Correlation of the excretion of mycoplasma and kinetics of antibodies detected by complement fixation, passive hemagglutination and rapid seroagglutination in Mycoplasma mycoides subsp. mycoides SC experimental infection in cattle

During an experimental reproduction of CBPP, 5 inoculated cows and 5 contacts cows were bled twice a week and antibodies research was performed using complement fixation test (CFT), passive haemagglutination test (PHAT) and slide agglutination test (SAT). In the same period, trachobronchial washings were performed weekly to detect and to count Mycoplasma mycoides subsp. mycoides SC. For four months these tests were used to compare antibodies kinetics with kinetics of mycoplasmas excretion. With titer over 40 as threshold of positivity, PHAT detects antibodies earlier than CFT and SAT at the beginning of infection, but fails to detect chronic carriers. In contacts cows no failure has been observed with CFT and SAT except during prodromic phase of infection. These results are valid for natural infections. However, for inoculated animals, most serious failures to detect infected cows occur.     

Susceptibility of goats and calves after experimental inoculation or contact exposure to a Canadian strain of Mycoplasma mycoides subsp. mycoides isolated from a goat.

Transmissibility of Mycoplasma mycoides subsp. mycoides infection from experimentally inoculated goats to other goats and calves was studied. Eight goats and six calves were housed in an 18 m2 room. Six of the goats were inoculated endobronchially with strain D44 isolated from a natural case of polyarthritis in Ontario. These six goats died within a week of Mycoplasma septicemia. The two contact goats or the six calves never showed signs of disease and M. mycoides subsp. mycoides was not recovered from these animals. The contact goats and four calves were killed 25 days after exposure. They were all seronegative, M. mycoides subsp. mycoides was not recovered at necropsy and none had pathomorphological changes attributable to this Mycoplasma. The two remaining calves were inoculated endobronchially with 10(9) CFU of strain D44 and observed for 20 days. They never showed signs of disease and did not have significant lesions at necropsy. Both developed a significant serological response to M. mycoides subsp. mycoides, although this organism was not recovered during the experimental period or at necropsy. This study did not provide evidence for transmission of M. mycoides subsp. mycoides from endobronchially inoculated goats to contact goats or calves and endobronchially inoculated calves did not develop pneumonia. This would suggest that the infection of the goat population in Canada with this pathogen would not be a significant threat to the cattle population.     

Contagious bovine pleuropneumonia (CBPP) caused by vaccine strain T1/44 of Mycoplasma mycoides subsp. mycoides SC

A study was carried out on four adult cattle to assess the pathogenicity of Mycoplasma mycoides subsp. mycoides SC strain T1/44, currently used as a vaccine for the control of contagious bovine pleuropneumonia (CBPP) in Namibia. Post mortem examination 9 weeks after endobronchial inoculation of the vaccine strain to three of the four animals revealed unilateral pleuropneumonic lesions, pleuritis and well-developed sequesters in two of the three inoculated animals and several small sequesters surrounded by pleuropneumonic lesions in the diaphragmatic and apical lobes in one animal. The fourth animal, which was not directly inoculated but was in close contact with the inoculated animals, revealed only an adhesion area of the lung to the ribcage. Serological examination carried out using the complement fixation test (CFT) detected positive titres in all three intubated animals and the indirect CBPP-LppQ-ELISA was positive for two of the three inoculated animals. The contact animal showed no seroconversion. M. mycoides subsp. mycoides SC was isolated from the sequesters of two of the inoculated animals. Isolation of mycoplasmas was not possible from the third inoculated animal due to heavy contamination of the samples by other bacteria, but the presence of M. mycoides subsp. mycoides SC could be evidenced by PCR from clinical samples. The identity of the T1/44 vaccine strain isolated from the sequesters of two animals was confirmed by T1/44-specific PCR analysis and by IS1296 typing using Southern blot. These results clearly show that inoculation of T1/44 vaccine via the endobronchial route can lead to CBPP.     

Indirect infection of cattle with contagious bovine pleuropneumonia.

Hay infected with Mycoplasma mycoides sub-species mycoides (M mycoides) was fed to six animals on three occasions. Five animals developed complement-fixing antibody in their sera and gave a positive reaction to the comparative intradermal allergic test. No animal died from contagious bovine pleuropneumonia (CBPP) but when they were killed, three had unequivocal lesions of the disease. M mycoides was isolated from the animals with these lesions but not from the others. CBPP was transmitted to two animals put in contact with those animals showing disease, and M mycoides was recovered from lesions in both animals. It is suggested that when unexplained outbreaks of CBPP occur, the possibility of indirect transmission should be considered.     

Arthrogryposis, hydranencephaly and cerebellar hypoplasia syndrome in neonatal calves resulting from intrauterine infection with Aino virus

To determine the teratogenic potential of Aino virus (AINOV) in cattle, pregnant cows and fetal cattle were infected with a fresh isolate of AINOV. Five pregnant cows were inoculated intravenously with the virus at 122 to 162 days of gestation and allowed to give birth. All of the cows developed neutralizing antibodies to the virus, indicating that the cows had been infected with the virus; however, no clinical abnormalities were seen in their six newborn calves, and no specific antibodies to the virus were detected in the precolostral serum of calves. Five fetuses with fetal ages ranging from 132 to 156 days were inoculated in utero with the virus. One weak newborn and four stillborn calves were delivered at gestation days 256 to 263, i.e., less than the standard gestation term; they had congenital abnormalities including arthrogryposis, hydranencephaly and cerebellar hypoplasia. Antibodies specific to AINOV were detected in their precolostral serum. These results demonstrate that AINOV is a potential etiological agent of congenital malformation of cattle.     

Experimental Infection with Mycobacterium Avium,Serotype 2, in Pigs: 4. Contact Infection from Orally Inoculated Pigs*

In 3 experiments, 13 pigs were inoculated orally with 0.5 mg Mycobacterium avium daily for 5 days (1 mg = 32–68×10⁶ viable units). Five to 8 days after inoculation, 16 non-inoculated pigs were added to the inoculated pigs.Cultures from faeces showed excretion of M. avium from all the inoculated pigs for some time within the period 16 to 65 days after the last inoculation; the greatest numbers of organisms (62000 per 100 g faeces) were found at about the middle of the excretion period (Table 2). Two of the contact pigs were found to excrete M. avium in small numbers, 1 at 15, the other at 37 and 44 days after contact.All the inoculated pigs and 13 of the contact pigs showed positive intradermal tuberculin reactions. Post-mortem examination showed tuberculous lesions in all the inoculated pigs (Table 3). M. avium was transmitted to 15 of 16 pigs by contact. Five of these pigs showed gross lesions, 10 of them microscopic lesions only; in 9 of them the infection was proved by culture.     

A reassessment of the dual vaccine against rinderpest and contagious bovine pleuropneumonia

In the light of the recent outbreaks of rinderpest in Africa a further assessment of the efficacy of the simultaneous inoculation of rinderpest virus vaccine and contagious bovine pleuropneumonia vaccine was undertaken. Groups of cattle were inoculated with a dual preparation of rinderpest vaccine virus and Mycoplasma mycoides subspecies mycoides or M mycoides alone. These groups were then challenged with M mycoides, first unsuccessfully by an in-contact challenge method and then by subcutaneous challenge. All animals were examined clinically after challenge for evidence of contagious bovine pleuropneumonia and serologically for rinderpest virus and M mycoides mycoides antibodies. There was no evidence that the serological response to the dual vaccine was in any way less than that to either agent given alone and no clinical disease was detected in these animals after in-contact challenge. However, after subcutaneous challenge, the dual vaccinated groups reacted similarly to an unvaccinated control group and unlike the group vaccinated only with M mycoides. This would indicate that the rinderpest virus component of the dual vaccine interfered with the ability of the M mycoides component to induce a fully effective immune response. In the pan African rinderpest campaign the use of the dual vaccine in areas where contagious bovine pleuropneumonia occurs should be carefully considered; in areas where the disease does not occur it is contraindicated.     

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.     

Experimental infections of goats with Mycoplasma mycoides subspecies mycoides, LC type

In five experiments 29 goats were infected experimentally by five different routes with a strain of Mycoplasma mycoides subspecies mycoides, LC type, isolated from a contagious caprine pleuropneumonia-like outbreak on a farm in northern Sweden. All the goats were colonised except those inoculated subcutaneously with small doses. In its pattern of pathogenicity this strain was similar to other experimentally tested strains except that peroral infection in kids produced no clinical signs. A 'contact' goat was also colonised but the clinical signs seen in it were probably due to a concomitant infection with Pasteurella haemolytica.     

Effects of urinary excretion of nitrogen, potassium and sodium on urine volume in dairy cows

Data of 50 balance measurements were collected from dry and lactating Holstein cows to clarify the effects of urinary excretion of nitrogen (N), potassium (K) and sodium (Na) on urine volume in cows. In dry cows, orchardgrass silage, timothy hay, alfalfa silage and corn silage were offered to meet the TDN requirement. Orchardgrass silage or alfalfa silage diets were offered in switch back trials in lactating cows. There were no relationships between urinary excretion and plasma concentrations of K or Na in cows, but urinary N excretion increased with the increase of plasma urea nitrogen. There was positive correlation between urinary excretion and urinary content of Na, but urinary K contents increased rapidly by 1.3% with the increasing urinary K excretion and thereafter remained almost constant. The increasing urinary K and N excretion enhanced urine volume in cows, but urine production in cows was accurately estimated from the regression equation of urine K excretion on urine volume. Urine volume was not affected by urinary Na excretion. These results suggest that the increase of urine volume in cows affected by the increasing urinary excretion of K and N may be due to the maintenance of urine or plasma osmolality.     

Confirmation of the presence of Mycoplasma bovis in Hungarian cattle with pneumonia resembling pleuropneumonia.

Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.     

Infectious bovine rhinotracheitis virus: studies on the venereal carrier status in range cattle.

Genital samples collected at autopsy from 193 beef cows representing 22 different herds in Northern Australia yielded only one isolate of infectious bovine rhinotracheitis (IBR) virus. Serological evidence showed 59 per cent of similar cows had prior infection with this virus and of 19 sero-positive cows tested, 11 (58-2 per cent) shed detectable IBR virus from the vagina after treatment with corticosteroids. Transitory lesions of the vagina and vulva developed in five of the treated cows. Twenty-six (65 per cent) of 40 sero-positive bulls shed detectable IBR virus into the prepuce after corticosteroid treatment. Except for one bull, virus was not isolated after corticosteroid treatment of sero-negative animals. IBR virus and mucosal disease (MD) virus were not isolated from nasal swabs before or after corticosteroid administration. No correlation was observed between initial circulating antiboyd titre and virus excretion after treatment. There were no significant changes in levels of serum antibody during the virus excretion period.     

Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4 induce subclinical mastitis

In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.     

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.     

The effect of vitamin C supplementation on plasma concentration and urinary excretion of vitamin C in cattle

We investigated the plasma concentration and urinary excretion of vitamin C in cows supplemented with vitamin C. Five cows (mean BW = 597 kg) were allocated to a 5 x 5 Latin square design and supplemented with a vitamin C preparation coated with hydrogenated soybean oil at 0, 10, 20, 40, or 60 mg of vitamin C per kg of BW per day for 9 d. Plasma and urine samples were collected for measuring vitamin C concentration. Urinary excretion of vitamin C was expressed as the ratio of vitamin C to creatinine. Plasma vitamin C concentration and urinary vitamin C excretion increased quadratically as dietary vitamin C increased (P < 0.001); that is, the lowest dose affected neither plasma vitamin C concentration nor urinary vitamin C excretion but the plasma vitamin C concentration and urinary vitamin C excretion increased (P < 0.05) with increasing supplementation of vitamin C at greater doses. This suggests that plasma vitamin C concentration affects urinary excretion of vitamin C in cattle and that plasma vitamin C concentration exceeded the renal threshold for vitamin C in the cows receiving vitamin C at 20 mg/kg of BW per day. Furthermore, increased urinary excretion of vitamin C appears to limit plasma vitamin C concentration in response to vitamin C intake. The daily excretion of vitamin C was estimated by the reported value of daily creatinine excretion, indicating that the daily amount of vitamin C excreted into urine was more than half of supplied vitamin C. Therefore, a large part of supplied vitamin C probably escapes ruminal degradation and is absorbed but excreted into urine.     

Detection of Mycoplasma mycoides subspecies mycoides SC in bovine lung and lymph node tissues by culture, sandwich ELISA and polymerase chain reaction systems

Cattle from Northern Portugal, many with pulmonary lesions typical of contagious bovine pleuropneumonia, were investigated for the presence of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), which is the causative agent of CBPP, with several detection tests. Sandwich ELISA that included a culture enrichment stage, and 2 different PCR diagnostic systems were used to detect MmmSC in lung and mediastinal lymph node tissues from these animals. The comparison of typical CBPP pathology with the results of detection revealed that no single one of these methods provided a perfect match to the pathological data. Best performing tests were the PCR with laser induced fluorescence and PCR with pleuroTRAP kit (Chemicon, Australia), which are diagnostic systems based on amplification of genomic MmmSC DNA followed by sensitive detection of the amplified products. These were followed by the broth-enriched sandwich ELISA, which uses a monoclonal antibody specific to the M. mycoides cluster, to capture the antigen.     

Abattoir-based survey of contagious bovine pleuropneumonia in cattle in Turkey

Blood samples collected from 945 cattle at four local abattoirs in Turkey were examined for contagious bovine pleuropneumonia (CBPP) by the complement fixation test (CFT) and competitive ELISA (cELISA). In addition, the carcases of the animals were examined macroscopically at the abattoirs and 62 lung samples which had lesions suggestive of CBPP were collected for bacteriological culture. To identify suspicious isolates the PCR was used in addition to the routine biochemical tests. By the CFT, two of the 945 serum samples were seropositive, and by the cELISA, four of them were seropositive. In the bacteriological culture of the lungs, growth was observed in 18 (29 per cent) of the samples by the observation of turbidity in the broths. However, when these broths were inoculated into an agar base, growth was observed in only three (4.8 per cent) samples. These isolates were identified as Mycoplasma species on the basis of biochemical tests. In the PCR analysis of DNA extracted from the broths, none of the isolates was identified as Mycoplasma mycoides subspecies mycoides small colony or one of the members of the M mycoides cluster, but amplification was obtained in only eight (44.4 per cent) of 18 samples, using Mycoplasma-genus specific primers. These DNA samples were examined further with primers specific to 16S rRNA and were then sequenced and compared with the databanks; DNA homologies at different levels were observed in five samples, with Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovis and Mycoplasma bovigenitalium.     

Frequency of excretion of group B streptococci (Streptococcus agalactiae) in the milk during subclinical forms of mammary gland diseases in cows

The milk excretion of group B streptococci (Streptococcus agalactiae) from the udder quarters was examined in thirty cows of a heavily infected herd. Six samplings were performed in ten- to fourteen-day intervals. With respect to excretion rate, the set of cows could be divided into three groups: 1. group of cows excreting S. agalactiae from all udder quarters permanently and absolutely regularly; 2. cows excreting S. agalactiae regularly only from some quarters, certain quarter being negative at all samplings; 3. cows excreting streptococci from all quarters absolutely irregularly, without any conclusive order or dependence. The cytological picture of all samples exhibited no signs of inflammation. The discussion deals with some factors that may influence the excretion of streptococci with milk.     

Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes

The efficacy of an adjuvanted bovine rotavirus vaccine in pregnant cattle (15 heifers and 2 cows) was studied. Each of 4 animals was inoculated IM at 8, 5, and 2 weeks before parturition with a water-in-oil emulsion containing live purified bovine rotavirus, mineral oil, and a mannide oleate compound. Four other animals were treated identically, except that muramyl dipeptide was added to the virus preparation. Five additional animals were inoculated orally at the same time intervals with adjuvant-free viral suspension, and 4 other pregnant animals inoculated only with buffer served as uninoculated controls. Kinetic studies of the specific immune responses were determined by quantification of the rotavirus-neutralizing antibodies and by a rotavirus lymphocyte stimulation test in vitro. Results showed that only the emulsions induced marked enhancement of rotavirus antibody titers in the serum, colostrum, and milk of inoculated cows. Colostral and milk lymphocytes isolated from these cows had a positive in vitro proliferative response to rotavirus stimulation, which lasted at least 21 days after parturition. The values of the stimulation index obtained with the colostral/milk lymphocytes were higher than those of the blood lymphocytes, reflecting increased lymphocyte activity in the colostrum/milk. However, addition of muramyl dipeptide to the emulsion preparation did not exert any potentiating effect on the immune response to rotavirus. Calves fed for the first 5 days after birth with a rotavirus-immune cell-free colostrum supplement were protected from a rotavirus challenge exposure on the third day after birth. Virus was not detectable in their feces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elimination of erythromycin in milk after intramammary administration in cows with specific mastitis: relation to dose,milking frequency and udder health

Elimination of erythromycin in milk following intramammary therapy of specific mastitis in cows was studied. Five cows received therapy in one quarter (G1), and eight in two quarters with five milked twice (G2) and three thrice a day (G3). Dose infused was 300 mg/quarter 12 h × 5 times. The drug concentrations in milk were determined using microbial assay technique with Micrococcus luteus as the test organism. Considerable variations occurred in the excretion of drug; levels for treated quarters being 8.25 to 37.61 μg/ml at first milking that declined rapidly at 24 h and no drug activity was observed beyond 36 h post treatment. In total, about 6–25% of the last infused dose appeared in the milk. Drug crossed to 1/15 quarter (G1), 6/10 quarters (G2) and all the six untreated quarters (G3). Crossover levels were significantly higher in mastitic quarters and for G3 cows, but duration of excretion remained same in all cases. It seems that crossover of erythromycin to untreated quarters is related to the udder health and dose infused.