Comparison of distal forelimb conformations between Japanese Black and Holstein-Friesian newborn calves.

Flexural and hyperextension deformities are congenital problems in calves. We, therefore, aimed to investigate the distal limb conformation in 1 day- and 28-day-old female Holstein-Friesian (HF) calves (n=21), male Japanese Black (JB) calves (n=15), and female JB calves (n=15). The claw angle of the forelimb dorsal claw wall in a standing position and recorded other parameters, including body weight, withers height, circumference of forelimbs, and flexor tendon thickness in the forelimbs, were measured and compared these between the three groups. At 1 day old, the mean claw angles were 51.1° in female HF calves, 47.0° in male JB calves, and 41.8° in female JB calves; the 95% confidence intervals (CIs) of the claw angles showed large distributions in all three groups. One female HF and one male JB calves showed mild flexural deformity, whereas four JB calves showed hyperextension deformity. At 28 days old, the mean claw angles were 51.7° in female HF calves, 51.2° in male JB calves, and 48.4° in female JB calves; the 95% CIs of the claw angles showed smaller distributions than those at 1 day old in all groups. For all groups, the limb deformities had improved without treatment at 28 days old. As a feature of the breed, female JB calves were apt to show hyperextended deformities inversely proportional to the body weight. These limb deformities healed spontaneously and were thought to be physiological.

Changes in mRNA of immune factors expressed by milk somatic cells of Holstein cows with hypocalcemia after calving.

Changes in immune factors expressed by milk somatic cells from Holstein cows with hypocalcemia after calving were investigated in this study. Fourteen multiparous Holstein cows after their 3rd or 4th calving in one farm were used. The cows were divided into 2 groups: 7 cows needing treatment due to onset of hypocalcemia (hypocalcemia group; age = 5.53 ± 0.27 years, parity = 3.14 ± 0.14) and 7 cows without health problems (control group; age = 5.88 ± 0.31 years, parity = 3.57 ± 0.26). Milk samples were collected aseptically using a cannula and mRNA of immune factors expressed by milk somatic cells were analyzed. Milk samples (50 mL) were collected from the right rear mammary gland of cows before milking at day 1 and weeks 1, 2, 4, and 8 after calving. All milk samples showed a negative reaction to the California Mastitis Test. Levels of relative interleukin (IL)-6 and cathelicidin in the hypocalcemia group were lower than those in the control group in weeks 1 to 8. A significant difference in relative IL-6 levels was found in week 4 (P < 0.05). These results suggest that levels of IL-6 expressed by milk somatic cells may be affected by hypocalcemia in dairy cows.

Dans la présente étude les modifications des facteurs immunitaires exprimées par les cellules somatiques du lait de vaches Holstein présentant une hypocalcémie après le vêlage ont été examinées. Quatorze vaches Holstein multipares après leur 3e ou 4e vêlage provenant d'une ferme ont été utilisées. Les vaches ont été réparties en deux groupes : sept vaches nécessitant un traitement en raison de l'apparition d'une hypocalcémie (groupe hypocalcémie; âge = 5,53 ± 0,27 ans, parité = 3,14 ± 0,14) et sept vaches sans problème de santé (groupe témoin; âge = 5,88 ± 0,31 ans, parité = 3,57 ± 0,26). Des échantillons de lait ont été prélevés de manière aseptique à l'aide d'une canule et l'ARNm des facteurs immunitaires exprimés par les cellules somatiques du lait a été analysé. Des échantillons de lait (50 mL) ont été prélevés dans la glande mammaire arrière droite des vaches avant la traite au jour 1 et aux semaines 1, 2, 4 et 8 après le vêlage. Tous les échantillons de lait ont montré une réaction négative au California Mastitis Test. Les niveaux relatifs d'interleukine (IL)-6 et de cathélicidine dans le groupe hypocalcémie étaient inférieurs à ceux du groupe témoin au cours des semaines 1 à 8. Une différence significative des taux relatifs d'IL-6 a été observée à la semaine 4 (P < 0,05). Ces résultats suggèrent que les taux d'IL-6 exprimés par les cellules somatiques du lait peuvent être affectés par l'hypocalcémie chez les vaches laitières.(Traduit par Docteur Serge Messier).

The Suppression of Th1 Response by Inducing TGF-ß1 From Regulatory T Cells in Bovine Mycoplasmosis.

Regulatory T cells (Tregs) regulate immune responses and maintain host immune homeostasis. Tregs contribute to the disease progression of several chronic infections by oversuppressing immune responses via the secretion of immunosuppressive cytokines, such as transforming growth factor (TGF)-ß and interleukin-10. In the present study, we examined the association of Tregs with Mycoplasma bovis infection, in which immunosuppression is frequently observed. Compared with uninfected cattle, the percentage of Tregs, CD4+CD25highFoxp3+ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-ß1, and M. bovis infection induced TGF-ß1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-ß1 on bovine immune cells. Treatment with TGF-ß1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-ß1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection.

Flexor tendon transection and post-surgical external fixation in calves affected by severe metacarpophalangeal flexural deformity.

This study aimed to evaluate the transection of superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT) in calves with severe metacarpophalangeal flexural deformities (MPFD). The study comprised 17 forelimbs of 10 calves that were diagnosed at the Animal Medical Centre, Rakuno Gakuen University. The calves were treated via transection of the SDFT and DDFT with retention of the suspensory ligament, followed by external fixation according to a post-surgical gait test. The post-procedural prognosis was determined at 14 days post-surgery. Of the 17 limbs, 14 (82%) achieved non-lameness and a good prognosis. Surgical complications were not observed in any treated calves. The transection of SDFT and DDFT is an effective first-line surgical option for calves with severe MPFD.

Upregulation of PD-L1 Expression by Prostaglandin E2 and the Enhancement of IFN-γ by Anti-PD-L1 Antibody Combined With a COX-2 Inhibitor in Mycoplasma bovis Infection.

Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.

Cytokine levels of peripheral blood mononuclear cells in the clinical cases of Holstein calves infected with Mycoplasma bovis.

The immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed in the clinical cases with Mycoplasma (M.) bovis infection. Seventy-eight Holstein calves in one farm were used. These calves were divided into three groups; the calves with M. bovis infection of poor outcome after treatment (Non-Recovery Group), the calves with M. bovis infection recovered (Recovery Group) and clinically healthy calves (Control Group). Blood samples were collected at days of the first medical treatment and the final treatment or euthanasia. IL-17A levels in the Non-Recovery Group were higher than those in the Recovery Group on both days. Our result suggested that the IL-17A of PBMC is an important factor to affect outcome of the calves with M. bovis infection.

Relationship Between mRNA of Immune Factors Expressed by Milk Somatic Cells and Bacteria Present in Healthy Lactating Holstein Cows.

INTRODUCTION: The characteristics of immune factors in somatic cells from lactating dairy cows and their association with commensal bacteria in normal milk have not been clarified. This study investigated the relationship between the pathogenic bacteria in milk and somatic cell immune factors in healthy lactating cows. MATERIAL AND METHODS: In total 44 healthy Holstein cows were studied on one farm. Milk samples were collected aseptically using a cannula and these samples were cultured for detection of bacteria and analysis of mRNA of immune factors expressed by somatic cells. Cows were divided into two groups based on the microbial status of their milk samples: 12 cows showed bacteria in cultures (positive group), and the other 32 cows did not (negative group). RESULTS: The mRNA levels of IL-6, lactotransferrin, and cathelicidin expressed by somatic cells after milking decreased significantly compared to those before milking in both groups (P < 0.05). There were significantly lower mRNA levels of IL-6 and cathelicidin in the positive group compared to those in the negative group before milking. CONCLUSION: These results suggest that mRNA levels of IL-6 and cathelicidin expressed by the somatic cells may be affected by the presence of bacteria in healthy lactating dairy cows.

Radiographic analysis of the angle in the lateromedial projection of the metacarpophalangeal joint and the distal interphalangeal joint in metacarpophalangeal flexural deformities in calves.

The bone alignment of the metacarpophalangeal joint (MPJ) of the distal interphalangeal joint (DIPJ) in metacarpophalangeal flexural deformity (MPFD) in calves was evaluated by radiography. This study was designed by retrospective study of radiographs. Lateral to medial radiographs of distal forelimbs were taken from 19 MPFD affected calves (35 forelimbs) and 21 normal calves (42 forelimbs). Based on the radiographs, the lateral angles of MPJ were measured from the metacarpal bone axis and proximal phalanx axis, and lateral angles of DIPJ were measured from the middle phalanx axis and distal phalanx axis. Mean lateral angle of MPJ in the normal limbs was 175.9 (95% CI 174.5 to 177.4). Mean lateral angles of MPJ in MPFD were as follows: mild: 167.1 (158.9-175.2), moderate: 165.1 (158.5-171.7) and severe: 150.6 (146-155.1). MPJ angle in MPFD limbs was narrower than that in the normal limbs (mild, moderate and severe: P=0.017, P=0.003 and P<0.001, respectively). Mean lateral angle of DIPJ in the normal limbs was 211.9 (210.7-213.2). Mean lateral angles of DIPJ in moderate: 200.6 (195.2-206.1) and severe: 204.9 (203.3-206.5) MPFD were narrower than that in the normal limbs (both P<0.001). There was no significant difference between the normal limbs and mild: 210.3 (206.9-213.7) MPFD limbs (P=0.7). The clinical severity of MPFD corresponded well with the lateral angle of MPJ. The flexion of DIPJ in moderate and severe MPFD was similar to the flexion of MPJ in MPFD. This suggested that the lateral to medial radiographs accurately reflected the MPJ flexion and the DIPJ in MPFD in calves, providing useful information for the treatment of MPFD.

Relationship between bovine endometrial thickness and plasma progesterone and estradiol concentrations in natural and induced estrus.

The objective of this study was to investigate cyclical changes in endometrial thickness in relation to progesterone (P4) and estradiol-17ß (E2) concentrations during natural and induced estrus in 15 cows. In the prostaglandin (PG) F2α-induced estrus group, ultrasonography (USG) at 6-h intervals was used to determine endometrial thickness 48-24 h before the PGF2α treatment until 24 h after ovulation (ovulation = Day 0). In the natural estrus group, USG was performed every 48 h from Day 3 to Days 15-18 after the first ovulation, and then every 6 h until 24 h after ovulation. Endometrial thickness was standardized using Day 13 as a reference day. Blood was collected during every USG examination and plasma P4 and E2 concentrations were determined. Endometrial thickness of the induced estrus group (n = 11) was greater than that of the natural estrus group (n = 9) between 60 and 12 h before ovulation (P < 0.05). In the natural estrus group, prior to an increase in endometrial thickness, a decrease in P4 and an increase in E2 were detected. In the induced estrus group, based on the time of ovulation, an increase in endometrial thickness was detected at the same time of a decrease in P4 before an increase in E2. These results suggest that decreases in P4 concentrations may be a cue to changes in endometrial thickness, while increases in E2 concentrations appear to sustain and/or enhance these changes.

Intrauterine infection with bovine leukemia virus in pregnant dam with high viral load.

Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.

Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.

INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.

Sero-epidemiological analysis of vertical transmission relative risk of Borna disease virus infection in dairy herds.

Borna disease virus (BDV) is a virus that causes a neurological disease in domestic animals, including a variety of animal species in Japan. Few studies have examined the mode of transmission of this virus in cattle, and the exact mechanisms underlying the transmission of the virus need to be elucidated. This study aimed to examine the contribution of vertical transmission of the virus, which occurs when the virus is transmitted from the mother to offspring during gestation or birth. We used an epidemiological approach. The relative risk (RR) was calculated for cattle born to BDV sero-positive cows from farms with a higher within-herd prevalence of BDV (56.8%). We tested the sera of 1,122 dairy cattle from 24 dairy herds in Hokkaido Prefecture, Japan, for BDV infection using the ELISA and western blotting method. The overall level of BDV sero-prevalence was 22.1%. Seroprevalence was significantly higher in closed-breeding herds that do not have buying in cows (39.7%) than in farms that restock cattle by buying in cows (4.4%, P<0.01). The overall RR of BDV vertical transmission from infected mothers to their daughters was 1.86 (95% confidence interval (CI): 1.54-2.56). Our results show that vertical transmission contributes significantly to BDV transmission in the farms tested in this study.

C-X-C chemokine receptor type 4 and cytokine expressions in cows of a dairy herd with high prevalence of calves persistently infected with bovine viral diarrhea virus.

Animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) play an important role in the spread of BVDV. Alteration of maternal C-X-C chemokine receptor type 4 (CXCR4) expression has been suspected as closely concerned with the production of PI calves. It is not clear what the influence of CXCR4 response to the prevalence of PI calves. We have previously reported a dairy herd with high prevalence of PI calves within a short period having a single origin of infection. CXCR4 and cytokine expressions in cows of this herd were investigated. There were no significant differences in CXCR4 and cytokine expressions between the dams of PI calves and the dams of non PI calves in the herd. In the comparison among the herds, CXCR4 expressions in the PI producing herds were significantly lower than the BVDV-free herd. Moreover, CXCR4 expressions in the high prevalence herd and the low prevalence herd were similar. These findings among herds corresponded with the previously reported experimental production of persistent infection with BVDV in cows. Based on the cytokine profile of these herds, IL-10 was significantly higher in the high prevalence herd and the BVDV-free herd. The combination of low expression of CXCR4 and high expression of IL-10 might be closely concerned with some bias for the production of PI calves.

Effects of interferon-tau on cattle persistently infected with bovine viral diarrhea virus.

In this study, the antiviral effects of bovine interferon-tau (boIFN-tau) on bovine viral diarrhea virus (BVDV) were examined in vitro and in vivo. In the in vitro experiments, the replication of cytopathic and non-cytopathic BVDV was inhibited in the bovine cells treated with boIFN-tau. The replication of BVDV was completely suppressed by boIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the effect of boIFN-tau on virus propagation in cattle persistently infected (PI) with non-cytopathic BVDV, boIFN-tau was subcutaneously administered to PI cattle at 10(5) U/kg or 10(6) U/kg body weight 5 times per week for 2 weeks. No physical abnormality such as depression was observed in the cattle during the experiment. The mean BVDV titers in the serum of the PI cattle decreased slightly during the boIFN-tau administration period with the dose of 10(6) U/kg. However, the BVDV titers in the serum returned to the pre-administration level after the final boIFN-tau administration. These results suggest that boIFN-tau demonstrates an anti-BVDV effect, reducing the BVDV level in serum transiently when injected into PI cattle.

Bovine viral diarrhea virus infection in a dairy herd with high prevalence of persistently infected calves.

A dairy herd including approximately 50 milking cows and 40 heifers and calves was investigated. This herd was detected with high prevalence of calves persistently infected (PI) with bovine viral diarrhea virus (BVDV). Nine PI animals including a milking cow and 8 newborn calves were detected in the herd within 4 months. Prevalence of PI animals in this herd was estimated 7.0% which was very high compared to that estimated in previous reports. All newborn PI calves were strongly suspected to have a single origin of infection as estimated from the homology of the virus genes. The cause of high prevalence could not be clarified. Removal of PI animals and continuous examination of newborn calves were important for the elimination of BVDV from the herd.

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks.

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.

A case control study of Bovine Viral Diarrhea Virus (BVDV) Persistent Infection (PI) in Betsukai, Hokkaido, Japan.

The Betsukai town office implemented bovine viral diarrhea virus (BVDV) preventive activities (i.e., vaccination and surveillance) in 2006. Using bulk tank milk screening followed by individual blood tests using a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method, persistent infection (PI) cattle were detected and eliminated from the population. Based on data for PI cattle detected between 2006 and 2007, we conducted a case control study to find risk factors associated with the presence of PI cattle. Significantly associated farm level factors for increasing risk of producing PI cattle include; 1) no recent purchase of cattle (between 2004 and 2007) and 2) no prevention of people/animals entering the premises. This study suggests that not only vertical transmission from dam to calf but also indirect contact with people and animals play an important role in transmitting BVDV infection and subsequent production of PI animals.

Classification of new BVDV isolates from Philippine water buffalo using the viral E2 region.

Characterization of bovine viral diarrhea virus (BVDV) isolates has been focused of several studies this last decade. Until now lots of new strains are being unfolded maybe due to the viral fast mutation ability. As we focused our research on water buffalo immunology, we were able to identify a probable new BVDV isolates. RNA was extracted from water buffalo blood in the Philippines. The extracted RNA was reverse-transcribed and synthesized cDNA. Oligonucleotide primers from the viral E2 region were used to amplify the target viral gene and later purified, cloned and sequenced. The E2 region with 420 bp nucleotides long was compared with existing published sequences in the GenBank. Based on the constructed phylogenetic tree, the isolated strain showed to be a BVDV type 1b along with Osloss and CP7 strains. Further classification of the new isolates was done within the BVDV type 1b1 group, which was compared with other strains in the sub-group. The analysis revealed that Lamspringe/738, KE9 and 2543/87 were the closest with 92% homology. Additional study is being done to further qualify and quantify the extent of the existence of this new BVDV isolates in water buffalo in the Philippines. This is the first report of BVDV in the Philippines and first concerning BVDV in Philippine water buffalo.

Availability of oral swab sample for the detection of bovine viral diarrhea virus (BVDV) gene from the cattle persistently infected with BVDV.

Bovine nasal and oral discharges were used as samples for bovine viral diarrhea virus (BVDV) gene detection. Viral genes in serum (S), nasal discharge (N) and oral discharge (O) were quantified with real-time polymerase chain reaction using SYBR Green by the relative quantification method, and findings were compared among samples. Although the quantity of the BVDV gene in S was greater than those in N and O, all samples were available to identify persistently infected (PI) cattle with BVDV by reverse transcription polymerase chain reaction (RT-PCR). The swab samples were able to be stored for a few days at 4 degrees C with a little decrease of amplification signal in RT-PCR. Oral swab sampling was easier than nasal swab sampling, and was also less uncomfortable for the cattle than other sampling methods without pain or unnecessary retention. This sampling method can be performed without any special technique and equipment. Therefore, the oral swab sampling method is useful for screening to detect BVDV PI cattle by RT-PCR.