Use of a Quantitative TaqMan‐PCR for the Fast Quantification of Mycobacteria in Broth Culture,Eukaryotic Cell Culture and Tissue

The quantification of slow‐growing mycobacteria such as Mycobacterium tuberculosis or M. bovis from in vitro and in vivo samples is complicated by their long generation time, their ability to form aggregates, and their capacity to persist in a state of dormancy. We compared different methods for the establishment of growth curves for broth cultures of M. bovis bacille Calmette‐Guérin (BCG). A quantitative TaqMan‐PCR yielded results comparable with those obtained by protein quantification and measurement of the ATP content of the cultures. The quantitative TaqMan‐PCR furthermore turned out to be particularly suitable for the measurement of multiplication of BCG within eukaryotic cells. Furthermore, it is a fast method allowing an estimation of the mycobacterial load in tissue long before colony counts can be obtained.     

Oral vaccination of brushtail possums with BCG: Investigation into factors that may influence vaccine efficacy and determination of duration of protection

AIMS: To determine factors that may influence the efficacy of an oral pelleted vaccine containing Mycobacterium bovis bacille Calmette-Guérin (BCG) to induce protection of brushtail possums against tuberculosis. To determine the duration of protective immunity following oral administration of BCG.

METHODS: In Study 1, a group of possums (n=7) was immunised by feeding 10 pellets containing dead Pasteur BCG, followed 15 weeks later with a single pellet of live Pasteur BCG. At that time, four other groups of possums (n=7 per group) were given a single pellet of live Pasteur BCG orally, a single pellet of live Danish BCG orally, 10 pellets of live Pasteur BCG orally, or a subcutaneous injection of live Pasteur BCG. For the oral pelleted vaccines, BCG was formulated into a lipid matrix, and each pellet contained approximately 10⁷ colony forming units (cfu) of BCG, while the vaccine injected subcutaneously contained 10⁶ cfu of BCG. A sixth, non-vaccinated, group (n=7) served as a control. All possums were challenged by the aerosol route with a low dose of virulent M. bovis 7 weeks after vaccination, and killed 7–8 weeks after challenge. Protection against challenge with M. bovis was assessed from pathological and bacteriological findings.

In Study 2, lipid-formulated live Danish BCG was administered orally to three groups of possums (10–11 per group), and these possums were challenged with virulent M. bovis 8, 29 or 54 weeks later. The possums were killed 7 weeks after challenge, to assess protection in comparison to a non-vaccinated group.

RESULTS: The results from Study 1 showed that vaccine efficacy was not adversely affected by feeding dead BCG prior to live BCG. Feeding 10 vaccine pellets induced a level of protection similar to feeding a single pellet. Protection was similar when feeding possums a single pellet containing the Pasteur or Danish strains of BCG. All vaccinated groups had significantly reduced pathological changes or bacterial counts when compared to the non-vaccinated group. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis.

CONCLUSIONS: Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.     

Nodular presentation of Dirofilaria repens infection in a cat mimicking a fibrosarcoma

A cat with multiple subcutaneous nodules suggesting a soft tissue sarcoma by physical and computed tomographic examination was diagnosed as being affected by subcutaneous filariosis based on cytologic and ultrasonographic assessments. Nodules were surgically removed and extracted nematodes were identified by PCR as Dirofilaria repens. Furthermore, DNA of Dipetalonema dracunculoides (syn. Acantocheilonema dracunculoides) was detected by PCR, with no evidence of circulating microfilariae. To the best of the authors' knowledge, this represents the first report describing adults of D repens in multiple subcutaneous nodules in a cat. Cytopathologic examination allowed characterization of the parasitic nature of the nodules. Veterinary practitioners should be aware of the possible nodular presentation of D repens in cats and should include D repens in the differential diagnosis of subcutaneous neoformations in the cat.     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection.

METHODS: Possums were inoculated with 5 x 10⁶ colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7), orally (n=10), via the endo-bronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 10⁶ cfu and half of which received 5 x 10⁷ cfu of BCG). The possums were humanely killed between 1–4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically.

RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endo-bronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i.

CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intra-dermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis.

METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 10⁵ colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis.

RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05).

CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

Detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in tissue samples of cattle and buffaloes

Tissue samples were collected at random from cattle (Bos taurus) and buffalo (Bubalus bubalis) from an abattoir of the district of Lahore and were analyzed for the presence of Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis through acid-fast staining and polymerase chain reaction (PCR). Body condition of animals and diarrhea were recorded. Most of the animals were emaciated. Diarrhea was noticed in 15.6% of buffaloes and 19.2% of cattle. Intestinal pathology was observed in 29% of buffaloes and 32.8% of cattle. Number of mesenteric lymph node (MLN) showing gross lesions was a bit higher (35.6%) in cattle than buffalo (31.2%). Acid-fast staining of tissue scraping smears revealed the presence of acid-fast bacilli (AFB) in 17.4% intestinal and 16.4% MLN tissue samples in buffalo, while in cattle 19.2% intestinal and 17.8% MLN were found positive for AFB. In buffaloes, PCR confirmed 12.8% intestinal and 12.4% MLN positive samples for M. avium subsp. paratuberculosis. However, in cattle, PCR analysis demonstrated 14.2% positive results for M. avium subsp. paratuberculosis in both MLN and intestinal tissue samples. PCR also confirmed M. bovis in 5.8% of cattle and 5% of buffalo MLN and intestinal tissues. PCR positive tissue samples for M. avium subsp. paratuberculosis were from those animals which were emaciated, having diarrhea, and severe gross lesions. AFB were also detected in tissue scraping smears of these animals. It is concluded that infection by various mycobacterium species can be differentiated by PCR, which is not possible by acid-fast staining technique.     

Molecular Typing of Mycobacterium bovis Isolates in Argentina: First Description of a Person‐to‐Person Transmission Case

Bovine tuberculosis is caused by Mycobacterium bovis, a mycobacterium highly similar to M. tuberculosis that belongs to the M. tuberculosis complex. The main host of M. bovis is cattle but it also affects many other mammalians including humans. Tuberculosis in humans caused by either M. bovis or M. tuberculosis is clinically hard to distinguish. During 2004–2005, samples from 448 patients with diagnosis of TB were collected from different regions of Argentina. The PRA technique identified 400 isolates with representative patterns of mycobacterium. The predominant ones were the M. tuberculosis complex, the M. avium–M. intracellulare complex and M. gordonae. Samples with M. tuberculosis complex PRA restriction profiles were analyzed with a multiplex PCR to differentiate between M. tuberculosis and M. bovis. Multiplex PCR identified nine M. bovis. The results allowed the possibility to establish that 2% of pulmonary tuberculosis was due to M. bovis. Isolates of M. bovis from humans were examined using spoligotyping. These isolates presented five different spoligotypes. The main spoligotype was also the most frequently one found in cattle. The remaining human spoligotypes (grouped in clusters) are occasionally found in cattle. Variable number tandem repeat (VNTR) analysis identified five different patterns. By combining the results of spoligotyping and VNTR analysis, we were able to differentiate seven M. bovis isolates. The remaining two M. bovis samples showed the same spoligotype and VNTR profile and belonged to household contacts. An MDR‐M. bovis was isolated from the samples of these household contacts. The identification of two epidemiologically linked cases of human M. bovis infection suggests person‐to‐person transmission of an MDR‐M. bovis.     

Cavernous tuberculosis of the lungs and secondary hypertrophic osteo-arthropathy in a Siberian tiger (Panthera tigrus)

In a colony of large felines, a Siberian tiger died after having shown lameness for 4 months. X-ray examination 3 months prior to death revealed extensive bone proliferations around the left carpus and cavitations in the lungs. In spite of tuberculostatic therapy the animal died and a final diagnosis of cavernous lung tuberculosis(M. bovis) and a related osteo-arthropathy was made. A 7-year-old Siberian tiger owned by the Royal Rotterdam Zoo ‘Blijdorp’, was brought for examination to the veterinary surgery clinic of the State University of Utrecht. The patient walked with difficulty and showed thickening of the left and right carpi. The weight of the animal was 180 kg. After sedation by means of an intramuscular injection of 45 mg of phencyclidine (Sernylan-Parke Davis) and 90 mg of acepromazine (Ventranquil-Philips Duphar), combined with 10 mg of atropine, it was possible to inject 19 ml of 5 % thiopentone sodium (B.P. Boots Pure Drug Go. Ltd) into the tail vein. Narcosis was maintained using nitrous oxide and halothane (Fluothane-ICI Ltd). Résumé. Un tigre de Sibérie, appartenant à un groupe de gros félins est mort après avoir présenté pendant 4 mois des phénomènes paralytiques. Un examen radiologique, effectué 3 mois avant la mort a révélé d'importantes proliférations osseuses autour du carpe gauche et la présence de cavernes pulmonaires. Malgré l'institution d'un traitement anti-tuberculeux, l'animal mourut, et le diagnostic final était celui de tuberculose pulmonaire à Mycobacterium bovis avec ostéo-arthropathie de la même origine. Zusammenfassung. In einer Grosskatzenkolonie starb ein sibirischer Tiger, nachdem er 4 Monate lang Lahmheit gezeigt hatte. Die Röntgenuntersuchung 3 Monate vor dem Tode zeigte ausgedehnte Knochenwucherungen um den linken Carpus und Cavitationen in der Lunge. Trotz tuberculostatischer Therapie starb das Tier, und die abschliessende Diagnose lautete auf cavernöse Lungentuberkulose (M. bovis) und eine damit in Beziehung stehende Osteo-arthropathie.     

Pancytopenia in a cat with visceral leishmaniasis

Abstract: A 4‐year‐old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune‐mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.     

Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd

This report describes an outbreak of Mycoplasma bovis mastitis affecting 45 cows in a herd of 122 dairy cattle in Northern Italy. Clinically, the outbreak was characterized by agalactia, multiple swollen and painless quarters, high milk somatic cell count and unresponsiveness to conventional antibiotic therapy. M. bovis was isolated from the milk samples of all the 32 affected cows tested and from the mammary tissue of three affected cows that underwent necropsy. No other pathogens were isolated from these samples. Lesions in two of the necropsied cows were characterized by mild chronic suppurative mastitis and galactophoritis. The other necropsied cow showed a chronic necrosuppurative and pyogranulamaous galactophoritis, a condition not previously associated with M. bovis. M. bovis was detected immunohistochemically in the lumen of the affected mammary ducts suggesting that ascending infection via the teat canal was the likely route of transmission. No other intralesional pathogens were demonstrated microscopically.     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Design,optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil

Background: ‘Candidatus Mycoplasma turicensis’ (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and ‘Candidatus M. haemominutum’ (CMhm) but not for CMtc. Although real‐time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries. Objectives: The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false‐negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Methods: Species‐specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10‐fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica‐based protocol and tested using the PCR assay. Results: Primer concentration, annealing temperature, and MgCl₂ concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. Conclusion: These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.     

Cutaneous Mycobacterium avium Infection in a Cat

Abstract— A 6-year-old spayed female domestic shorthair cat with a paranasal cutaneous mass caused by Mycobacterium avium is reported. Recurrence after cryosurgery and nonresponsiveness to dapsone necessitated surgical removal. The cat remained clinically normal for 14 months when the occurrence of multiple facial nodules with acid-fast bacilli present caused the owner to elect euthanasia. Résumé— Le cas d'une chatte européenne castrée de six ans présentant une masse périnasale due àMycobacterium avium est rapporté. La rechute après cryochirurgie et l'absence de réponse à la dapsone a nécessité une ablation chirurgicale. Le chat est resté apparemment sain 14 mois après l'opération, jusqu'à l'appartion de nodules multiples sur la face avec des baciles acid-alcolorésistants. L'animal a été euthanasié. Zusammenfassung— Beschrieben wird eine kurzhaarige Hauskatze, sechs Jahre alt, weiblich, kastriert, mit einer paranasalen kutanen Zubildung, die durch Mycobacterium avium verursacht wurde. Eine Wiederauftreten nach Kryochirurgie und ein Nichtansprechen auf Dapson zwang zu einer chirurgischen Entfernung. Die Katze blieb 14 Monate lang klinisch gesund bis das Auftreten von multiplen fazialen Knoten mit histologisch nachweisbaren säurefesten Bazillen dazu führte, daß der Besitzer sich für die Euthanasié des Tieres entschied. Resumen En este artículo se estudia una gata castrada de seis años de raza moméstica de pelo corto, con una masa cutánea paranasal causada por el Mycobacterium avium. La masa fue removida quirürgicamente después del tratamiento con dapsone y la aplicación de criocirugía con la consiguiente reaparición de la misma. Por un periodo de 14 meses la gata permaneció clínicamente normal, pero después el animal desarrolló nódulos faciales multiples con la aparición de bacilos que podian ser teñidos con tinturas ácidas, ante lo cual el dueño decidió terminar con la vida del animal.     

Prevalence ofBabesia bovis andAnaplasma marginale at selected localities in Sri Lanka

Summary Sera were collected from a minimum of 20 cattle aged nine to 36 months at each of 14 localities in five climatic zones of Sri Lanka. Sera were tested for antibodies toBabesia bovis andAnaplasma by an indirect fluorescent antibody test and a card agglutination test, respectively. Antibodies toB. bovis andAnaplasma were detected in all samples tested from each of 14 and 12 localities respectively. In general, prevalences were consistently high among localities below 1,200m and lower and more variable in the hill country. Management practice rather than climate was considered to be the main factor influencing prevalences. Mangement is discussed in relation to the risk of disease outbreak.

---

Resumen Se colectó suero de un minimo de 20 bovinos, en edades comprendidas entre 9–36 meses, en cada una de 14 localidades en 5 zonas climáticas de Sri Lanka. El suero fue examinado para detectar anticuerpos contraB. bovis yAnaplasma mediante la prueba indirecta de anticuerpos fluorescentes y la prueba de la tarjeta, respectivamente. Se detectaron anticuerpos contraB. bovis yAnaplasma en todas las muestras examinadas en 14 y 12 localidades respectivamente. En general, las prevalencias fueron mayores por debajo de los 1200 m.s.n.m. y menores en zonas monta?osas. Se consideraron las prácticas de manejo, más que el clima, como el factor principal que influenció las prevalencias. Se discute el manejo en relación con el riesgo de un brote de la enfermedad.

---

Résumé Des sérums one été sur un minimum de 20 bovins agés de 9 à 36 mois pour chacune des 14 localités de 5 zones climatiques du Sri Lanka. Les sérums ont été testés pour la recherche des anticorps deBabesia bovis etAnaplasma respectivement, par fluorescence indirecte et test d'agglutination sur carte. Les anticorps àB. bovis et àAnaplasma ont été détectés respectivement dans 14 et 12 localités. En général, la prévalence était constamment plus élevée dans les localités au-dessous de 1200m et plus basse et plus variable dans les zones de collines. Les méthodes d'élevage, plus que le climat, sont considérées comme le facteur influan?ant le plus les prévalences. La gestion des troupeaux et la relation avec le risque d'apparition d'un foyer sont discutées.

     

Seroprevalence of bovine tuberculosis infection in yaks (Bos grunniens) on the Qinghai-Tibetan Plateau of China

Bovine tuberculosis (BTB), a chronic disease caused by Mycobacterium bovis, has been widely reported in bovines in developing countries, but there is little information on this infection in domestic yaks. Seroprevalence of antibodies to M. bovis in yaks from six and three regions of Tibet and Qinghai plateau, China, respectively, was investigated in 2011 by enzyme-linked immunosorbent assay kits. A total of 1,244 (Tibet 938, Qinghai 306) blood samples was collected and the results showed that 24 (2.6 %) of Tibetan samples and 3 (1 %) of Qinghai's samples were positive for BTB. The findings of the present study indicated that M. bovis infection is prevalent in Chinese yaks in Qinghai and Tibet. These observations should raise a serious public health concern considering the extent to which the herdsmen of the study areas are in contact with their animals and the levels at which they use untreated livestock products. This is the first study showing the infection of M. bovis in domestic yaks.     

Parasitemia due to Sarcocystis neurona‐like infection in a clinically ill domestic cat

An 8‐year‐old, 6‐kg, male neutered Domestic Shorthair cat was presented to The Ohio State University Veterinary Medical Center (OSU‐VMC) for difficulty breathing. Physical examination and thoracic radiographs indicated pneumonia, a soft‐tissue mass in the left caudal lung lobe, and diffuse pleural effusion. The effusion was classified as modified transudate. Rare extracellular elongated (~5–7 μm × 1–2 μm) zoites with a central round to oval‐shaped purple to deep purple vesicular nucleus with coarsely stippled chromatin and light blue cytoplasm were seen on a peripheral blood smear. Serum IgG and IgM were positive for Sarcocystis sp. antibodies and negative for Toxoplasma gondii antibodies, suggesting that the infection was acute rather than a recrudescence of prior infection. This organism was most consistent with either Sarcocystis neurona or Sarcocystis dasypi based on DNA sequence analysis of PCR products using COC ssRNA, ITS‐1, snSAG2, and JNB25/JD396 primer sets. This is the first report to visualize by light microscopy circulating Sarcocystis sp. merozoites in the peripheral blood of a domestic cat. Therefore, Sarcocystis should be considered as a differential diagnosis in cats with suspected systemic protozoal infection.     

First report of the use of mucosal swabs of the palatine tonsillar crypt for detection of Mycoplasma bovis in naturally infected calves

ABSTRACT

Aims: To compare detection by real-time PCR of DNA from Mycoplasma bovis on mucosal swabs taken from the palatine tonsillar crypt and the mainstem bronchi of clinically asymptomatic calves after slaughter.

Methods: We compared the sensitivity of mucosal swabs taken from two sites: the palatine tonsillar crypt and the mainstem bronchi. Paired samples were taken post-mortem at slaughter from 55 clinically well calves from an infected herd and were tested by real-time PCR for the presence of M. bovis-specific DNA.

Results: Mycoplasma bovis DNA was detected in 51 palatine tonsillar crypt swabs (92.7 (95% CI?=?82.4–98.0)%) and seven mainstem bronchial swabs (12.7 (95% CI?=?5.3–24.5)%). All seven calves with positive mainstem bronchial swabs also had positive palatine tonsillar crypt swabs.

Conclusions: When compared to mucosal swabs of the mainstem bronchi, mucosal swabs of the palatine tonsillar crypt were seven times more sensitive for the post-mortem detection of M. bovis DNA. The viability of detected M. bovis was not assessed, because any cattle carrying viable or non-viable M. bovis DNA were determined to be a potential risk to eradication. Palatine tonsillar crypt mucosa may be a useful anatomical site for real-time PCR detection of M. bovis DNA in naturally infected calves. More work is needed to define the persistence and viability of M. bovis at this anatomical site.

Clinical relevance: The results of this study helped form the basis of surveillance tools used in M. bovis control and eradication efforts. Familiarity with these results may help veterinarians better communicate with their clients about the science behind the eradication efforts.     

Selection for high immune response: an alternative approach to animal health maintenance?

To test the hypothesis that variation in ability to respond immunologically correlates with health, Yorkshire pigs were bred for high (HIR) and low (LIR) antibody (Ab) and cell-mediated immune response (CMI). Selection was based on standardized measures of Ab (secondary response to hen egg white lysozyme, serum IgG concentration) and CMI (cutaneous delayed-type hypersenstivity to purified protein derivative of tuberculin after immunization with bacillus Calmette-Guérin and in vitro lymphocyte response to Con-A). Differences in Ab and CMI by line were not restricted to the antigens used in the selection. Antibody response to vaccines was highest in HIR and non-responders were restricted to LIR pigs. The HIR pigs had the best rate of weight gain. After infection with Mycoplasma hyorhinis, HIR developed more severe arthritis and less polyserositis. Differences were associated with variation in cytokine message in joint-related cells. Following exposure to attenuated transmissible gastroenteritis virus, natural killer cells of the LIR pigs but not of HIR or control lines, were unresponsive. Genetic selection for Ab and CMI may provide health and productivity advantages and complement traditional health-maintenance methods.     

Development of real-time PCR assay for the detection of Mycoplasma bovis

Mycoplasma bovis is one of the important bovine mycoplasma involved in economically important clinical conditions like respiratory diseases, otitis media, and mastitis. The present study was undertaken with the objective of developing a SYBR Green dye-based real-time PCR assay targeting uvrC gene for the diagnosis of M. bovis. The analytical sensitivity and specificity of the assay were evaluated. The test showed 10³-fold more sensitivity than conventional PCR and detected down to 100 fg level of DNA. It was found to be specific, as no cross reactivity was shown with other related bacteria and Mycoplasma species. The developed assay was able to detect down to 40 copies of uvrC gene from spiked bovine milk samples. At present, this developed assay may be used as a valuable diagnostic tool for the detection of Mycoplasma bovis.