Onderstepoort Journal of Veterinary Research

**Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa**

Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories) in North West Province, South Africa, were examined by an enzyme-linked immunosorbent assay (ELISA) for antibodies to the Bacillus anthracis toxin protective antigen (PA). As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63 %) wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a whole and the other groups (P < 0.001) and no significant difference between the South African and control groups (P > 0.05). Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheres, six out of ten White-backed Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypius tracheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. It is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and the issue of the role of vultures in transmission of anthrax.

Ixodid ticks on dogs belonging to people in rural communities and villages in Maputo Province, Mozambique

The species composition and geographic distribution of ixodid ticks infesting domestic dogs owned by people in rural communities and villages in Maputo Province was established by collecting ticks from dogs at each of 27 localities spread throughout the province. Ticks were collected from a total of 132 dogs, and nine species belonging to four genera were identified. One dog was infested with six species, three with five and 13 with four species. Haemaphysalis elliptica followed by Rhipicephalus simus were present on dogs at most localities, and their geographic distribution in Maputo Province has been mapped for the first time.

**Bovine intestinal cellular responses following primary and challenge infections with Calicophoron microbothrium metacercariae**

This study was carried out to establish whether cattle can develop resistance to re-infection by Calicophoron microbothrium by assessing the response of intestinal mucosal globule leukocytes, eosinophils, mast cells and basophils, and the establishment of the parasite in the host. A total of 24 1-year-old Tuli steers were randomly divided into four groups of six animals each and infected with C. microbothrium metacercariae. On the first day of the study, animals in Groups I and II were immunized with 5 000 metacercariae and then challenged with 15 000 metacercariae on Day 150 post-immunization. Animals in Group III were immunized with 15 000 metacercariae at the same time that Groups I and II animals were challenged to act as a positive control group. Animals in Group IV were left uninfected and acted as a negative control group. Three animals from each group were slaughtered on Day 28 post-challenge and the remainder were slaughtered on Day 42 post-challenge. The established amphistomes were recovered and histopathological and cytological examinations were done on the jejunum, duodenum, abomasum and the rumen. The establishment rates of the challenge infection in the immunized and challenged groups were lower and ranged from 0 to 0.2 % as compared to 6 %> from naive animals infected as positive controls. Animals immunized and then challenged with C. microbothrium had significantly higher eosinophil, mast cell and globule leukocytes counts in the intestinal mucosa (P < 0.05) as compared to those of the control group. The study indicates that cattle can develop resistance to C. microbothrium re-infection and that eosinophils and mast cells may be important cells in the rejection of the parasite.

In vitro **isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures**

Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10 % foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.

Acute and long-term effects of exposure to sodium monofluoroacetate (1080) in sheep

Acute and long-term effects of a single, relatively high oral dose (0.25 and 0.30 mg/kg) of sodium monofluoroacetate (1080) on the survival and productivity of sheep were evaluated to establish a better understanding of 1080 poisoning and identify more specific changes diagnostic of toxicosis. In survivors, clinical signs of acute 1080 toxicosis such as salivation and lethargy were generally very mild. Fasted animals were more prone to 1080 toxicity. In animals that died, more severe signs, including tachypnoea, dyspnoea, and tremors occurred for 15-20 min prior to death. 1080 concentrations were highest in the blood > heart > skeletal muscle > liver. 1080 could not be detected in any of these organs of the animals that survived. Serum citrate concentrations were elevated for 4 days after dosing. No clinical or biochemical abnormalities were found in any animal after 4 days. Histopathological lesions were most marked in the heart and lung with inflammation, necrosis, and scattered foci of fibrous tissue in the myocardium, pulmonary oedema and inflammation of the lung. No adverse long-term effects on general health or reproductive performance were observed in any sheep that survived the first 4 days following exposure to 1080. The most reliable diagnostic indicators of 1080 exposure in sheep were measurement of its residues in blood, skeletal muscle and ruminal contents, increased serum citrate concentration, elevated heart rate, and characteristic electrocardiograph changes (up to 4 days after exposure). Death from 1080 is most likely to occur within 96 h, and animals that survived this period appeared normal.

**Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa**

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5 %), 20/21 (95.2 %) and 42/42 (100 %) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2 %>), 13/21 (61.9 %>) and 30/42 (71.4 %>) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.

Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006

Pigeon paramyxovirus type 1 (PPMV-1), a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbill (Bucorvus leadbeateri) which became acutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

Prevalence of serotype specific antibody to equine encephalosis virus in Thoroughbred yearlings in South Africa (1999-2004)

Cohorts of yearlings were sampled over a period of 6 years in a retrospective serological survey to establish the annual prevalence of serotype specific antibody to equine encephalosis virus on Thoroughbred stud farms distributed within defined geographical regions of South Africa. Seasonal seroprevalence varied between 3.6 % and 34.7 %, revealing both single and multiple serotype infections in an individual yearling. During the course of this study serotypes 1 and 6 were most frequently and extensively identified while the remaining serotypes 2, 3, 4, 5 and 7 were all identified as sporadic and localized infections affecting only individual horses. This study of the seasonal prevalence of equine encephalosis virus has a corollary and serves as a useful model in the seasonal incidence of the serotypes of African horse sickness and bluetongue in regions where the respective diseases are endemic.

**Studies on effects of lactose on experimental Trypanosoma vivax infection in Zebu cattle. 1. Plasma kinetics of intravenously administered lactose at onset of infection and pathology**

Lactose in normal saline was administered intravenously to a group of Zebu cattle infected with Trypanosoma vivax to determine the blood plasma kinetics at onset of an experimental infection and its ability to protect tissues against damage as part of preliminary studies to determine its suitability for use in the treatment of trypanosomosis. Significantly (P < 0.01) higher lactose concentrations were observed in the T. vivax-infected bulls at 30 min and 1 h (P < 0.05) post-infection (p.i.) and by 4 h p.i. the plasma lactose remained above the level prior to infusion, after which it fell slightly below the pre-infusion level in the uninfected group. Calculated pharmacokinetic parameters revealed delayed excretion of lactose in the T. w'vax-infected group soon after infection. The total body clearance (CI B) was significantly (P < 0.05) reduced. The biological half-life (t ½), elimination rate constant (k e) and apparent volume of distribution (Vd) were relatively decreased (P > 0.05) as a result of the T. vivax infection. Retention of lactose in the plasma was attributed to decreased plasma clearance. It is suggested that the presence of trypanosomes in circulation rather than organic lesions could have been responsible for the delay observed in the excretion of lactose. At 12 weeks p.i., when the experiment was terminated, the group infected and given lactose infusion (despite higher parasitaemia) had no gross or histopathological lesions in the brain, spleen, lymph nodes, heart, kidneys, liver and testes. However, the group infected but not infused with lactose were emaciated, had pale mucosae, watery blood, general muscular atrophy, serous atrophy of coronary fat and other adipose tissue, hepatomegaly, splenomegaly, swollen and oedematous lymph nodes, all of which are suggestive of trypanosomosis. Histopathological lesions included narrowing of Bowman's space and hypercellularity of glomerular tufts in the kidneys with the mean glomerular tuft nuclear indices (GTNs) in the group significantly higher (P< 0.01) than the mean GTNs of the lactose-infused and control bulls. Degenerative changes occurred in the myocardium, spleen, testes and epididymides. The tesicular and epididymal lesions are indicative of male reproductive dysfunction.

**Experimental infections of baboons (Papio spp.) and vervet monkeys (Cercopithecus aethiops) with Trichinella zimbabwensis and successful treatment with ivermectin**

Experimental Trichinella zimbabwensis infections were established in three baboons (Papio sp.) and four vervet monkeys (Cercopithecus aethiops) and the clinical-pathological manifestations assessed. The infected animals showed clinical signs ranging from fever, diarrhoea, periorbital oedema and muscular pain in varying degrees. One baboon became blind due to the infection. Levels of creatinine phosphokinase and lactate dehydrogenase increased to reach a peak on Day 42 post-infection (pi) for both baboons and monkeys. Blood parameters such as packed cell volume, levels of red blood cells and white blood cells did not change significantly from the normal ranges except for the levels of eosinophils which peaked above the normal ranges at Day 28 and 56 pi in baboons and at Day 56 pi in monkeys. Two baboons and two monkeys died during the course of the experiment. They were emaciated and showed lesions such as ascites, hydropericardium, congested liver and enlarged gall bladder. Histopathological findings of various muscles included a basophilic transformation of muscle cells, the disappearance of sarcomere myofibrils and basophilic sarcoplasm with the presence of Trichinella larvae in the sarcoplasm. These changes were mainly in the massetter and were of various intensities in the tail, gastrocnemius and biceps muscles. Five consecutive treatments with an oxfendazole-levamisole combination on surviving animals failed to clear the infection whereas ivermectin cleared the infection after one treatment in two monkeys and after two treatments in a baboon.

Onderstepoort Journal of Veterinary Research

Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

Ixodid ticks on dogs belonging to people in rural communities and villages in Maputo Province, Mozambique

Bovine intestinal cellular responses following primary and challenge infections with Calicophoron microbothrium metacercariae

In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures