Comparison of the Johne''s absorbed EIA and the complement-fixation test for the diagnosis of Johne''s disease in cattle

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.     

A role for DNA characterisation in ovine footrot

Bacteroides nodosus involved in several outbreaks of ovine footrot over a number of years were subjected to DNA restriction endonuclease analysis. Individual isolates were found to have characteristic Bam HI profiles which permitted their accurate identification and differentiation from other isolates. Bam HI profiles of B. nodosus isolates were used in epidemiological investigations involving consecutive outbreaks of footrot on individual and neighbouring farms. The relationship of given isolates to a common source could be established by this means. Restriction endonuclease analysis provides an additional epidemiological tool in ovine footrot investigations as it accurately identifies interstrain differences in a manner not possible by conventional bacteriological and serological means.     

Evaluation of the gamma-interferon assay for eradication of tuberculosis in a goat herd

Objective To evaluate the usefulness of the gamma-interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence.
Design An immunological study involving four serial comparative gamma-interferon and single intradermal tuberculin tests.
Animals A herd of 87 goats of Guadarrama breed.
Procedure Serial testing and segregation of animals.
Results We found that the number of infections detected by the gamma-interferon test was considerably greater than the number detected by the single intradermal tuberculin test. A group of 10 animals was negative to both tests in two consecutive rounds and three kids were negative in the last round of testing.
Conclusions Gamma-interferon assay is appropriate for diagnosis and eradication of tuberculosis in goats. This test is able to detect early Mycobacterium bovis infection. Avian reactors with simultaneous increased reaction to bovine PPD in the gamma-interferon assay (designated as avian^B reactors) should be considered test positive for M bovis . By serial testing with the gamma-interferon and the single intradermal tuberculin tests, and a policy of segregation of kids at birth, it is possible to achieve a group of animals test negative for tuberculosis from a herd of goats with high immunoreactivity to this infection.     

Identification and isolation of a novel herpesvirus in a captive mob of eastern grey kangaroos (Macropus giganteus)

A novel herpesvirus was detected in a captive mob of eastern grey kangaroos (Macropus giganteus) during diagnostic workup for individuals with ulcerative cloacitis. Virus was initially detected in tissues using a consensus herpesvirus PCR. No viral inclusions or particles had been evident in routine histologic or transmission electron microscopic sections of cloacal lesions. Virus was isolated from samples and transmission electron microscopy of the resulting isolates confirmed that the virus was morphologically consistent with a herpesvirus. Nucleotide sequencing of the PCR product from tissue samples and from the isolates revealed that the virus was in the subfamily Gammaherpesvirinae and was distinct from other known herpesviruses. The correlation between the lesions and the novel virus remains unknown. Two herpesviruses, both in the subfamily Alphaherpesvirinae, have previously been described in macropods and are known to cause systemic clinical disease. This is the first reported gammaherpesvirus within the order Marsupialia, and may provide valuable information regarding the evolution and phylogeny of this virus family. Based on current herpesvirus nomenclature convention, the authors propose the novel herpesvirus be named Macropodid herpesvirus 3 (MaHV-3).     

Two herpesviruses associated with disease in wild Atlantic loggerhead sea turtles (Caretta caretta)

Herpesviruses are associated with lung-eye-trachea disease and gray patch disease in maricultured green turtles (Chelonia mydas) and with fibropapillomatosis in wild sea turtles of several species. With the exception fibropapillomatosis, no other diseases of wild sea turtles of any species have been associated with herpesviral infection. In the present study, six necropsied Atlantic loggerhead sea turtles (Caretta caretta) had gross and histological evidence of viral infection, including oral, respiratory, cutaneous, and genital lesions characterized by necrosis, ulceration, syncytial cell formation, and intranuclear inclusion bodies. Nested polymerase chain reaction targeting a conserved region of the herpesvirus DNA-dependent-DNA polymerase gene yielded two unique herpesviral sequences referred to as loggerhead genital-respiratory herpesvirus and loggerhead orocutaneous herpesvirus. Phylogenetic analyses indicate that these viruses are related to and are monophyletic with other chelonian herpesviruses within the subfamily alpha-herpesvirinae. We propose the genus Chelonivirus for this monophyletic group of chelonian herpesviruses.     

A Study of Endometritis Causing Repeat Breeding of Cycling Iraqi Buffalo Cows

The objectives of this study were to determine the non‐specific aerobic and anaerobic bacterial causes of endometritis causing repeat breeding of cycling Iraqi buffalo cows at Nineveh province, validate diagnostic criteria for endometritis and to evaluate the treatment efficiency of using systemic or intra‐uterine infusion of antibiotics for the treatment of endometritis. Data were collected from 60 buffalo cows with history of repeat breeding in different herds. All buffaloes were subjected to detailed clinical examination including external inspection, vaginoscopy and transrectal palpation of the cervix, uterus and ovaries. Swabs for bacteriology and biopsies for histopathology were collected from the uterine lumen from each cow. Character, odour and estimation of polymorphonuclear cells (PMN) of the vaginal mucus were scored. Blood samples were collected from cows for creatine kinase (CK) and aspartate aminotransferase (AST) measurement. Treatment conducted using oxytetracycline with tylosin in local intrauterine infusion or systemically with hormonal treatment. The most pre‐disposing factor for uterine infection was retained placenta (13.3%). The most prevalent bacteria in uterine lumen were E. coli (23%), Archanobacterium pyogenes (13%) and Staphylococcus aureus (10%) were mostly isolated from buffaloes with repeat breeding. Vaginal mucus character score was associated with the bacterial growth density score. The difference in PMN was highly significant (p < 0.01) in animals with repeat breeding than control groups. In addition, PMNs was significantly (p < 0.01) correlated r = 0.894 with the character of vaginal discharge. High level of PMNs observed in buffaloes infected with A. pyogenes. Buffalo cows with endometritis had higher CK (321.47 ± 39.06 vs 162.01 ± 16.41 U/l) and AST (133.93 ± 12.43 vs 97.01 ± 6.86 U/l) activities (p < 0.05) than control‐heifers, but no significant difference was observed between buffalo cows with endometritis in CK (321.47 ± 39.06 vs 208.33 ± 5.84) and AST (133.93 ± 12.43 vs 156.17 ± 9.65) activities than control‐pluriparious. It could be concluded that A. pyogenes was the only non‐specific uterine pathogen directly associated with severe endometrial lesions. Vaginoscopy examination combined with palpation of uterus increase the accuracy of diagnosing endometritis and cytogenic examination of uterine discharge is more reliable method of establishing the presence or absence of uterine inflammation in buffalo cows. Animals with repeat breeding (endometritis) showed clinical cure and improved pregnancy in all treatment groups with no significant difference. The use of oestradiol in repeat breeder cases has no effect in improving neither clinical cure rate nor pregnancy rate.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.     

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types.

LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR.

A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%).

DIAGNOSIS: Bovine haemoplasmosis.

CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.     

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO₂ and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H₂DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca²⁺ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.