Leigh A. Jones

Dissecting the impact of protein versus energy host nutrition on the expression of immunity to gastrointestinal parasites during lactation

Many mammals exhibit a periparturient relaxation of previously established immune responses (PPRI) to gastrointestinal nematodes culminating in increased worm burdens. It has been suggested that the extent of PPRI may have a nutritional basis as it is considerably augmented when protein supply is scarce. Subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. However, this effect is often confounded with increased food intake and thus increased energy levels. Herein, we aimed to dissect the effects of protein and energy nutrition on the immune status and resistance to re-infection with gastrointestinal nematodes in the periparturient host. The lactating, Nippostrongylus brasiliensis re-infected rat was utilised as an established model for mammalian PPRI. Experimental animals were assigned to restricted feeding regimens designed to achieve four pre-determined levels of crude protein (CP) at one of two levels of metabolisable energy (ME) and parasitological and immunological measurements taken at either day 6 or day 9 post re-infection. We clearly show that increased supply of dietary CP, but not increased dietary ME, significantly reduced worm burdens. The increased magnitude of worm expulsion with increased dietary CP supply strongly correlated with mucosal mast cell accumulation in the small intestine. In addition, increased CP and not ME supply increased mucosal eosinophil numbers. Furthermore, increased CP led to higher levels of total IgG at high ME only and there were interactive effects of CP and ME on serum levels of IgG1 and IgG2a. Perhaps surprisingly, CP nutrition did not affect expression of either Th1 (IFN-γ) or Th2 (IL-4, IL-13) cytokines in the mesenteric lymph nodes. These data emphasise the role of immunonutrition, and particularly dietary protein, in combating infectious disease such as gastrointestinal parasitism.

Immunomodulatory effects of dietary protein during Nippostrongylus brasiliensis re-infection in lactating rats.

Periparturient relaxation of immunity (PPRI) to secondary infection with nematodes is believed to have a nutritional basis due to differential partitioning of scarce nutrient resources, particularly protein, to reproductive rather than immune functions. At times of protein scarcity, an increase in protein supply has been reported to assuage this phenomenon. The Nippostrongylus brasiliensis reinfected lactating rat model is now being utilized to investigate the immune reactions underlying the modifying role of dietary protein on PPRI. Herein, we demonstrate that lactating rats reinfected with N. brasiliensis under high protein (HP) dietary conditions exhibit decreased worm burdens and reduced colon egg counts compared to their low protein (LP) counterparts. These reductions correlated with increased mastocytosis and greater goblet cell hyperplasia. Additionally, the local antibody profile revealed that HP reinfected lactating rats developed a stronger antigen specific IgG2b response earlier in infection in comparison with their LP counterparts. Our study provides evidence that increased dietary protein content reduces the PPRI to N. brasiliensis re‐infection in the lactating rat through improved mucosal immune responses.

Genome-wide transcriptomic analysis of intestinal tissue to assess the impact of nutrition and a secondary nematode challenge in lactating rats.

Background Gastrointestinal nematode infection is a major challenge to the health and welfare of mammals. Although mammals eventually acquire immunity to nematodes, this breaks down around parturition, which renders periparturient mammals susceptible to re-infection and an infection source for their offspring. Nutrient supplementation reduces the extent of periparturient parasitism, but the underlying mechanisms remain unclear. Here, we use a genome wide approach to assess the effects of protein supplementation on gene expression in the small intestine of periparturient rats following nematode re-infection. Methodology/Principal Findings The use of a rat whole genome expression microarray (Affymetrix Gene 1.0ST) showed significant differential regulation of 91 genes in the small intestine of lactating rats, re-infected with Nippostrongylus brasiliensis compared to controls; affected functions included immune cell trafficking, cell-mediated responses and antigen presentation. Genes with a previously described role in immune response to nematodes, such as mast cell proteases, and intelectin, and others newly associated with nematode expulsion, such as anterior gradient homolog 2 were identified. Protein supplementation resulted in significant differential regulation of 64 genes; affected functions included protein synthesis, cellular function and maintenance. It increased cell metabolism, evident from the high number of non-coding RNA and the increased synthesis of ribosomal proteins. It regulated immune responses, through T-cell activation and proliferation. The up-regulation of transcription factor forkhead box P1 in unsupplemented, parasitised hosts may be indicative of a delayed immune response in these animals. Conclusions/Significance This study provides the first evidence for nutritional regulation of genes related to immunity to nematodes at the site of parasitism, during expulsion. Additionally it reveals genes induced following secondary parasite challenge in lactating mammals, not previously associated with parasite expulsion. This work is a first step towards defining disease predisposition, identifying markers for nutritional imbalance and developing sustainable measures for parasite control in domestic mammals.

Amelioration of the periparturient relaxation of immunity to parasites through a reduction in mammalian reproductive effort

The degree of periparturient relaxation of immunity to gastrointestinal parasites has a nutritional basis, as overcoming protein scarcity through increased protein supply improves lactational performance, enhances local immune responses and reduces worm burdens. Herein lactating rats, re-infected with Nippostrongylus brasiliensis, are used to test the hypothesis that a similar and rapid improvement of immunity can be achieved through reducing nutrient demand at times of dietary protein scarcity. Reducing litter size from 12 to three pups during lactation resulted, as expected, in cessation of maternal body weight loss and increased pup body weight gain compared with dams which continued to nurse 12 pups. This increase in performance concurred with a rapid decrease in parasitism; within 3 days post nutrient reduction, a 87% reduction in the number of worm eggs found in the colon and 83% reduction in worm burdens was observed, which concurred with increased local immune responses, i.e. 70% more mast cells and 44% more eosinophils in the small intestinal mucosa, to levels similar to those in dams nursing three pups throughout. However, there were no concurrent changes in goblet cell hyperplasia, serum anti-N. brasiliensis-specific antibody levels or mRNA expression of IL-4, IL-10 or IL-13 in the mesenteric lymph nodes. To our knowledge the current study is the first to employ a litter reduction strategy to assess the rate of immune improvement upon overcoming nutrient scarcity in a non-ruminant host. These data support the hypothesis that periparturient relaxation of immunity to gastrointestinal nematodes can be reduced by restoring nutrient adequacy and, importantly, that this improvement can occur very rapidly.

Dietary protein and energy supplies differentially affect resistance to parasites in lactating mammals.

Periparturient relaxation of immunity (PPRI) to parasites in mammals results in higher worm burden and worm egg excretion and may have a nutritional basis. Nippostrongylus brasiliensis re-infected lactating rats fed low-crude protein (CP) diets show an augmented degree of PPRI compared with their high CP-fed counterparts. However, such effects of CP scarcity have been confounded by metabolisable energy (ME) scarcity due to increased intake of the high-CP foods. Here, we independently assessed the effects of dietary CP and ME scarcity on the degree of PPRI. Second, parity rats were infected with N. brasiliensis larvae before mating. Upon parturition, dams were allocated to one of six feeding treatments (1-6), consisting of two levels of dietary ME supply, each with three levels of CP supply. On day 2 of lactation, dams were either re-infected with 1600 N. brasiliensis larvae or sham-infected with PBS, while litter size was standardised at ten pups. Dams and litters were weighed daily until either day 8 or 11 of lactation, when worm burdens were assessed as a proxy for PPRI. Increased CP and ME supply independently improved lactational performance. While ME supply did not affect parasitism, increasing CP supply reduced worm burden and the percentage of female worms in the small intestine; the latter was especially pronounced at the lower level of ME supply. The present results support the view that PPRI to parasites may be sensitive to CP scarcity, but not to moderate ME scarcity.

Leucine and methionine deficiency impairs immunity to gastrointestinal parasites during lactation.

Lactating rats reinfected with Nippostrongylus brasiliensis fed low-crude protein (CP) foods show reduced lactational performance and less resistance to parasites compared with their high-CP counterparts. Here, we hypothesised that feeding high-CP foods deficient in specific essential amino acids (AA) would result in similar penalties. Second-parity lactating rats, immunised with 1600 N. brasiliensis infective larvae before mating, were fed foods with either 250 (high protein; HP) or 150 (low protein; LP) g CP/kg, or were HP deficient in either leucine (HP-Leu) or methionine (HP-Met). On day 1 of lactation, litter size was standardised at twelve pups. On day 2, dams were either reinfected with 1600 N. brasiliensis larvae or sham-infected with PBS. Dams and litters were weighed daily until either day 8 or 11, when worm burdens, and inflammatory cells and systemic levels of N. brasiliensis-specific Ig isotypes were assessed. Data from five out of sixteen HP-Met rats were omitted due to very high levels of food refusals from parturition onwards. Relative to feeding HP foods, feeding LP, HP-Met and HP-Leu foods reduced dam weight gain and, to a lesser extent, litter weight gain, and increased the number of worm eggs in the colon, indicative of a reduction in resistance to parasites. However, only feeding LP and HP-Leu foods resulted in increased worm numbers, while none of the feeding treatments affected systemic Ig, mast and goblet cells, and eosinophil numbers. The present results support the view that resistance to parasites during lactation may be sensitive to specific essential AA scarcity.

Resistance to gastrointestinal nematodes during the periparturient period is sensitive to specific amino acid deficiency

Resistance to gastrointestinal nematodes during the periparturient period is sensitive to specific amino acid deficiency P Sakkas, L A Jones, J G M. Houdijk, D P Knox, I Kyriazakis Animal Health, SAC, Edinburgh, EH9 3JG, United Kingdom, Parasitology Division, Moredun Research Institute, Edinburgh, Penicuik EH26 0PZ, United Kingdom, Veterinary Faculty, University of Thessaly, Karditsa 43100, Greece Email: Panos.Sakkas@sac.ac.uk

Rapid restoration of immunity to parasites in lactating rats by changing nutrient demand

The periparturient relaxation of immunity to parasites in mammals may have a nutritional basis. Indeed, at times of protein scarcity, resistance and immunity to the intestinal parasite Nippostrongylus brasiliensis in lactating rats improves with increased protein supply and reduced litter size and thus reduced nutrient demand. Here, we use the latter observation to assess the rate at which improved host nutritional status can improve periparturient resistance and immunity. Second parity rats were infected with 1600 N. brasiliensis larvae prior to mating (primary infection). Upon parturition (d0), dams were fed ad libitum a low-protein food (100 g CP/kg DM) and were either nursing 12 pups (LS12) or 3 pups (LS3) throughout, or nursed 12 pups until day 5 when their litter was adjusted to 3 pups (LS12-3). Rats were re-infected with 1600 larvae on d2 (secondary infection). Food intake, and dam and litter weight were assessed daily until either d5 (for LS12 only), or d8 and d11 (all treatments) when the number of worms, worm eggs in the colon contents (EIC) and small intestinal mucosal inflammatory cells per villus-crypt unit (vcu) were assessed. These data were log-transformed prior to statistical analysis through ANOVA.

Transcriptomic analysis of host intestinal tissue to assess the consequences of dietary protein and secondary infection on immunity to nematodes

Periparturient breakdown of immunity to nematodes renders animals susceptible to infection and a source of infection for their offspring. Protein supplementation can reduce associated elevated parasite burdens; however, the underlying mechanism is unclear. Here, we used a genome-wide approach to assess the effects of protein supplementation and secondary infection, on gene expression in the small intestine of lactating rats during nematode expulsion. Previously immune, lactating rats (n 6) were either sham infected or re-infected with 1600 Nippostrongylus brasiliensis infective larvae, and were restrictedly fed iso-energetic foods with low (130 g CP/kg DM) or high (270 g CP/kg DM) protein content. At post-mortem intestinal tissue was fixed for RNA extraction. The Affymetrix Rat (Gene 1.0 ST) array was used for gene expression analysis. Data normalisation, clustering and statistical analysis was performed in Genespring GX 11.0; function, network and pathway analysis was performed with Ingenuity Pathway Analysis. Statistical significance was determined by a two-way ANOVA, with dietary protein and secondary infection as factors. Secondary infection significantly regulated the expression of 91 genes; top affected functions included immune cell trafficking and cell-mediated immune responses. Genes with a previously described role in immunity to nematodes in various infection models were identified. Other genes, such as AGR2 and G6PC, were for the first time associated with worm expulsion.