A Study of Complement-fixation and Serum Agglutination Tests on Vibrio fetus Infected and Immunized Cattle

Complement-fixation (CF) and tube agglutination (TA) tests for demonstration of Vibrio fetus antibodies were conducted on the sera of three groups of ten heifers. One group was vaccinated subcutaneously with a commercial V. fetus var venerealis bacterin and challenged intra-utero, at the external os cervicus one month later; the second was infected only and the third vaccinated only.

The vaccinated cattle developed high CF serum titers, but no such increase was observed in animals infected only. A moderate increase in serum antibody titers was demonstrated by the TA test following either infection or vaccination; although titers observed were not higher than those observed in the sera of some apparently normal uninfected animals. The group receiving both vaccine and challenge was the only one in which significant serum antibody titers were demonstrable by the TA test. The sera of these animals also had significant titers in the CF tests.

The CF and TA tests detected serum antibodies produced by the parenteral inoculation of V. fetus antigen. These two tests were of limited value in detecting serum antibodies from animals with genital V. fetus var venerealis infection, although the formation of local antibodies was demonstrable by the vaginal mucus agglutination test.

     

Some Effects of Experimental Vibrio Fetus (Venerealis) Infection on Cattle Inoculated with a Commercial Bacterin

Four groups of ten heifers were used to study the effect of a commercial Vibrio fetus bacterin on the development of vaginal mucus agglutination (VMA) reactions and subsequent reproductive performance in the presence of active V. fetus infection. Two groups were vaccinated and one of these was infected one month later. The other two groups were not vaccinated and one of these was infected. It was found that in the absence of infection the bacterin did not produce diagnostically significant VMA reactions but in the presence of infection reactions comparable with the control group were detected. The infected control group required 4.9 services per conception, the first service-conception interval was 126.9 days and 50 per cent of the animals aborted. The corresponding figures in the infected vaccinates were 3.5 services, 68.9 days and 12.5 per cent abortions. It was concluded that although this bacterin has a beneficial effect on reproductive performance in the presence of genital vibrosis, maximum breeding efficiency will not be obtained unless V. fetus infection is eliminated. Persistent local reactions appeared at the site of inoculation in the majority of the vaccinated animals.     

Bovine Vibriosis: The Distribution and Specificity of Antibodies Induced by Vaccination and Infection and the Immunofluorescent Localization of the Organism in infected Heifers

Two groups of three Holstein heifers were immunized respectively with Vibrio fetus venerealis and Vibrio fetus intestinalis incorporated in Freund's complete adjuvant. Both serum and vaginal mucus agglutination titers increased following immunization. Vaginal mucus samples were more frequently positive when the homologous cells were used as antigen in the agglutination test.

Ten non-immunized heifers were inoculated with another strain of V. fetus venerealis and slaughtered at periods of 30 to 40 and 60 to 70 days post-inoculation (DPI). Agglutinating antibodies were present in the vaginal mucus of some infected individuals by five weeks post-inoculation. In the course of the experiment 11 vaginal mucus samples were obtained which agglutinated heated cells of the infecting strain; one aggglutinated whole cells. Precipitins toward homologous antigens could not be demonstrated in vaginal mucus but four of six samples tested precipitated a heat stable extract from an intestinal strain of the same O-serotype. Bacterial antigen was detected by immunofluorescence on the surface, as well as within and beneath the epithelium at all levels of the reproductive tract regardless of time of slaughter. Lesions in infected animals consisted of focal and diffuse lymphocytosis, plasmacytosis, and epithelial vacuolation. Diffuse neutrophilic infiltration of the oviducts was observed.

Agglutinins appeared in the serum of each of nine heifers immunized with whole cells of same venereal strain. Group mean serum titers for whole and heated cells were 1/28,000 and 1/1,300 respectively. Vaginal mucus samples agglutinated whole cells in 48% of tests while 6.3% reacted with heated cells. Serum, but not vaginal mucus, of immunized animals precipitated soluble antigens of the immunizing strain. The immunizing strain of V. fetus did not infect the reproductive tract of any of six immunized heifers upon challenge.

     

Pathogenicity of Vibrio Coli for Swine II. Experimental Infection of Conventional Pigs with Vibrio Coli

The pathogenicity of V. coli for conventional swine was studied by inoculating pigs with cultures of V. coli and V. coli infected gut of gnotobiotic pigs. Thus, six conventional pigs were inoculated with strains of V. coli freshly isolated from infected gnotobiotic pigs. The cultures were grown in simulated sows milk, and added to the feed. Two other groups, of three pigs each, were infected by administration of minced intestine from two gnotobiotic pigs, heavily infected with the organism. Vibrio was isolated from all pigs, including five of the six controls, but larger numbers were isolated from the inoculated groups, especially from those fed macerated gut. Clinical signs of disease were not observed.     

Incident Light Immunofluorescence of Alcohol-fixed Colonies of Ruminant Mycoplasma

Two species of ruminant mycoplasma colonies had to be fixed in ethyl alcohol so that incident immunofluorescence method could be applied. In addition, the stain reaction had to be kept for 90 minutes at 37°C.

This fluorescent antibody (FA) method was developed to identify colonies of Vom strain of Mycoplasma mycoides var. capri, V-5 strain of M. mycoides var. mycoides, and PG-2 strain of M. agalactiaeon agar, using fluorescent ultraviolet light. Fluorescence was not demonstrated when heterologous conjugates or normal rabbit serum conjugate were applied but the reaction appeared to be specific for each strain of mycoplasma.

The FA method was able to differentiate specific mycloplasma colonies in mixed cultures.

     

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke' Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987.

METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939–2945) from the NZRM, representing restriction types a–g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE.

RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a–g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study.

CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke' Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.     

Experimental infections with Campylobacter fetus in bulls of different ages

Factors affecting the establishment of a carrier state of Campylobactor fetus subsp. fetus (venerealis) in preputial cavities of bulls were investigated by following infection in bulls of two different age groups until slaughter, 9–18 weeks post infection. Each of the four older bulls (66–74 months at infection) and three of the four younger bulls (41–49 months at infection) were culturally positive until slaughter, indicating no increased susceptibility with age. Relative proportions of immunoglobulin classes and albumin in preputial fluids were generally similar to those determined prior to infection, although protein concentrations decreased and ratios of IgG/IgA and IgG₁/IgG₂ increased in most of the bulls. An unexplained diminution of sample volumes occurred with progressive sampling. Changes in concentrations of proteins and in sample volumes bore no apparent relationship to infection of bulls with C. fetus. Low levels of C. fetus agglutinins were detected in sample obtained both before and after infection, but no appreciable rise in antibody titers occurred following infection. Alterations in superficial antigens of C. fetus isolates obtained during the course of infection were demonstrated in the majority of animals. The capacity of the organism to undergo antigenic variation and to provoke a minimal immune response may contribute to its prolonged survival in the preputial cavity.     

Enzootic Pneumonia of Pigs: Identification of a Causative Mycoplasma in Infected Pigs and in Cultures by Immunofluorescent Staining

Immunofluorescent staining has been used to identify Mycoplasma hyopneumoniae in smears of broth cultures, in infected pig testicle cell cultures, and in frozen cut sections of pneumonic lungs from field and experimentally produced cases of enzootic pneumonia. In the pneumonic pig lung, fluorescent staining was limited to the surface of the bronchial and bronchiolar epithelium and to the contained exudate. In a series of trials using experimentally infected pigs fluorescence was not detected until 25 days post-infection and was regularly seen in pigs killed thereafter. Porcine immune globulin precipitated from the serum of experimentally infected pigs and conjugated with fluorescein isothiocyanate was reactive and specific for the detection of M. hyopneumoniae. Immune globulin conjugates prepared from the serum of hyperimmunized rabbits were reactive but in some cases produced a faint non-specific staining of frozen tissue sections. No such non-specific reactions were noted on stained culture smears or cell cultures.

Fluorescence was not seen in known positive preparations stained with non-immune pig globulin conjugates or in preparations from uninoculated cell cultures or pigs, stained with non-immune or immune globulin conjugates.

Mycoplasma hyorhinis was detected by immunofluorescent staining with homologous conjugates, in smears of broth cultures and in tissue sections from pigs with polyserositis.

Immunofluorescent staining was found to be species specific and useful for the early species identification of mycoplasma isolated from pigs.

     

Bovine Infectious Keratoconjunctivitis: Serological Aspects of Moraxella bovis Infection

A modification of a gel diffusion precipitin test (GDPT) was used to detect antibodies for Moraxella bovis (M. bovis) in the sera of cattle affected with bovine infectious keratoconjunctivitis (BIK). The test was also used for the detection of sequential antibody development in cattle vaccinated with cultures of M. bovis. Also, strains of M. bovis isolated from cattle herds affected with BIK were characterized serologically as a part of an identification scheme using the test.

A comparison of the antigenic properties of various strains of M. bovis and M. bovis-like organisms was conducted using the test. The results indicated that there might be antigenic relationships between M. bovisand M. bovis-like organisms such as Moraxella liquefaciens, Moraxella nonliquefaciens, an unidentified hemolytic diplococcus, Mima polymorpha, Mima polymorpha var. oxidans and Herellea vaginicola

The authors suggest that the GDPT can be used for serological studies of BIK, and the identification and antigenic analysis of M. bovis. They indicate, however, that a more definitive study is needed to evaluate the reliability of the test for quantitative work.

     

A detection assay forCampylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA

A rapid screening assay forCampylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C₁/C₂) from the hypervariable region of 16S rRNA ofC. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as threeC. fetus subsp.venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. sputorum subsp.bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C₃/C₂) and two temperature PCR cycling conditions, increased the probability of detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C₁/C₂ PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI artificial insemination - bp base pair - Cff Campylobacter fetus subsp.fetus - Cfv Campylobacter fetus subsp.venerealis - EYT egg yolk Tris - MH Mueller-Hinton - PCR polymerase chain reaction - REA restriction endonuclease analysis - TE Tris-EDTA     

Comparative Study of Agar Mediums for Propagation of Ruminant Mycoplasma

A brain heart infusion agar supplemented with 16.7% rabbit serum (BHIR) was found the most suitable for the culturing of ruminant mycoplasma. Gourlay medium and Perreau medium (4, 5) were not suitable for growth of Mycoplasma mycoides var. mycoides or M. agalactiae, but were satisfactory for M. mycoides var. capri.

Four strains of M. mycoides var. mycoides, three strains of M. agalactiae and three strains of M. mycoides var. capri were grown in our laboratory.

     

Diagnosis and control of Vibrio foetus infection in the bovine

Extract

During the past thirty years Vibrio foetus infection in cattle has been reported from most countries of the world. Sporadic abortions, usually in mid-gestation, have been accepted until recently as the most important aspect of the infection. Of late years, however, it has become apparent that V. foetus can be responsible also for poor conception rates-associated probably with early embryonic mortality. It has been shown further that V. foetus can be transmitted not only by the bull at the time of service but also in semen used for artificial in semi nation.     

Serological Analysis of Thermolabile Antigens of Vibrio anguillarum

Abstract

Serological analysis of thermolabile antigens of representative strains belonging to the J-O-1, J-O-2, and J-O-3 serotypes of Vibrio anguillarum, which are the major serotypes of thermostable antigens (O-antigens) in Japan, showed that the strains belonging to each O-serotype possessed a common thermolabile antigen designated as k-1. In addition to the k-1 antigen, strains of the J-O-1 serotype of biovar II V. anguillarum possessed another antigen designated as k-2. These findings were confirmed by examining additional strains belonging to each O-serotype. The k-1 antigen was found in all the strains of J-O-4, J-O-5, J-O-6, J-O-7, and J-O-8 serotypes, which are minor serotypes in Japan. The same antigen was also found in some strains of V. metschnikovii, Aeromonas hydrophila, and A. salmonicida.     

Isolation and Characterization of Large Marine Bacteriophage (Myoviridae), VhKM4 Infecting Vibrio harveyi

Abstract

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7.

Received June 16, 2016; accepted October 7, 2016 Published online February 6, 2017     

Bovine Campylobacteriosis: A Review

Campylobacteriosis (vibriosis) is a venereal disease of cattle caused by the organism Campylobacter fetus subspecies fetus previously known as Vibrio fetus subspecies venerealis. Characteristically the disease causes infertility in the female with an increased number of services necessary for conception. Abortions late in gestation are also occasionally seen. Most cases or outbreaks occur after the recent introduction of an infected bull or cow into a susceptible breeding herd. Often the disease remains undetected until late fall when the livestock owner recognizes that he has a number of females exhibiting estrus. A tentative diagnosis can be made by a study of the herd history and can often be confirmed by laboratory means.

In recent years many advances have been made towards establishing an understanding of the immune response that occurs with infection and systemic immunization. In this review, recommendations are made regarding the appropriate time to immunize the breeding herd against campylobacteriosis.

     

Prevalence of bovine campylobacteriosis in indigenous cattle of three states in Nigeria

Summary A survey of bovine campylobacteriosis in breeding bulls and cows was carried out in the states of Kaduna, Kano and Borno. Six hundred and eighty nine cattle composed of 585 and 104 breeding bulls and cows respectively were sampled.Campylobacter fetus subsp.venerealis was isolated from 12 bulls whileCampylobacter fetus subsp.fetus was isolated from three of them. Campylobacter fetus subsp.fetus was isolated fromfour cows while Campylobacter fetus subsp.venerealis was isolated from one cow. The overall prevalence of campylobacteriosis in the three states was 2.9% (20/689). The result of the study identifiesCampylobacter fetus subsp.venerealis as the agent of enzootic infertility in Nigeria and suggests that it may be a significant problem.

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Resumen Un estudio sobre la prevalencia de campilobacteriosis en reproductores machos y hembras bovinos se llevó a cabo en los estados de Kaduna, Kano y Borno. Seiscientos ochenta y nueve animales, compuestos de 585 y 104 toros reproductores y vacas respectivamente, fueron examinados.Campylobacter fetus subsp.venerealis fue aislado de 12 toros, mientras queC. fetus subsp.fetus, se aisló de tres de ellos.C. fetus subsp.fetus se aisló de cuatro vacas, mientras queC. fetus subsp.venerealis, se aisló de una vaca. La prevalencia general de campilobacteriosis en los tres estados fue de 2.9% (20/689). El resultado de este estudio identifica alC. fetus subsp.venerealis, como el agente de infertilidad enzoótica en Nigeria, y sugiere que este puede ser un problema significativo.

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Résumé Une enquête sur la campylobactériose bovine a été menée sur du bétail reproducteur dans les états de Kaduna, Kano et Borno au Nigeria. Elle a porté sur 689 bovins dont 585 taureaux et 104 femelles.C. foetus subsp.veneralis a été isolé sur 12 taureaux etC foetus subsp.foetus sur 3 d'entre eux.C. foetus subsp.foetus a été décelé sur 4 vaches tandis queC. foetus subsp.foetus ne 1'a été que sur une seule. L'a prévalence globale de la maladie dans les 3 états représente 2,9 p. 100, soit cas sur 689 examinés. Cette enquête a permis d'identifierC. foetus subsp.veneralis comme étant responsable d'une infertilité enzootique au Nigeria. Elle indique que cette affection peut constituer un important problème.

     

Detection of Campylobacter Antibodies in Sheep Sera by a Dot‐ELISA Using Acid Extracts from C. fetus ssp. fetus and C. jejuni Strains and Comparison with a Complement Fixation Test

In this study, a dot‐enzyme‐linked immunosorbent assay (Dot‐ELISA) was evaluated in comparison with a complement fixation test (CFT) for the detection of Campylobacter antibodies in sheep sera. Acid glycine extracts (AGE) of both Campylobacter fetus ssp. fetus and Campylobacter jejuni strains that had been isolated from the gall‐bladder of slaughtered sheep was used as antigen in both tests. A total of 153 sheep sera from aborted (74) and slaughtered (79) sheep were examined by both Dot‐ELISA and CFT. Twenty‐two sera showed anti‐complementary activity were not suitable for CFT. Of the 22 sera showing anti‐complementary activity, two sera were found to be positive in Dot‐ELISA. Eighty‐eight (67.2%) of the remaining 131 sera were negative by both Dot‐ELISA and CFT using AGE of both Campylobacter strains whereas 43 sera (32.8%) gave different reaction patterns in Dot‐ELISA and CFT with the extracts of both Campylobacter strains. Twelve sera were positive by both tests using AGE of C. fetus ssp. fetus but CFT failed to detect antibodies in nine of these sera when AGE of C. jejuni was used. Twelve sera were positive by both tests only when AGE of C. fetus ssp. fetus was used. Eleven sera were positive only by CFT. Seven of these reacted only with the AGE of C. fetus ssp. fetus and four sera were positive by using AGE of both Campylobacter strains. The remaining eight sera were found to be positive only by dot‐immunobinding assay either with the AGE of both Campylobacter strains or with the AGE of one of the Campylobacter strains. It is concluded that Dot‐ELISA using AGE from C. fetus ssp. fetus could be employed for the detection of Campylobacter antibodies in sheep sera and the additional use of AGE from C. jejuni as antigen appeared not to be profitable for this purpose.     

Differentiation Among Strains of Goat Mycoplasma by Incident Light Immunofluorescence

Antigenic differentiation between strains of goat mycoplasma was studied by direct fluorescent antibody reactions employing incident (vertical) ultraviolet light. Agar colonies of the mycoplasma grown in petri dishes were fixed by alcohol in situ, and stained with conjugated globulin before examination with ultraviolet light.

The fluorescent antibody (FA) conjugate against Vom strain of Mycoplasma mycoides var. capri was Vom strain-specific, no cross reaction with Mexico, Connecticut, or Maryland strains. Similarly, the Mexico strain conjugate was specific for colonies of Mexico, and did not cross with the Vom, strain. Additionally, the conjugate of the PG-2 strain of Mycoplasma agalactiae, which was specific for the colonies of PG-2 was refractory for the strain #99 of M. agalactiae.

It was therefore possible to utilize an immunofluorescent technique (incident ultraviolet light) to demonstrate differences among strains of M. mycoides var. capri and M. agalactiae.

     

副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用

为建立同时快速检测海产品中的副溶血弧菌(VP)和溶藻弧菌(VA)的双重PCR方法,本研究根据VP和VA的toxR基因序列设计针对这两种细菌的两对特异性引物,建立能够快速同时检测这两种细菌的双重PCR方法,并对该反应体系的特异性和灵敏度进行检测。结果显示纯培养细菌VP和VA的检测灵敏度分别为2.32×103cfu/mL和2.56×103cfu/mL,临床病料检测灵敏度分别为2 cfu和3 cfu;与枸橼酸杆菌、沙门氏菌、美人鱼弧菌、霍乱弧菌、麦氏弧菌无交叉反应。研究表明本实验方法操作简便快速、特异性强、灵敏度高、稳定性好,并且经济实惠,值得推广应用。