Miriam Maas

Assessing the impact of feline immunodeficiency virus and bovine tuberculosis co-infection in African lions

Bovine tuberculosis (BTB), caused by Mycobacterium bovis, is a disease that was introduced relatively recently into the Kruger National Park (KNP) lion population. Feline immunodeficiency virus (FIVple) is thought to have been endemic in lions for a much longer time. In humans, co-infection between Mycobacterium tuberculosis and human immunodeficiency virus increases disease burden. If BTB were to reach high levels of prevalence in lions, and if similar worsening effects would exist between FIVple and BTB as for their human equivalents, this could pose a lion conservation problem. We collected data on lions in KNP from 1993 to 2008 for spatio-temporal analysis of both FIVple and BTB, and to assess whether a similar relationship between the two diseases exists in lions. We found that BTB prevalence in the south was higher than in the north (72 versus 19% over the total study period) and increased over time in the northern part of the KNP (0–41%). No significant spatio-temporal differences were seen for FIVple in the study period, in agreement with the presumed endemic state of the infection. Both infections affected haematology and blood chemistry values, FIVple in a more pronounced way than BTB. The effect of co-infection on these values, however, was always less than additive. Though a large proportion (31%) of the lions was co-infected with FIVple and M. bovis, there was no evidence for a synergistic relation as in their human counterparts. Whether this results from different immunopathogeneses remains to be determined.

Repeatability evaluation of a dairy farm management questionnaire

Abstract The purpose of the research reported in this paper was to examine the repeatability of a dairy farm management questionnaire. The questionnaire was used in a study of the effects of dairy farm management and bulk tank milk protein concentration (BTMPC). The repeatability was studied by a simple test-retest design and was incorporated into the overall design of the management-bulk tank milk protein project. The source population was farms in a specific region of the Netherlands that delivered milk without interruption from February 1987 onward, and had annual milk quotas of at least 250 000 kg. Repeatabilities of categoricaly scaled questionnaire items were estimated by the intraclass kappa coefficient. Repeatabilities of continuous-scaled items were estimated by Pearsons intraclass correlation coefficient. The standard error of each coefficient was estimated via a jackknife procedure. Influence of the following factors on question repeatability was examined: method of questionnaire administration, type of question, category of dairy farm management, and stratum from which the farms were sampled. The study also investigated the effect of dichotomization of polytomous response data on repeatability. Seventy-one percent of the questionnaire items ultimately introduced into the crude analysis of the farm management-BTMPC study had chance-corrected repeatability coefficients greater than or equal to 0.40. Sixty-nine percent of the items had coefficients greater than or equal to 0.50. Repeatability quality of the questionnaire was judged to be adequate and recommendations are made for dairy farm management questionnaire formulation.

The elephant interferon gamma assay: a contribution to diagnosis of tuberculosis in elephants

Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other species including elephants and vice versa, in case of prolonged intensive contact. An accurate diagnostic approach covering all phases of the infection in elephants is required. As M. tb is an intracellular pathogen and cell-mediated immune (CMI) responses are elicited early after infection, the skin test is the CMI assay of choice in humans and cattle. However, this test is not applicable in elephants. The interferon gamma (IFN-γ) assay is considered a good alternative for the skin test in general, validated for use in cattle and humans. This study was aimed at development of an IFN-γ assay applicable for diagnosis of tuberculosis in elephants. Recombinant elephant IFN-γ (rEpIFN-γ) produced in eukaryotic cells was used to immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-γ-specific monoclonal antibody production and subcloned, and antibodies were isotyped and affinity purified. Western blot confirmed recognition of the rEpIFN-γ. The optimal combination of capture and detection antibodies selected was able to detect rEpIFN-γ in concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native elephant IFN-γ, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a M. tb infection-suspected elephant and a culture-confirmed M. tb-infected elephant. The latter showed measurable production of IFN-γ after stimulation with ESAT6/CFP10 PPDB and PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex (MTBC)-specific antigens in other species. Hence, the IFN-γ assay presented potential as a diagnostic tool for the detection of elephant tuberculosis. Validation of the assay will require its application in large populations of non-infected and infected elephants.

Hematologic and serum chemistry reference intervals for free-ranging lions (Panthera leo).

Hematologic and serum chemistry values are used by veterinarians and wildlife researchers to assess health status and to identify abnormally high or low levels of a particular blood parameter in a target species. For free-ranging lions (Panthera leo) information about these values is scarce. In this study 7 hematologic and 11 serum biochemistry values were evaluated from 485 lions from the Kruger National Park, South Africa. Significant differences between sexes and sub-adult (≤ 36 months) and adult (>36 months) lions were found for most of the blood parameters and separate reference intervals were made for those values. The obtained reference intervals include the means of the various blood parameter values measured in captive lions, except for alkaline phosphatase in the subadult group. These reference intervals can be utilized for free-ranging lions, and may likely also be used as reference intervals for captive lions.

Development of a lion-specific interferon-gamma assay

The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species.

Lion (Panthera leo) and cheetah (Acinonyx jubatus) IFN-γ sequences.

Cloning and sequencing of the full length lion and cheetah interferon-gamma (IFN-gamma) transcript will enable the expression of the recombinant cytokine, to be used for production of monoclonal antibodies and to set up lion and cheetah-specific IFN-gamma ELISAs. These are relevant in blood-based diagnosis of bovine tuberculosis, an important threat to lions in the Kruger National Park. Alignment of nucleotide and amino acid sequences of lion and cheetah and that of domestic cats showed homologies of 97-100%.

Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

Prevalence of Leptospira spp. and Seoul hantavirus in brown rats (Rattus norvegicus) in four regions in the Netherlands, 2011-2015

ABSTRACT Background: Brown rats (Rattus norvegicus) may carry pathogens that can be a risk for public health. Brown rats in the Netherlands were tested for the zoonotic pathogens Leptospira spp. and Seoul hantavirus (SEOV), in order to obtain insight in their prevalence. Methods and results: Cross-sectional studies were performed at four locations from 2011 to 2015. The rats were tested for Leptospira spp. using real-time PCR and/or culture resulting in a prevalence ranging between 33–57%. Testing for SEOV was done through an adapted human Seoul hantavirus ELISA and real-time RT-PCR. Although at several locations the ELISA indicated presence of SEOV antibodies, none could be confirmed by focus reduction neutralization testing. Conclusion: The results indicate a widespread presence of Leptospira spp. in brown rats in the Netherlands, including areas with a low leptospirosis incidence in humans. No evidence for circulation of SEOV was found in this study.

Seroprevalence of hantaviruses and Leptospira in muskrat and coypu trappers in the Netherlands, 2016

ABSTRACT Aims: Seoul orthohantavirus (SEOV) and Leptospira spp. are zoonotic pathogens with rats as main reservoir. Recently, the presence of SEOV in brown rats was reported in one region in the Netherlands. Brown rats are a frequent bycatch in traps placed to catch muskrats (Ondatra zibethicus) and coypus (Myocastor coypus), and thus are a potential health risk for trappers. It was our aim to determine the seroprevalence of orthohantavirus, specifically SEOV, and Leptospira spp in Dutch trappers. Methods and results: Participating trappers provided serum samples and completed an online questionnaire. The serum was tested for the presence of antibodies against six orthohantaviruses and eight Leptospira serovars. Two hundred-sixty trappers completed the online questionnaire (65%), and 246 (61%) and 162 (40%) serum samples were tested for relevant orthohantaviruses and Leptospira spp., respectively. The seroprevalence of Puumala orthohantavirus in Dutch trappers was 0.4% (95% CI: 0.1–2.3%). None of the participants tested positive for SEOV. The seroprevalence of leptospirosis was 1.2% (95% CI: 0.3–4.4%), although Leptospira spp. are present in brown rats in the Netherlands.Significance of study: The results indicate that the infections with orthohantaviruses and leptospires is low for muskrat and coypu trappers.

Autochthonous Human Case of Seoul Virus Infection, the Netherlands

Orthohantaviruses are a group of rodentborne viruses with a worldwide distribution. The orthohantavirus Seoul virus (SEOV) can cause hemorrhagic fever with renal syndrome in humans and is distributed worldwide, like its reservoir host, the rat. Cases of SEOV in wild and pet rats have been described in several countries, and human cases have been reported in the United Kingdom, France, Canada, and the United States. In the Netherlands, SEOV has previously been found in wild brown rats. We describe an autochthonous human case of SEOV infection in the Netherlands. This patient had nonspecific clinical symptoms of an orthohantavirus infection (gastrointestinal symptoms and distinct elevation of liver enzymes). Subsequent source investigation revealed 2 potential sources, the patient’s feeder rats and a feeder rat farm. At both sources, a high prevalence of SEOV was found in the rats. The virus closely resembled the Cherwell and Turckheim SEOV strains that were previously found in Europe.