV2/PRRSV-inoculated pigs had severe dyspnea, lethargy, and occasional icterus; after 10 dpi, mortality in this group was 10/11 (91%), and all PCV2/ PRRSV-inoculated pigs were dead by 20 dpi. PCV2-inoculated pigs developed lethargy and sporadic icterus, and 8/19 (42%) developed exudative epidermitis; mortality was 5/19 (26%). PRRSV-inoculated pigs developed dyspnea and mild lethargy that resolved by 28 dpi. Microscopic lesions consistent with postweaning multisystemic wasting syndrome (PMWS) were present in both PCV2- and PCV2/PRRSV-inoculated pigs and included lymphoid depletion, necrotizing hepatitis, mild necrotizing bronchiolitis, and infiltrates of macrophages that occasionally contained basophilic intracytoplasmic inclusion bodies in lymphoid and other tissues. PCV2/ PRRSV-inoculated pigs also had severe proliferative interstitial pneumonia and more consistent hepatic lesions. The most severe lesions contained the greatest number of PCV2 antigen-containing cells. PRRSV-inoculated pigs had moderate proliferative interstitial pneumonia but did not develop bronchiolar or hepatic lesions or lymphoid depletion. All groups remained seronegative to porcine parvovirus. The results indicate that 1) PCV2 coinfection increases the severity of PRRSV-induced interstitial pneumonia in CD/CD pigs and 2) PCV2 but not PRRSV induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS.     

Identification and cornification-related gene expression of canine keratinocyte differentiation-associated protein, Kdap

The outermost layer of skin, the epidermis, is cornified epithelial tissue composed of keratinocytes. To maintain the structure and function of the epidermis, the regulation of proliferation, differentiation, and cornification of keratinocytes is crucial, and various soluble factors secreted by keratinocytes are involved in these regulations. Previously, work has shown that keratinocytes secreted the protein Kdap (keratinocyte differentiation-associated protein) associated with the formation of cornified cell envelopes, a specialized protective barrier structure on the periphery of terminally differentiating keratinocytes. In the present report, the canine counterpart of human Kdap is identified and an attempt has been made to define its physiological role in canine keratinization. Canine Kdap (cKdap) showed structural features commonly observed in other counterparts and is secreted from transfected cells. The expression profile of cKdap mRNA, which was restrictively expressed in cornified epithelial tissues besides skin has also been determined. These findings indicate that there is a strong association between cKdap expression and cornification, which supports previous observations that Kdap is involved in the synthesis and/or degradation of cornified cell envelopes in humans and mice.     

Efficacy of cobalt-60 radiation therapy for the treatment of nasal cavity nonkeratinizing squamous cell carcinoma in the dog

The purposes of this study were to evaluate the efficacy of cobalt-60 radiotherapy in the treatment of nonkeratinizing squamous cell carcinoma of the nasal cavity in dogs and to compare this treatment group to historical controls. Six dogs with histopathologically confirmed nasal cavity nonkeratinizing squamous cell carcinoma were treated with cobalt-60 radiotherapy to a total dose of either 63 Gy or 54 Gy. Overall survival times ranged from 30 days to 330 days, with a median survival time of 165 days. Nasal cavity nonkeratinizing squamous cell carcinoma in the dog is an aggressive tumor that responds poorly to radiotherapy.     

Hanging Drop Monoculture for Selection of Optimal Antioxidants During In Vitro Maturation of Porcine Oocytes

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H₂O₂ supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H₂O₂. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.     

Effects of road transportation on metabolic and immunological responses in Holstein heifers

This study examined the effects of road transportation on metabolic and immunological responses in dairy heifers. Twenty Holstein heifers in early pregnancy were divided into non‐transported (NT; n = 7) and transported (T; n = 13) groups. Blood was collected before transportation (BT), immediately after transportation for 100 km (T1) and 200 km (T2), and 24 h after transportation (AT). The T heifers had higher (P < 0.05) blood cortisol and non‐esterified fatty acid concentrations after T1 and T2 than did NT heifers. By contrast, the T heifers had lower (P < 0.05) serum triglyceride concentrations after T1 and T2 than had the NT heifers. The serum cortisol and triglyceride concentrations returned (P > 0.05) to the BT concentrations at 24 h AT in the T heifers. The granulocyte‐to‐lymphocyte ratio and the percentage of monocytes were higher (P < 0.05) after T2 in the T heifers than in the NT heifers, suggesting that transportation stress increased the numbers of innate immune cells. T heifers had higher (P < 0.01) plasma haptoglobin concentrations than NT heifers 24 h AT. In conclusion, transportation increased cortisol secretion and was correlated with increased metabolic responses and up‐regulation of peripheral innate immune cells in dairy heifers.